OXFORD IB DIplOm a pROgRam m e 2 0 1 4 ED I TI O N BIOLO GY C O U R S E C O M PA N I O N Andrew Allott David Mindorf 3 Great Clarendon Street, Oxford, OX2 6DP, United Kingdom Oxford University Press is a department of the University of Oxford. It furthers the Universitys objective of excellence in research, scholarship, and education by publishing worldwide. Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Oxford University Press 2014 The moral rights of the authors have been asserted First published in 2014 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, by licence or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above. You must not circulate this work in any other form and you must impose this same condition on any acquirer British Library Cataloguing in Publication Data Data available 978-0-19-839211-8 1 3 5 7 9 10 8 6 4 2 Paper used in the production of this book is a natural, recyclable product made from wood grown in sustainable forests. The manufacturing process conforms to the environmental regulations of the country of origin. Printed in Malaysia Acknowledgements The publishers would like to thank the following for permissions to use their photographs: Cover image: Paul Souders/Corbis; p1: Sulston & Horvitz; p2: DR YORGOS NIKAS/SCIENCE PHOTO LIBRARY; p3a: DR.JEREMY BURGESS/SCIENCE PHOTO LIBRARY; p3b: Shutterstock; p6: Ferran Garcia-Pichel, Max Planck Institute of Marine Biology, Bermen Germany; p7a: Prof. P.Motta & T. Naguro/ SPL; p7b: Andrew Allot; p7c: Andrew Allot; p7d: MICHAEL ABBEY/ SCIENCE PHOTO LIBRARY; p8a: Carolina Biological Supply Co/Visuals Unlimited, Inc.; p8b: ASTRID & HANNS-FRIEDER MICHLER/SCIENCE PHOTO LIBRARY; p9: MICHAEL ABBEY/SCIENCE PHOTO LIBRARY; p10a: DR. PETER SIVER, VISUALS UNLIMITED /SCIENCE PHOTO LIBRARY; p10b: Sulston & Horvitz; p12: JAMES CAVALLINI/SCIENCE PHOTO LIBRARY; p14a: CHRIS BARRY/VISUALS UNLIMITED, INC. /SCIENCE PHOTO LIBRARY; p14b: SIMON FRASER/DEPARTMENT OF HAEMATOLOGY, RVI, NEWCASTLE/SCIENCE PHOTO LIBRARY; p16a: TEK IMAGE/SCIENCE PHOTO LIBRARY; p17: LAWRENCE BERKELEY NATIONAL LABORATORY/ SCIENCE PHOTO LIBRARY; p19: A B Dowsett/SPL; p20a: Eye of Science/SPL; p20b: CNRI/SCIENCE PHOTO LIBRARY; p21a: BIOPHOTO ASSOCIATES/SCIENCE PHOTO LIBRARY; p21b: MICROSCAPE/SCIENCE PHOTO LIBRARY; p22a: BIOPHOTO ASSOCIATES/ SCIENCE PHOTO LIBRARY; p22b: DR GOPAL MURTI/SCIENCE PHOTO LIBRARY; p22c: DR GOPAL MURTI/SCIENCE PHOTO LIBRARY; p22d: MICROSCAPE/SCIENCE PHOTO LIBRARY; p22e: DR KARI LOUNATMAA/ SCIENCE PHOTO LIBRARY; p22f: MICROSCAPE/SCIENCE PHOTO LIBRARY; p23a: DON W. FAWCETT/SCIENCE PHOTO LIBRARY; p23b: DR. GOPAL MURTI/SCIENCE PHOTO LIBRARY; p23c: Andrew Allot; p24a: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p24b: DR.JEREMY BURGESS/ SCIENCE PHOTO LIBRARY; p25a: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p25b: DAVID M. PHILLIPS/SCIENCE PHOTO LIBRARY; p25c: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p27: Author Image; p28: NIBSC/ SCIENCE PHOTO LIBRARY; p29: Author Image; p32: Janaka Dharmasena/ Shutterstock; p43a: OUP; p43b: Andrew Allot; p44: Herve Conge/SPL; p45: David Mayer, Consultant and CSL Liver Surgery, Queen Elizabeth Hospital, Birmingham; p46a: THOMAS DEERINCK, NCMIR/SCIENCE PHOTO LIBRARY; p46b: The VRoma Project (www.vroma.org); p48: GEORGETTE DOUWMA/ SCIENCE PHOTO LIBRARY; p49: DAVID MCCARTHY/SCIENCE PHOTO LIBRARY; p51: M.I. Walker/SPL; p53a,b,c,d: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p54a,b: STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY; p55a: Dharam M Ramnani; p55b: MANFRED KAGE/SCIENCE PHOTO LIBRARY; p55c: MANFRED KAGE/SCIENCE PHOTO LIBRARY; p57: MOREDUN ANIMAL HEALTH LTD/SCIENCE PHOTO LIBRARY; p58: OUP; p54: Andrew Allot; p60: J Herve Conge, ISM/ SPL; p61: OUP; p62: Vasiliy Koval/Shutterstock; p66: LAGUNA DESIGN/SCIENCE PHOTO LIBRARY; p69a-p69b: OUP; p70: CLAIRE PAXTON & JACQUI FARROW/SCIENCE PHOTO LIBRARY; p71: DR KEITH WHEELER/SCIENCE PHOTO LIBRARY; p72: OUP; p73a: Dr. Elena Kiseleva/SPL; p73b: Dr. Gopal Murti/SPL; p73c: Dr. Elena Kiseleva/SPL; p75a: LAGUNA DESIGN/SCIENCE PHOTO LIBRARY; p75b: LAGUNA DESIGN/ SCIENCE PHOTO LIBRARY; p75c: LAGUNA DESIGN/SCIENCE PHOTO LIBRARY; p79: OUP; p80a: Andrew Allot; p80b-81: OUP; p83a: OUP; p83b: Giles Bell; p90a: OUP; p90b: www.rcsb.org; p91: www.rcsb.org; p92a: Yikrazuul/Wikipedia; p92b: OUP; p95: JAMES KING-HOLMES/SCIENCE PHOTO LIBRARY; p101-102: OUP; p110: SPL; p116: Author Image; p122: Tony Rusecki / Alamy; p123a: OUP; p123b: Glenn Tattersall; p124a: MATTHEW OLDFIELD/SCIENCE PHOTO LIBRARY; p124b: Author Image; p152: OUP; p126a: OUP; p126b: Petrov Andrey/Shutterstock; p130a: OUP; p130b: OUP; p130c: Andrew Allott; p131c: Andrew Allott; p132a: OUP; p133: William Allott; p134: OUP; p141: OUP; p143a: Jax.org; p143b: Jax.org; p143c: Jax.org; p144: www.ncbi.nlm.nih.gov/pubmed; p146a: Eye of Science/SPL; p146b: Eye of Science/SPL; p148: MAURO FERMARIELLO/SCIENCE PHOTO LIBRARY; p150a: M .Wurtz/Biozentrum/University o fBasel/SPL; p150b: Kwangshin Kim/SPL; p151: www.ncbi.nlm.nih.gov; p152: Dr. Oscar Lee Miller, Jr of the University of Virginia; p155a: OUP; p155b: Andrew Allot; p156: OUP; p158a: DEPT. OF CLINICAL CYTOGENETICS, ADDENBROOKES HOSPITAL/SCIENCE PHOTO LIBRARY; p158b: Tomasz Markowski/ Dreamstime; p159: L. WILLATT, EAST ANGLIAN REGIONAL GENETICS SERVICE/SCIENCE PHOTO LIBRARY; p160-161b: OUP; p162a: Andrew Allot; p164a,b,c,d: Andrew Allot; p165a,b,c,d: Andrew Allot; p166a: OUP; p166b: OUP; p166c: OUP; p169: OUP; p171a: OUP; p171b: OUP; p172: William Allott; p176: Enrico Coen; p177-184a: OUP; p184b: OUP; p186: RIA NOVOSTI/ SCIENCE PHOTO LIBRARY; p188: VOLKER STEGER/SCIENCE PHOTO LIBRARY; p189: OUP; p190a: Andrew Allot; p190b: DAVID PARKER/SCIENCE PHOTO LIBRARY; p190c-196c: OUP; p197: WALLY EBERHART, VISUALS UNLIMITED /SCIENCE PHOTO LIBRARY; p198a: GERARD PEAUCELLIER, ISM /SCIENCE PHOTO LIBRARY; p198b: GERARD PEAUCELLIER, ISM /SCIENCE PHOTO LIBRARY; p198c: Author Image; p199: PHILIPPE PLAILLY/SCIENCE PHOTO LIBRARY; p201: OUP; p202: Parinya Hirunthitima/Shutterstock; p203a: OUP; p203b: OUP; p203c: ERIC GRAVE/SCIENCE PHOTO LIBRARY; p203d: OUP; p204a,b,c,d: Andrew Allot; p205a: Author Image; p205b: CreativeNature.nl/Shutterstock; p205c: Author Image; p206: OUP; p207: OUP; p207b: Author Image; p209: Author Image; p210: OUP; p211: OUP; p212a: OUP; p212b: Andrew Allott; p214: Andrew Allott; p215a: OUP; p215b: Andrew Allott; p215c: Andrew Allott; p215d: Rich Lindie/Shutterstock; p215e: OUP; p217a: OUP; p217b: Andrew Allott; p217d: OUP; p221: Giorgiogp2/Wikipedia; p223a: Andrew Allott; p223b: Andrew Allott; p224: OUP; p225a: OUP; p225b: Andrew Allott; p225c: Andrew Allott; p228-242b: OUP; p243: Erik Lam/Shutterstock; p244: Sinclair Stammers/SPL; p246a: Wikipedia; p246b: Daiju AZUMA; p246c: Wikipedia; p246d: Shutterstock; p248a: Andrew Allott; p248b Andrew Allott; p250a: OUP; p250b: OUP; p251a: OUP; p251b: OUP; p251c: OUP; p251d: OUP; p251e: PETER CHADWICK/SCIENCE PHOTO LIBRARY; p253: OUP; p259: Author Image; p261: OUP; p262a: OUP; p262b: OUP; p264: Andrew Allot; p265: Kipling Brock/Shutterstock; p270a: Author Image; p270b: Author Image; p272: OUP; p276a: OUP; p276b: BOB GIBBONS/SCIENCE PHOTO LIBRARY; p279: BSIP VEM/SCIENCE PHOTO LIBRARY; p281: Dennis Kunkel/Photolibrary; p282: Author Image; p283a: Andrew Allot; p283b: OUP; p286: Author Image; p290: Public Domain/Wikipedia; p292a: OUP; p292b: OUP; p294a: OUP; p294b: BIOPHOTO ASSOCIATES/SCIENCE PHOTO LIBRARY; p298: Andrew Allot; p299: OUP; p302: OUP; p303a: OUP; p303b: Andrew Allot; p304a: OUP; p304b: OUP; p305: JAMES CAVALLINI/SCIENCE PHOTO LIBRARY; p306: ST MARYS HOSPITAL MEDICAL SCHOOL/SCIENCE PHOTO LIBRARY; p307: OUP; p308: Wikipedia; p309: OUP; p315: OUP; p317: DU CANE MEDICAL IMAGING LTD/SCIENCE PHOTO LIBRARY; p318: OUP; p320a: OUP; p320b: THOMAS DEERINCK, NCMIR/SCIENCE PHOTO LIBRARY; p323: OUP; p325: BSIP VEM/SCIENCE PHOTO LIBRARY; p327: OUP; p328a: SCIENCE VU, VISUALS UNLIMITED /SCIENCE PHOTO LIBRARY; p328b: OUP; p330: J. ZBAEREN/EURELIOS/SCIENCE PHOTO LIBRARY; p331: OUP; p332: OAK RIDGE NATIONAL LABORATORY/US DEPARTMENT OF ENERGY/SCIENCE PHOTO LIBRARY; p333: OUP; p334: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p339: CHASSENET/BSIP/SCIENCE PHOTO LIBRARY; p340: Author Image; p343: SIMON FRASER/SCIENCE PHOTO LIBRARY; p344: LEE D. SIMON/SCIENCE PHOTO LIBRARY; p346: SPL; p348: Image of PDB ID 1aoi (K. Luger, A.W. Mader, R.K. Richmond, D.F. Sargent, T.J. Richmond (1997) structure of the core particle at 2.8 A resolution Nature 389: 251-260) created with Chimera (UCSF Chimera--a visualization system for exploratory research and analysis. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. J Comput Chem. 2004 Oct;25(13):160512. ); p349: Public Domain/Wikipedia; p351: SCIENCE PHOTO LIBRARY; p352: Andrew Allot; p353: Charvosi/Wikipedia; p357: Axel Bueckert/ Shutterstock; p358: PNAS.Org; p359: DR ELENA KISELEVA/SCIENCE PHOTO LIBRARY; p363a: Jmol; p363b: RCSB.org; p367: 1970 American Association for the Advancement of Science. Miller, O. L. et al. Visualization of bacterial genes in action. Science 169,392395 (1970). All rights reserved; p368a: Nobelprize.org; p368b: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p368c: SINCLAIR STAMMERS/SCIENCE PHOTO LIBRARY; p370a: Andrew Allot; p373: Shutterstock; p375: RAMON ANDRADE 3DCIENCIA/SCIENCE PHOTO LIBRARY; p387a: CNRI/SCIENCE PHOTO LIBRARY; p387b: Petrov Andrey/Shutterstock; p387c: Prof. Kenneth R Miller/ SPL; p387d: Andrew Allot; p387e: Andrew Allot; p388: Dr. Carmen Manella, Wadsworth Center,New York State Department of Health; p390: Prof. Kenneth R Miller/ SPL; p392: Andrew Allot; p398: Andrew Allot; p399: Barrie Juniper; p403: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p404: SINCLAIR STAMMERS/ SCIENCE PHOTO LIBRARY; p405a: Smugmug.Com; p405b: SCIENCE PHOTO LIBRARY; p406a: POWER AND SYRED/SCIENCE PHOTO LIBRARY; p406b: DR KEITH WHEELER/SCIENCE PHOTO LIBRARY; p410: SIDNEY MOULDS/ SCIENCE PHOTO LIBRARY; p411: DR KEITH WHEELER/SCIENCE PHOTO Continued on back page. Contents 1 Cell Biology Introduction to cells Ultrastructure o cells Membrane structure Membrane transport The origin o cells C ell division 7 Nucleic acids (AHL) 1 16 25 33 45 51 2 Molecular Biology Molecules to metabolism 61 Water 68 C arbohydrates and lipids 73 Proteins 87 E nzymes 96 S tructure o D NA and RNA 1 05 D NA replication, transcription and translation 111 C ell respiration 1 22 Photosynthesis 1 29 3 Genetics Genes C hromosomes Meiosis Inheritance Genetic modication and biotechnology 1 41 1 49 1 59 1 68 1 87 4 Ecology S pecies, communities and ecosystems E nergy fow C arbon cycling C limate change 2 01 21 3 220 229 2 41 2 49 258 2 63 6 Human physiology D igestion and absorption The blood system D eence against inectious diseases Gas exchange Neurones and synapses Hormones, homeostasis and reproduction 3 43 355 3 62 8 Metabolism, cell respiration and photosynthesis (AHL) Metabolism C ell respiration Photosynthesis 3 73 3 80 3 89 9 Plant biology (AHL) Transport in the xylem o plants Transport in the phloem o plants Growth in plants Reproduction in plants 403 41 2 42 2 42 9 10 Genetics and evolution (AHL) Meiosis Inheritance Gene pool and speciation 2 79 2 89 3 02 31 0 31 9 329 Antibody production and vaccination Movement The kidney and osmoregulation S exual reproduction 5 75 5 82 5 91 C Ecology and conservation Species and communities C ommunities and ecosystems Impacts o humans on ecosystems C onservation o biodiversity Population ecology The nitrogen and phosphorous cycles 603 61 3 62 5 63 5 642 649 D Human physiology 43 9 445 45 5 11 Animal physiology (AHL) 5 Evolution and biodiversity E vidence or evolution Natural selection C lassication and biodiversity C ladistics D NA structure and replication Transcription and gene expression Translation Environmental protection Medicine B ioormatics Human nutrition D igestion Functions o the liver The heart Hormones and metabolism Transport o respiratory gases 65 9 671 678 684 694 699 Internal Assessment (with thanks to Mark Headlee for his assistance with this chapter) 708 Index 71 3 465 476 485 499 A Neurobiology and behaviour Neural development The human brain Perception o stimuli Innate and learned behaviour Neuropharmacology Ethology 513 518 526 533 5 41 5 48 B Biotechnology and bioinformatics Microbiology: organisms in industry 557 B iotechnology in agriculture 5 65 iii Course book defnition The IB Learner Profle The IB D iploma Programme course books are resource materials designed to support students throughout their two- year D iploma Programme course o study in a particular subj ect. They will help students gain an understanding o what is expected rom the study o an IB D iploma Programme subj ect while presenting content in a way that illustrates the purpose and aims o the IB . They refect the philosophy and approach o the IB and encourage a deep understanding o each subj ect by making connections to wider issues and providing opportunities or critical thinking. The aim o all IB programmes to develop internationally minded people who work to create a better and more peaceul world. The aim o the programme is to develop this person through ten learner attributes, as described below. The books mirror the IB philosophy o viewing the curriculum in terms o a whole- course approach; the use o a wide range o resources, international mindedness, the IB learner prole and the IB D iploma Programme core requirements, theory o knowledge, the extended essay, and creativity, action, service ( C AS ) . E ach book can be used in conj unction with other materials and indeed, students o the IB are required and encouraged to draw conclusions rom a variety o resources. Suggestions or additional and urther reading are given in each book and suggestions or how to extend research are provided. In addition, the course companions provide advice and guidance on the specic course assessment requirements and on academic honesty protocol. They are distinctive and authoritative without being prescriptive. IB mission statement The International B accalaureate aims to develop inquiring, knowledgeable and caring young people who help to create a better and more peaceul world through intercultural understanding and respect. To this end the organization works with schools, governments and international organizations to develop challenging programmes o international education and rigorous assessment. These programmes encourage students across the world to become active, compassionate and lielong learners who understand that other people, with their dierences, can also be right. iv Inquirers: They develop their natural curiosity. They acquire the skills necessary to conduct inquiry and research and snow independence in learning. They actively enj oy learning and this love o learning will be sustained throughout their lives. Knowledgeable: They explore concepts, ideas, and issues that have local and global signicance. In so doing, they acquire in-depth knowledge and develop understanding across a broad and balanced range o disciplines. Thinkers: They exercise initiative in applying thinking skills critically and creatively to recognize and approach complex problems, and make reasoned, ethical decisions. C ommunicators: They understand and express ideas and inormation condently and creatively in more than one language and in a variety o modes o communication. They work eectively and willingly in collaboration with others. Princip led: They act with integrity and honesty, with a strong sense o airness, j ustice and respect or the dignity o the individual, groups and communities. They take responsibility or their own action and the consequences that accompany them. O p en-minded: They understand and appreciate their own cultures and personal histories, and are open to the perspectives, values and traditions o other individuals and communities. They are accustomed to seeking and evaluating a range o points o view, and are willing to grow rom the experience. C aring: They show empathy, compassion and respect towards the needs and eelings o others. They have a personal commitment to service, and to act to make a positive dierence to the lives o others and to the environment. Risk-takers: They approach unamiliar situations and uncertainty with courage and orethought, and have the independence o spirit to explore new roles, ideas, and strategies. They are brave and articulate in deending their belies. B alanced: They understand the importance o intellectual, physical and emotional ballance to achieve personal well- being or themselves and others. Refective: They give thoughtul consideration to their own learning and experience. They are able to assess and understand their strengths and limitations in order to support their learning and personal development. What constitutes malpractice? Malpractice is behaviour that results in, or may result in, you or any student gaining an unair advantage in one or more assessment component. Malpractice includes plagiarism and collusion. Plagiarism is defned as the representation o the ideas or work o another person as your own. The ollowing are some o the ways to avoid plagiarism: words and ideas o another person to support ones arguments must be acknowledged passages that are quoted verbatim must be enclosed within quotation marks and acknowledged C D -Roms, email messages, web sites on the Internet and any other electronic media must be treated in the same way as books and j ournals the sources o all photographs, maps, illustrations, computer programs, data, graphs, audio- visual and similar material must be acknowledged i they are not your own work works o art, whether music, flm dance, theatre arts or visual arts and where the creative use o a part o a work takes place, the original artist must be acknowledged. A note on academic honesty It is o vital importance to acknowledge and appropriately credit the owners o inormation when that inormation is used in your work. Ater all, owners o ideas ( intellectual property) have property rights. To have an authentic piece o work, it must be based on your individual and original ideas with the work o others ully acknowledged. Thereore, all assignments, written or oral, completed or assessment must use your own language and expression. Where sources are used or reerred to, whether in the orm o direct quotation or paraphrase, such sources must be appropriately acknowledged. How do I acknowledge the work of others? The way that you acknowledge that you have used the ideas o other people is through the use o ootnotes and bibliographies. Footnotes ( placed at the bottom o a page) or endnotes ( placed at the end o a document) are to be provided when you quote or paraphrase rom another document, or closely summarize the inormation provided in another document. You do not need to provide a ootnote or inormation that is part o a body o knowledge. That is, defnitions do not need to be ootnoted as they are part o the assumed knowledge. B ibliograp hies should include a ormal list o the resources that you used in your work. Formal means that you should use one o the several accepted orms o presentation. This usually involves separating the resources that you use into dierent categories ( e.g. books, magazines, newspaper articles, internet-based resources, C ds and works o art) and providing ull inormation as to how a reader or viewer o your work can fnd the same inormation. A bibliography is compulsory in the E xtended Essay. C ollusion is defned as supporting malpractice by another student. This includes: allowing your work to be copied or submitted or assessment by another student duplicating work or dierent assessment components and/or diploma requirements. O ther orms o malp ractice include any action that gives you an unair advantage or aects the results o another student. Examples include, taking unauthorized material into an examination room, misconduct during an examination and alsiying a C AS record. v Using your IB Biology Online Resources What is Kerboodle? Kerboodle is an online learning platorm. I your school has a subscription to IB B iology Kerboodle O nline Resources you will be able to access a huge bank o resources, assessments, and presentations to guide you through this course. What is in your Kerboodle Online Resources? There are three main areas or students on the IB B iology Kerboodle: planning, resources, and assessment. Resources There a hundreds o extra resources available on the IB B iology Kerboodle O nline. You can use these at home or in the classroom to develop your skills and knowledge as you progress through the course. Watch videos and animations o experiments, difcult concepts, and science in action. Hundreds o worksheets read articles, perorm experiments and simulations, practice your skills, or use your knowledge to answer questions. Look at galleries o images rom the book and see their details close up. Find out more by looking at recommended sites on the Internet, answer questions, or do more research. Planning B e prepared or the practical work and your internal assessment with extra resources on the IB B iology Kerboodle online. Learn about the dierent skills that you need to perorm an investigation. Plan and prepare experiments o your own. Learn how to analyse data and draw conclusions successully and accurately. One of hundreds of worksheets. vi Practical skills presentation. Assessment C lick on the assessment tab to check your knowledge or revise or your examinations. Here you will fnd lots o interactive quizzes and examstyle practice questions. Formative tests: use these to check your comprehension, theres one auto-marked quiz or every sub-topic. E valuate how confdent you eel about a sub-topic, then complete the test. You will have two attempts at each question and get eedback ater every question. The marks are automatically reported in the markbook, so you can see how you progress throughout the year. Summative tests: use these to practice or your exams or as revision, theres one auto- marked quiz or every topic. Work through the test as i it were an examination go back and change any questions you arent sure about until you are happy, then submit the test or a fnal mark. The marks are automatically reported in the markbook, so you can see where you may need more practice. Assessment practice: use these to practice answering the longer written questions you will come across when you are examined. These worksheets can be printed out and perormed as a timed test. Don't forget! You can also fnd extra resources on our ree website www.oxfordsecondary.co.uk/ib-biology Here you can fnd all o the answers and even more practice questions. vii Introduction This book is a companion or students o B iology in the International B accalaureate D iploma Programme. B iology is the most popular choice o science subj ect as part o the IB diploma. The study o biology should lead students to appreciate the interconnectedness o lie within the biosphere. With a ocus on understanding the nature o science, IB B iology will allow you to develop a level o scientifc literacy that will better prepare you to act on issues o local and global concern, with a ull understanding o the scientifc point o view. The structure o this book is closely based on the biology programme in the S ubj ect Guide. S ubheadings restate the specifc assessment statements. Topics 1 6 explain in detail the C ore material that is common to both S L and HL courses. Topics 7 1 1 explain the AHL ( additional higher level material) . Topics A, B , C and D cover the content o the options. All topics include the ollowing elements: Understanding The specifcs o the content requirements or each sub- topic are covered in detail. C oncepts are presented in ways that will promote enduring understanding. Applications These sections help you to develop your understanding by studying a specifc illustrative example or learning about a signifcant experiment in the history o biology. Skills topics These sections encourage you to apply your understanding through practical activities and analysis o results rom classic biological research. In some cases this involves instructions or handling data rom experiments and also use o IC T. Some o the skills sections involve experiments with known outcomes, aimed at promoting understanding through doing and seeing. O thers involve ideas or experimental work with unknown outcomes, where you can defne the problem and the methods. These are a valuable opportunities to build the skills that are assessed in IA ( see page 708) . viii Nature of science Here you can explore the methods o science and some o the knowledge issues that are associated with scientifc endeavour. This is done using careully selected examples, including biological research that led to paradigm shits in our understanding o the natural world. Theory of Knowledge These short sections have headings that are equivocal ` knowledge questions. The text that follows often details one possible answer to the knowledge question. We encourage you draw on these examples of knowledge issues in your TOK essays. Of course, much of the material elsewhere in the book, particularly in the nature of science sections, can be used to prompt TOK discussions. activity A variety of short topics are included under this heading with the focus in all cases on active learning. We encourage you research these topics yourself, using information available in textbooks or on the Internet. The aim is to promote an independent approach to learning. We believe that the optimal approach to learning is to be active the more that you do for yourself, guided by your teacher, the better you will learn. Data-based questions These questions involve studying and analysing data from biological research this type of question appears in both Paper 2 and Paper 3 for SL and HL IB Biology. Answers to these questions can be found at www.oxfordsecondary.co.uk/ib-biology End -of-Topic Questions At the end o each topic you will fnd a range o questions, including both past IB B iology exam questions and new questions. Answers can be ound at www.oxordsecondary. co.uk/ib- biology 1 CE LL B I O LO GY Introduction There is an unbroken chain o lie rom the rst cells on Earth to all cells ound in organisms alive today. Eukaryotes have a much more complex cell structure than prokaryotes. The evolution o multicellular organisms allowed cell specialization and cell replacement. C ell division is essential but is carried out dierently in prokaryotes and eukaryotes. While evolution has resulted in a biological world o enormous diversity, the study o cells shows us that there are also universal eatures. For example, the fuid and dynamic structure o biological membranes allows them to control the composition o cells. 1.1 Introduction to cells Understanding According to the cell theory, living organisms are composed o cells. Organisms consisting o only one cell carry out all unctions o lie in that cell. Surace area to volume ratio is important in the limitation o cell size. Multicellular organisms have properties that emerge rom the interaction o their cellular components. Specialized tissues can develop by cell dierentiation in multicellular organisms. Dierentiation involves the expression o some genes and not others in a cells genome. The capacity o stem cells to divide and dierentiate along dierent pathways is necessary in embryonic development. It also makes stem cells suitable or therapeutic uses. Nature of science Applications Questioning the cell theory using atypical examples, including striated muscle, giant algae and aseptate ungal hyphae. Investigation o unctions o lie in Paramecium and one named photosynthetic unicellular organism. Use o stem cells to treat Stargardts disease and one other named condition. Ethics o the therapeutic use o stem cells rom specially created embryos, rom the umbilical cord blood o a new-born baby and rom an adults own tissues. Skills Looking or trends and discrepancies: although Use o a light microscope to investigate the most organisms conorm to cell theory, there are exceptions. Ethical implications o research: research involving stem cells is growing in importance and raises ethical issues. structure o cells and tissues. Drawing cell structures as seen with the light microscope. Calculation o the magnifcation o drawings and the actual size o structures shown in drawings or micrographs. 1 1 C E LL B I O LO G Y The cell theory Living organisms are composed of cells. The internal structure of living organisms is very intricate and is built up from very small individual parts. O rgans such as the kidney and the eye are easily visible. If they are dissected we can see that large organs are made of a number of different tissues, but until microscopes were invented little or nothing was discovered about the structure of tissues. From the 1 7th century onwards biologists examined tissues from both plants and animals using microscopes. Although there was much variation, certain features were seen again and again. A theory was developed to explain the basic features of structure the cell theory. This states that cells are the fundamental building blocks of all living organisms. The smallest organisms are unicellular they consist of j ust one cell. Larger organisms are multicellular they are composed of many cells. C ells vary considerably in size and shape but they share certain common features: Every living cell is surrounded by a membrane, which separates the cell contents from everything else outside. C ells contain genetic material which stores all of the instructions needed for the cells activities. Many of these activities are chemical reactions, catalysed by enzymes produced inside the cell. C ells have their own energy release system that powers all of the cells activities. S o, cells can be thought of as the smallest living structures nothing smaller can survive. Figure 1 Coloured scanning electron micrograph (SEM) 2 of a human embryo on the tip of a pin 1 .1 I n tro d u ctI o n to ce lls Exceptions to the cell theory Looking for trends and discrepancies: although most organisms conform to cell theory, there are exceptions. An early stage in scientifc investigation is to look or trends things that appear to be ound generally rather than j ust in specifc cases. These trends can lead to the development o a theory. A scientifc theory is a way o interpreting the natural world. Theories allow us to make predictions. S ometimes exceptions to a general trend are ound. These are called discrepancies. S cientists have to j udge whether the discrepancies are common or serious enough to make predictions too unreliable to be useul. The theory is then discarded. The cell theory is an example o where scientists have looked or trends and discrepancies. Robert Hooke was the frst to use the word cell or structures in living organisms. He did this in 1 665 ater examining cork and other parts o plants. Ater describing cells in cork he wrote this: Figure 2 Robert Hookes drawing of cork cells Aiviy Nor is this kind of texture peculiar to cork only, for upon examination with my microscope I have found that the pith of the Elder or almost any other tree, the inner pith of the Cany hollow stems of several other vegetables: as of Fennel, Carrets, Daucus, Bur-docks, Teasels, Fearn, some kind of Reeds etc. have much such a kind of Schematisme, as I have lately shown that of cork. S o Hooke wasnt content with looking at j ust one type o plant tissue he looked at many and discovered a general trend. S ince Hookes day biologists have looked at tissues rom a huge variety o living organisms. Many o these tissues have been ound to consist o cells, so the cell theory has not been discarded. However, some discrepancies have been discovered organisms or parts o organisms that do not consist o typical cells. More discrepancies may be discovered, but it is extremely unlikely that the cell theory will ever be discarded, because so many tissues do consist o cells. Figure 3 What is the unit of life: the boy or his cells? These two answers represent the holistic and the reductionist approach in biology. image viewed here Using light microscopes eyepiece lens Use of a light microscope to investigate the structure of cells and tissues. Try to improve your skill at using microscopes as much as you can. Learn the names o parts o the microscope. Understand how to ocus the microscope to get the best possible image. Look ater your microscope so it stays in perect working order. Know how to troubleshoot problems. turret coarse-focusing knob ne-focusing knob objective lens specimen stage light from mirror or light bulb Figure 4 Compound light microscope 3 1 C E LL B I O LO G Y Focusing Put the slide on the stage, with the most promising region exactly in the middle o the hole in the stage that the light comes through. Always ocus at low power rst even i eventually you need high power magnication. Focus with the larger coarse- ocusing knobs rst, then when you have nearly got the image in ocus make it really sharp using the smaller ne- ocusing knobs. I you want to increase the magnication, move the slide so the most promising region is exactly in the middle o the eld o view and then change to a higher magnication lens. Looking after your microscope Always ocus by moving the lens and the specimen urther apart, never closer to each other. Make sure that the slide is clean and dry beore putting it on the stage. Never touch the suraces o the lenses with your ngers or anything else. C arry the microscope careully with a hand under it to support its weight securely. Troubleshooting Problem: Nothing is visible when I try to ocus. Solution: Make sure the specimen is actually under the lens, by careully positioning the slide. It is easier to nd the specimen i you ocus at low power rst. Problem: A circle with a thick black rim is visible. Solution: There is an air bubble on the slide. Ignore it and try to improve your technique or making slides so that there are no air bubbles. Problem: There are blurred parts o the image Types of slide The slides that we examine with a microscope can be permanent or temporary. Making permanent slides is very skilled and takes a long time, so these slides are normally made by experts. Permanent slides o tissues are made using very thin slices o tissue. Making temporary slides is quicker and easier so we can do this or ourselves. Examining and drawing plant and animal cells Almost all cells are too small to be seen with the naked eye, so a microscope is needed to study them. It is usually easy to see whether a cell is rom a plant or an animal, even though there are many dierent cell types in both the plant and animal kingdoms. Place the cells on the slide in a layer not more than one cell thick. Add a drop o water or stain. C areully lower a cover slip onto the drop. Try to avoid trapping any air bubbles. Remove excess fuid or stain by putting the slide inside a olded piece o paper towel and pressing lightly on the cover slip. It is best to examine the slide rst using low power. Move the slide to get the most promising areas in the middle o the eld o view and then move up to high power. D raw a ew cells, so you remember their structure. cover slip cells carefully lower the cover slip stain or water even when I ocus it as well as I can. gently squeeze to remove exces uid Solution: Either the lenses or the slide have dirt on them. Ask your teacher to clean it. Problem: The image is very dark. Solution: Increase the amount o light passing through the specimen by adj usting the diaphragm. Problem: The image looks rather bleached. Solution: D ecrease the amount o light passing through the specimen by adj usting the diaphragm. 4 cover slip folded a er towel slide Figure 5 Making a temporary mount 1 .1 I n tro d u ctI o n to ce lls 1 Moss leaf 2 B anana fruit cell 10 m 3 Mammalian liver cell 5 m 20 m Use a moss plant with very thin leaves. Mount a single lea in a drop o water or methylene blue stain. Scrape a small amount o the sot tissue rom a banana and place on a slide. Mount in a drop o iodine solution. S crape cells rom a reshly cut surace o liver ( not previously rozen) . S mear onto a slide and add methylene blue to stain. 4 Leaf lower epidermis 5 Human cheek cell 6 White blood cell 20 m 2 m 10 m Peel the lower epidermis o a lea. The cell drawn here was rom Valeriana. Mount in water or in methylene blue. Figure 6 Plant and animal cell drawings A thin layer o mammalian blood can be smeared over a slide and stained with Leishmans stain. S crape cells rom the inside o your cheek with a cotton bud. S mear them on a slide and add methylene blue to stain. Drawing cells Drawing cell structures as seen with the light microscope. C areul drawings are a useul way o recording the structure o cells or other biological structures. Usually the lines on the drawing represent the edges o structures. D o not show unnecessary detail and only use aint shading. D rawings o structures seen using a microscope will be larger than the structures actually are the drawing shows them magnifed. O n page 6 the method or calculating the magnifcation o a drawing is explained. E verything on a drawing should be shown to the same magnifcation. a) Use a sharp pencil with a hard lead to draw single sharp lines. b) Join up lines careully to orm continuous structures such as cells c) D raw lines reehand, but use a ruler or labelling lines. cell bad good bad good bad cell good Figure 7 Examples of drawing styles 5 1 C E LL B I O LO G Y Calculation o magnifcation and actual size Calculation o the magnifcation o drawings and the actual size o structures shown in drawings or micrographs. When we look down a microscope the structures that we see appear larger than they actually are. The microscope is magniying them. Most microscopes allow us to magniy specimens by two or three dierent actors. This is done by rotating the turret to switch rom one obj ective lens to another. A typical school microscope has three levels o magnifcation: 40 ( low power) 1 00 ( medium power) 400 ( high power) I we take a photo down a microscope, we can magniy the image even more. A photo taken down a microscope is called a micrograph. There are many micrographs in this book, including electron micrographs taken using an electron microscope. It is very important when using this ormula to make sure that the units or the size o the image and actual size o the specimen are the same. They could both be millimetres ( mm) or micrometres ( m) but they must not be dierent or the calculation will be wrong. Millimetres can be converted to micrometres by multiplying by one thousand. Micrometres can be converted to millimetres by dividing by one thousand. S cale bars are sometimes put on micrographs or drawings, or j ust alongside them. These are straight lines, with the actual size that the scale bar represents. For example, i there was a 1 0 mm long scale bar on a micrograph with a magnifcation o 1 0, 000 the scale bar would have a label o 1 m. EXAMPLE: When we draw a specimen, we can make the drawing larger or smaller, so the magnifcation o the drawing isnt necessarily the same as the magnifcation o the microscope. The length o an image is 3 0 mm. It represents a structure that has an actual size o 3 m. D etermine the magnifcation o the image. To fnd the magnifcation o a micrograph or a drawing we need to know two things: the size o the image ( in the drawing or the micrograph) and the actual size o the specimen. This ormula is used or the calculation: 3 0 mm = 3 0 1 0 - 3 m 3 m = 3 1 0 - 6 m Either: size o image magnifcation = ___ actual size o specimen 30 1 0 - 3 Magnifcation = _ 3 1 0-6 = 1 0, 000 Or: 3 0 mm = 3 0, 000 m I we know the size o the image and the magnifcation, we can calculate the actual size o a specimen. 3 0, 000 Magnifcation = _ 3 = 1 0, 000 Data-based questions 1 a) D etermine the magnifcation o the string o Thiomargarita cells in fgure 8, i the scale bar represents 0.2 mm [3 ] b) Determine the width o the string o cells. [2] Figure 8 6 Thiomargarita 1 .1 I n tro d u ctI o n to ce lls 2 b) D etermine the length o the cheek cell. In fgure 9 the actual length o the mitochondrion is 8 m. a) D etermine the magnifcation o this electron micrograph. [2 ] [2 ] b) C alculate how long a 5 m scale bar would be on this electron micrograph. [2 ] c) Determine the width o the mitochondrion. [1 ] Figure 10 4 a) Human cheek cell Using the width o the hens egg as a guide, estimate the actual length o the ostrich egg ( fgure 1 1 ) . [2 ] b) E stimate the magnifcation o the image. Figure 9 3 [2 ] Mitochondrion The magnifcation o the human cheek cell rom a compound microscope ( fgure 1 0) is 2 , 000 . a) C alculate how long a 2 0 m scale bar would be on the image. [2 ] Figure 11 Ostrich egg Testing the cell theory Questioning the cell theory using atypical examples, including striated muscle, giant algae and aseptate fungal hyphae. To test the cell theory you should look at the structure o as many living organisms as you can, using a microscope. Instructions or microscope use are given on page 4. In each case you should ask the question, D oes the organism or tissue ft the trend stated in the cell theory by consisting o one or more cells? In humans they have an average length o about 3 0 mm, whereas other human cells are mostly less than 0.03 mm in length. Instead o having one nucleus they have many, sometimes as many as several hundred. Three atypical examples are worth considering: Striated muscle is the type o tissue that we use to change the position o our body. The building blocks o this tissue are muscle fbres, which are similar in some ways to cells. They are surrounded by a membrane and are ormed by division o pre-existing cells. They have their own genetic material and their own energy release system. However muscle fbres are ar rom typical. They are much larger than most animal cells. Figure 12 Striated muscle fbres 7 1 C E LL B I O LO G Y Fungi consist o narrow thread-like structures called hyphae. These hyphae are usually white in colour and have a fuy appearance. They have a cell membrane and, outside it, a cell wall. In some types o ungi the hyphae are divided up into small cell-like sections by cross walls called septa. However, in aseptate ungi there are no septa. Each hypha is an uninterrupted tube-like structure with many nuclei spread along it. Algae are organisms that eed themselves by photosynthesis and store their genes inside nuclei, but they are simpler in their structure and organization than plants. Many algae consist o one microscopic cell. There are vast numbers o these unicellular algae in the oceans and they orm the basis o most marine ood chains. Less common are some algae that grow to a much larger size, yet they still seem to be single cells. They are known as giant algae. Acetabularia is one example. It can grow to a length o as much as 1 00 mm, despite only having one nucleus. I a new organism with a length o 1 00 mm was discovered, we would certainly expect it to consist o many cells, not just one. Figure 13 Aseptate hypha Figure 14 Giant alga Unicellular organisms Organisms consisting of only one cell carry out all functions of life in that cell. The unctions o lie are things that all organisms must do to stay alive. S ome organisms consist o only one cell. This cell thereore has to carry out all the unctions o lie. B ecause o this the structure o unicellular organisms is more complex than most cells in multicellular organisms. Unicellular organisms carry out at least seven unctions o lie: Nutrition obtaining ood, to provide energy and the materials needed or growth. Metabolism chemical reactions inside the cell, including cell respiration to release energy. Growth an irreversible increase in size. Response the ability to react to changes in the environment. Excretion getting rid o the waste products o metabolism. Homeostasis keeping conditions inside the organism within tolerable limits. Reproduction producing ospring either sexually or asexually. Many unicellular organisms also have a method o movement, but some remain in a xed position or merely drit in water or air currents. 8 1 .1 I n tro d u ctI o n to ce lls Limitations on cell size Surface area to volume ratio is important in the limitation of cell size. In the cytoplasm of cells, large numbers of chemical reactions take place. These reactions are known collectively as the metabolism of the cell. The rate of these reactions ( the metabolic rate of the cell) is proportional to the volume of the cell. For metabolism to continue, substances used in the reactions must be absorbed by the cell and waste products must be removed. S ubstances move into and out of cells through the plasma membrane at the surface of the cell. The rate at which substances cross this membrane depends on its surface area. The surface area to volume ratio of a cell is therefore very important. If the ratio is too small then substances will not enter the cell as quickly as they are required and waste products will accumulate because they are produced more rapidly than they can be excreted. Surface area to volume ratio is also important in relation to heat production and loss. If the ratio is too small then cells may overheat because the metabolism produces heat faster than it is lost over the cells surface. same cube unfolded Figure 15 Volume and surace area o a cube Functions of life in unicellular organisms Investigation of functions of life in Paramecium and one named photosynthetic unicellular organism. Paramecium is a unicellular organism that can be cultured quite easily in the laboratory. Alternatively collect some pond water and use a centrifuge to concentrate the organisms in it to see if Paramecium is present. Place a drop of culture solution containing Paramecium on a microscope slide. Add a cover slip and examine the slide with a microscope. The nucleus o the cell can divide to produce the extra nuclei that are needed when the cell reproduces. Oten the reproduction is asexual with the parent cell dividing to orm two daughter cells. Food vacuoles contain smaller organisms that the Paramecium has consumed. These are gradually digested and the nutrients are absorbed into the cytoplasm where they provide energy and materials needed or growth. The cell membrane controls what chemicals enter and leave. It allows the entry o oxygen or respiration. Excretion happens simply by waste products difusing out through the membrane. The contractile vacuoles at each end o the cell ll up with water and then expel it through the plasma membrane o the cell, to keep the cells water content within tolerable limits. Metabolic reactions take place in the cytoplasm, including the reactions that release energy by respiration. Enzymes in the cytoplasm are the catalysts that cause these reactions to happen. Beating o the cilia moves the Paramecium through the water and this can be controlled by the cell so that it moves in a particular direction in response to changes in the environment. Figure 16 Paramecium 9 1 C E LL B I O LO G Y Chlamydomonas is a unicellular alga that lives in soil and freshwater habitats. It has been used widely for research into cell and molecular biology. Although it is green in colour and carries out photosynthesis it is not a true plant and its cell wall is not made of cellulose. The nucleus o the cell can divide to produce genetically identical nuclei or asexual reproduction. Nuclei can also use and divide to carry out a sexual orm o reproduction. In this image, the nucleus is concealed by chloroplasts. The contractile vacuoles at the base o the fagella ll up with water and then expel it through the plasma membrane o the cell, to keep the cells water content within tolerable limits. Photosynthesis occurs inside chloroplasts in the cytoplasm. Carbon dioxide can be converted into the compounds needed or growth here, but in the dark carbon compounds rom other organisms are sometimes absorbed through the cell membrane i they are available. Metabolic reactions take place in the cytoplasm, with enzymes present to speed them up. The cell wall is reely permeable and it is the membrane inside it that controls what chemicals enter and leave. Oxygen is a waste product o photosynthesis and is excreted by diusing out through the membrane. Figure 17 Beating o the two fagella moves the Chlamydomonas through the water. A lightsensitive eyespot allows the cell to sense where the brightest light is and respond by swimming towards it. Chlamydomonas Multicellular organisms Multicellular organisms have properties that emerge from the interaction of their cellular components. S ome unicellular organisms live together in colonies, for example a type of alga called Volvox aureus. E ach colony consists of a ball made of a protein gel, with 5 00 or more identical cells attached to its surface. Figure 1 8 shows two colonies, with daughter colonies forming inside them. Although the cells are cooperating, they are not fused to form a single cell mass and so are not a single organism. Figure 18 Volvox colonies O rganisms consisting of a single mass of cells, fused together, are multicellular. O ne of the most intensively researched multicellular organisms is a worm called Caenorhabditis elegans. The adult body is about one millimetre long and it is made up of exactly 95 9 cells. This might seem like a large number, but most multicellular organisms have far more cells. There are about ten million million cells in an adult human body and even more in organisms such as oak trees or whales. Although very well known to biologists, Caenorhabditis elegans has no common name and lives unseen in decomposing organic matter. It feeds on the bacteria that cause decomposition. C. elegans has a mouth, pharynx, intestine and anus. It is hermaphrodite so has both male and female reproductive organs. Almost a third of the cells are neurons, or 10 1 .1 I n tro d u ctI o n to ce lls nerve cells. Most o these neurons are located at the ront end o the worm in a structure that can be regarded as the animals brain. Although the brain in C. elegans coordinates responses to the worms environment, it does not control how individual cells develop. The cells in this and other multicellular organisms can be regarded as cooperative groups, without any cells in the group acting as a leader or supervisor. It is remarkable how individual cells in a group can organize themselves and interact with each other to orm a living organism with distinctive overall properties. The characteristics o the whole organism, including the act that it is alive, are known as emergent properties. E mergent properties arise rom the interaction o the component parts o a complex structure. We sometimes sum this up with the phrase: the whole is greater than the sum o its parts. A simple example o an emergent property was described in a C hinese philosophical text written more than 2 , 5 00 years ago: Pots are fashioned from clay. But its the hollow that makes the pot work. S o, in biology we can carry out research by studying component parts, but we must remember that some bigger things result rom interactions between these components. Cell diferentiation in multicellular organisms Specialized tissues can develop by cell dierentiation in multicellular organisms. In multicellular organisms dierent cells perorm dierent unctions. This is sometimes called division o labour. In simple terms, a unction is a job or a role. For example the unction o a red blood cell is to carry oxygen, and the unction o a rod cell in the retina o the eye is to absorb light and then transmit impulses to the brain. Oten a group o cells specialize in the same way to perorm the same unction. They are called a tissue. B y becoming specialized, the cells in a tissue can carry out their role more efciently than i they had many dierent roles. They can develop the ideal structure, with the enzymes needed to carry out all o the chemical reactions associated with the unction. The development o cells in dierent ways to carry out specifc unctions is called dierentiation. In humans, 2 2 0 distinctively dierent highly specialized cell types have been recognized, all o which develop by dierentiation. toK Hw a w i wh m i b ha ah? An emergent property o a system is not a property o any one component o the system, but it is a property o the system as a whole. Emergence reers to how complex systems and patterns arise rom many small and relatively simple interactions. We cannot thereore necessarily predict emergent properties by studying each part o a system separately (an approach known as reductionism) . Molecular biology is an example o the success that a reductionist approach can have. Many processes occurring in living organisms have been explained at a molecular level. However, many argue that reductionism is less useul in the study o emergent properties including intelligence, consciousness and other aspects o psychology. The interconnectivity o the components in cases like these is at least as important as the unctioning o each individual component. One approach that has been used to study interconnectivity and emergent properties is computer modelling. In both animal behaviour and ecology, a programme known as the Game o Lie has been used. It was devised by John Conway and is available on the Internet. Test the Game o Lie by creating initial confgurations o cells and seeing how they evolve. Research ways in which the model has been applied. Gene expression and cell diferentiation Dierentiation involves the expression o some genes and not others in a cells genome. There are many dierent cell types in a multicellular organism but they all have the same set o genes. The 2 2 0 cell types in the human body have the same set o genes, despite large dierences in their structure and activities. To take an example, rod cells in the retina o the eye produce a pigment that absorbs light. Without it, the rod cell would not be able to do its j ob o sensing light. A lens cell in the eye produces no pigments and is transparent. I it did contain pigments, less light would 11 1 C E LL B I O LO G Y pass through the lens and our vision would be worse. While they are developing, both cell types contain the genes for making the pigment, but these genes are only used in the rod cell. This is the usual situation cells do not j ust have genes with the instructions that they need, they have genes needed to specialize in every possible way. There are approximately 2 5 , 000 genes in the human genome, and these genes are all present in a body cell. However, in most cell types less than half of the genes will ever be needed or used. When a gene is being used in a cell, we say that the gene is being expressed. In simple terms, the gene is switched on and the information in it is used to make a protein or other gene product. The development of a cell involves switching on particular genes and expressing them, but not others. C ell differentiation happens because a different sequence of genes is expressed in different cell types. The control of gene expression is therefore the key to development. An extreme example of differentiation involves a large family of genes in humans that carry the information for making receptors for odorants smells. These genes are only expressed in cells in the skin inside the nose, called olfactory receptor cells. Each of these cells expresses j ust one of the genes and so makes one type of receptor to detect one type of odorant. This is how we can distinguish between so many different smells. Richard Axel and Linda B uck were given the Nobel Prize in 2 004 for their work on this system. Stem cells The capacity o stem cells to divide and diferentiate along diferent pathways is necessary in embryonic development. It also makes stem cells suitable or therapeutic uses. A new animal life starts when a sperm fertilizes an egg cell to produce a zygote. An embryo is formed when the zygote divides to give two cells. This two- cell embryo divides again to produce a four- cell embryo, then eight, sixteen and so on. At these early stages in embryonic development the cells are capable of dividing many times to produce large amounts of tissue. They are also extremely versatile and can differentiate along different pathways into any of the cell types found in that particular animal. In the 1 9th century, the name stem cell was given to the zygote and the cells of the early embryo, meaning that all the tissues of the adult stem from them. S tem cells have two key properties that have made them one of the most active areas of research in biology and medicine today. Figure 19 12 Embryonic stem cells S tem cells can divide again and again to produce copious quantities of new cells. They are therefore useful for the growth of tissues or the replacement of cells that have been lost or damaged. S tem cells are not fully differentiated. They can differentiate in different ways, to produce different cell types. 1 .1 I n tro d u ctI o n to ce lls Embryonic stem cells are thereore potentially very useul. They could be used to produce regenerated tissue, such as skin or people who have suered burns. They could provide a means o healing diseases such as type 1 diabetes where a particular cell type has been lost or is malunctioning. They might even be used in the uture to grow whole replacement organs hearts or kidneys, or example. These types o use are called therapeutic, because they provide therapies or diseases or other health problems. There are also non-therapeutic uses or embryonic stem cells. One possibility is to use them to produce large quantities o striated muscle fbres, or meat, or human consumption. The bee burgers o the uture may thereore be produced rom stem cells, without the need to rear and slaughter cattle. It is the early stage embryonic stem cells that are the most versatile. Gradually during embryo development the cells commit themselves to a pattern o dierentiation. This involves a series o points at which a cell decides whether to develop along one pathway or another. Eventually each cell becomes committed to develop into one specifc cell type. Once committed, a cell may still be able to divide, but all o these cells will dierentiate in the same way and they are no longer stem cells. Small numbers o cells remain as stem cells, however, and they are still present in the adult body. They are present in many human tissues, including bone marrow, skin and liver. They give some human tissues considerable powers o regeneration and repair. The stem cells in other tissues only allow limited repair brain, kidney and heart or example. Therapeutic uses of stem cells Use of stem cells to treat Stargardts disease and one other named condition. There are a ew current uses o stem cells to treat diseases, and a huge range o possible uture uses, many o which are being actively researched. Two examples are given here: one involving embryonic stem cells and one using adult stem cells. Stargardts disease The ull name o this disease is S targardts macular dystrophy. It is a genetic disease that develops in children between the ages o six and twelve. Most cases are due to a recessive mutation o a gene called AB C A4. This causes a membrane protein used or active transport in retina cells to malunction. As a consequence, photoreceptive cells in the retina degenerate. These are the cells that detect light, so vision becomes progressively worse. The loss o vision can be severe enough or the person to be registered as blind. Researchers have developed methods or making embryonic stem cells develop into retina cells. This was done initially with mouse cells, which were then injected into the eyes o mice that had a condition similar to Stargardts disease. The injected cells were not rejected, did not develop into tumours or cause any other problems. The cells moved to the retina where they attached themselves and remained. Very encouragingly, they caused an improvement in the vision o the mice. In November 2 01 0, researchers in the United S tates got approval or trials in humans. A woman in her 5 0s with S targardts disease was treated by having 5 0, 000 retina cells derived rom embryonic stem cells inj ected into her eyes. Again the cells attached to the retina and remained there during the our- month trial. There was an improvement in her vision, and no harmul side eects. 13 1 C E LL B I O LO G Y Further trials with larger numbers o patients are needed, but ater these initial trials at least, we can be optimistic about the development o treatments or S targardts disease using embryonic stem cells. can be done by treating the patient with chemicals that kill dividing cells. The procedure is known as chemotherapy. However, to remain healthy in the long term the patient must be able to produce the white blood cells needed to ght disease. S tem cells that can produce blood cells must be present, but they are killed by chemotherapy. The ollowing procedure is thereore used: A large needle is inserted into a large bone, usually the pelvis, and fuid is removed rom the bone marrow. S tem cells are extracted rom this fuid and are stored by reezing them. They are adult stem cells and only have the potential or producing blood cells. A high dose o chemotherapy drugs is given to the patient, to kill all the cancer cells in the bone marrow. The bone marrow loses its ability to produce blood cells. The stem cells are then returned to the patients body. They re- establish themselves in the bone marrow, multiply and start to produce red and white blood cells. Figure 20 Stargardts disease leukemia This disease is a type o cancer. All cancers start when mutations occur in genes that control cell division. For a cancer to develop, several specic mutations must occur in these genes in one cell. This is very unlikely to happen, but as there are huge numbers o cells in the body, the overall chance becomes much larger. More than a quarter o a million cases o leukemia are diagnosed each year globally and there are over 2 00, 000 deaths rom the disease. In many cases this procedure cures the leukemia completely. Once the cancer-inducing mutations have occurred in a cell, it grows and divides repeatedly, producing more and more cells. Leukemia involves the production o abnormally large numbers o white blood cells. In most cancers, the cancer cells orm a lump or tumour but this does not happen with leukemia. White blood cells are produced in the bone marrow, a sot tissue in the hollow centre o large bones such as the emur. They are then released into the blood, both in normal conditions and when excessive numbers are produced with leukemia. A normal adult white blood cell count is between 4, 000 and 1 1 ,000 per mm 3 o blood. In a person with leukemia this number rises higher and higher. C ounts above 30,000 per mm 3 suggest that a person may have leukemia. I there are more than 1 00, 000 per mm 3 it is likely that the person has acute leukemia. To cure leukemia, the cancer cells in the bone marrow that are producing excessive numbers o white blood cells must be destroyed. This 14 Figure 21 Removal of stem cells from bone marrow 1 .1 I n tro d u ctI o n to ce lls The ethics of stem cell research Ethical implications o research: research involving stem cells is growing in importance and raises ethical issues. S tem cell research has been very controversial. Many ethical obj ections have been raised. S cientists should always consider the ethical implications o their research beore doing it. S ome o the research that was carried out in the past would not be considered ethically acceptable today, such as medical research carried out on patients without their inormed consent. D ecisions about whether research is ethically acceptable must be based on a clear understanding o the science involved. S ome people dismiss all stem cell research as unethical, but this shows a misunderstanding o the dierent possible sources o the stem cells being used. In the next section, three possible sources o stem cells and the ethics o research involving them are discussed. Sources of stem cells and the ethics of using them Ethics o the therapeutic use o stem cells rom specially created embryos, rom the umbilical cord blood o a new-born baby and rom an adults own tissues. Stem cells can be obtained rom a variety o sources. E mbryos can be deliberately created by ertilizing egg cells with sperm and allowing the resulting zygote to develop or a ew days until it has between our and sixteen cells. All o the cells are embryonic stem cells. B lood can be extracted rom the umbilical cord o a new- born baby and stem cells obtained rom it. The cells can be rozen embyi m Almost unlimited growth potential. Can dierentiate into any type in the body. More risk o becoming tumour cells than with adult stem cells, including teratomas that contain dierent tissue types. Less chance o genetic damage due to the accumulation o mutations than with adult stem cells. Likely to be genetically dierent rom an adult patient receiving the tissue. Removal o cells rom the embryo kills it, unless only one or two cells are taken. and stored or possible use later in the babys lie. S tem cells can be obtained rom some adult tissues such as bone marrow. These types o stem cell vary in their properties and thereore in their potential or therapeutic use. The table below gives some properties o the three types, to give the scientifc basis or an ethical assessment. c b m Easily obtained and stored. Commercial collection and storage services already available. Fully compatible with the tissues o the adult that grows rom the baby, so no rejection problems occur. Limited capacity to dierentiate into dierent cell types only naturally develop into blood cells, but research may lead to production o other types. Limited quantities o stem cells rom one babys cord. The umbilical cord is discarded whether or not stem cells are taken rom it. A m Difcult to obtain as there are very ew o them and they are buried deep in tissues. Less growth potential than embryonic stem cells. Less chance o malignant tumours developing than rom embryonic stem cells. Limited capacity to dierentiate into dierent cell types. Fully compatible with the adults tissues, so rejection problems do not occur. Removal o stem cells does not kill the adult rom which the cells are taken. 15 1 C E LL B I O LO G Y Stem cell research has been very controversial. Many ethical obj ections have been raised. There are most obj ections to the use of embryonic stem cells, because current techniques usually involve the death of the embryo when the stem cells are taken. The main question is whether an early stage embryo is as much a human individual as a new- born baby, in which case killing the embryo is undoubtedly unethical. When does a human life begin? There are different views on this. Some consider that when the sperm fertilizes the egg, a human life has begun. Others say that early stage embryos have not yet developed human characteristics and cannot suffer pain, so they should be thought of simply as groups of stem cells. Some suggest that a human life truly begins when there is a heartbeat, or bone tissue or brain activity. These stages take place after a few weeks of development. Another view is that it is only when the embryo has developed into a fetus that is capable of surviving outside the uterus. Some scientists argue that if embryos are specially created by in vitro fertilization (IVF) in order to obtain stem cells, no human that would otherwise have lived has been denied its chance of living. However, a counterargument is that it is unethical to create human lives solely for the purpose of obtaining stem cells. Also, IVF involves hormone treatment of women, with some associated risk, as well as an invasive surgical procedure for removal of eggs from the ovary. If women are paid for supplying eggs for IVF this could lead to the exploitation of vulnerable groups such as college students. We mu st no t fo rge t e thical argume nts in favo ur o f the u se o f e mb ryo nic ste m ce lls. The y have the p o te ntial to allo w me tho ds o f tre atme nt fo r dise ase s and disab ilitie s that are cu rre ntly incu rab le , so the y co u ld gre atly re du ce the su ffe ring o f so me individuals. Figure 22 Harvesting umbilical cord blood 1.2 ultrastrctre of cells Understanding Prokaryotes have a simple cell structure without compartments. Eukaryotes have a compartmentalized cell structure. Prokaryotes divide by binary fssion. Electron microscopes have a much higher resolution than light microscopes. Nature of science 16 Applications The structure and unction o organelles within exocrine gland cells o the pancreas. The structure and unction o organelles within palisade mesophyll cells o the lea. Skills Developments in scientifc research ollow Drawing the ultrastructure o prokaryotic cells improvements in apparatus: the invention o electron microscopes led to greater understanding o cell structure. based on electron micrographs. Drawing the ultrastructure o eukaryotic cells based on electron micrographs. Interpretation o electron micrographs to identiy organelles and deduce the unction o specialized cells. 1 . 2 u lt r A s t r u c t u r e o f c e l l s th invnin h n mip Developments in scientifc research ollow improvements in apparatus: the invention o electron microscopes led to greater understanding o cell structure. Much o the progress in biology over the last 1 50 years has ollowed improvements in the design o microscopes. In the second hal o the 1 9th century improved light microscopes allowed the discovery o bacteria and other unicellular organisms. C hromosomes were seen or the rst time and the processes o mitosis, meiosis and gamete ormation were discovered. The basis o sexual reproduction, which had previously eluded William Harvey and many other biologists, was seen to be the usion o gametes and subsequent development o embryos. The complexity o organs such as the kidney was revealed and mitochondria, chloroplasts and other structures were discovered within cells. There was a limit to the discoveries that could be made though. For technical reasons that are explained later in this sub-topic, light microscopes cannot produce clear images o structures smaller than 0.2 micrometres (m) . (A micrometre is a thousandth o a millimetre.) Many biological structures are smaller than this. For example, membranes in cells are about 0.01 m thick. Progress was hampered until a dierent type o microscope was invented the electron microscope. Electron microscopes were developed in Germany during the 1 930s and came into use in research laboratories in the 1 940s and 5 0s. They allowed images to be produced o things as small as 0.001 m 2 00 times smaller than with light microscopes. The structure o eukaryotic cells was ound to be ar more intricate than most biologists had expected and many previous ideas were shown to be wrong. For example, in the 1 890s the light microscope had revealed darker green areas in the chloroplast. They were called grana and interpreted as droplets o chlorophyll. The electron microscope showed that grana are in act stacks o fattened membrane sacs, with the chlorophyll located in the membranes. Whereas mitochondria appear as tiny structureless rods or spheres under the light microscope, the electron microscope revealed them to have an intricate internal membrane structure. The electron microscopes revealed what is now called the ultrastructure o cells, including previously unknown eatures. Ribosomes, lysosomes and the endoplasmic reticulum were all discovered and named in the 1 95 0s, or example. It is unlikely that there are structures as signicant as these still to be discovered, but improvements in the design o electron microscopes continue and each improvement allows new discoveries to be made. A recent example, described in subtopic 8.2 , is electron tomography a method o producing 3 - D images by electron microscopy. The resolution of electron microscopes Electron microscopes have a much higher resolution than light microscopes. I we look at a tree with unaided eyes we can see its individual leaves, but we cannot see the cells within its leaves. The unaided eye can see things with a size o 0.1 mm as separate objects, but no smaller. To see the cells within the lea we need to use a light microscope. This allows us to see things with a size o down to about 0.2 m as separate objects, so cells can become individually visible they can be distinguished. Making the separate parts o an obj ect distinguishable by eye is called resolution. The maximum resolution o a light microscope is 0. 2 m, which is 2 00 nanometres ( nm) . However powerul the lenses o a light microscope are, the resolution cannot be higher than this because it is limited by the wavelength o light ( 400700 nm) . I we try to resolve smaller obj ects by Figure 1 An electron microscope in use 17 1 C E LL B I O LO G Y making lenses with greater magnifcation, we fnd that it is impossible to ocus them properly and get a blurred image. This is why the maximum magnifcation with light microscopes is usually 400. Beams o electrons have a much shorter wavelength, so electron microscopes have a much higher resolution. The resolution o modern electron microscopes is 0.001 m or 1 nm. Electron microscopes thereore have a resolution that is 200 times greater than light microscopes. This is why light microscopes reveal the structure o cells, but electron microscopes reveal the ultrastructure. It explains why light microscopes were needed to see bacteria with a size o 1 micrometre, but viruses with a diameter o 0.1 micrometres could not be seen until electron microscopes had been invented. resolutio Unaided eyes Light microscopes Ativity commee ad siee While still a young student in Berlin in the late 1920s Ernst Ruska developed magnetic coils that could ocus beams o electrons. He worked on the idea o using these lenses to obtain an image as in a light microscope, but with electron beams instead o light. During the 1930s he developed and refned this technology. By 1939 Ruska had designed the frst commercial electron microscope. In 1986 he was awarded the Nobel Prize in Physics or this pioneering work. Ruska worked with the German frm Siemens. Other companies in Britain, Canada and the United States also developed and manuactured electron microscopes. 18 Scientists in dierent countries usually cooperate with each other but commercial companies do not. What are the reasons or this dierence? Electron microscopes Millimetes (mm) Miometes (m) naometes (m) 0.1 100 100,000 0.0002 0.2 200 0.000001 0.001 1 Prokaryotic cell structure Prokaryotes have a simple cell structure without compartments . All organisms can be divided into two groups according to their cell structure. Eukaryotes have a compartment within the cell that contains the chromosomes. It is called the nucleus and is bounded by a nuclear envelope consisting o a double layer o membrane. Prokaryotes do not have a nucleus. Prokaryotes were the frst organisms to evolve on Earth and they still have the simplest cell structure. They are mostly small in size and are ound almost everywhere in soil, in water, on our skin, in our intestines and even in pools o hot water in volcanic areas. All cells have a cell membrane, but some cells, including prokaryotes, also have a cell wall outside the cell membrane. This is a much thicker and stronger structure than the membrane. It protects the cell, maintains its shape and prevents it rom bursting. In prokaryotes the cell wall contains peptidoglycan. It is oten reerred to as being extracellular. As no nucleus is present in a prokaryotic cell its interior is entirely flled with cytoplasm. The cytoplasm is not divided into compartments by membranes it is one uninterrupted chamber. The structure is thereore simpler than in eukaryotic cells, though we must remember that it is still very complex in terms o the biochemicals that are present, including many enzymes. O rganelles are present in the cytoplasm o eukaryotic cells that are analogous to the organs o multi- cellular organisms in that they are distinct structures with specialized unctions. Prokaryotes do not have cytoplasmic organelles apart rom ribosomes. Their size, measured in S vedberg units ( S ) is 70S, which is smaller than those o eukaryotes. 1 . 2 u lt r A s t r u c t u r e o f c e l l s Part o the cytoplasm appears lighter than the rest in many electron micrographs. This region contains the DNA o the cell, usually in the orm o one circular DNA molecule. The DNA is not associated with proteins, which explains the lighter appearance compared with other parts o the cytoplasm that contain enzymes and ribosomes. This lighter area o the cell is called the nucleoid meaning nucleus-like as it contains DNA but is not a true nucleus. Cell division in prokaryotes Prokaryotes divide by binary fssion. All living organisms need to produce new cells. They can only do this by division o pre- existing cells. C ell division in prokaryotic cells is called binary fssion and it is used or asexual reproduction. The single circular chromosome is replicated and the two copies o the chromosome move to opposite ends o the cell. D ivision o the cytoplasm o the cell quickly ollows. E ach o the daughter cells contains one copy o the chromosome so they are genetically identical. dawing pkayi Draw the ultrastructure o prokaryotic cells based on electron micrographs. B ecause prokaryotes are mostly very small, their internal structure cannot be seen using a light microscope. It is only with much higher magnifcation in electron micrographs that we can see the details o the structure, called the ultrastructure. D rawings o the ultrastructure o prokaryotes are thereore based on electron micrographs. Shown below and on the next page are two electron micrographs o E. coli, a bacterium ound in our intestines. One o them is a thin section and shows the internal structure. The other has been prepared by a dierent technique and shows the external structure. A drawing o each is also shown. B y comparing the drawings with the electron micrographs you can learn how to identiy structures within prokaryotic cells. E lectron micrograp h of Escherichia coli (1 2 m in length) D rawing to help interp ret the electron micrograp h ribosomes cell wall plasma membrane cytoplasm nucleoid (region containing naked DNA) Aiviy oh nam pkay Biologists sometimes use the term bacteria instead o prokaryote. This may not always be appropriate because the term prokaryote encompasses a larger group o organisms than true bacteria (Eubacteria) . It also includes organisms in another group called the Archaea. There is a group o photosynthetic organisms that used to be called blue-green algae, but their cell structure is prokaryotic and algae are eukaryotic. This problem has been solved by renaming them as Cyanobacteria. What problems are caused by scientists using dierent words or things than nonscientists? 19 1 C E LL B I O LO G Y Electron micrograph of Escherichia coli showing surface features pili agellum Shown below is another micrograph o a prokaryote. You can use it to practice your skill at drawing the ultrastructure o prokaryotic cells. You can also fnd other electron micrographs o prokaryotic cells on the internet and try drawing these. There is no need to spend a long time drawing many copies o a particular structure, such as the ribosomes. You can indicate their appearance in one small representative part o the cytoplasm and annotate your drawing to say that they are ound elsewhere. Activity Garlic cells and compartmentalization Garlic cells store a harmless sulphur-containing compound called alliin in their vacuoles. They store an enzyme called alliinase in other parts o the cell. Alliinase converts alliin into a compound called allicin, which has a very strong smell and favour and is toxic to some herbivores. This reaction occurs when herbivores bite into garlic and damage cells, mixing the enzyme and its substrate. Perhaps surprisingly, many humans like the favour, but to get it garlic must be crushed or cut, not used whole. 20 You can test this by smelling a whole garlic bulb, then cutting or crushing it and smelling it again. Figure 2 Brucella abortus (Bangs bacillus) , 2 m in length Eukaryotic cell structure Eukaryotes have a compartmentalized cell structure. Eukaryotic cells have a much more complicated internal structure than prokaryotic cells. Whereas the cytoplasm o a prokaryotic cell is one undivided space, eukaryotic cells are compartmentalized. This means that they are divided up by partitions into compartments. The partitions are single or double membranes. The most important o these compartments is the nucleus. It contains the cells chromosomes. The compartments in the cytoplasm are known as organelles. Just as each organ in an animals body is specialized 1 . 2 u lt r A s t r u c t u r e o f c e l l s to perform a particular role, each organelle in a eukaryotic cell has a distinctive structure and function. There are several advantages in being compartmentalized: Enzymes and substrates for a particular process can be much more concentrated than if they were spread throughout the cytoplasm. S ubstances that could cause damage to the cell can be kept inside the membrane of an organelle. For example, the digestive enzymes of a lysosome could digest and kill a cell, if they were not safely stored inside the lysosome membrane. C onditions such as pH can be maintained at an ideal level for a particular process, which may be different to the levels needed for other processes in a cell. O rganelles with their contents can be moved around within the cell. dawig kayi Draw the ultrastructure o eukaryotic cells based on electron micrographs. The ultrastructure of eukaryotic cells is very complex and it is often best to draw only part of a cell. Your drawing is an interpretation of the structure, so you need to understand the structure of the organelles that might be present. n double nuclear membrane nuclear pores dense chromatin chromatin rgh pami im ribosomes The table below contains an electron micrograph of each of the commonly occurring organelles, with a drawing of the structure. B rief notes on recognition features and the function of each organelle are included. The nuclear membrane is double and has pores through it. The nucleus contains the chromosomes, consisting o DNA associated with histone proteins. Uncoiled chromosomes are spread through the nucleus and are called chromatin. There are oten densely staining areas o chromatin around the edge o the nucleus. The nucleus is where DNA is replicated and transcribed to orm mRNA, which is exported via the nuclear pores to the cytoplasm. The rER consists o fattened membrane sacs, called cisternae. Attached to the outside o these cisternae are ribosomes. They are larger than in prokaryotes and are classied as 80S. The main unction o the rER is to synthesize protein or secretion rom the cell. Protein synthesized by the ribosomes o the rER passes into its cisternae and is then carried by vesicles, which bud o and are moved to the Golgi apparatus. cisterna 21 1 C E LL B I O LO G Y Gogi apparatus cisterna vesicles lysosome digestive enzymes This organelle consists o fattened membrane sacs called cisternae, like rER. However the cisternae are not as long, are oten curved, do not have attached ribosomes and have many vesicles nearby. The Golgi apparatus processes proteins brought in vesicles rom the rER. Most o these proteins are then carried in vesicles to the plasma membrane or secretion. These are approximately spherical with a single membrane. They are ormed rom Golgi vesicles. They contain high concentrations o protein, which makes them densely staining in electron micrographs. They contain digestive enzymes, which can be used to break down ingested ood in vesicles or break down organelles in the cell or even the whole cell. lysosome membrane Mitohondrion inner membrane outer membrane crista matrix free ribosomes These appear as dark granules in the cytoplasm and are not surrounded by a membrane. They have the same size as ribosomes attached to the rER about 20nm in diameter, and known as 80S. Free ribosomes synthesize protein, releasing it to work in the cytoplasm, as enzymes or in other ways. Ribosomes are constructed in a region o the nucleus called the nucleolus. choropast A double membrane surrounds the chloroplast. Inside are stacks o thylakoids, which are fattened sacs o membrane. The shape o chloroplasts is variable but is usually spherical or ovoid. They produce glucose and a wide variety o other organic compounds by photosynthesis. Starch grains may be present inside chloroplasts i they have been photosynthesizing rapidly. starch grain stroma double membrane thylakoid Vauoes and vesies vacuole containing food vesicles 22 A double membrane surrounds mitochondria, with the inner o these membranes invaginated to orm structures called cristae. The fuid inside is called the matrix. The shape o mitochondria is variable but is usually spherical or ovoid. They produce ATP or the cell by aerobic cell respiration. Fat is digested here i it is being used as an energy source in the cell. large vacuole These are organelles that consist simply o a single membrane with fuid inside. Many plant cells have large vacuoles that occupy more than hal o the cell volume. Some animals absorb oods rom outside and digest them inside vacuoles. Some unicellular organisms use vacuoles to expel excess water. Vesicles are very small vacuoles used to transport materials inside the cell. 1 . 2 u lt r A s t r u c t u r e o c e l l s In the cytoplasm o cells there are small cylindrical bres called microtubules that have a variety o roles, including moving chromosomes during cell division. Animal cells have structures called centrioles, which consist o two groups o nine triple microtubules. Centrioles orm an anchor point or microtubules during cell division and also or microtubules inside cilia and fagella. Mib and ni triple microtubules These are whip-like structures projecting rom the cell surace. They contain a ring o nine double microtubules plus two central ones. Flagella are larger and usually only one is present, as in a sperm. Cilia are smaller and many are present. Cilia and fagella can be used or locomotion. Cilia can be also be used to create a current in the fuid next to the cell. ciia and faga double plasma microtubule membrane The electron micrograph below shows a liver cell with labels to identify some of the organelles that are present. mitochondrion Using your understanding of these organelles, draw the whole cell to show its ultrastructure. nucleus free ribosomes FPO <839211_ph1.2.15> rough endoplasmic reticulum Figure 3 Golgi a aratus lysosome Electron micrograph of part of a liver cell 23 1 C E LL B I O LO G Y Exocrine gland cells of the pancreas The structure and function of organelles within exocrine gland cells of the pancreas. Gland cells secrete substances they release them through their plasma membrane. There are two types of gland cells in the pancreas. E ndocrine cells secrete hormones into the bloodstream. E xocrine gland cells in the pancreas secrete digestive enzymes into a duct that carries them to the small intestine where they digest foods. FPO <Insert 839211_ ph1.2.16> Enzymes are proteins, so the exocrine gland cells have organelles needed to synthesize proteins in large quantities, process them to make them ready for secretion, transport them to the plasma membrane and then release them. The electron micrograph on the right shows these organelles: plasma membrane mitochondrion nucleus rough ER Golgi apparatus vesicles lysosomes Figure 4 Electron micrograph of pancreas cell Palisade mesophyll cells The structure and function of organelles within palisade mesophyll cells of the leaf. The function of the leaf is photosynthesis producing organic compounds from carbon dioxide and other simple inorganic compounds, using light energy. The cell type that carries out most photosynthesis in the leaf is palisade mesophyll. The shape of these cells is roughly cylindrical. Like all living plant cells the cell is surrounded by a cell wall, with a plasma membrane inside it. The electron micrograph on the right shows the organelles that a palisade mesophyll cell contains: cell wall plasma membrane chloroplasts mitochondrion vacuole nucleus 24 Figure 5 Electron micrograph of palisade mesophyll cell 1 . 3 M e M b rAn e s tru ctu r e Ipig h of kayoi ll Interpret electron micrographs to identiy organelles and deduce the unction o specialized cells. I the organelles in a eukaryotic cell can be identifed and their unction is known, it is oten possible to deduce the overall unction o the cell. S tudy the electron micrographs in fgures 6, 7 and 8. Identiy the organelles that are present and try to deduce the unction o each cell. Figure 7 Figure 6 Figure 8 1.3 Mma Understanding Phospholipids orm bilayers in water due to the amphipathic properties o phospholipid molecules. Membrane proteins are diverse in terms o structure, position in the membrane and unction. Cholesterol is a component o animal cell membranes. Nature of science Applications Cholesterol in mammalian membranes reduces membrane fuidity and permeability to some solutes. Skills Using models as representations o the Drawing the fuid mosaic model. real world: there are alternative models o membrane structure. Falsication o theories with one theory being superseded by another: evidence alsied the DavsonDanielli model. Analysis o evidence rom electron microscopy that led to the proposal o the DavsonDanielli model. Analysis o the alsication o the DavsonDanielli model that led to the SingerNicolson model 25 1 C E LL B I O LO G Y OH hydrophilic phosphate head P O O H C H H C O C O O H H H H H H H H H H H H H H H H C O H C O C H C H C H H C H C H C H H H C H C H C H C H C H C H C H H H C H C H H H C C C H C H C H H H C H C H C H C H C H C H H H H H H H H H H Phospholipid bilayers Phospholipids form bilayers in water due to the amphipathic properties of phospholipid molecules. Some substances are attracted to water they are hydrop hilic. O ther substances are not attracted to water they are hydrop hobic. C H C H C H Phospholipids are unusual because part o a phospholipid molecule is hydrophilic and part is hydrophobic. Substances with this property are described as amp hip athic. hydrophobic hydrocarbon tails C H C H C H C H The hydrophilic part o a phospholipid is the phosphate group. The hydrophobic part consists o two hydrocarbon chains. The chemical structure o phospholipids is shown in fgure 1 . The structure can be represented simply using a circle or the phosphate group and two lines or the hydrocarbon chains. C H H Figure 1 The molecular structure o a phospholipid. The phosphate oten has other hydrophilic groups attached to it, but these are not shown in this diagram Figure 2 Simplifed diagram o a phospholipid molecule The two parts o the molecule are oten called phosphate heads and hydrocarbon tails. When phospholipids are mixed with water the phosphate heads are attracted to the water but the hydrocarbon tails are attracted to each other, but not to water. B ecause o this the phospholipids become arranged into double layers, with the hydrophobic hydrocarbon tails acing inwards towards each other and the hydrophilic heads acing the water on either side. These double layers are called phospholipid bilayers. They are stable structures and they orm the basis o all cell membranes. hydrophilic phosphate head hydrophobic hydrocarbon tails phospholipid bilayer Figure 3 Simplifed diagram o a phospholipid bilayer Models of membrane structure Using models as representations of the real world: there are alternative models of membrane structure. In the 1 9 2 0 s, Gorter and Grendel extracted phospholipids rom the plasma membrane o red blood cells and calculated that the area that the phospholipids occupied when 26 arranged in a monolayer was twice as large as the area o plasma membrane. They deduced that the membrane contained a bilayer o phospholipids. There were several errors in 1 . 3 M e M b rAn e s tru ctu r e their methods but luckily these cancelled each other out and there is now very strong evidence or cell membranes being based on phospholipid bilayers. band between. Proteins appear dark in electron micrographs and phospholipids appear light, so this appearance tted the D avson- D anielli model. Membranes also contain protein and Gorter and Grendels model did not explain where this is located. In the 1 9 3 0s D avson and D anielli proposed layers o protein adj acent to the phospholipid bilayer, on both sides o the membrane. They proposed this sandwich model because they thought it would explain how membranes, despite being very thin, are a very eective barrier to the movement o some substances. High magnication electron micrographs o membranes were made in the 1 9 5 0s, which showed a railroad track appearance two dark lines with a lighter Another model o membrane structure was proposed in 1 966 by Singer and Nicolson. In this model the proteins occupy a variety o positions in the membrane. Peripheral proteins are attached to the inner or outer surace. Integral proteins are embedded in the phospholipid bilayer, in some cases with parts protruding out rom the bilayer on one or both sides. The proteins are likened to the tiles in a mosaic. B ecause the phospholipid molecules are ree to move in each o the two layers o the bilayer, the proteins are also able to move. This gives the model its name the fuid mosaic model. Polm wih h davodailli mol Falsifcation o theories with one theory being superseded by another: evidence alsifed the DavsonDanielli model. The D avsonD anielli model o membrane structure was accepted by most cell biologists or about 3 0 years. Results o many experiments tted the model including X- ray diraction studies and electron microscopy. In the 1 95 0s and 60s some experimental evidence accumulated that did not t with the D avson D anielli model: Freeze-etched electron micrograp hs. This technique involves rapid reezing o cells and then racturing them. The racture occurs along lines o weakness, including the centre o membranes. Globular structures scattered through reeze- etched images o the centre o membranes were interpreted as transmembrane proteins. S tructure of membrane p roteins. Improvements in biochemical techniques allowed proteins to be extracted rom membranes. They were ound to be very varied in size and globular in shape so were unlike the type o structural protein that would orm continuous layers on the Figure 4 Freeze-etched electron micrograph of nuclear membranes, with nuclear pores visible and vesicles in the surrounding cytoplasm. The diagram on page 28 shows the line of fracture through the centre of the inner and outer nuclear membranes. Transmembrane proteins are visible in both of the membranes 27 1 C E LL B I O LO G Y periphery o the membrane. Also the proteins were hydrophobic on at least part o their surace so they would be attracted to the hydrocarbon tails o the phospholipids in the centre o the membrane. Fluorescent antibody tagging. Red or green fuorescent markers were attached to antibodies that bind to membrane proteins. The membrane proteins o some cells were tagged with red markers and other cells with green markers. The cells were used together. Within 40 minutes the red and green markers were mixed throughout the membrane o the used cell. This showed that membrane proteins are ree to move within the membrane rather than being xed in a peripheral layer. Taken together, this experimental evidence alsied the D avsonD anielli model. A replacement was needed that tted the evidence and the model that became widely accepted was the S ingerNicolson fuid mosaic model. It has been the leading model or over ty years but it would be unwise to assume that it will never be superseded. There are already some suggested modications o the model. An important maxim or scientists is Think it possible that you might be mistaken. Advances in science happen because scientists rej ect dogma and instead search continually or better understanding. cytoplasm nucleus inner membrane outer membrane Evidence for and against the DavsonDanielli model of membrane structure Analysis of evidence from electron microscopy that led to the proposal of the DavsonDanielli model. Figure 5 shows the plasma membrane o a red blood cell and some o the cytoplasm near the edge o the cell. 1 . D escribe the appearance o the plasma membrane. [2 ] 2 . Explain how this appearance suggested that the membrane had a central region o phospholipid with layers o protein on either side. [2 ] 3 . Suggest reasons or the dark grainy appearance o the cytoplasm o the red blood cell. [2 ] 4. C alculate the magnication o the electron micrograph assuming that the thickness o the membrane is 1 0 nanometres. [3 ] The two sets o data- based questions that ollow are based on the types o data that were used to alsiy the D avsonD anielli model o membrane structure. 28 Figure 5 TEM of plasma membrane of a red blood cell 1 . 3 M e M b rAn e s tru ctu r e daa-a qio: Membranes in Difusion o proteins in membranes reeze-etched electron micrographs Frye and Edidin used an elegant technique to obtain evidence or the fuid nature o membranes. They attached fuorescent markers to membrane proteins green markers to mouse cells and red markers to human cells. In both cases, spherical cells growing in tissue culture were used. The marked mouse and human cells were then used together. At rst, the used cells had one green hemisphere and one red one, but over the minutes ollowing usion, the red and green markers gradually merged, until they were completely mixed throughout the whole o the cell membrane. B locking o ATP production did not prevent this mixing ( ATP supplies energy or active processes in the cell) . Figure 6 shows a reeze- etched electron micrograph image o part o a cell. It was prepared by Proessor Horst Robenek o Mnster University. tim af cll wih mak flly mix/% fio / rl rl rl rl Ma mi 1 2 3 4 Figure 6 1 In all o the ractured membranes in the micrograph small granules are visible. 4 0 10 3 0 25 40 54 40 87 88 93 100 120 100 C alculate the mean percentage o cells with markers ully mixed or each time ater usion. [4] 2 Plot a graph o the results, including range bars or times where there was variation in the results. To do this you plot the highest and lowest results with a small bar and j oin these bars with a ruled line. You should also plot the mean result with a cross. This will lie on the range bar. [4] 3 D escribe the trend shown by the graph. [1 ] Identiy three mitochondria visible in the micrograph, either using labels or by describing their positions. [2 ] 4 Explain whether the results t the D avsonD anielli model or the S ingerNicolson model more closely. [2 ] Explain the evidence rom the micrograph that this cell was processing proteins in its cytoplasm. [2 ] 5 Explain the benet o plotting range bars on graphs. [2 ] 6 During this experiment the cells were incubated at 37 C . Suggest a reason or the researchers choosing this temperature. [1 ] b) E xplain the signicance o these granules in the investigation o membrane structure. 3 0 1 a) S tate what these granules are. 2 5 [2 ] [3 ] O ne o the membranes that surround the nucleus is visible on the let o the micrograph. D educe whether it is the inner or outer nuclear membrane. ( Always give your reasons when asked to deduce something.) [2 ] Extension questions on this topic can be ound at www.oxordsecondary. co.uk/ib- biology 29 1 7 8 9 The experiment was repeated at dierent temperatures. Figure 7 shows the results. Explain the trends shown in the graph or temperatures between 1 5 and 3 5 C . [2 ] Explain the trends shown in the graph or temperatures below 1 5 C . [2 ] When ATP synthesis was blocked in the cells, the mixing o the red and green markers still occurred. E xplain what conclusion can be drawn rom this. [1 ] 1 0 Predict, with reasons, the results o the experiment i it was repeated using cells rom arctic fsh rather than rom mice or humans. % of cells with markers fully mixed after 40 minutes C E LL B I O LO G Y 100 1 1 1 1 1 1 50 0 1 1 1 1 1 1 5 15 25 35 incubation temperature (C) Figure 7 Eect o temperature on the rate o diusion o fuorescent markers in membranes [1 ] Membrane proteins Membrane proteins are diverse in terms o structure, position in the membrane and unction. C ell membranes have a wide range o unctions. The primary unction is to orm a barrier through which ions and hydrophilic molecules cannot easily pass. This is carried out by the phospholipid bilayer. Almost all other unctions are carried out by proteins in the membrane. Six examples are listed in table 1 . functions o membrane proteins Hormone binding sites (also called hormone receptors) , or example the insulin receptor. Figure 8 shows an example. Immobilized enzymes with the active site on the outside, or example in the small intestine. Cell adhesion to orm tight junctions between groups o cells in tissues and organs. Cell-to-cell communication, or example receptors or neurotransmitters at synapses. Channels or passive transport to allow hydrophilic particles across by acilitated difusion. Pumps or active transport which use ATP to move particles across the membrane. Table 1 Figure 8 Hormone receptor ( purple) embedded in phospholipid bilayer (grey) . The hormone (blue/red) is thyroid stimulating hormone. G-protein (brown) conveys the hormone's message to the interior o the cell 30 B ecause o these varied unctions, membrane proteins are very diverse in structure and in their position in the membrane. They can be divided into two groups. Integral proteins are hydrophobic on at least part o their surace and they are thereore embedded in the hydrocarbon chains in the centre o the membrane. Many integral proteins are transmembrane they extend across the membrane, with hydrophilic parts proj ecting through the regions o phosphate heads on either side. 1 . 3 M e M b rAn e s tru ctu r e Peripheral proteins are hydrophilic on their surace, so are not embedded in the membrane. Most o them are attached to the surace o integral proteins and this attachment is oten reversible. Some have a single hydrocarbon chain attached to them which is inserted into the membrane, anchoring the protein to the membrane surace. Figure 9 includes examples o both types o membrane protein. Membranes all have an inner ace and an outer ace and membrane proteins are orientated so that they can carry out their unction correctly. For example, pump proteins in the plasma membranes o root cells in plants are orientated so that they pick up potassium ions rom the soil and pump them into the root cell. The protein content o membranes is very variable, because the unction o membranes varies. The more active a membrane, the higher is its protein content. Membranes in the myelin sheath around nerve fbres j ust act as insulators and have a protein content o only 1 8% . The protein content o most plasma membranes on the outside o the cell is about 5 0% . The highest protein contents are in the membranes o chloroplasts and mitochondria, which are active in photosynthesis and respiration. These have protein contents o about 75 % . dawig mma Draw the fuid mosaic model o membrane structure. The structure o membranes is ar too complicated or us to show all o it in ull detail in a drawing, but we can show our understanding o it using symbols to represent the molecules present. A diagram o membrane structure is shown in fgure 9. Figure 9 The diagram shows these components o a membrane: phospholipids; integral proteins; peripheral proteins; cholesterol. Membrane structure 31 1 C E LL B I O LO G Y Identiy which each component in the diagram is. Using similar symbols to represent the components draw the structure o a membrane, according to the fuid mosaic model, that contains these proteins: channels or acilitated diusion, pumps or active transport, immobilized enzymes and receptors or hormones or neurotransmitters. It is worth thinking about what you have been doing when you draw the fuid mosaic model o membrane structure. D rawings simpliy and interpret a structure or process. They are used in science as visual explanations. They show our understanding o a structure or process and not merely what it looks like. D rawings are based on models, hypotheses or theories. For example, when we show an animal tissue as a group o cells with lines to represent the plasma membranes, we are basing our drawing on the cell theory. A diagram in a book or scientic paper usually starts out as a drawing on paper by the author, which is tidied up to make it suitable or printing. It is now possible to use computer sotware, but a pencil and paper are perhaps still the best way to draw. No artistic ability is needed or scientic drawing, and all biologists can develop and improve their drawing skills. O course some biologists produce particularly good drawings. Some examples are shown in gure 1 0. Figure 10 Anatomical drawings by Leonardo da Vinci Cholesterol in membranes Cholesterol is a component of animal cell membranes. The two main components o cell membranes are phospholipids and proteins. Animal cell membranes also contain cholesterol. CH 3 CH 2 CH 2 CH 3 cholesterol CH 3 CH CH 2 CH CH 3 CH 3 C holesterol is a type o lipid, but it is not a at or oil. Instead it belongs to a group o substances called steroids. Most o a cholesterol molecule is hydrophobic so it is attracted to the hydrophobic hydrocarbon tails in the centre o the membrane, but one end o the cholesterol molecule has a hydroxyl ( - O H) group which is hydrophilic. This is attracted to the phosphate heads on the periphery o the membrane. C holesterol molecules are thereore positioned between phospholipids in the membrane. HO hydrophilic Figure 11 32 hydrophobic The structure of cholesterol The amount o cholesterol in animal cell membranes varies. In the membranes o vesicles that hold neurotransmitters at synapses as much o 3 0% o the lipid in the membrane is cholesterol. 1 . 4 M e M b r An e trAn s Po r t The role of cholesterol in membranes Cholesterol in mammalian membranes reduces membrane fuidity and permeability to some solutes. C ell membranes do not correspond exactly to any o the three states o matter. The hydrophobic hydrocarbon tails usually behave as a liquid, but the hydrophilic phosphate heads act more like a solid. O verall the membrane is fuid as components o the membrane are ree to move. The fuidity o animal cell membranes needs to be careully controlled. I they were too fuid they would be less able to control what substances pass through, but i they were not fuid enough the movement o the cell and substances within it would be restricted. C holesterol disrupts the regular packing o the hydrocarbon tails o phospholipid molecules, so prevents them crystallizing and behaving as a solid. However it also restricts molecular motion and thereore the fuidity o the membrane. It also reduces the permeability to hydrophilic particles such as sodium ions and hydrogen ions. D ue to its shape cholesterol can help membranes to curve into a concave shape, which helps in the ormation o vesicles during endocytosis. 1.4 Mma ap Understanding Particles move across membranes by simple diusion, acilitated diusion, osmosis and active transport. The fuidity o membranes allows materials to be taken into cells by endocytosis or released by exocytosis. Vesicles move materials within cells. Nature of science Experimental design: accurate quantitative measurements in osmosis experiments are essential. Applications Structure and unction o sodiumpotassium pumps or active transport and potassium channels or acilitated diusion in axons. Tissues or organs to be used in medical procedures must be bathed in a solution with the same osmolarity as the cytoplasm to prevent osmosis. Skills Estimation o osmolarity in tissues by bathing samples in hypotonic and hypertonic solutions. 33 1 C E LL B I O LO G Y outside of cell endocytosis Endocytosis The fuidity o membranes allows materials to be taken into cells by endocytosis or released by exocytosis. cell interior A vesicle is a small sac o membrane with a droplet o fuid inside. Vesicles are spherical and are normally present in eukaryotic cells. They are a very dynamic eature o cells. They are constructed, moved around and then deconstructed. This can happen because o the fuidity o membranes, which allows structures surrounded by a membrane to change shape and move. To orm a vesicle, a small region o a membrane is pulled rom the rest o the membrane and is pinched o. Proteins in the membrane carry out this process, using energy rom ATP. Vesicles can be ormed by pinching o a small piece o the plasma membrane o cells. The vesicle is ormed on the inside o the plasma membrane. It contains material that was outside the cell, so this is a method o taking materials into the cell. It is called endocytosis. Figure 1 shows how the process occurs. vesicle Figure 1 Endocytosis Vesicles taken in by endocytosis contain water and solutes rom outside the cell but they also oten contain larger molecules needed by the cell that cannot pass across the plasma membrane. For example, in the placenta, proteins rom the mothers blood, including antibodies, are absorbed into the etus by endocytosis. S ome cells take in large undigested ood particles by endocytosis. This happens in unicellular organisms including Amoeba and Paramecium. S ome types o white blood cells take in pathogens including bacteria and viruses by endocytosis and then kill them, as part o the bodys response to inection. Vesicle movement in cells Vesicles move materials within cells. Vesicles can be used to move materials around inside cells. In some cases it is the contents o the vesicle that need to be moved. In other cases it is proteins in the membrane o the vesicle that are the reason or vesicle movement. An example o moving the vesicle contents occurs in secretory cells. Protein is synthesized by ribosomes on the rough endoplasmic reticulum ( rE R) and accumulates inside the rE R. Vesicles containing the proteins bud o the rE R and carry them to the Golgi apparatus. The vesicles use with the Golgi apparatus, which processes the protein into its nal orm. When this has been done, vesicles bud o the Golgi apparatus and move to the plasma membrane, where the protein is secreted. In a growing cell, the area o the plasma membrane needs to increase. Phospholipids are synthesized next to the rER and become inserted into the rER membrane. Ribosomes on the rER synthesize membrane proteins which also become inserted into the membrane. Vesicles bud o the rE R and move to the plasma membrane. They use with it, each 34 1 . 4 M e M b r An e trAn s Po r t increasing the area of the plasma membrane by a very small amount. This method can also be used to increase the size of organelles in the cytoplasm such as lysosomes and mitochondria. Proteins are synthesized by ribosomes and then enter the rough endoplasmic reticulum ENDOCYTOSIS Part of the plasma membrane is pulled inwards A droplet of uid becomes enclosed when a vesicle is pinched o Vesicles can then move through the cytoplasm carrying their contents Vesicles bud o from the rER and carry the proteins to the Golgi apparatus The Golgi apparatus modies the proteins outside of cell exocytosis Vesicles bud o from the Golgi apparatus and carry the modied proteins to the plasma membrane vesicle EXOCYTOSIS Vesicles fuse with the plasma membrane The contents of the vesicle are expelled The membrane then attens out again Figure 2 Exocytosis The fuidity o membranes allows materials to be taken into cells by endocytosis or released by exocytosis. Vesicles can be used to release materials from cells. If a vesicle fuses with the plasma membrane, the contents are then outside the membrane and therefore outside the cell. This process is called exocytosis. D igestive enzymes are released from gland cells by exocytosis. The polypeptides in the enzymes are synthesized by the rER, processed in the Golgi apparatus and then carried to the membrane in vesicles for exocytosis. In this case the release is referred to as secretion, because a useful substance is being released, not a waste product. E xocytosis can also be used to expel waste products or unwanted materials. An example is the removal of excess water from the cells of unicellular organisms. The water is loaded into a vesicle, sometimes called a contractile vacuole, which is then moved to the plasma membrane for expulsion by exocytosis. This can be seen quite easily in Paramecium, using a microscope. Figure 4 shows a drawing of Paramecium showing a contractile vesicle at each end of the cell. cell interior Figure 3 Exocytosis contractile vesicle Simple difusion mouth Particles move across membranes by simple diusion, acilitated diusion, osmosis and active transport. endoplastule S imple diffusion is one of the four methods of moving particles across membranes. D iffusion is the spreading out of particles in liquids and gases that happens because the particles are in continuous random motion. More particles move from an area of higher concentration to an area of lower concentration than move in the opposite direction. There is therefore a net movement from the higher to the lower concentration a movement down the concentration gradient. Living endoplast contractile vesicle Figure 4 Drawing of Paramecium 35 1 C E LL B I O LO G Y toK can he same aa jusify muually exlusive nlusins? In an experiment to test whether NaCl can difuse through dialysis tubing, a 1% solution o NaCl was placed inside a dialysis tube and the tube was clamped shut. The tube containing the solution was immersed in a beaker containing water. A conductivity meter was inserted into the water surrounding the tubing. I the conductivity o the solution increases, then the NaCl is difusing out o the tubing. time /s 1 cnuiviy 10 mg l - 1 0 81.442 30 84.803 60 88.681 90 95.403 120 99.799 Noting the uncertainty o the conductivity probe, discuss whether the data supports the conclusion that NaCl is difusing out o the dialysis tubing. organisms do not have to use energy to make diusion occur so it is a passive process. S imple diusion across membranes involves particles passing between the phospholipids in the membrane. It can only happen i the phospholipid bilayer is permeable to the particles. Non- polar particles such as oxygen can diuse through easily. I the oxygen concentration inside a cell is reduced due to aerobic respiration and the concentration outside is higher, oxygen will pass into the cell through the plasma membrane by passive diusion. An example is shown in fgure 6 . Figure 5 Model o difusion with dots representing particles The centre o membranes is hydrophobic, so ions with positive or negative charges cannot easily pass through. Polar molecules, which have partial positive and negative charges over their surace, can diuse at low rates between the phospholipids o the membrane. Small polar particles such as urea or ethanol pass through more easily than large particles. the cornea has no blood supply so its cells obtain oxygen by simple diusion from the air high concentration of oxygen in the air air high concentration of oxygen in the tears that coat the cornea uid (tears) cell on outer surface of the cornea oxygen passes through the plasma membrane by simple diusion lower concentration of oxygen in the cornea cells due to aerobic respiration Figure 6 Passive difusion daa-base quesins: Difusion o oxygen in the cornea Oxygen concentrations were measured in the cornea o anesthetized rabbits at dierent distances rom the outer surace. These measurements were continued into the aqueous humor behind the cornea. The rabbits cornea is 400 micrometres (400 m) thick. The graph (fgure 7) shows the measurements. You may need to look at a diagram o eye structure beore answering the questions. The oxygen concentration in normal air is 2 0 kilopascals ( 2 0 kPa) . 36 1 C alculate the thickness o the rabbit cornea in millimetres. [1 ] 2 a) D escribe the trend in oxygen concentrations in the cornea rom the outer to the inner surace. [2 ] b) Suggest reasons or the trend in oxygen concentration in the cornea. [2 ] 3 a) C ompare the oxygen concentrations in the aqueous humor with the concentrations in the cornea. [2 ] 1 . 4 M e M b r An e trAn s Po r t 20 [2 ] 4 Using the data in the graph, evaluate diffusion as a method of moving substances in large multicellular organisms. [2 ] 5 a) Predict the effect of wearing contact lenses on oxygen concentrations in the cornea. [1 ] b) S uggest how this effect could be minimized. [1 ] 6 The range bars for each data point indicate how much the measurements varied. Explain the reason for showing range bars on the graph. [2 ] Concentration of oxygen/kPa b) Using the data in the graph, deduce whether oxygen diffuses from the cornea to the aqueous humor. 15 10 5 0 0 100 200 300 400 distance from outer surface of cornea/m Figure 7 Facilitated difusion Particles move across membranes by simple difusion, acilitated difusion, osmosis and active transport. Facilitated diffusion is one of the four methods of moving particles across membranes. Ions and other particles that cannot diffuse between phospholipids can pass into or out of cells if there are channels for them through the plasma membrane. These channels are holes with a very narrow diameter. The walls of the channel consist of protein. The diameter and chemical properties of the channel ensure that only one type of particle passes through, for example sodium ions, or potassium ions, but not both. B ecause these channels help particles to pass through the membrane, from a higher concentration to a lower concentration, the process is called facilitated diffusion. C ells can control which types of channel are synthesized and placed in the plasma membrane and in this way they can control which substances diffuse in and out. Figure 8 shows the structure of a channel for magnesium ions, viewed from the side and from the outside of the membrane. The structure of the protein making up the channel ensures that only magnesium ions are able to pass through the hole in the centre. (a) (b) Membrane Cytoplasm Osmosis Particles move across membranes by simple difusion, acilitated difusion, osmosis and active transport. Osmosis is one of the four methods of moving particles across membranes. Figure 8 Magnesium channel 37 1 C E LL B I O LO G Y Water is able to move in and out o most cells reely. S ometimes the number o water molecules moving in and out is the same and there is no net movement, but at other times more molecules move in one direction or the other. This net movement is osmosis. Figure 9 O smosis is due to dierences in the concentration o substances dissolved in water ( solutes) . Substances dissolve by orming intermolecular bonds with water molecules. These bonds restrict the movement o the water molecules. Regions with a higher solute concentration thereore have a lower concentration o water molecules ree to move than regions with a lower solute concentration. B ecause o this there is a net movement o water rom regions o lower solute concentration to regions with higher solute concentration. This movement is passive because no energy has to be expended directly to make it occur. O smosis can happen in all cells because water molecules, despite being hydrophilic, are small enough to pass though the phospholipid bilayer. Some cells have water channels called aquaporins, which greatly increase membrane permeability to water. E xamples are kidney cells that reabsorb water and root hair cells that absorb water rom the soil. At its narrowest point, the channel in an aquaporin is only slightly wider than water molecules, which thereore pass through in single fle. Positive charges at this point in the channel prevent protons (H+ ) rom passing through. Active transport Particles move across membranes by simple difusion, acilitated difusion, osmosis and active transport. Active transport is one o the our methods o moving particles across membranes. C ells sometimes take in substances, even though there is already a higher concentration inside than outside. The substance is absorbed against the concentration gradient. Less commonly, cells sometimes pump substances out, even though there is already a larger concentration outside. This type o movement across membranes is not diusion and energy is needed to carry it out. It is thereore called active transport. Most active transport uses a substance called ATP as the energy supply or this process. E very cell produces its own supply o ATP by cell respiration. Active transport is carried out by globular proteins in membranes, usually called pump proteins. The membranes o cells contain many dierent pump proteins allowing the cell to control the content o its cytoplasm precisely. Figure 10 38 Action of a pump protein Figure 1 0 illustrates how a pump protein works. The molecule or ion enters the pump protein and can reach as ar as a central chamber. A conormational change to the protein takes place using energy rom ATP. Ater this, the ion or molecule can pass to the opposite side o the membrane and the pump protein returns to its original conormation. The pump protein shown transports Vitamin B 1 2 into E. coli. 1 . 4 M e M b r An e trAn s Po r t daa-a qui: Phosphate absorption in barley roots Roots were cut off from barley plants and were used to investigate phosphate absorption. Roots were placed in phosphate solutions and air was bubbled through. The phosphate concentration was the same in each case, but the percentage of oxygen and nitrogen was varied in the air bubbled through. The rate of phosphate absorption was measured. Table 1 shows the results. 1 2 Describe the effect of reducing the oxygen concentration below 21 .0% on the rate of phosphate absorption by roots. You should only use information from the table in your answer. [3 ] Explain the effect of reducing the oxygen percentage from 2 1 .0 to 0.1 on phosphate absorption. In your answer you should use as much biological understanding as possible of how cells absorb mineral ions. 4 Table 1 0 .4 0 .3 [3 ] Phosphate absorption /mol g2 1 h 2 1 0 .2 0 .1 0 An experiment was done to test which method of membrane transport was used by the roots to absorb phosphate. Roots were placed in the phosphate solution as before, with 2 1 .0% oxygen bubbling through. Varying concentrations of a substance called D NP were added. D NP blocks the production of ATP by aerobic cell respiration. Figure 1 1 shows the results of the experiment. 3 oxyg nig Phpha /% /% api/ml g1 h 1 0.1 99.9 0.07 0.3 99.7 0.15 0.9 99.1 0.27 2.1 97.1 0.32 21.0 79.0 0.33 0 2 4 6 8 10 DNP concentration / mmol dm 2 3 Figure 11 Efect o DNP concentration on phosphate absorption D educe, with a reason, whether the roots absorbed the phosphate by diffusion or active transport. [2 ] D iscuss the conclusions that can be drawn from the data in the graph about the method of membrane transport used by the roots to absorb phosphate. [2 ] Active transport of sodium and potassium in axons Structure and function of sodiumpotassium pumps for active transport. An axon is part of a neuron ( nerve cell) and consists of a tubular membrane with cytoplasm inside. Axons can be as narrow as one micrometre in diameter, but as long as one metre. Their function is to convey messages rapidly from one part of the body to another in an electrical form called a nerve impulse. A nerve impulse involves rapid movements of sodium and then potassium ions across the axon membrane. These movements occur by facilitated diffusion through sodium and potassium channels. They occur because of concentration gradients between the inside and outside of the axon. The concentration gradients are built up by active transport, carried out by a sodium potassium pump protein. The sodiumpotassium pump follows a repeating cycle of steps that result in three sodium ions being pumped out of the axon and two potassium ions being pumped in. Each time the pump goes round this cycle it uses one ATP. The cycle consists of these steps: 1 The interior of the pump is open to the inside of the axon; three sodium ions enter the pump and attach to their binding sites. 2 ATP transfers a phosphate group from itself to the pump; this causes the pump to change shape and the interior is then closed. 3 The interior of the pump opens to the outside of the axon and the three sodium ions are released. 39 1 C E LL B I O LO G Y 4 Two potassium ions from outside can then enter and attach to their binding sites. 5 B inding of potassium causes release of the phosphate group; this causes the pump to change shape again so that it is again only open to the inside of the axon. 1 6 The interior of the pump opens to the inside of the axon and the two potassium ions are released; sodium ions can then enter and bind to the pump again ( stage 1 ) . 2 3 p p ATP ADP 4 5 6 p p Figure 12 Active transport in axons Facilitated difusion o potassium in axons Structure and unction o sodiumpotassium pumps or active transport and potassium channels or acilitated difusion in axons. A nerve impulse involves rapid movements of sodium and then potassium ions across the axon membrane. These movements occur by facilitated diffusion through sodium and potassium channels. Potassium channels will be described here as a special example of facilitated diffusion. E ach potassium channel consists of four protein subunits with a narrow pore between them that allows potassium ions to pass in either direction. The pore is 0.3 nm wide at its narrowest. 40 Potassium ions are slightly smaller than 0 . 3 nm, but when they dissolve they become bonded to a shell of water molecules that makes them too large to pass through the pore. To pass through, the bonds between the potassium ion and the surrounding water molecules are broken and bonds form temporarily between the ion and a series of amino acids in the narrowest part of the pore. After the potassium ion has passed through this part of the pore, 1 . 4 M e M b r An e trAn s Po r t it can again become associated with a shell o water molecules. Other positively charged ions that we might expect to pass through the pore are either too large to t through or are too small to orm bonds with the amino acids in the narrowest part o the pore, so they cannot shed their shell o water molecules. This explains the specicity o the pump. Potassium channels in axons are voltage gated. Voltages across membranes are due to an imbalance o positive and negative charges across the membrane. I an axon has relatively more positive charges outside than inside, potassium channels are closed. At one stage during a nerve impulse there are relatively more positive charges inside. This causes potassium channels to open, allowing potassium ions to diuse through. However, the channel rapidly closes again. This seems to be due to an extra globular protein subunit or ball, attached by a fexible chain o amino acids. The ball can t inside the open pore, which it does within milliseconds o the pore opening. The ball remains in place until the potassium channel returns to its original closed state. This is shown in gure 1 3 . 1 channel closed + + + 2 channel briey open - - - + + + + + +++ - - - - - + + + + + + + + + ++ + + - - - chain ball net negative charge inside the axon and net positive charge outside net negative charge K+ ions - - - + + + + + + + inside of axon outside net positive charge 3 channel closed by ball and chain - - - + + + + + + + + hydrophobic core of the membrane - + + + + + + - + - + hydrophilic outer parts of the membrane Figure 13 eimai f mlaiy Estimation of osmolarity in tissues by bathing samples in hypotonic and hypertonic solutions. O smosis is due to solutes that orm bonds with water. These solutes are osmotically active. Glucose, sodium ions, potassium ions and chloride ions are all osmotically active and solutions o them are oten used in osmosis experiments. C ells contain many dierent osmotically active solutes. The osmolarity o a solution is the total concentration o osmotically active solutes. The units or measuring it are osmoles or milliosmoles ( mO sm) . The normal osmolarity o human tissue is about 3 00 mO sm. An isotonic solution has the same osmolarity as a tissue. A hypertonic solution has a higher osmolarity and a hypotonic solution has a lower osmolarity. I samples o a tissue are bathed in hypertonic and hypotonic solutions, and 41 1 C E LL B I O LO G Y measurements are taken to fnd out whether water enters or leaves the tissue, it is possible to deduce what concentration o solution would be data-base questions: Osmosis in plant tissues isotonic and thereore fnd out the osmolarity o the tissue. The data- based questions below give the results rom an experiment o this type. 4 I samples o plant tissue are bathed in salt or sugar solutions or a short time, any increase or decrease in mass is due almost entirely to water entering or leaving the cells by osmosis. Figure 1 4 shows the percentage mass change o our tissues, when they were bathed in salt solutions o dierent concentrations. 1 a) S tate whether water moved into or out o the tissues at 0.0 mol dm 3 sodium chloride solution. [1 ] 40 + 3 The experiment in the data- based question can be repeated using potato tubers, or any other plant tissue rom around the world that is homogeneous and tough enough to be handled without disintegrating. D iscuss with a partner or group how you could do the ollowing things: 42 1 D ilute a 1 mol dm 3 sodium chloride solution to obtain the concentrations shown on the graph. 2 O btain samples o a plant tissue that are similar enough to each other to give comparable results. 3 Ensure that the surace o the tissue samples is dry when fnding their mass, both at the start and end o the experiment. 4 Ensure that all variables are kept constant, apart rom salt concentration o the bathing solution. + + + + + + 20 % Mass change PINE KERNEL + Sodium chloride concentration / mol dm 2 3 10 0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .7 0 .8 0 .9 1.0 BUTTERNUT SQUASH SWEET POTATO 2 10 2 20 2 30 D educe which tissue had the lowest solute concentration in its cytoplasm. Include how you reached your conclusion in your answer. [2 ] S uggest reasons or the dierences in solute concentration between the tissues. [3 ] + 30 b) State whether water moved into or out o the tissues at 1 .0 mol dm 3 sodium chloride solution. [2 ] 2 Explain the reasons or using percentage mass change rather than the actual mass change in grams in this type o experiment. [2 ] 2 40 CACTUS 2 50 Figure 14 Mass changes in plant tissues bathed in salt solutions 5 Leave the tissue in the solutions or long enough to get a signifcant mass change, but not so long that another actor aects the mass, such as decomposition! 6 You might choose to be more inventive in your experimental approach. Figure 1 5 gives one idea or measuring changes to the turgidity o plant tissue, but other methods could be used. plant tissue angle gives measure of turgidity weight Figure 15 Method of plant tissue of assessing turgidity 1 . 4 M e M b r An e trAn s Po r t expimal dig Experimental design: accurate quantitative measurements in osmosis experiments are essential. An ideal experiment gives results that have only one reasonable interpretation. C onclusions can be drawn from the results without any doubts or uncertainties. In most experiments there are some doubts and uncertainties, but if the design of an experiment is rigorous, these can be minimized. The experiment then provides strong evidence for or against a hypothesis. This checklist can be used when designing an experiment: Results should if possible be quantitative as these give stronger evidence than descriptive results. Measurements should be as accurate as possible, using the most appropriate and best quality meters or other apparatus. Repeats are needed, because however accurately quantitative measurements are taken biological samples are variable. All factors that might affect the results of the experiment must be controlled, with only the factors under investigation being allowed to vary and all other factors remaining constant. After doing an experiment the design can be evaluated using this checklist. The evaluation might lead to improvements to the design that would have made the experiment more rigorous. Figure 16 Replicates are needed for each treatment in a rigorous experiment If you have done an osmosis experiment in which samples of plant tissue are bathed in solutions of varying solute concentration, you can evaluate its design. If you did repeats for each concentration of solution, and the results were very similar to each other, your results were probably reliable. Designing osmosis experiments Rigorous experimental design is needed to produce reliable results: how can accurate quantitative measurements be obtained in osmosis experiments? The osmolarity of plant tissues can be investigated in many ways. Figure 1 7 shows some red onion cells that had been placed in a sodium chloride solution. The following method can be used to observe the consequences of osmosis in red onion cells. 1 Peel off some epidermis from the scale of a red onion bulb. 2 C ut out a sample of it, about 5 5 mm. 3 Mount the sample in a drop of distilled water on a microscope slide, with a cover slip. Figure 17 Micrograph of red onion cells placed in salt solution 43 1 C E LL B I O LO G Y 4 O bserve using a microscope. The cytoplasm should fll the space inside the cell wall, with the plasma membrane pushed up against it. 5 Mount another sample o epidermis in sodium chloride solutions with concentration o 0.5 mol dm - 3 or 3 % . I water leaves the cells by osmosis and the volume o cytoplasm is reduced, the plasma membrane pulls away rom the cell wall, as shown in Figure 1 7. Plant cells with their membranes pulled away rom their cell walls are plasmolysed and the process is plasmolysis. This method can be used to help design an experiment to fnd out the osmolarity o onion cells or other cells in which the area occupied by the cytoplasm can easily be seen. The checklist in the previous section can be used to try to ensure that the design is rigorous. Preventing osmosis in excised tissues and organs Tissues or organs to be used in medical procedures must be bathed in a solution with the same osmolarity as the cytoplasm to prevent osmosis. Animal cells can be damaged by osmosis. Figure 1 8 shows blood cells that have been a) b) Figure 18 c) Blood cells bathed in solutions o diferent solute concentration In a solution with higher osmolarity ( a hypertonic solution) , water leaves the cells by osmosis so their cytoplasm shrinks in volume. The area o plasma membrane does not change, so it develops indentations, which are sometimes called crenellations. In a solution with lower osmolarity ( hypotonic) , the cells take in water by osmosis and swell up. They may eventually burst, leaving ruptured plasma membranes called red cell ghosts. B oth hypertonic and hypotonic solutions thereore damage human cells, but in a solution with same osmolarity as the cells ( isotonic) , water molecules enter and leave the cells at the same rate so they remain healthy. It is thereore important or any human tissues and organs to be bathed in an isotonic solution during medical procedures. Usually an isotonic sodium chloride solution is 44 bathed in solutions with ( a) the same osmolarity, ( b) higher osmolarity and ( c) lower osmolarity. used, which is called normal saline. It has an osmolarity o about 3 00 mO sm ( milliO smoles) . Normal saline is used in many medical procedures. It can be: saely introduced to a patients blood system via an intravenous drip. used to rinse wounds and skin abrasions. used to keep areas o damaged skin moistened prior to skin grats. used as the basis or eye drops. rozen to the consistency o slush or packing hearts, kidneys and other donor organs that have to be transported to the hospital where the transplant operation is to be done. 1 . 5 tH e o rI GI n o f ce lls Figure 19 Donor liver packed in an isotonic medium, surrounded by isotonic slush. There is a worldwide shortage of donor organs in most countries it is possible to register as a possible future donor 1.5 th igi Understanding Cells can only be ormed by division o pre-existing cells. The frst cells must have arisen rom non-living material. The origin o eukaryotic cells can be explained by the endosymbiotic theory. Applications Evidence rom Pasteurs experiments that spontaneous generation o cells and organisms does not now occur on Earth. Nature of science Testing the general principles that underlie the natural world: the principle that cells only come rom pre-existing cells needs to be verifed. Cell division and the origin of cells Cells can only be ormed by division o pre-existing cells. Since the 1 880s there has been a theory in biology that cells can only be produced by division of a pre-existing cell. The evidence for this hypothesis is very strong and is discussed in the nature of science panel below. The implications of the hypothesis are remarkable. If we consider the trillions of cells in our bodies, each one was formed when a previously 45 1 C E LL B I O LO G Y toK Wha d we gain, and wha d we le, when we name mehing? When Dr Craig Venters team announced that they had succeeded in transplanting the synthetic genome rom one bacterium into another bacterium in the journal Science some ethicists responded by questioning the language o calling it the creation o a synthetic cell: The science is ying 30,000 eet over the publics understanding ... Scientists can be their own worst enemy by using words like clone or synthetic lie. Glenn Mcgee, funde f Ameican Junal f biehic Frankly, hes describing it in a way thats drumming up controversy more than characterising it accurately. His claim that weve got the frst selreplicating lie orm whose parent is a computer, thats just silly. It misuses the word parent. The advance here needs to be described in sane and accurate ways. What he's managed to do is synthesise a genome much larger than any genome thats been synthesised rom scratch beore. Gegy Kaenick, Haing Iniue reeach schla existing cell divided in two. B eore that all o the genetic material in the nucleus was copied so that both cells ormed by cell division had a nucleus with a ull complement o genes. We can trace the origin o cells in the body back to the frst cell the zygote that was the start o our lives, produced by the usion o a sperm and an egg. S perm and egg cells were produced by cell division in our parents. We can trace the origins o all cells in our parents bodies back to the zygote rom which they developed, and then continue this process over the generations o our human ancestors. I we accept that humans evolved rom pre- existing ancestral species, we can trace the origins o cells back through hundreds o millions o years to the earliest cells on Earth. There is thereore a continuity o lie rom its origins on Earth to the cells in our bodies today. In 2 01 0 there were reports that biologists had created the frst artifcial cell, but this cell was not entirely new. The base sequence o the D NA o a bacterium ( Mycoplasma mycoides) was synthesized artifcially, with a ew deliberate changes. This D NA was transerred to pre- existing cells o a dierent type o bacterium ( Mycoplasma capricolum) , which was eectively converted into Mycoplasma mycoides. This process was thereore an extreme orm o genetic modifcation and the creation o entirely new cells remains an insuperable challenge at the moment. Aciviy the l f silphium The Greek coin in fgure 2 depicts a Silphium plant, which grew in a small part o what is now Libya and was highly prized or its medicinal uses, especially as a birth control agent. It seems to have been so widely collected that within a ew hundred years o the ancient Greeks colonizing North Arica it had become extinct. Rather than arising again spontaneously, Silphium has remained extinct and we cannot now test its contraceptive properties scientifcally. How can we prevent the loss o other plants that could be o use to us? Figure 2 Figure 1 46 Synthetic Mycoplasma bacteria An ancient Greek coin, showing Silphium 1 . 5 tH e o rI GI n o f ce lls Spontaneous generation and the origin of cells Veriying the general principles that underlie the natural world: the principle that cells only come rom pre-existing cells needs to be verifed. Spontaneous generation is the ormation o living organisms rom non-living matter. The Greek philosopher and botanist Theophrastus reported that a plant called Silphium had sprung up rom soil where it was not previously present and described this as an example o spontaneous generation. Aristotle wrote about insects being ormed rom the dew alling on leaves or rom the hair, fesh or aeces o animals. In the 1 6th century the GermanSwiss botanist and astrologer Paracelsus quoted observations o spontaneous generation o mice, rogs and eels rom water, air or decaying matter. It is easy to see how ideas o spontaneous generation could have persisted when cells and microorganisms had not been discovered and the nature o sexual reproduction was not understood. From the 1 7th century onwards biologists carried out experiments to test the theory that lie could arise rom non-living matter. Francesco Redi showed that maggots only developed in rotting meat i fies were allowed to come into contact with it. Lazzaro Spallanzani boiled soup in eight containers, then sealed our o them and let the others open to the air. Organisms grew in the containers let open but not in the others. S ome biologists remained convinced that spontaneous generation could occur i there was access to the air. Louis Pasteur responded by carrying out careully designed experiments with swan- necked fasks, which established beyond reasonable doubt that spontaneous generation o lie does not now occur. Pasteurs experiments are described in the next section o this sub- topic. Apart rom the evidence rom the experiments o Pasteur and others, there are other reasons or biologists universally accepting that cells only come rom pre- existing cells: A cell is a highly complex structure and no natural mechanism has been suggested or producing cells rom simpler subunits. No example is known o increases in the number o cells in a population, organism or tissue without cell division occurring. Viruses are produced rom simpler subunits but they do not consist o cells, and they can only be produced inside the host cells that they have inected. Spontaneous generation and Pasteurs experiments Evidence rom Pasteurs experiments that spontaneous generation o cells and organisms does not now occur on Earth. Louis Pasteur made a nutrient broth by boiling water containing yeast and sugar. He showed that i this broth was kept in a sealed fask, it remained unchanged, and no ungi or other organisms appeared. He then passed air though a pad o cotton wool in a tube, to lter out microscopic particles rom the air, including bacteria and the spores o ungi. I the pad o cotton wool was placed in broth in a sealed fask, within 3 6 hours, there were large number o microorganisms in the broth and mould grew over its surace. The most amous o Pasteurs experiments involved the use o swan-necked fasks. He placed samples o broth in fasks with long necks and then melted the glass o the necks and bent it into a variety o shapes, shown in gure 3 . Pasteur then boiled the broth in some o the fasks to kill any organisms present but let others unboiled as controls. Fungi and other organisms soon appeared in the unboiled fasks but not in the boiled ones, even ater long periods o time. The broth in the fasks was in contact with air, which it had been suggested was needed or spontaneous generation, yet no spontaneous generation occurred. Pasteur snapped the necks o some o the fasks to leave a shorter vertical neck. Organisms were soon apparent in these fasks and decomposed the broth. 47 1 C E LL B I O LO G Y Pasteur published his results in 1 860 and subsequently repeated them with other liquids including urine and milk, with the same results. He concluded that the swan necks prevented organisms rom the air getting into the broth or other liquids and that no organisms appeared spontaneously. His experiments convinced most biologists, both at the time o publication and since then. Origin o the frst cells The frst cells must have arisen rom non-living material. I we trace back the ancestry o cells over billions o years, we must eventually reach the earliest cells to have existed. These were the frst living things on Earth. Unless cells arrived on E arth rom somewhere else in the universe, they must have arisen rom non- living material. This is a logical conclusion, but it gives perhaps the hardest question o all or biologists to answer: how could a structure as complex as the cell have arisen by natural means rom non-living material? It has sometimes been argued that complex structures cannot arise by evolution, but there is evidence that this can happen in a series o stages over long periods o time. Living cells may have evolved over hundreds o millions o years. There are hypotheses or how some o the main stages could have occurred. Figure 3 Drawings o Pasteurs swan-necked fasks 1. Production of carbon compounds such as sugars and amino acids 2. Assembly of carbon compounds into polymers S tanley Miller and Harold Urey passed steam through a mixture o methane, hydrogen and ammonia. The mixture was thought to be representative o the atmosphere o the early E arth. E lectrical discharges were used to simulate lightning. They ound that amino acids and other carbon compounds needed or lie were produced. A possible site or the origin o the frst carbon compounds is around deep- sea vents. These are cracks in the Earths surace, characterized by gushing hot water carrying reduced inorganic chemicals such as iron sulphide. These chemicals represent readily accessible supplies o energy, a source o energy or the assembly o these carbon compounds into polymers. water vapour ammonia (NH 3 ) methane (CH 4) hydrogen (H 2 ) electrode condenser cold water in cooled water containing organic compounds Figure 5 Deep sea sample taken for chemical analysis Figure 4 Miller and 48 Ureys apparatus vents 1 . 5 tH e o rI GI n o f ce lls 3. Formation of membranes I phospholipids or other amphipathic carbon compounds were among the frst carbon compounds, they would have naturally assembled into bilayers. Experiments have shown that these bilayers readily orm vesicles resembling the plasma membrane o a small cell. This would have allowed dierent internal chemistry rom that o the surroundings to develop. 4. Development of a mechanism for inheritance Living organisms currently have genes made o D NA and use enzymes as catalysts. To replicate D NA and be able to pass genes on to ospring, enzymes are needed. However, or enzymes to be made, genes are needed. The solution to this conundrum may have been an earlier phase in evolution when RNA was the genetic material. It can store inormation in the same way as D NA but it is both sel- replicating and can itsel act as a catalyst. Figure 6 Liposomes Endosymbiosis and eukaryotic cells The origin o eukaryotic cells can be explained by the endosymbiotic theory. The theory o endosymbiosis helps to explain the evolution o eukaryotic cells. It states that mitochondria were once ree- living prokaryotic organisms that had developed the process o aerobic cell respiration. Larger prokaryotes that could only respire anaerobically took them in by endocytosis. Instead o killing and digesting the smaller prokaryotes they allowed them to continue to live in their cytoplasm. As long as the smaller prokaryotes grew and divided as ast as the larger ones, they could persist indefnitely inside the larger cells. According to the theory o endosymbiosis they have persisted over hundreds o millions o years o evolution to become the mitochondria inside eukaryotic cells today. The larger prokaryotes and the smaller aerobically respiring ones were in a symbiotic relationship in which both o them benefted. This is known as a mutualistic relationship. The smaller cell would have been supplied with ood by the larger one. The smaller cell would have carried out aerobic respiration to supply energy efciently to the larger cell. Natural selection thereore avoured cells that had developed this endosymbiotic relationship. The endosymbiotic theory also explains the origin o chloroplasts. I a prokaryote that had developed photosynthesis was taken in by a larger cell and was allowed to survive, grow and divide, it could have developed into the chloroplasts o photosynthetic eukaryotes. Again, both o the organisms in the endosymbiotic relationship would have benefted. Aiviy Wh did i bgi? Erasmus Darwin was Charles Darwins grandather. In a poem entitled The Temple o Nature, published in 1803, he tells us how and where he believed lie to have originated: Organic Lie began beneath the waves ... Hence without parent by spontaneous birth Rise the frst specks o animated earth Has Erasmus Darwins hypothesis that lie began in the sea been alsifed? 49 1 C E LL B I O LO G Y original ancestral prokaryote Activity evolution of the nucleus Bangiomorpha and the origins of sex. The frst known eukaryote and frst known multicellular organism is Bangiomorpha pubescens. Fossils o this red alga were discovered in 1,200 million year old rocks rom northern Canada. It is the frst organism known to produce two dierent types o gamete a larger sessile emale gamete and a smaller motile male gamete. Bangiomorpha is thereore the frst organism known to reproduce sexually. It seems unlikely that eukaryote cell structure, multicellularity and sexual reproduction evolved simultaneously. What is the most likely sequence or these landmarks in evolution? evolution of photosynthesis evolution of aerobic respiration evolution of linear chromosomes, mitosis and meiosis endocytosis produces mitochondria endocytosis to produce chloroplasts evolution of plant cells plant cell (eukaryotic) Figure 7 evolution of animal cells animal cell (eukaryotic) Endosymbiosis Although no longer capable of living independently, chloroplasts and mitochondria both have features that suggest they evolved from independent prokaryotes: 50 They have their own genes, on a circular D NA molecule like that of prokaryotes. They have their own 70S ribosomes of a size and shape typical of some prokaryotes. They transcribe their D NA and use the mRNA to synthesize some of their own proteins. They can only be produced by division of pre- existing mitochondria and chloroplasts. 1 . 6 ce ll d I VI s I o n 1.6 c ivii Understanding Mitosis is division o the nucleus into two genetically identical daughter nuclei. Chromosomes condense by supercoiling during mitosis. Cytokinesis occurs ater mitosis and is dierent in plant and animal cells. Interphase is a very active phase o the cell cycle with many processes occurring in the nucleus and cytoplasm. Cyclins are involved in the control o the cell cycle. Mutagens, oncogenes and metastasis are involved in the development o primary and secondary tumours. Applications The correlation between smoking and incidence o cancers. Skills Identifcation o phases o mitosis in cells viewed with a microscope. Determination o a mitotic index rom a micrograph. Nature of science Serendipity and scientifc discoveries: the discovery o cyclins was accidental. The role of mitosis Mitosis is division o the nucleus into two genetically identical daughter nuclei. The nucleus of a eukaryotic cell can divide to form two genetically identical nuclei by a process called mitosis. Mitosis allows the cell to divide into two daughter cells, each with one of the nuclei and therefore genetically identical to the other. B efore mitosis can occur, all of the D NA in the nucleus must be replicated. This happens during interphase, the period before mitosis. E ach chromosome is converted from a single D NA molecule into two identical D NA molecules, called chromatids. D uring mitosis, one of these chromatids passes to each daughter nucleus. Mitosis is involved whenever cells with genetically identical nuclei are required in eukaryotes: during embryonic development, growth, tissue repair and asexual reproduction. Although mitosis is a continuous process, cytologists have divided the events into four phases: prophase, metaphase, anaphase and telophase. The events that occur in these phases are described in a later section of this sub- topic. Hydra viridissima with a small new polyp attached, produced by asexual reproduction involving mitosis Figure 1 51 1 C E LL B I O LO G Y Interphase Activity There is a limit to how many times most cells in an organism can undergo mitosis. Cells taken rom a human embryo will only divide between 40 and 60 times, but given that the number o cells doubles with each division, it is easily enough to produce an adult human body. There are exceptions where much greater numbers o divisions can occur, such as the germinal epithelium in the testes. This is a layer o cells that divides to provide cells used in sperm production. Discuss how many times the cells in this layer might need to divide during a man's lie. Mitosis in esi Cy t o k I N TE R PH A SE s Each of the 1 chromosomes is duplicated Cellular contents, apart from the chromosomes are duplicated. G G0 Figure 2 The cell cycle is the seque nce o events b etween one cell division and the next. It has two main phases: interphase and cell division. Interphase is a very active phase in the lie o a cell when many metabolic reactions occur. S ome o these, such as the reactions o cell respiration, also occur during cell division, b ut D NA replication in the nucleus and protein synthesis in the cytoplasm only happen during interphase. During interphase the numbers o mitochondria in the cytoplasm increase. This is due to the growth and division o mitochondria. In plant cells and algae the numbers o chloroplasts increase in the same way. They also synthesize cellulose and use vesicles to add it to their cell walls. Interphase consists o three phases, the G 1 phase, S phase and G 2 phase. In the S phase the cell replicates all the genetic material in its nucleus, so that ater mitosis both the new cells have a complete set o genes. Some do not progress beyond G 1 , because they are never going to divide so do not need to prepare or mitosis. They enter a phase called G 0 which may be temporary or permanent. Supercoiling of chromosomes G2 S Interphase is a very active phase o the cell cycle with many processes occurring in the nucleus and cytoplasm. The cell cycle Chromosomes condense by supercoiling during mitosis. During mitosis, the two chromatids that make up each chromosome must be separated and moved to opposite poles o the cell. The DNA molecules in these chromosomes are immensely long. Human nuclei are on average less than 5 m in diameter but D NA molecules in them are more than 5 0,000 m long. It is thereore essential to package chromosomes into much shorter structures. This process is known as condensation o chromosomes and it occurs during the frst stage o mitosis. Condensation occurs by means repeatedly coiling the DNA molecule to make the chromosome shorter and wider. This process is called supercoiling. Proteins called histones that are associated with DNA in eukaryote chromosomes help with supercoiling and enzymes are also involved. Phases of mitosis Identifcation o phases o mitosis in cells viewed with a microscope. There are large numbers o dividing cells in the tips o growing roots. I root tips are treated chemically to allow the cells to be separated, they can be squashed to orm a single layer o cells on a microscope slide. S tains that bind to D NA are used to make the chromosomes visible and stages o mitosis can then be observed using a microscope. 52 To be able to identiy the our stages o mitosis, it is necessary to understand what is happening in them. Ater studying the inormation in this section you should be able to observe dividing cells using a microscope or in a micrograph and assign them to one o the phases. 1 . 6 ce ll d I VI s I o n Prophase The chromosomes become shorter and atter by coiling. To become short enough they have to coil repeatedly. This is called supercoiling. The nucleolus breaks down. Microtubules grow rom structures called microtubule organizing centres (MTOC) to orm a spindle-shaped array that links the poles o the cell. At the end o prophase the nuclear membrane breaks down. Interphase chromosomes are Prophase nucleoli visible visible inside the nuclear membrane centromere MTOC in the nucleus but no individual chromosomes microtubules nuclear envelope disintegrates chromosome consisting of two sister chromatids Early prophase spindle microtubules Late prophase Metaphase Microtubules continue to grow and attach to the centromeres on each chromosome. The two attachment points on opposite sides o each centromere allow the chromatids o a chromosome to attach to microtubules rom diferent poles. The microtubules are all put under tension to test whether the attachment is correct. This happens by shortening o the microtubules at the centromere. I the attachment is correct, the chromosomes remain on the equator o the cell. Metaphase plate equator mitotic spindle Metaphase chromosomes Metaphase aligned on the equator and not inside a nuclear membrane Anaphase At the start o anaphase, each centromere divides, allowing the pairs o sister chromatids to separate. The spindle microtubules pull them rapidly towards the poles o the cell. Mitosis produces two genetically identical nuclei because sister chromatids are pulled to opposite poles. This is ensured by the way that the spindle microtubules were attached in metaphase. Daughter chromosomes separate Anaphase two groups of V-shaped chromatids pointing to the two poles Anaphase 53 1 C E LL B I O LO G Y Telophase The chromatids have reached the poles and are now called chromosomes. At each pole the chromosomes are pulled into a tight group near the MTOC and a nuclear membrane reforms around them. The chromosomes uncoil and a nucleolus is formed. By this stage of mitosis the cell is usually already dividing and the two daughter cells enter interphase again. Telophase tight groups of chromosomes at each pole, new cell wall forming at the equator Interphase nucleoli visible inside the nuclear membranes but not individual chromosomes Cleavage furrow Nuclear envelope forming Telophase data-base questions: Centromeres and telomeres Figure 3 and the other micrographs on the preceeding pages show cells undergoing mitosis. In gure 3 , D NA has been stained blue. The centromeres have been stained with a red fuorescent dye. At the ends o the chromosomes there are structures called telomeres. These have been stained with a green fuorescent dye. 1 D educe the stage o mitosis that the cell was in, giving reasons or your answer. [3 ] 2 The cell has an even number o chromosomes. a) S tate how many chromosomes there are in this cell. b) E xplain the reason or body cells in plants and animals having an even number o chromosomes. Figure 3 Cell in mitosis c) [1 ] [2 ] In the micrograph o a cell in interphase, the centromeres are on one side o the nucleus and the telomeres are on the other side. S uggest reasons or this. [2 ] d) An enzyme called telomerase lengthens the telomeres, by adding many short repeating base sequences o DNA. This enzyme is only active in the germ cells that are used to produce gametes. When DNA is replicated during the cell cycle in body cells, the end o the telomere cannot be replicated, so the telomere becomes shorter. Predict the consequences or a plant or animal o the shortening o telomeres. [2 ] 54 1 . 6 ce ll d I VI s I o n The mitotic index Determination o a mitotic index rom a micrograph. The mitotic index is the ratio between the number o cells in mitosis in a tissue and the total number o observed cells. It can be calculated using this equation: number o cells in mitosis Mitotic index = ___ total number o cells Figure 4 is a micrograph o cells rom a tumour that has developed rom a Leydig cell in the testis. The mitotic index or this tumour can be calculated i the total number o cells in the micrograph is counted and also the number o cells in meiosis. To fnd the mitotic index o the part o a root tip where cells are prolierating rapidly, these instructions can be used: Obtain a prepared slide o an onion or garlic root tip. Find and examine the meristematic region, i.e. a region o rapid cell division. C reate a tally chart. C lassiy each o about a hundred cells in this region as being either in interphase or in any o the stages o mitosis. Use this data to calculate the mitotic index. Figure 4 Cells undergoing mitosis in a Leydig cell tumour Cytokinesis Cytokinesis occurs ater mitosis and is diferent in plant and animal cells. C ells can divide ater mitosis when two genetically identical nuclei are present in a cell. The process o cell division is called cytokinesis. It usually begins beore mitosis has actually been completed and it happens in a dierent way in plant and animal cells. In animal cells the plasma membrane is pulled inwards around the equator o the cell to orm a cleavage urrow. This is accomplished using a ring o contractile protein immediately inside the plasma membrane at the equator. The proteins are actin and myosin and are similar to proteins that cause contraction in muscle. When the cleavage urrow reaches the centre, the cell is pinched apart into two daughter cells. In plant cells vesicles are moved to the equator where they use to orm tubular structures across the equator. With the usion o more vesicles these tubular structures merge to orm two layers o membrane across the whole o the equator, which develop into the plasma membranes o the two daughter cells and are connected to the existing plasma membranes at the sides o the cell, completing the division o the cytoplasm. The next stage in plants is or pectins and other substances to be brought in vesicles and deposited by exocytosis between the two new membranes. This orms the middle lamella that will link the new cell walls. B oth o the daughter cells then bring cellulose to the equator and deposit it by exocytosis adj acent to the middle lamella. As a result, each cell builds its own cell wall adj acent to the equator. Figure 5 Cytokinesis in (a) fertilized sea urchin egg (b) cell from shoot tip of Coleus plant 55 1 C E LL B I O LO G Y Cyclins and the control of the cell cycle Cyclins are involved in the control o the cell cycle. Each o the phases o the cell cycle involves many important tasks. A group o proteins called cyclins is used to ensure that tasks are perormed at the correct time and that the cell only moves on to the next stage o the cycle when it is appropriate. C yclins bind to enzymes called cyclin- dependent kinases. These kinases then become active and attach phosphate groups to other proteins in the cell. The attachment o phosphate triggers the other proteins to become active and carry out tasks specifc to one o the phases o the cell cycle. concentration There are our main types o cyclin in human cells. The graph in fgure 6 shows how the levels o these cyclins rise and all. Unless these cyclins reach a threshold concentration, the cell does not progress to the next stage o the cell cycle. Cyclins thereore control the cell cycle and ensure that cells divide when new cells are needed, but not at other times. G 1 phase S phase G 2 phase mitosis Cyclin D triggers cells to move from G 0 to G 1 and from G 1 into S phase. Cyclin E prepares the cell for DNA replication in S phase. Cyclin A activates DNA replication inside the nucleus in S phase. Cyclin B promotes the assembly of the mitotic spindle and other tasks in the cytoplasm to prepare for mitosis. Figure 6 Discovery of cyclins Serendipity and scientifc discoveries: the discovery o cyclins was accidental. During research into the control o protein synthesis in sea urchin eggs, Tim Hunt discovered a protein that increased in concentration ater ertilization then decreased in concentration, unlike other proteins which continued to increase. The protein was being synthesized over a period o about 30 minutes and then soon ater was being broken down. Further experiments showed that the protein went through repeated increases and decreases in concentration that coincided with the phases o the cell cycle. The breakdown occurred about ten minutes ater the start o mitosis. Hunt named the protein cyclin. 56 Further research revealed other cyclins and confrmed what Hunt had suspected rom an early stage that cyclins are a key actor in the control o the cell cycle. Tim Hunt was awarded a Nobel Prize or Physiology in 2 001 to honour his work in the discovery o cyclins. His Nobel Lecture can be downloaded rom the internet and viewed. In it he mentions the importance o serendipity several times because he had not set out to discover how the cell cycle is controlled. This discovery is an example o serendipity a happy and unexpected discovery made by accident. 1 . 6 ce ll d I VI s I o n tumur frmai a ar Mutagens, oncogenes and metastasis are involved in the development o primary and secondary tumours. Tumours are abnormal groups o cells that develop at any stage o lie in any part o the body. In some cases the cells adhere to each other and do not invade nearby tissues or move to other parts o the body. These tumours are unlikely to cause much harm and are classifed as benign. In other tumours the cells can become detached and move elsewhere in the body and develop into secondary tumours. These tumours are malignant and are very likely to be lie- threatening. D iseases due to malignant tumours are commonly known as cancer and have diverse causes. C hemicals and agents that cause cancer are known as carcinogens, because carcinomas are malignant tumours. There are various types o carcinogens including some viruses. All mutagens are carcinogenic, both chemical mutagens and also high energy radiation such as X- rays and short- wave ultraviolet light. This is because mutagens are agents that cause gene mutations and mutations can cause cancer. Aiviy car rarh Tumours can orm in any tissue at any age, but the skin, lung, large intestine (bowel) , breast and prostate gland are particularly vulnerable. Cancer is a major cause o death in most human populations so there is a pressing need to fnd methods o prevention and treatment. This involves basic research into the control o the cell cycle. Great progress has been made but more is needed. Who should pay or research into cancer? Mutations are random changes to the base sequence o genes. Most genes do not cause cancer i they mutate. The ew genes that can become cancer-causing ater mutating are known as oncogenes. In a normal cell oncogenes are involved in the control o the cell cycle and cell division. This is why mutations in them can result in uncontrolled cell division and thereore tumour ormation. S everal mutations must occur in the same cell or it to become a tumour cell. The chance o this happening is extremely small, but because there are vast numbers o cells in the body, the total chance o tumour ormation during a lietime is signifcant. When a tumour cell has been ormed it divides repeatedly to orm two, then our, then eight cells and so on. This group o cells is called a primary tumour. Metastasis is the movement o cells rom a primary tumour to set up secondary tumours in other parts o the body. Smoking and cancer The correlation between smoking and incidence o cancers. A correlation in science is a relationship between two variable actors. The relationship between smoking and cancer is an example o a correlation. There are two types o correlation. With a positive correlation, when one actor increases the other one also increases; they also decrease together. With a negative correlation, when one actor increases the other decreases. There is a positive correlation between cigarette smoking and the death rate due to cancer. This has been shown repeatedly in surveys. table 1 shows the results o one o the largest surveys, and the longest 57 1 C E LL B I O LO G Y continuous one. The data shows that the more cigarettes smoked per day, the higher the death rate due to cancer. They also show a higher death rate among those who smoked at one time but had stopped. The results o the survey also show huge increases in the death rate due to cancers o the mouth, pharynx, larynx and lung. This is expected as smoke rom cigarettes comes into contact with each o these parts o the body, but there is also a positive correlation between smoking and cancers o the esophagus, stomach, kidney, bladder, pancreas and cervix. Although the death rate due to other cancers is not signifcantly dierent in smokers and non- smokers, table 1 shows smokers are several times more likely to die rom all cancers than non- smokers. It is important in science to distinguish between a correlation and a cause. Finding that there is a positive correlation between smoking and cancer does not prove that smoking causes cancer. However, in this case the causal links are well established. C igarette smoke contains many dierent chemical substances. Twenty o these have been shown in experiments to cause tumours in the lungs o laboratory animals or humans. There is evidence that at least orty other chemicals in cigarette smoke are carcinogenic. This leaves little doubt that smoking is a cause o cancer. caue o death etween 1951 and 2001 current moker (igarette/day) lieong non-moker former igarette moker 114 1524 25 All cancers 360 466 588 747 1,061 Lung cancer 17 68 131 233 417 Cancer of mouth, pharynx, larynx and esophagus 9 26 36 47 106 334 372 421 467 538 (sampe ize: 34,439 mae dotor in britain) All other cancers Table 1 58 Mortaity rate per 100,000 men/year from British Medical Journal 328(7455) June 24 2004 1 . 6 ce ll d I VI s I o n daa-ba qui: The efect o smoking on health One o the largest ever studies o the eect o smoking on health involved 34,439 male British doctors. Inormation was collected on how much they smoked rom 1 951 to 2001 and the cause o n-mkr 114 igar pr ay 1524 igar pr ay >25 igar pr ay 107 237 310 471 1,037 1,447 1,671 1,938 Stomach and duodenal ulcers 8 11 33 34 Cirrhosis o the liver 6 13 22 68 Parkinsons disease 20 22 6 18 typ f ia Respiratory (diseases o the lungs and airways) Circulatory (diseases o the heart and blood vessels) 1 death was recorded or each o the doctors who died during this period. The table below shows some o the results. The fgures given are the number o deaths per hundred thousand men per year. D educe whether there is a positive correlation between smoking and the mortality rate due to all types o disease. [2 ] 4 2 Using the data in the table, discuss whether the threat to health rom smoking is greater with respiratory or with circulatory diseases. [4] 5 3 Discuss whether the data suggests that smoking a small number o cigarettes is sae. [3] D iscuss whether the data p roves that smoking is a cause o cirrhosis o the liver. [3 ] The table does not include deaths due to cancer. The survey showed that seven types o cancer are linked with smoking. Suggest three cancers that you would expect smoking to cause. [3 ] 59 1 C E LL B I O LO G Y Questions 1 c) E xplain the dierence in area o the inner and outer mitochondrial membranes. [3 ] Figure 7 represents a cell rom a multicellular organism. d) Using the data in the table, identiy two o the main activities o liver cells. [2 ] 3 In human secretory cells, or example in the lung and the pancreas, positively charged ions are pumped out, and chloride ions ollow passively through chloride channels. Water also moves rom the cells into the liquid that has been secreted. prokaryotic or eukaryotic; [1 ] In the genetic disease cystic brosis, the chloride channels malunction and too ew ions move out o the cells. The liquid secreted by the cells becomes thick and viscous, with associated health problems. ( ii) part o a root tip or a nger tip; [1 ] a) S tate the names o the processes that: Figure 7 a) Identiy, with a reason, whether the cell is ( i) ( iii) in a phase o mitosis or in interphase. [1 ] b) The magnication o the drawing is 2 ,5 00 . ( i) C alculate the actual size o the cell. ( ii) move chloride ions out o the secretory cells. [2 ] ( ii) C alculate how long a 5 m scale bar should be i it was added to the drawing. [1 ] c) Predict what would happen to the cell i it was placed in a concentrated salt solution or one hour. Include reasons or your answer. [3] Plasma membrane 4 The amount o D NA present in each cell nucleus was measured in a large number o cells taken rom two dierent cultures o human bone marrow ( gure 8) . a) For each label ( I, II and III) in the S ample B graph, deduce which phase o the cell cycle the cells could be in; i.e. G1 , G2 or S . [3 ] Area (m 2 ) 1,780 Rough endoplasmic reticulum 30,400 Mitochondrial outer membrane 7,470 Mitochondrial inner membrane 39,600 Nucleus 280 Lysosomes 100 Other components 18,500 Table 2 a) C alculate the total area o membranes in the liver cell. [2 ] b) C alculate the area o plasma membrane as a percentage o the total area o membranes in the cell. S how your working. [3 ] 60 b) Explain why the fuid secreted by people with cystic brosis is thick and viscous. [4] Table 2 shows the area o membranes in a rat liver cell. Membrane component [1 ] ( iii) move water out o the secretory cells. [1 ] b) Estimate the approximate amount o D NA per nucleus that would be expected in the ollowing human cell types: ( i) bone marrow at prophase ( ii) bone marrow at telophase. Number of cells (in thousands) 2 move positively charged ions out o the secretory cells [1 ] 3 Sample A (non-dividing cell culture) 2 1 5 10 15 DNA/pg per nucleus Figure 8 Number of cells (in thousands) ( i) [2 ] Sample B 3 (rapidly dividing cell culture) I 2 III 1 II 5 10 15 DNA/pg per nucleus 2 M o le cu lar B I o lo GY Intdtin Water is the medium for life. Living organisms control their composition by a complex web of chemical reactions that occur within this medium. Photosynthesis uses the energy in sunlight to supply the chemical energy needed for life and cell respiration releases this energy when it is needed. C ompounds of carbon, hydrogen and oxygen are used to supply and store energy. Many proteins act as enzymes to control the metabolism of the cell and others have a diverse range of biological functions. Genetic information is stored in D NA and can be accurately copied and translated to make the proteins needed by the cell. 2.1 Molecules to metabolism undstnding Molecular biology explains living processes in terms o the chemical substances involved. Carbon atoms can orm our bonds allowing a diversity o compounds to exist. Lie is based on carbon compounds including carbohydrates, lipids, proteins and nucleic acids. Metabolism is the web o all the enzyme catalysed reactions in a cell or organism. Anabolism is the synthesis o complex molecules rom simpler molecules including the ormation o macromolecules rom monomers by condensation reactions. Catabolism is the breakdown o complex molecules into simpler molecules including the hydrolysis o macromolecules into monomers. appitins Urea as an example o a compound that is produced by living organisms but can also be artifcially synthesized. Skis Drawing molecular diagrams o glucose, ribose, a saturated atty acid and a generalized amino acid. Identifcation o biochemicals such as carbohydrate, lipid or protein rom molecular diagrams. Nt f sin Falsifcation o theories: the artifcial synthesis o urea helped to alsiy vitalism. 61 2 M O L E C U L AR B I O LO G Y Molecular biology Molecular biology explains living processes in terms o the chemical substances involved. Figure 1 A molecular biologist at work in the laboratory The discovery o the structure o D NA in 1 95 3 started a revolution in biology that has transormed our understanding o living organisms. It raised the possibility o explaining biological processes rom the structure o molecules and how they interact with each other. The structures are diverse and the interactions are very complex, so although molecular biology is more than 5 0 years old, it is still a relatively young science. Many molecules are important in living organisms including one as apparently simple as water, but the most varied and complex molecules are nucleic acids and proteins. Nucleic acids comprise D NA and RNA. They are the chemicals used to make genes. Proteins are astonishingly varied in structure and carry out a huge range o tasks within the cell, including controlling chemical reactions o the cell by acting as enzymes. The relationship between genes and proteins is at the heart o molecular biology. The approach o the molecular biologist is reductionist as it involves considering the various biochemical processes o a living organism and breaking down into its component parts. This approach has been immensely productive in biology and has given us insights into whole organisms that we would not otherwise have. Some biologists argue that the reductionist approach o the molecular biologist cannot explain everything though, and that when component parts are combined there are emergent properties that cannot be studied without looking at the whole system together. Synthesis of urea Urea as an example o a compound that is produced by living organisms but can also be artifcially synthesized. Urea is a nitrogen- containing compound with a relatively simple molecular structure ( fgure 2 ) . It is a component o urine and this was where it was frst discovered. It is produced when there is an excess o amino acids in the body, as a means o excreting the nitrogen rom the amino acids. A cycle o reactions, catalysed by enzymes, is used to produce it ( fgure 3 ) . This happens in the liver. Urea is then transported by the blood stream to the kidneys where it is fltered out and passes out o the body in the urine. Urea can also be synthesized artifcially. The chemical reactions used are dierent rom those in the liver and enzymes are not involved, but the urea that is produced is identical. O ammonia + carbon dioxide ammonium carbamate urea + water C H 2N Figure 2 62 NH 2 Molecular diagram of urea About 1 00 million tonnes are produced annually. Most o this is used as a nitrogen ertilizer on crops. 2 .1 M o le c u le s to M e tab o li s M CO 2 + NH 3 enzyme 1 carbamoyl phosphate ornithine urea enzyme 2 arginase citrulline arginine aspartate fumarate enzyme 3 enzyme 4 argininosuccinate Figure 3 The cycle of reactions occurring in liver cells that is used to synthesize urea urea and the alsifcation o vitalism Falsifcation o theories: the artifcial synthesis o urea helped to alsiy vitalism. Urea was discovered in urine in the 1 720s and was assumed to be a product o the kidneys. At that time it was widely believed that organic compounds in plants and animals could only be made with the help o a vital principle. This was part o vitalism the theory that the origin and phenomena o lie are due to a vital principle, which is dierent rom purely chemical or physical orces. Aristotle used the word psyche or the vital principle a Greek word meaning breath, lie or soul. In 1 82 8 the German chemist Friedrich Whler synthesized urea artifcially using silver isocyanate and ammonium chloride. This was the frst organic compound to be synthesized artifcially. It was a very signifcant step, because no vital principle had been involved in the synthesis. Whler wrote this excitedly to the S wedish chemist Jns Jacob B erzelius: In a manner of speaking, I can no longer hold my chemical water. I must tell you that I can make urea without the kidneys of any animal, be it man or dog. An obvious deduction was that i urea had been synthesized without a vital principle, other organic compounds could be as well. Whlers achievement was evidence against the theory o vitalism. It helped to alsiy the theory, but it did not cause all biologists to abandon vitalism immediately. It usually requires several pieces o evidence against a theory or most biologists to accept that it has been alsifed and sometimes controversies over a theory continue or decades. Although biologists now accept that processes in living organisms are governed by the same chemical and physical orces as in non- living matter, there remain some organic compounds that have not been synthesized artifcially. It is still impossible to make complex proteins such as hemoglobin, or example, without using ribosomes and other components o cells. Four years ater his synthesis o urea, Whler wrote this to B erzelius: Organic chemistry nowadays almost drives one mad. To me it appears like a primeval tropical forest full of the most remarkable things; a dreadful endless jungle into which one dare not enter, for there seems no way out. 63 2 M O L E C U L AR B I O LO G Y carbon ompounds ativity Carbon atoms can orm our bonds allowing a diversity o compounds to exist. crbon ompounds Can you fnd an example o a biological molecule in which a carbon atom is bonded to atoms o three other elements or even our other elements? C arbon is only the 1 5 th most abundant element on Earth, but it can be used to make a huge range of different molecules. This has given living organisms almost limitless possibilities for the chemical composition and activities of their cells. The diversity of carbon compounds is explained by the properties of carbon. Titin is a giant protein that acts as a molecular spring in muscle. The backbone o the titin molecule is a chain o 100,000 atoms, linked by single covalent bonds. Carbon atoms form covalent bonds with other atoms. A covalent bond is formed when two adjacent atoms share a pair of electrons, with one electron contributed by each atom. Covalent bonds are the strongest type of bond between atoms so stable molecules based on carbon can be produced. Each carbon atom can form up to four covalent bonds more than most other atoms, so molecules containing carbon can have complex structures. The bonds can be with other carbon atoms to make rings or chains of any length. Fatty acids contain chains of up to 2 0 carbon atoms for example. The bonds can also be with other elements such as hydrogen, oxygen, nitrogen or phosphorus. Can you fnd an example o a molecule in your body with a chain o over 1,000,000,000 atoms? C arbon atoms can bond with just one other element, such as hydrogen in methane, or they can bond to more than one other element as in ethanol ( alcohol found in beer and wine) . The four bonds can all be single covalent bonds or there can be two single and one double covalent bond, for example in the carboxyl group of ethanoic acid (the acid in vinegar) . H H C H methane classifying arbon ompounds H H H H C C H H H H C H ethanol Living organisms use four main classes of carbon compound. They have different properties and so can be used for different purposes. O C Carbohydrates are characterized by their composition. They are composed of carbon, hydrogen and oxygen, with hydrogen and oxygen in the ratio of two hydrogen atoms to one oxygen, hence the name carbohydrate. ethanoic acid O H H O Lie is based on carbon compounds including carbohydrates, lipids, proteins and nucleic acids. H H H H H H H H H H H H H H H H H H C C C C C C C C C C C C C C C C C H H H H H H H H H H H linolenic acid an omega-3 fatty acid Figure 4 Some common naturally-occurring carbon compounds O C OH Lip ids are a broad class of molecules that are insoluble in water, including steroids, waxes, fatty acids and triglycerides. In common language, triglycerides are fats if they are solid at room temperature or oils if they are liquid at room temperature. Proteins are composed of one or more chains of amino acids. All of the amino acids in these chains contain the elements carbon, hydrogen, oxygen and nitrogen, but two of the twenty amino acids also contain sulphur. Nucleic acids are chains of subunits called nucleotides, which contain carbon, hydrogen, oxygen, nitrogen and phosphorus. There are two types of nucleic acid: ribonucleic acid ( RNA) and deoxyribonucleic acid ( D NA) . 64 2 .1 M o le c u le s to M e tab o li s M Drawing molecules Drawing molecular diagrams of glucose, ribose, a saturated fatty acid and a generalized amino acid. There is no need to memorize the structure o many dierent molecules but a biologist should be able to draw diagrams o a ew o the most important molecules. Each atom in a molecule is represented using the symbol o the element. For example a carbon Name of group hydroxyl Full structure O atom is represented with C and an oxygen atom with O . S ingle covalent bonds are shown with a line and double bonds with two lines. S ome chemical groups are shown with the atoms together and bonds not indicated. Table 1 gives examples. Simplied notation OH H H amine NH 2 N H O carboxyl COOH C O H H methyl C H CH 3 H Table 1 Ribose The ormula or ribose is C 5 H 1 0 O 5 The molecule is a fve- membered ring with a side chain. OH 5 H C 4 Four carbon atoms are in the ring and one orms the side chain. The carbon atoms can be numbered starting with number 1 on the right. The hydroxyl groups ( O H) on carbon atoms 1 , 2 and 3 point up, down and down respectively. H O H C1 C C2 OH OH H H 3 Ribose Glucose The ormula or glucose is C 6 H 1 2 O 6 The molecule is a six- membered ring with a side chain. 6 CH 2 OH 5 C O H OH H C C H 4C HO 3 Five carbon atoms are in the ring and one orms the side chain. The carbon atoms can be numbered starting with number 1 on the right. The hydroxyl groups ( O H) on carbon atoms 1 , 2 , 3 and 4 point down, down, up and down respectively, although in a orm o glucose used by plants to make cellulose the hydroxyl group on carbon atom 1 points upwards. OH CH 1 C OH 2 H C OH Glucose 65 2 M O L E C U L AR B I O LO G Y O Saturated fatty acids OH C The carbon atoms form an unbranched chain. In saturated fatty acids they are bonded to each other by single bonds. H C H H C H The number of carbon atoms is most commonly between 1 4 and 2 0. H C H At one end of the chain the carbon atom is part of a carboxyl group H C H At the other end the carbon atom is bonded to three hydrogen atoms. All other carbon atoms are bonded to two hydrogen atoms. H C H H C H H C H H C H Amino acids H C H A carbon atom in the centre of the molecule is bonded to four different things: an amine group, hence the term amino acid; a carboxyl group which makes the molecule an acid; a hydrogen atom; the R group, which is the variable part of amino acids. H C H H C H H C H H C H H C H H C H H O R N H C C O H full molecular diagram R O H N 2N C COOH CH 3 (CH 2 ) n H H simplied molecular diagram C Full molecular diagram o a saturated atty acid OH Molecular diagrams o an amino acid Simplifed molecular diagram o a saturated atty acid Identifying molecules Identifcation o biochemicals as carbohydrate, lipid or protein rom molecular diagrams. The molecules of carbohydrates, lipids and proteins are so different from each other that it is usually quite easy to recognize them. 66 Proteins contain C , H, O and N whereas carbohydrates and lipids contain C , H and O but not N. Many proteins contain sulphur ( S ) but carbohydrates and lipids do not. C arbohydrates contain hydrogen and oxygen atoms in a ratio of 2 :1 , for example glucose is C 6 H 1 2 O 6 and sucrose ( the sugar commonly used in baking) is C 1 2 H 22 O 1 1 Lipids contain relatively less oxygen than carbohydrates, for example oleic acid ( an unsaturated fatty acid) is C 1 8 H 34O 2 and the steroid testosterone is C 1 9 H 28 O 2 Figure 5 A commonly-occurring biological molecule 2 .1 M o le c u le s to M e tab o li s M Metbolism Metabolism is the web of all the enzyme catalysed reactions in a cell or organism. All living organisms carry out large numbers o dierent chemical reactions. These reactions are catalysed by enzymes. Most o them happen in the cytoplasm o cells but some are extracellular, such as the reactions used to digest ood in the small intestine. Metabolism is the sum o all reactions that occur in an organism. Metabolism consists o pathways by which one type o molecule is transormed into another, in a series o small steps. These pathways are mostly chains o reactions but there are also some cycles. An example is shown in fgure 3. E ven in relatively simple prokaryote cells, metabolism consists o over 1 , 000 dierent reactions. Global maps showing all reactions are very complex. They are available on the internet, or example in the Kyoto E ncyclopedia o Genes and Genomes. anbolism Anabolism is the synthesis of complex molecules from simpler molecules including the formation of macromolecules from monomers by condensation reactions. Metabolism is oten divided into two parts, anabolism and catabolism. Anabolism is reactions that build up larger molecules rom smaller ones. An easy way to remember this is by recalling that anabolic steroids are hormones that promote body building. Anabolic reactions require energy, which is usually supplied in the orm o ATP. Anabolism includes these processes: Protein synthesis using ribosomes. D NA synthesis during replication. Photosynthesis, including production o glucose rom carbon dioxide and water. Synthesis o complex carbohydrates including starch, cellulose and glycogen. ctbolism Catabolism is the breakdown of complex molecules into simpler molecules including the hydrolysis of macromolecules into monomers. C atabolism is the part o metabolism in which larger molecules are broken down into smaller ones. C atabolic reactions release energy and in some cases this energy is captured in the orm o ATP, which can then be used in the cell. C atabolism includes these processes: D igestion o ood in the mouth, stomach and small intestine. C ell respiration in which glucose or lipids are oxidized to carbon dioxide and water. D igestion o complex carbon compounds in dead organic matter by decomposers. 67 2 M O L E C U L AR B I O LO G Y 2.2 Water understnding Water molecules are polar and hydrogen bonds form between them. Hydrogen bonding and dipolarity explain the adhesive, cohesive, thermal and solvent properties of water. Substances can be hydrophilic or hydrophobic. applictions Comparison of the thermal properties of water with those of methane. Use of water as a coolant in sweat. Methods of transport of glucose, amino acids, cholesterol, fats, oxygen and sodium chloride in blood in relation to their solubility in water. Ntre of science Use theories to explain natural phenomena: the theory that hydrogen bonds form between water molecules explains waters properties. H H Water molecules are polar and hydrogen bonds form between them. O tends to small pull the positive electrons charge + on each slightly hydrogen in this atom direction Corresponding negative charge 2 - on oxygen atom Figure 1 Water molecules water molecule hydrogen bond Figure 2 The dotted line indicates the presence of an intermolecular force between the molecules. This is called a hydrogen bond 68 Hydrogen bonding in wter A water molecule is ormed by covalent bonds between an oxygen atom and two hydrogen atoms. The bond between hydrogen and oxygen involves unequal sharing o electrons it is a polar covalent bond. This is because the nucleus o the oxygen atom is more attractive to electrons than the nuclei o the hydrogen atoms ( fgure 1 ) . B ecause o the unequal sharing o electrons in water molecules, the hydrogen atoms have a partial positive charge and oxygen has a partial negative charge. B ecause water molecules are bent rather than linear, the two hydrogen atoms are on the same side o the molecule and orm one pole and the oxygen orms the opposite pole. Positively charged particles ( positive ions) and negatively charged particles ( negative ions) attract each other and orm an ionic bond. Water molecules only have partial charges, so the attraction is less but it is still enough to have signifcant eects. The attraction between water molecules is a hydrogen bond. S trictly speaking it is an intermolecular orce rather than a bond. A hydrogen bond is the orce that orms when a hydrogen atom in one polar molecule is attracted to a slightly negative atom o another polar covalent molecule. Although a hydrogen bond is a weak intermolecular orce, water molecules are small, so there are many o them per unit volume o water and large numbers o hydrogen bonds ( fgure 2 ) . C ollectively they give water its unique properties and these properties are, in turn, o immense importance to living things. 2 . 2 W at e r Hydrogen bonds and the properties of water Use theories to explain natural phenomena: the theory that hydrogen bonds form between water molecules explains waters properties. There is strong experimental evidence or hydrogen bonds, but it remains a theory that they orm between water molecules. Scientists cannot prove without doubt that they exist as they are not directly visible. However, hydrogen bonds are a very useul way o explaining the properties o water. They explain the cohesive, adhesive, thermal and solvent properties o water. It is these distinctive properties that make water so useul to living organisms. It might seem unwise to base our understanding o the natural world on something that has not been proven to exist. However this is the way that science works we can assume that a theory is correct i there is evidence or it, i it helps to predict behaviour, i it has not been alsifed and i it helps to explain natural phenomena. Properties of water Hydrogen bonding and dipolarity explain the cohesive, adhesive, thermal and solvent properties of water. Cohesive properties C ohesion reers to the binding together o two molecules o the same type, or instance two water molecules. Water molecules are cohesive they cohere, which means they stick to each other, due to hydrogen bonding, described in the previous section. This property is useul or water transport in plants. Water is sucked through xylem vessels at low pressure. The method can only work i the water molecules are not separated by the suction orces. D ue to hydrogen bonding this rarely happens and water can be pulled up to the top o the tallest trees over a hundred metres. Adhesive properties Hydrogen bonds can orm between water and other polar molecules, causing water to stick to them. This is called adhesion. This property is useul in leaves, where water adheres to cellulose molecules in cell walls. I water evaporates rom the cell walls and is lost rom the lea via the network o air spaces, adhesive orces cause water to be drawn out o the nearest xylem vessel. This keeps the walls moist so they can absorb carbon dioxide needed or photosynthesis. Thermal properties Water has several thermal properties that are useul to living organisms: High specifc heat capacity. Hydrogen bonds restrict the motion o water molecules and increases in the temperature o water require hydrogen bonds to be broken. Energy is needed to do this. As a result, the amount o energy needed to raise the temperature o water is relatively large. To cool down, water must lose relatively large amounts o energy. Waters temperature remains relatively stable in comparison to air or land, so it is a thermally stable habitat or aquatic organisms. High latent heat o vap orization. When a molecule evaporates it separates rom other molecules in a liquid and becomes a vapour molecule. The heat needed to do this is called the latent heat o 69 2 M O L E C U L AR B I O LO G Y vaporization. Evaporation therefore has a cooling effect. C onsiderable amounts of heat are needed to evaporate water, because hydrogen bonds have to be broken. This makes it a good evaporative coolant. S weating is an example of the use of water as a coolant. High boiling point. The boiling point of a substance is the highest temperature that it can reach in a liquid state. For the same reasons that water has a high latent heat of vaporization, its boiling point is high. Water is therefore liquid over a broad range of temperatures from 0 C to 1 00 C. This is the temperature range found in most habitats on Earth. Solvent properties Water has important solvent properties. The polar nature of the water molecule means that it forms shells around charged and polar molecules, preventing them from clumping together and keeping them in solution. Water forms hydrogen bonds with polar molecules. Its partially negative oxygen pole is attracted to positively charged ions and its partially positive hydrogen pole is attracted to negatively charged ions, so both dissolve. C ytoplasm is a complex mixture of dissolved substances in which the chemical reactions of metabolism occurs. toK Hydrophilic and hydrophobic How do scientic explanations difer rom pseudo-scientic explanations? Substances can be hydrophilic or hydrophobic. Homeopathy is a practice where remedies are prepared by dissolving things like charcoal, spider venom or deadly nightshade. This mother tincture o harmul substance is diluted again and again to the point where a sample rom the solution is unlikely to contain a single molecule o the solute. It is this ultra-dilute solution that is claimed to have medicinal properties. The properties are reerred to as the memory o water. Despite the large number o practitioners o this practice, no homeopathic remedy has ever been shown to work in a large randomized placebo-controlled clinical trial. The literal meaning of the word hydrophilic is water-loving. It is used to describe substances that are chemically attracted to water. All substances that dissolve in water are hydrophilic, including polar molecules such as glucose, and particles with positive or negative charges such as sodium and chloride ions. S ubstances that water adheres to, cellulose for example, are also hydrophilic. S ome substances are insoluble in water although they dissolve in other solvents such as propanone ( acetone) . The term hydrophobic is used to describe them, though they are not actually water-fearing. Molecules are hydrophobic if they do not have negative or positive charges and are nonpolar. All lipids are hydrophobic, including fats and oils Figure 3 70 When two nonpolar molecules in water come into contact, weak interactions form between them and more hydrogen bonds form between water molecules 2 . 2 W at e r I a nonpolar molecule is surrounded by water molecules, hydrogen bonds orm between the water molecules, but not between the nonpolar molecule and the water molecules. I two nonpolar molecules are surrounded by water molecules and random movements bring them together, they behave as though they are attracted to each other. There is a slight attraction between nonpolar molecules, but more signifcantly, i they are in contact with each other, more hydrogen bonds can orm between water molecules. This is not because they are water-earing: it is simply because water molecules are more attracted to each other than to the nonpolar molecules. As a result, nonpolar molecules tend to join together in water to orm larger and larger groups. The orces that cause nonpolar molecules to join together into groups in water are known as hydrophobic interactions. comparing water and methane Comparison o the thermal properties o water with those o methane. The properties o water have already been described. Methane is a waste product o anaerobic respiration in certain prokaryotes that live in habitats where oxygen is lacking. Methanogenic prokaryotes live in swamps and other wetlands and in the guts o animals, including termites, cattle and sheep. They also live in waste dumps and are deliberately encouraged to produce methane in anaerobic digesters. Methane can be used as a uel but i allowed to escape into the atmosphere it contributes to the greenhouse eect. Water and methane are both small molecules with atoms linked by single covalent bonds. However water molecules are polar and can orm hydrogen bonds, whereas methane molecules are nonpolar and do not orm hydrogen bonds. As a result their physical properties are very dierent. The data in table 1 shows some o the physical properties o methane and water. The density and specifc heat capacity are given or methane and water in a liquid state. The data shows that water has a higher specifc heat capacity, higher latent heat o vaporization, higher melting point and higher boiling point. Whereas methane is liquid over a range o only 2 2 C , water is liquid over 1 00 C . Popy Mhn W Formula CH 4 H 2O Molecular mass 16 Density Specifc heat capacity 0.46g per cm 18 3 1g per cm 3 2.2 J per g per C 4.2 J per g per C Latent heat o vaporization 760 J/g 2,257 J/g Melting point 182 C 0 C Boiling point 160 C 100 C Table 1 Comparing methane and water Figure 4 Bubbles of methane gas, produced by prokaryotes decomposing organic matter at the bottom of a pond have been trapped in ice when the pond froze 71 2 M O L E C U L AR B I O LO G Y cooling the body with sweat Use of water as a coolant in sweat. Sweat is secreted by glands in the skin. The sweat is carried along narrow ducts to the surace o the skin where it spreads out. The heat needed or the evaporation o water in sweat is taken rom the tissues o the skin, reducing their temperature. B lood fowing through the skin is thereore cooled. This is an eective method o cooling the body because water has a high latent heat o vaporization. S olutes in the sweat, especially ions such as sodium, are let on the skin surace and can sometimes be detected by their salty taste. There are methods o cooling other than sweating, though many o these also rely on heat loss due to evaporation o water. Panting in dogs and birds is an example. Transpiration is evaporative loss o water rom plant leaves; it has a cooling eect which is useul in hot environments. Sweat secretion is controlled by the hypothalamus o the brain. It has receptors that monitor blood temperature and also receives sensory inputs rom temperature receptors in the skin. I the body is overheated the hypothalamus stimulates the sweat glands to secrete up to two litres o sweat per hour. Usually no sweat is secreted i the body is below the target temperature, though when adrenalin is secreted we sweat even i we are already cold. This is because adrenalin is secreted when our brain anticipates a period o intense activity that will tend to cause the body to overheat. Transport in blood plasma Methods of transport of glucose, amino acids, cholesterol, fats, oxygen and sodium chloride in blood in relation to their solubility in water. B lood transports a wide variety o substances, using several methods to avoid possible problems and ensure that each substance is carried in large enough quantities or the bodys needs. S odium chloride is an ionic compound that is reely soluble in water, dissolving to orm sodium ions ( Na + ) and chloride ions ( C l - ) , which are carried in blood plasma. Amino acids have both negative and positive charges. B ecause o this they are soluble in water but their solubility varies depending on the R group, some o which are hydrophilic while others are hydrophobic. All amino acids are soluble enough to be carried dissolved in blood plasma. 72 Glucose is a polar molecule. It is reely soluble in water and is carried dissolved in blood plasma. O xygen is a nonpolar molecule. B ecause o the small size o the molecule it dissolves in water but only sparingly and water becomes saturated with oxygen at relatively low concentrations. Also, as the temperature o water rises, the solubility o oxygen decreases, so blood plasma at 3 7 C can hold much less dissolved oxygen than water at 2 0 C or lower. The amount o oxygen that blood plasma can transport around the body is ar too little to provide or aerobic cell respiration. This problem is overcome by the use o hemoglobin in red blood cells. Hemoglobin has binding sites or oxygen and greatly increases the capacity o the blood or oxygen transport. 2 . 3 c a r b o h y d r at e s a n d l i P i d s Fats molecules are entirely nonpolar, are larger than oxygen and are insoluble in water. They are carried in blood inside lipoprotein complexes. These are groups of molecules with a single layer of phospholipid on the outside and fats inside. The hydrophilic phosphate heads of the phospholipids face outwards and are in contact with water in the blood plasma. The hydrophobic hydrocarbon tails face inwards and are in contact with the fats. There are also proteins in the phospholipid monolayer, hence the name lipoprotein. Cholesterol molecules are hydrophobic, apart from a small hydrophilic region at one end. This is not enough to make cholesterol dissolve in water and instead it is transported with fats in lipoprotein complexes. The cholesterol molecules are positioned in the phospholipid monolayers, with the hydrophilic region facing outwards in the region with the phosphate heads of the phospholipids. phospholipid protein cholesterol triglyceride Figure 5 Arrangement of molecules in a lipoprotein complex 2.3 c p understnding Monosaccharide monomers are linked together by condensation reactions to orm disaccharides and polysaccharide polymers. Fatty acids can be saturated, monounsaturated or polyunsaturated. Unsaturated atty acids can be cis or trans isomers. Triglycerides are ormed by condensation rom three atty acids and one glycerol. Ntre of science Evaluating claims: health claims made about lipids need to be assessed. applictions Structure and unction o cellulose and starch in plants and glycogen in humans. Scientifc evidence or health risks o trans-ats and saturated ats. Lipids are more suitable or long-term energy storage in humans than carbohydrates. Evaluation o evidence and the methods used to obtain evidence or health claims made about lipids. Skills Use o molecular visualization sotware to compare cellulose, starch and glycogen. Determination o body mass index by calculation or use o a nomogram. 73 2 M O L E C U L AR B I O LO G Y toK carbohydrates i w cmpeng paradgms gve dferen explanans a phenmenn, hw can we decde whch s crrec? Monosaccharide monomers are linked together by condensation reactions to orm disaccharides and polysaccharide polymers. Thomas Kuhn, in his book The Structure o Scientifc Revolutions adopted the word paradigm to reer to the rameworks that dominate the interpretation o inormation in a scientifc discipline at a particular point in time. The paradigm impacts the kinds o questions that are supposed to be asked. Nutritionism is the reductionist paradigm that the presence o indicator nutrients are the key determinant o healthy ood. Even highly processed ood may be advertised as healthy depending on the degree to which it contains healthy nutrients. Words like carbs, vitamins and polyunsaturated at have entered the everyday lexicon. Some argue that this aligns consumer anxiety with the commercial interests o ood manuacturers. An alternative paradigm or determining the healthiness o ood is argued or by Michael Pollan in his book In Deense o Food. It argues that ood quality should be determined by cultural tradition which tended to look at ood more holistically: Glucose, ructose and ribose are all examples o monosaccharides. The structure o glucose and ribose molecules was shown in sub-topic 2 .1 . Monosaccharides can be linked together to make larger molecules. Monosaccharides are single sugar units. D isaccharides consist o two monosaccharides linked together. For example, maltose is made by linking two glucose molecules together. S ucrose is made by linking a glucose and a ructose. Polysaccharides consist o many monosaccharides linked together. S tarch, glycogen and cellulose are polysaccharides. They are all made by linking together glucose molecules. The dierences between them are described later in this sub-topic. When monosaccharides combine, they do so by a process called condensation ( fgure 1 ) . This involves the loss o an O H rom one molecule and an H rom another molecule, which together orm H 2 O . Thus, condensation involves the combination o subunits and yields water. Linking together monosaccharides to orm disaccharides and polysaccharides is an anabolic process and energy has to be used to do it. ATP supplies energy to the monosaccharides and this energy is then used when the condensation reaction occurs. H H HO The sheer novelty and glamor o the Western diet, with its seventeen thousand new ood products every year and the marketing power thirty-two billion dollars a year used to sell us those products, has overwhelmed the orce otradition and let us where we now fnd ourselves: relying on science and journalism and government and marketing to help us decide what to eat H H OH HO OH Monosaccharides, C 6 H 12 O 6 e.g. glucose, fructose, galactose H 2O Condensation Hydrolysis (water removed) (water added) H HO Michael Pollan, In Deense oFood: An Eater's Maniesto H O Glycosidic bond Condensation Figure 1 e.g. maltose, sucrose, lactose OH Hydrolysis H H HO Disaccharide, C 12 H 22 O 11 O O O OH Polysaccharide e.g. starch, glycogen Condensation and hydrolysis reactions between monosaccharides and disaccharides 74 2 . 3 c a r b o h y d r at e s a n d l i P i d s Imaging carbohydrate molecules Use of molecular visualization software to compare cellulose, starch and glycogen. The most widely used molecular visualization software is JMol, which can be downloaded free of charge. There are also many websites that use JMol, which are easier to use. S uggestions of suitable websites are available with the electronic resources that accompany this book. When JMol software is being used, you should be able to make these changes to the image of a molecule that you see on the screen: Use the scroll function on the mouse to make the image larger or smaller. Left click and move the mouse to rotate the image. Right click to display a menu that allows you to change the style of molecular model, label the atoms, make the molecule rotate continuously or change the background colour. S pend some time developing your skill in molecular visualization and then try these questions to test your skill level and learn more about the structure of polysaccharides. Questions 1 Select glucose with the ball and stick style with a black background. 2 4 [2 ] S elect sucrose with sticks style and a blue background. 3 What colours are used to show carbon, hydrogen and oxygen atoms? What is the difference between the glucose ring and the fructose ring in the sucrose molecule? [1 ] S elect amylose, which is the unbranched form of starch, with the wireframe style and a white background. If possible select a short amylose chain and then a longer one. What is the overall shape of an amylose molecule? [1 ] How many glucose molecules in the chain are linked to only one other glucose? [1 ] S elect amylopectin, with the styles and colours that you prefer. Amylopectin is the branched form of starch. Zoom in to look closely at a position where there is a branch. A glucose molecule must be linked to an extra third glucose to make the branch. What is different about this linkage, compared to the linkages between glucose molecules in unbranched parts of the molecule? [1 ] How many glucose molecules are linked to only one other glucose in the amylopectin molecule? [1 ] Figure 2 Images of sugars using molecular visualization software (a) fructose, (b) maltose, (c) lactose 75 2 M O L E C U L AR B I O LO G Y 5 Select glycogen. It is similar but not identical to the amylopectin orm o starch. 6 Select cellulose. 7 What is the dierence between glycogen and amylopectin? [1 ] How is it dierent in shape rom the other polysaccharides? [1 ] Look at the oxygen atom that orms part o the ring in each glucose molecule in the chain. What pattern do you notice in the position o these oxygen atoms along the chain? Polysaccharides Structure and function of cellulose and starch in plants and glycogen in humans. Starch, glycogen and cellulose are all made by linking together glucose molecules, yet their structure and unctions are very dierent. This is due to dierences in the type o glucose used to make them and in the type o linkage between glucose molecules. Glucose has fve O H groups, any o which could be used in condensation reactions, but only three o them are actually used to link to make polysaccharides. The most common link is between the O H on carbon atom 1 ( on the right hand side in molecular diagrams o glucose) and the O H on carbon atom 4 ( shown on the let hand side) . The O H on carbon atom 6 ( shown at the top o molecular diagrams) is used to orm side branches in some polysaccharides. Figure 3 Glucose molecule Glucose can have the OH group on carbon atom 1 pointing either upwards or downwards. In alpha glucose (-glucose) the OH group points downwards but in beta glucose (-glucose) it points upwards. This small dierence has major consequences or polysaccharides made rom glucose. Cellulose is made by linking together -glucose molecules. Condensation reactions link carbon atom 1 to carbon atom 4 on the next -glucose. The OH groups on carbon atom 1 and 4 point in opposite directions: up on carbon 1 and down on carbon 4. To bring these OH groups together and allow a condensation reaction to occur, each -glucose added to the chain has to be positioned at 1 80 to the previous one. The glucose subunits in the chain are oriented alternately upwards and downwards. The consequence o this is that the cellulose molecule is a straight chain, rather than curved. 76 Figure 4 Cellulose C ellulose molecules are unbranched chains o - glucose, allowing them to orm bundles with hydrogen bonds linking the cellulose molecules. These bundles are called cellulose microfbrils. They have very high tensile strength and are used as the basis o plant cell walls. The tensile strength o cellulose prevents plant cells rom bursting, even when very high pressures have developed inside the cell due to entry o water by osmosis. 2 . 3 c a r b o h y d r at e s a n d l i P i d s Starch is made by linking together -glucose molecules. As in cellulose, the links are made by condensation reactions between the OH groups on carbon atom 1 o one glucose and carbon atom 4 o the adjacent glucose. These OH groups both point downwards, so all the glucose molecules in starch can be orientated in the same way. The consequence o this is that the starch molecule is curved, rather than straight. There are two orms o starch. In amylose the chain o -glucose molecules is unbranched and orms a helix. In amylopectin the chain is branched, so has a more globular shape. Starch is only made by plant cells. Molecules o both types o starch are hydrophilic but they are too large to be soluble in water. They are thereore useul in cells where large amounts o glucose need to be stored, but a concentrated glucose solution would cause too much water to enter a cell by osmosis. Starch is used as a store o glucose and thereore o energy in seeds and storage organs such as potato cells. Starch is made as a temporary store in lea cells when glucose is being made aster by photosynthesis than it can be exported to other parts o the plant. Figure 5 Starch glycogen it is easy to add extra glucose molecules or remove them. This can be done at both ends o an unbranched molecule or at any o the ends in a branched molecule. S tarch and glycogen molecules do not have a fxed size and the number o glucose molecules that they contain can be increased or decreased. Glycogen is very similar to the branched orm o starch, but there is more branching, making the molecule more compact. Glycogen is made by animals and also some ungi. It is stored in the liver and some muscles in humans. Glycogen has the same unction as starch in plants: it acts as a store o energy in the orm o glucose, in cells where large stores o dissolved glucose would cause osmotic problems. With both starch and Figure 6 Glycogen lipids Triglycerides are formed by condensation from three fatty acids and one glycerol. Lipids are a diverse group o carbon compounds that share the property o being insoluble in water. Triglycerides are one o the principal groups o lipid. Examples o triglycerides are the at in adipose tissue in humans 77 2 M O L E C U L AR B I O LO G Y and the oil in sunfower seeds. Fats are liquid at body temperature ( 3 7 C ) but solid at room temperature ( 2 0 C ) whereas oils are liquid at both body temperature and room temperature. A triglyceride is made by combining three atty acids with one glycerol ( see gure 7) . Each o the atty acids is linked to the glycerol by a condensation reaction, so three water molecules are produced. The linkage ormed between each atty acid and the glycerol is an ester bond. This type o bond is ormed when an acid reacts with the O H group in an alcohol. In this case the reaction is between the C O O H group on a atty acid and an O H on the glycerol. Triglycerides are used as energy stores. The energy rom them can be released by aerobic cell respiration. B ecause they do not conduct heat well, they are used as heat insulators, or example in the blubber o Arctic marine mammals. Glycerol Fatty acids H C O H HO C (CH 2 ) n CH 3 Triglyceride (fat) H H H C O Condensation C (CH 2 ) n CH 3 (water removed) H C O O H C O H H C O H HO O O HO H Figure 7 C (CH 2 ) n CH 3 O C (CH 2 ) n CH 3 O 3H 2 O C (CH 2 ) n CH 3 H C H O C (CH 2 ) n CH 3 O Ester bond Formation of a triglyceride from glycerol and three fatty acids enrgy storag Lipids are more suitable for long term energy storage in humans than carbohydrates. Lipids and carbohydrates are both used or energy storage in humans, but lipids are normally used or long- term energy storage. The lipids that are used are ats. They are stored in specialized groups o cells called adipose tissue. Adipose tissue is located immediately beneath the skin and also around some organs including the kidneys. greater because ats orm pure droplets in cells with no water associated, whereas each gram o glycogen is associated with about two grams o water, so lipids are actually six times more ecient in the amount o energy that can be stored per gram o body mass. This is important, because we have to carry our energy stores around with us wherever we go. It is even more important or animals such as birds and bats that fy. There are several reasons or using lipids rather than carbohydrates or long- term energy storage: 78 The amount o energy released in cell respiration per gram o lipids is double the amount released rom a gram o carbohydrates. The same amount o energy stored as lipid rather than carbohydrate thereore adds hal as much to body mass. In act the mass advantage o lipids is even S tored lipids have some secondary roles that could not be perormed as well by carbohydrates. B ecause lipids are poor conductors o heat, they can be used as heat insulators. This is the reason or much o our stored at being in sub- cutaneous adipose tissue next to the skin. B ecause at 2 . 3 c a r b o h y d r at e s a n d l i P i d s is liquid at body temperature, it can also act as a shock absorber. This is the reason or adipose tissue around the kidneys and some other organs. Glycogen is the carbohydrate that is used or energy storage, in the liver and in some muscles. Although lipids are ideal or longterm storage o energy, glycogen is used or short- term storage. This is because glycogen can be broken down to glucose rapidly and then transported easily by the blood to where it is needed. Fats in adipose tissue cannot be mobilized as rapidly. Glucose can be used either in anaerobic or aerobic cell respiration whereas ats and atty acids can only be used in aerobic respiration. The liver stores up to 1 5 0 grams o glycogen and some muscles store up to 2 % glycogen by mass. d- qu: Emperor penguins 0.4 D uring the Antarctic winter emale E mperor 8.0 penguins live and eed at sea, but males have to stay on the ice to incubate the single egg the emale has laid. Throughout this time the males eat no ood. Ater 1 6 weeks the eggs hatch and the emales return. While the males are 12.0 incubating the eggs they stand in tightly packed groups o about 3 , 0 0 0 birds. To investigate the captive before reasons or standing in groups, 1 0 male birds were taken rom a colony at Pointe Geologie in 0.4 Antarctica. They had already survived 4 weeks 7.7 without ood. They were kept or 1 4 more weeks without ood in enced enclosures where they could not orm groups. All other conditions were kept the same as in the wild 11.8 colony. The mean air temperature was 1 6 . 4 C . The composition o the captive and the wild birds bodies was measured beore and ater the wild before 1 4- week period o the experiment. The results in kilograms are shown in fgure 8 . a) C alculate the total mass loss or each group o birds. [2 ] i) wild 0.5 6.8 18.2 14.3 0.8 captive after 0.4 6.9 14.4 17.3 2.2 wild after Key water lipid protein other substances Figure 8 ii) captive b) C ompare the changes in lipid content o the captive birds with those o the birds living ree in the colony. [2 ] c) B esides being used as an energy source, state another unction o lipid which might be important or penguin survival. [1 ] 79 2 M O L E C U L AR B I O LO G Y Body mass index Determination of body mass index by calculation or use of a nomogram. The body mass index, usually abbreviated to B MI, was developed by a B elgian statistician, Adolphe Quetelet. Two measurements are needed to calculate it: the mass o the person in kilograms and their height in metres. B MI is calculated using this ormula: mass in kilograms B MI = __2 ( height in metres) Units or B MI are kg m - 2 B MI can also be ound using a type o chart called a nomogram. A straight line between the height on the let hand scale and the mass on the right hand scale intersects the B MI on the central scale. The data based questions on page 81 include a B MI nomogram. B MI is used to assess whether a persons body mass is at a healthy level, or is too high or too low. Table 1 shows how this is done: bMi below 18.5 sttu underweight 18.524.9 normal weight 25.029.9 overweight 30.0 or more obese actvty etmtng ody ft prcntg To estimate body fat percentage, measure the thickness of a skinfold in millimetres using calipers in these four places: Front of upper arm Back of upper arm Below scapula Side of waist The measurements are added and then analysis tools available on the internet can be used to calculate the estimate. Figure 9 Measuring body fat with skinfold callipers 80 Table 1 In some parts o the world ood supplies are insufcient or are unevenly distributed and many people as a result are underweight. In other parts o the world a likelier cause o being underweight is anorexia nervosa. This is a psychological condition that involves voluntary starvation and loss o body mass. Obesity is an increasing problem in some countries. Excessive ood intake and insufcient exercise cause an accumulation o at in adipose tissue. The amount o body at can be estimated using skinold calipers (fgure 9) . Obesity increases the risk o conditions such as coronary heart disease and type 2 diabetes. It reduces lie expectancy signifcantly and is increasing the overall costs o health care in countries where rates o obesity are rising. Measuring body mass. What was this persons body mass index if their height was 1.80 metres? 2 . 3 c a r b o h y d r at e s a n d l i P i d s d qu: Nomograms and BMI b) S uggest two ways in which the woman could reduce her body mass. [2 ] Use fgure 1 1 to answer these questions. 1 a) S tate the body mass index o a man who has a mass o 75 kg and a height o 1 .45 metres. [1 ] 4. O utline the relationship between height and B MI or a fxed body mass. [1 ] b) Deduce the body mass status o this man. [1 ] 2 a) State the body mass o the person standing on the scales on the previous page. [1 ] b) The person has a height o 1 .8 metres. D educe their body mass status. [1 ] 3 a) A woman has a height o 1 5 0 cm and a B MI o 40. C alculate the minimum amount o body mass she must lose to reach normal body mass status. S how all o your working. [3 ] body mass/kg height/cm 150 140 130 120 125 body mass index 130 135 110 50 100 95 90 85 80 75 70 65 60 140 40 30 145 150 155 160 20 55 165 170 50 175 45 180 40 10 35 185 190 195 30 200 205 25 Figure 10 Jogger 210 Figure 11 Fatty acids Fatty acids can be saturated, monounsaturated or polyunsaturated. The basic structure o atty acids was described in sub- topic 2 .1 . There is a chain o carbon atoms, with hydrogen atoms linked to them by single covalent bonds. It is thereore a hydrocarbon chain. At one end o the chain is the acid part o the molecule. This is a carboxyl group, which can be represented as C O O H. The length o the hydrocarbon chain is variable but most o the atty acids used by living organisms have between 1 4 and 20 carbon atoms. Another variable eature is the bonding between the carbon atoms. In some atty 81 2 M O L E C U L AR B I O LO G Y OH O C H C H OH O C H C H H C H H C H H C H H C H H C H H C H OH O C H C H H C H H C H H C H H C H H C H H C H H C H H C H C H H C H H C H C H H C H H C H H C H H C H H C H C H C H C H H C H H C H H C H H C H C H H C H H C H H C H C H H C H H C H H C H H C H H C H H C H H H palmitic acid saturated non-essential Figure 12 linolenic acid polyunsaturated all cis essential omega 3 C H H C H H C H H palmitoleic acid monounsaturated cis non-essential omega 7 Examples of fatty acids acids all o the carbon atoms are linked by single covalent bonds, but in other atty acids there are one or more positions in the chain where carbon atoms are linked by double covalent bonds. I a carbon atom is linked to adj acent carbons in the chain by single bonds, it can also bond to two hydrogen atoms. I a carbon atom is linked by a double bond to an adj acent carbon in the chain, it can only bond to one hydrogen atom. A atty acid with single bonds between all o its carbon atoms thereore contains as much hydrogen as it possibly could and is called a saturated fatty acid. Fatty acids that have one or more double bonds are unsaturated because they contain less hydrogen than they could. I there is one double bond, the atty acid is monounsaturated and i it has more than one double bond it is p olyunsaturated. Figure 1 2 shows one saturated atty acid, one monounsaturated and one polyunsaturated atty acid. It is not necessary to remember names o specifc atty acids in IB B iology. unsatrated fatty acids Unsaturated fatty acids can be cis or trans isomers. In unsaturated atty acids in living organisms, the hydrogen atoms are nearly always on the same side o the two carbon atoms that are double bonded these are called cis- atty acids. The alternative is or the hydrogens to be on opposite sides called trans- atty acids. These two conormations are shown in fgure 1 4. In cis-atty acids, there is a bend in the hydrocarbon chain at the double bond. This makes triglycerides containing cis- unsaturated atty acids less good at packing together in regular arrays than saturated atty acids, so it lowers the melting point. Triglycerides with cis- unsaturated atty acids are thereore usually liquid at room temperature they are oils. Trans-atty acids do not have a bend in the hydrocarbon chain at the double bond, so they have a higher melting point and are solid at room temperature. Trans-atty acids are produced artifcially by partial hydrogenation o vegetable or fsh oils. This is done to produce solid ats or use in margarine and some other processed oods. H H H C C cis C C H trans Figure 13 Double bonds in fatty acids 82 Figure 14 Fatty acid stereochemistry (a) trans (b) cis 2 . 3 c a r b o h y d r at e s a n d l i P i d s Health risks of fats Scientifc evidence or health risks o trans-ats and saturated ats. There have been many claims about the eects o dierent types o at on human health. The main concern is coronary heart disease ( C HD ) . In this disease the coronary arteries become partially blocked by atty deposits, leading to blood clot ormation and heart attacks. A positive correlation has been ound between saturated atty acid intake and rates o C HD in many research programs. However, fnding a correlation does not prove that saturated ats cause the disease. It could be another actor correlated with saturated at intake, such as low amounts o dietary fbre, that actually causes C HD . There are populations that do not ft the correlation. The Maasai o Kenya or example have a diet that is rich in meat, at, blood and milk. They thereore have a high consumption o saturated ats, yet C HD is almost unknown among the Maasai. Figure 1 7 shows members o another Kenyan tribe that show this trend. Figure 15 Triglycerides in olive oil contain cis-unsaturated fatty acids D iets rich in olive oil, which contains cis- monounsaturated atty acids, are traditionally eaten in countries around the Mediterranean. The populations o these countries typically have low rates o C HD and it has been claimed that this is due to the intake o cis- monounsaturated atty acids. However, genetic actors in these populations, or other aspects o the diet such as the use o tomatoes in many dishes could explain the C HD rates. There is also a positive correlation between amounts o trans-at consumed and rates o C HD . Other risk actors have been tested, to see i they can account or the correlation, but none did. Trans-ats thereore probably do cause C HD . In patients who had died rom C HD , atty deposits in the diseased arteries have been ound to contain high concentrations o trans-ats, which gives more evidence o a causal link. narrowed lumen of artery fatty plaque causing thickening of the artery lining layer of muscle and elastic bres Figure 16 Artery outer coat of artery showing fatty plaque Figure 17 Samburu people of Northern Kenya. Like the Maasai, the Samburu have a diet rich in animal products but rates of heart disease are extremely low 83 2 M O L E C U L AR B I O LO G Y evaluating th halth risks of foods Evaluating claims: health claims made about lipids need to be assessed. Many health claims about oods are made. In some cases the claim is that the ood has a health benet and in other cases it is that the ood is harmul. Many claims have been ound to be alse when they are tested scientically. It is relatively easy to test claims about the eects o diet on health using laboratory animals. Large numbers o genetically uniorm animals can be bred and groups o them with the same age, sex and state o health can be selected or use in experiments. Variables other than diet, such as temperature and amount o exercise, can be controlled so that they do not infuence the results o the experiment. Diets can be designed so that only one dietary actor varies and strong evidence can thus be obtained about the eect o this actor on the animal. Results o animal experiments are oten interesting, but they do not tell us with certainty what the health eects are on humans o a actor in the diet. It would be very dicult to carry out similar controlled experiments with humans. It might be possible to select matched groups o experimental subj ects in terms o age, sex and health, but unless identical twins were used they would be genetically dierent. It would also be almost impossible to control other variables such as exercise and ew humans would be willing to eat a very strictly controlled diet or a long enough period. Researchers into the health risks o ood must thereore use a dierent approach. Evidence is obtained by epidemiological studies. These involve nding a large cohort o people, measuring their ood intake and ollowing their health over a period o years. S tatistical procedures can then be used to nd out whether actors in the diet are associated with an increased requency o a particular disease. The analysis has to eliminate the eects o other actors that could be causing the disease. Nature of science question: using volunteers in experiments. D uring the S econd World War, experiments were conducted both in England and in the US using conscientious obj ectors to military service as volunteers. The volunteers were willing to sacrice their health to help extend medical knowledge. A vitamin C trial in E ngland involved 2 0 volunteers. For six weeks they were all given a diet containing 70 mg o vitamin C . Then, or the next eight months, three volunteers were kept on the diet with 70 mg, seven had their dose reduced to 1 0 mg and ten were given no vitamin C . All o these ten volunteers developed scurvy. Three- centimetre cuts were made in their thighs, with the wounds closed up with ve stitches. These wounds ailed to heal. There was also bleeding rom hair ollicles and rom the gums. S ome o the volunteers developed more serious heart problems. The groups given 1 0 mg or 70 mg o vitamin C ared equally well and did not develop scurvy. Experiments on requirements or vitamin C have also been done using real guinea- pigs, which ironically are suitable because guinea-pigs, like 84 humans, cannot synthesize ascorbic acid. D uring trial periods with various intakes o vitamin C , concentrations in blood plasma and urine were monitored. The guinea- pigs were then killed and collagen in bone and skin was tested. The collagen in guinea- pigs with restricted vitamin C had less cross- linking between the protein bres and thereore lower strength. 1 Is it ethically acceptable or doctors or scientists to perorm experiments on volunteers, where there is a risk that the health o the volunteers will be harmed? 2 S ometimes people are paid to participate in medical experiments, such as drug trials. Is this more or less acceptable than using unpaid volunteers? 3 Is it better to use animals or experiments or are the ethical objections the same as with humans? 4 Is it acceptable to kill animals, so that an experiment can be done? 2 . 3 c a r b o h y d r at e s a n d l i P i d s anlysis of dt on helth risks of lipids Evaluation of evidence and the methods used to obtain the evidence for health claims made about lipids. An evaluation is defned in IB as an assessment o implications and limitations. Evidence or health claims comes rom scientifc research. There are two questions to ask about this research: 1 2 Implications do the results o the research support the health claim strongly, moderately or not at all? How widely spread is the data? This is shown by the spread o data points on a scattergraph or the size o error bars on a bar chart. The more widely spread the data, the less likely it is that mean dierences are signifcant. I statistical tests have been done on the data, do they show signifcant dierences? Limitations were the research methods used rigorous, or are there uncertainties about the conclusions because o weaknesses in methodology? The second question is answered by assessing the methods used. The points below reer to surveys and slightly dierent questions should be asked to assess controlled experiments. The frst question is answered by analysing the results o the research either experimental results or results o a survey. Analysis is usually easiest i the results are presented as a graph or other type o visual display. Is there a correlation between intake o the lipid being investigated and rate o the disease or the health beneft? This might be either a positive or negative correlation. How large is the dierence between mean ( average) rates o the disease with dierent levels o lipid intake? Small dierences may not be signifcant. How large was the sample size? In surveys it is usually necessary to have thousands o people in a survey to get reliable results. How even was the sample in sex, age, state o health and lie style? The more even the sample, the less other actors can aect the results. I the sample was uneven, were the results adjusted to eliminate the eects o other actors? Were the measurements o lipid intake and disease rates reliable? S ometimes people in a survey do not report their intake accurately and diseases are sometimes misdiagnosed. d- qu: Evaluating evidence from a health survey The Nurses Health S urvey is a highly respected survey into the health consequences o many actors. It began in 1 976 with 1 2 1 , 700 emale nurses in the US A and C anada, who completed a lengthy questionnaire about their liestyle actors and medical history. Follow- up questionnaires have been completed every two years since then. D etails o the methods used to assess diet and diagnose coronary heart disease can be ound by reading a research paper in the American Journal o Epidemiology, which is reely available on the internet: O h, K, Hu, FB , Manson, JE, S tamper, MJ and Willett, WC . ( 2 005 ) D ietary Fat Intake and Risk o C oronary Heart D isease in Women: 2 0 Years o Follow-up o the Nurses Health Study. American Journal of Epidemiology, 1 61 :672 679. doi:1 0.1 093 /aj e/kwi085 To asse ss the eects o trans- ats on rates o C HD , the participants in the survey were divide d into ive groups according to the ir trans- at intake. Q uintile 1 was the 2 0 % o participants with the lowest intake and quintile 5 was the 2 0 % with the highe st intake. The ave rage intake o trans- ats or each quintile was calculated, as a percentage o dietary energy intake. The re lative risk o C HD was o und or each quintile, with Q uintile 1 assigned a risk o 1 . The risk was adj usted or die rences b etween the quintiles in age , body mass index, smoking, alcohol intake , parental 85 2 M O L E C U L AR B I O LO G Y history o C HD , intake o other oods that aect C HD rate s and various othe r actors. Figure 1 8 is a graph showing the percentage o ene rgy rom trans- ats or e ach o the ive quintiles and the adj uste d relative risk o C HD . The e e ct o trans- at intake on relative risk o C HD is statistically signiicant with a conidence level o 9 9 % . 1 S tate the trend shown in the graph. 3 The mean age o nurses in the fve quintiles was not the same. E xplain the reasons or adj usting the results to compensate or the eects o age dierences. [2 ] 5 relative risk of CHD 1.4 1.2 1.0 0.8 0.6 0.4 0.2 S uggest reasons or using only emale nurses in this survey. [3 ] 2 4 1.6 0 1 1.5 2.0 2.5 percentage of energy from trans-fats [1 ] 3.0 Data for graph C alculate the chance, based on the statistical tests, o the dierences in C HD risk being due to actors other than trans- at intake. [2 ] % of energy from trans-fat 1.3 1.6 1.9 2.2 2.8 Relative risk of CHD 1.0 1.08 1.29 1.19 1.33 Figure 18 D iscuss evidence rom the graph that other actors were having some eect on rates o C HD . [2 ] Zutphen USA Slavonia Belgrade Crevalcor Zrenjanin Dalmatia Crete Montegiorgio Velika Rome Corfu Ushibuka Tanushimaru % Calories as saturated fat W. Finland Populations ranked by % calories as saturated fat E. Finland data-base questions: Saturated fats and coronary heart disease 22 19 19 18 14 12 10 10 9 9 9 9 8 7 3 3 Death CHD 992 351 420 574 214 288 248 152 86 rate/ 100,000 All yr 1 causes 1727 1318 1175 1088 1477 509 1241 1101 758 9 150 80 290 144 66 88 543 1080 1078 1027 764 1248 1006 Table 2 1 2 3 86 a) Plot a scattergraph o the data in table 2 . [5 ] b) O utline the trend shown by the scattergraph. [2 ] C ompare the results or: a) E ast and West Finland; [2 ] b) C rete and Montegiorgio. [2 ] Evaluate the evidence rom this survey or saturated ats as a cause o coronary heart disease. [4] 2 .4 Protein s 2.4 P understnding applictions Amino acids are linked together by Rubisco, insulin, immunoglobulins, rhodopsin, condensation to orm polypeptides. There are twenty diferent amino acids in polypeptides synthesized on ribosomes. Amino acids can be linked together in any sequence giving a huge range o possible polypeptides. The amino acid sequence o polypeptides is coded or by genes. A protein may consist o a single polypeptide or more than one polypeptide linked together. The amino acid sequence determines the threedimensional conormation o a protein. Living organisms synthesize many diferent proteins with a wide range o unctions. Every individual has a unique proteome. collagen and spider silk as examples o the range o protein unctions. Denaturation o proteins by heat or deviation o pH rom the optimum. Skills Draw molecular diagrams to show the ormation o a peptide bond. Ntre of science Patterns, trends and discrepancies: most but not all organisms assemble polypeptides rom the same amino acids. amino cids nd polypeptides Amino acids are linked together by condensation to orm polypeptides. Polypeptides are chains of amino acids that are made by linking together amino acids by condensation reactions. This happens on ribosomes by a process called translation, which will be described in sub- topic 2 .7. Polypeptides are the main component of proteins and in many proteins they are the only component. S ome proteins contain one polypeptide and other proteins contain two or more. The condensation reaction involves the amine group (- NH 2 ) of one amino acid and the carboxyl group (- C OOH) of another. Water is eliminated, as carboxyl group H H O H N C peptide bond amino group 1 C OH H O H N C condensation (water removed) N C H OH R R amino acid amino acid H O H H C C N C O H C OH H R R H2O Figure 1 Condensation joins two amino acids with a peptide bond 87 2 M O L E C U L AR B I O LO G Y in all condensation reactions, and a new bond is ormed between the two amino acids, called a peptide bond. A dipeptide is a molecule consisting o two amino acids linked by a peptide bond. A polypeptide is a molecule consisting o many amino acids linked by peptide bonds. Polypeptides can contain any number o amino acids, though chains o ewer than 2 0 amino acids are usually reerred to as oligopeptides rather than polypeptides. Insulin is a small protein that contains two polypeptides, one with 2 1 amino acids and the other with 3 0. The largest polypeptide discovered so ar is titin, which is part o the structure o muscle. In humans titin is a chain o 3 4, 3 5 0 amino acids, but in mice it is even longer with 3 5 , 2 1 3 amino acids. Drawing peptide bonds Draw molecular diagrams to show the ormation o a peptide bond. To orm a dipeptide, two amino acids are linked by a condensation reaction between the amine group o one amino acid and the carboxyl group o the other. This is shown in fgure 1 . The peptide bond is the same, whatever R group the amino acid carries. To test your skill at showing how peptide bonds are ormed, try showing the ormation o a peptide bond between two o the amino acids in fgure 2 . There are sixteen possible dipeptides that can be produced rom these our amino acids. You could also try to draw an oligopeptide o our amino acids, linked by three peptide bonds. I you do this correctly, you should see these eatures: There is chain o atoms linked by single covalent bonds orming the backbone o the oligopeptide, with a repeating sequence o - N- C- C- A hydrogen atom is linked by a single bond to each nitrogen atom in the backbone and an oxygen atom is linked by a double bond to one o the two carbon atoms. The amine ( - NH 2 ) and carboxyl ( - C O O H) groups are used up in orming the peptide bond and only remain at the ends o the chain. These are called the amino and carboxyl terminals o the chain. The R groups o each amino acid remain and proj ect outwards rom the backbone. COOH OH H C H H C H H H C H H C H H 2 N C COOH H 2 N C COOH H 2 N C COOH H H glutamic acid H alanine serine H H 2N C COOH H glycine Figure 2 Some common amino acids The diversity of amino acids There are twenty diferent amino acids in polypeptides synthesized on ribosomes. The amino acids that are linked together by ribosomes to make polypeptides all have some identical structural eatures: a carbon atom in the centre o the molecule is bonded to an amine group, a carboxyl group and a hydrogen atom. The carbon atom is also bonded to an R group, which is dierent in each amino acid. 88 2 .4 Protein s Twenty dierent amino acids are used by ribosomes to make polypeptides. The amine groups and the carboxyl groups are used up in orming the peptide bond, so it is the R groups o the amino acids that give a polypeptide its character. The repertoire o R groups allows living organisms to make and use an amazingly wide range o proteins. Some o the dierences are shown in table 1 . It is not necessary to try to learn these specifc dierences but it is important to remember that because o the dierences between their R groups, the twenty amino acids are chemically very diverse. S ome proteins contain amino acids that are not in the basic repertoire o twenty. In most cases this is due to one o the twenty being modifed ater a polypeptide has been synthesized. There is an example o modifcation o amino acids in collagen, a structural protein used to provide tensile strength in tendons, ligaments, skin and blood vessel walls. C ollagen polypeptides made by ribosomes contain proline at many positions, but at some o these positions it is converted to hydroxyproline, which makes the collagen more stable. Nine R groups are hydrophobic with between zero and nine carbon atoms Eleven R groups are hydrophilic Seven R groups can become charged Four hydrophilic Four R groups act as Three R groups act as Three R Six R groups R groups are an acid by giving up a a base by accepting a groups contain do not contain polar but never proton and becoming proton and becoming charged rings rings negatively charged positively charged Table 1 acvy scuvy Ascorbic acid (vitamin C) is needed to convert proline into hydroxyproline, so ascorbic acid deciency leads to abnormal collagen production. From your knowledge o the role o collagen, what efects do you expect this to have? Test your predictions by researching the symptoms o ascorbic acid deciency (scurvy) . Classifcation o amino acids amino cids nd origins Patterns, trends and discrepancies: most but not all organisms assemble polypeptides rom the same amino acids. It is a remarkable act that most organisms make proteins using the same 2 0 amino acids. In some cases amino acids are modifed ater a polypeptide has been synthesized, but the initial process o linking together amino acids on ribosomes with peptide bonds usually involves the same 2 0 amino acids. We can exclude the possibility that this trend is due to chance. There must be one or more reasons or it. S everal hypotheses have been proposed: These 20 amino acids were the ones produced by chemical processes on Earth beore the origin o lie, so all organisms used them and have continued to use them. Other amino acids might have been used, i they had been available. They are the ideal 2 0 amino acids or making a wide range o proteins, so natural selection will always avour organisms that use them and do not use other amino acids. All lie has evolved rom a single ancestral species, which used these 2 0 amino acids. B ecause o the way that polypeptides are made by ribosomes, it is difcult or any organism to change the repertoire o amino acids, either by removing existing ones or adding new ones. B iology is a complicated science and discrepancies are commonly encountered. Some species have been ound that use one o the three codons that normally signal the end o polypeptide synthesis ( stop codons) to encode an extra non- standard amino acid. For example, some species use UGA to code or selenocysteine and some use UAG to code or pyrrolysine. 89 2 M O L E C U L AR B I O LO G Y dt-bse questios: Commonality of amino acids 1 a) D iscuss which o the three hypotheses or use o the same 2 0 amino acids by most organisms is supported by the evidence. [3 ] b) S uggest ways o testing one o the hypotheses. 2 Figure 3 C ell walls o bacteria contain peptidoglycan, a complex carbon compound that contains sugars and short chains o amino acids. Some o these amino acids are dierent rom the usual repertoire o 2 0. Also, some o them are right-handed orms o amino acids, whereas the 2 0 amino acids made into polypeptides are always the let-handed orms. D iscuss whether this is a signifcant discrepancy that alsifes the theory that living organisms all make polypeptides using the same 2 0 amino acids. [5 ] Kohoutek Comet 26 diferent amino acids were ound in an articial comet produced by researchers at the Institut dAstrophysique Spatiale (CNRS/France) , which suggests that amino acids used by the rst living organisms on Earth may have come rom space Polypeptide diversity ativity Amino acids can be linked together in any sequence giving a huge range of possible polypeptides. clultig polypeptie iversity number of mio is number of possible mio i sequees 1 20 1 2 20 2 3 400 8,000 4 20 6 64 million 10.24 trillion Table 2 Calculate the missing values [2 ] Ribosomes link amino acids together one at a time, until a polypeptide is ully ormed. The ribosome can make peptide bonds between any pair o amino acids, so any sequence o amino acids is possible. The number o possible amino acid sequences can be calculated starting with dipeptides ( table 2 ) . B oth amino acids in a dipeptide can be any o the twenty so there are twenty times twenty possible sequences ( 2 0 2 ) . There are 2 0 2 0 2 0 possible tripeptide sequences ( 2 0 3 ) . For a polypeptide o n amino acids there are 2 0 n possible sequences. The number o amino acids in a polypeptide can be anything rom 2 0 to tens o thousands. Taking one example, i a polypeptide has 400 amino acids, there are 2 0 400 possible amino acid sequences. This is a mindbogglingly large number and some online calculators simply express it as infnity. I we add all the possible sequences or other numbers o amino acids, the number is eectively infnite. Genes and polypeptides The amino acid sequence of polypeptides is coded for by genes. The number o amino acid sequences that could be produced is immense, but living organisms only actually produce a small raction o these. Even so, a typical cell produces polypeptides with thousands o dierent sequences and must store the inormation needed to do this. The amino acid sequence o each polypeptide is stored in a coded orm in the base sequence o a gene. Figure 4 Lysozyme with nitrogen o amine groups shown blue, oxygen red and sulphur yellow. The active site is the clet upper let 90 S ome genes have other roles, but most genes in a cell store the amino acid sequence o a polypeptide. They use the genetic code to do this. Three bases o the gene are needed to code or each amino acid in the polypeptide. In theory a polypeptide with 400 amino acids should require a gene with a sequence o 1 , 2 00 bases. In practice genes are 2 .4 Protein s always longer, with extra base sequences at both ends and sometimes also at certain points in the middle. The base sequence that actually codes for a polypeptide is known to molecular biologists as the open reading frame. O ne puzzle is that open reading frames only occupy a small proportion of the total D NA of a species. Proteins and polypeptides A protein may consist o a single polypeptide or more than one polypeptide linked together. S ome proteins are single polypeptides, but others are composed of two or more polypeptides linked together. Integrin is a membrane protein with two polypeptides, each of which has a hydrophobic portion embedded in the membrane. Rather like the blade and handle of a folding knife the two polypeptides can either be adj acent to each other or can unfold and move apart when it is working. C ollagen consists of three long polypeptides wound together to form a rope- like molecule. This structure has greater tensile strength than the three polypeptides would if they were separate. The winding allows a small amount of stretching, reducing the chance of the molecule breaking. Hemoglobin consists of four polypeptides with associated non-polypeptide structures. The four parts of hemoglobin interact to transport oxygen more effectively to tissues that need it than if they were separate. num f plyppd exmpl bckgud 1 lysozyme Enzyme in secretions such as nasal mucus and tears; it kills some bacteria by digesting the peptidoglycan in their cell walls. 2 integrin Membrane protein used to make connections between structures inside and outside a cell. collagen Structural protein in tendons, ligaments, skin and blood vessel walls; it provides high tensile strength, with limited stretching. hemoglobin Transport protein in red blood cells; it binds oxygen in the lungs and releases it in tissues with a reduced oxygen concentration. 3 4 Table 3 Example o proteins with diferent numbers o polypeptides Protein conformations The amino acid sequence determines the three-dimensional conormation o a protein. The conformation of a protein is its three-dimensional structure. The conformation is determined by the amino acid sequence of a protein and its constituent polypeptides. Fibrous proteins such as collagen Figure 5 Integrin embedded in a membrane (grey) shown olded and inactive and open with binding sites inside and outside the cell indicated (red and purple) acvy Molecular biologists are investigating the numbers o open reading rames in selected species or each o the major groups o living organism. It is still ar rom certain how many genes in each species code or a polypeptide that the organism actually uses, but we can compare current best estimates: Drosophila melanogaster, the ruit fy, has base sequences or about 14,000 polypeptides. Caenorhabditis elegans, a nematode worm with less than a thousand cells, has about 19,000. Homo sapiens has base sequences or about 23,000 dierent polypeptides. Arabidopsis thaliana, a small plant widely used in research, has about 27,000. Can you nd any species with greater or lesser numbers o open reading rames than these? 91 2 M O L E C U L AR B I O LO G Y are elongated, usually with a repeating structure. Many proteins are globular, with an intricate shape that oten includes parts that are helical or sheet-like. Amino acids are added one by one, to orm a polypeptide. They are always added in the same sequence to make a particular polypeptide. In globular proteins the polypeptides gradually old up as they are made, to develop the fnal conormation. This is stabilized by bonds between the R groups o the amino acids that have been brought together by the olding. Figure 6 Lysozyme, showing how a polypeptide can be folded up to form a globular protein. Three sections that are wound to form a helix are shown red and a section that forms a sheet is shown yellow. Other parts of the polypeptide including both of its ends are green In globular proteins that are soluble in water, there are hydrophilic R groups on the outside o the molecule and there are usually hydrophobic groups on the inside. In globular membrane proteins there are regions with hydrophobic R groups on the outside o the molecule, which are attracted to the hydrophobic centre o the membrane. In fbrous proteins the amino acid sequence prevents olding up and ensures that the chain o amino acids remains in an elongated orm. Denaturation of proteins Denaturation of proteins by heat or pH extremes. The three- dimensional conormation o proteins is stabilized by bonds or interactions between R groups o amino acids within the molecule. Most o these bonds and interactions are relatively weak and they can be disrupted or broken. This results in a change to the conormation o the protein, which is called denaturation. A denatured protein does not normally return to its ormer structure the denaturation is permanent. S oluble proteins oten become insoluble and orm a precipitate. This is due to the hydrophobic R groups in the centre o the molecule becoming exposed to the water around by the change in conormation. Heat can cause denaturation because it causes vibrations within the molecule that can break intermolecular bonds or interactions. Proteins vary in their heat tolerance. S ome microorganisms that live in volcanic springs or in hot water near geothermal vents have proteins that are not denatured by temperatures o 80 C or higher. The best known example is D NA polymerase rom Thermus aquaticus, a prokaryote that was discovered in hot springs in Yellowstone National Park. It works best at 80 C and because o this it is widely used in biotechnology. Nevertheless, heat causes denaturation o most proteins at much lower temperatures. 92 E xtremes o pH, both acidic and alkaline, can cause denaturation. This is because charges on R groups are changed, breaking ionic bonds within the protein or causing new ionic bonds to orm. As with heat, the three-dimensional structure o the protein is altered and proteins that have been dissolved in water oten become insoluble. There are exceptions: the contents o the stomach are normally acidic, with a pH as low as 1 .5 , but this is the optimum pH or the protein-digesting enzyme pepsin that works in the stomach. Figure 7 When eggs are heated, proteins that were dissolved in both the white and the yolk are denatured. They become insoluble so both yolk and white solidify 2 .4 Protein s Protein functions Living organisms synthesize many diferent proteins with a wide range o unctions. O ther groups o carbon compounds have important roles in the cell, but none can compare with the versatility o proteins. They can be compared to the worker bees that perorm almost all the tasks in a hive. All o the unctions listed here are carried out by proteins. acvy du xpm A solution o egg albumen in a test tube can be heated in a water bath to nd the temperature at which it denatures. The efects o pH can be investigated by adding acids and alkalis to test tubes o egg albumen solution. To quantiy the extent o denaturation, a colorimeter can be used as denatured albumen absorbs more light than dissolved albumen. C atalysis there are thousands o dierent enzymes to catalyse specifc chemical reactions within the cell or outside it. Muscle contraction actin and myosin together cause the muscle contractions used in locomotion and transport around the body. C ytoskeletons tubulin is the subunit o microtubules that give animals cells their shape and pull on chromosomes during mitosis. Tensile strengthening fbrous proteins give tensile strength needed in skin, tendons, ligaments and blood vessel walls. B lood clotting plasma proteins act as clotting actors that cause blood to turn rom a liquid to a gel in wounds. bx Transp ort of nutrients and gases proteins in blood help transport oxygen, carbon dioxide, iron and lipids. Botox is a neurotoxin obtained rom Clostridium botulinum bacteria. C ell adhesion membrane proteins cause adj acent animal cells to stick to each other within tissues. 1 Membrane transp ort membrane proteins are used or acilitated diusion and active transport, and also or electron transport during cell respiration and photosynthesis. What are the reasons or injecting it into humans? 2 What is the reason or Clostridium botulinum producing it? 3 What are the reasons or injecting it rather than taking it orally? Hormones some such as insulin, FS H and LH are proteins, but hormones are chemically very diverse. Recep tors binding sites in membranes and cytoplasm or hormones, neurotransmitters, tastes and smells, and also receptors or light in the eye and in plants. Packing of D NA histones are associated with D NA in eukaryotes and help chromosomes to condense during mitosis. Immunity this is the most diverse group o proteins, as cells can make huge numbers o dierent antibodies. acvy There are many biotechnological uses or proteins including enzymes or removing stains, monoclonal antibodies or pregnancy tests or insulin or treating diabetics. Pharmaceutical companies now produce many dierent proteins or treating diseases. These tend to be very expensive, as it is still not easy to synthesize proteins artifcially. Increasingly, genetically modifed organisms are being used as microscopic protein actories. 93 2 M O L E C U L AR B I O LO G Y exampls of protins Rubisco, insulin, immunoglobulins, rhodopsin, collagen and spider silk as examples o the range o protein unctions. Six proteins which illustrate some o the unctions o proteins are described in table 4. rubo inuln This name is an abbreviation or ribulose bisphosphate carboxylase, which is arguably the most important enzyme in the world. The shape and chemical properties o its active site allow it to catalyse the reaction that xes carbon dioxide rom the atmosphere, which provides the source o carbon rom which all carbon compounds needed by living organisms can be produced. It is present at high concentrations in leaves and so is probably the most abundant o all proteins on Earth. This hormone is produced as a signal to many cells in the body to absorb glucose and help reduce the glucose concentration o the blood. These cells have a receptor or insulin in their cell membrane to which the hormone binds reversibly. The shape and chemical properties o the insulin molecule correspond precisely to the binding site on the receptor, so insulin binds to it, but not other molecules. Insulin is secreted by cells in the pancreas and is transported by the blood. immunoglobuln rhodopn These proteins are also known as antibodies. They have sites at the tips o their two arms that bind to antigens on bacteria or other pathogens. The other parts o the immunoglobulin cause a response, such as acting as a marker to phagocytes that can engul the pathogen. The binding sites are hypervariable. The body can produce a huge range o immunoglobulins, each with a diferent type o binding site. This is the basis o specic immunity to disease. Vision depends on pigments that absorb light. One o these pigments is rhodopsin, a membrane protein o rod cells o the retina. Rhodopsin consists o a light sensitive retinal molecule, not made o amino acids, surrounded by an opsin polypeptide. When the retinal molecule absorbs a single photon o light, it changes shape. This causes a change to the opsin, which leads to the rod cell sending a nerve impulse to the brain. Even very low light intensities can be detected. collagen spde lk There are a number o diferent orms o collagen but all are rope-like proteins made o three polypeptides wound together. About a quarter o all protein in the human body is collagen it is more abundant than any other protein. It orms a mesh o bres in skin and in blood vessel walls that resists tearing. Bundles o parallel collagen molecules give ligaments and blood vessel walls their immense strength. It orms part o the structure o teeth and bones, helping to prevent cracks and ractures. Diferent types o silk with diferent unctions are produced by spiders. Dragline silk is stronger than steel and tougher than Kevlar. It is used to make the spokes o spiders webs and the lielines on which spiders suspend themselves. When rst made it contains regions where the polypeptide orms parallel arrays. Other regions seem like a disordered tangle, but when the silk is stretched they gradually extend, making the silk extensible and very resistant to breaking. Protoms Every individual has a unique proteome. A proteome is all o the proteins produced by a cell, a tissue or an organism. B y contrast, the genome is all o the genes o a cell, a tissue or an organism. To fnd out how many dierent proteins are being produced, mixtures o proteins are extracted rom a sample and are then separated 94 2 .4 Protein s by gel electrophoresis. To identiy whether or not a particular protein is present, antibodies to the protein that have been linked to a fuorescent marker can be used. I the cell fuoresces, the protein is present. Whereas the genome o an organism is xed, the proteome is variable because dierent cells in an organism make dierent proteins. Even in a single cell the proteins that are made vary over time depending on the cells activities. The proteome thereore reveals what is actually happening in an organism, not what potentially could happen. Within a species there are strong similarities in the proteome o all individuals, but also dierences. The proteome o each individual is unique, partly because o dierences o activity but also because o small dierences in the amino acid sequence o proteins. With the possible exception o identical twins, none o us have identical proteins, so each o us has a unique proteome. E ven the proteome o identical twins can become dierent with age. Figure 8 Proteins rom a nematode worm have been separated by gel electrophoresis. Each spot on the gel is a diferent protein acvy acv cc: gm d pm We might expect the proteome of an organism to be smaller than its genome, as some genes do not code for polypeptides. In fact the proteome is larger. How could an organism produce more proteins than the number of genes that its genome contains? 95 2 M O L E C U L AR B I O LO G Y 2.5 enzyms understnding Enzymes have an active site to which specic substrates bind. Enzyme catalysis involves molecular motion and the collision o substrates with the active site. Temperature, pH and substrate concentration afect the rate o activity o enzymes. Enzymes can be denatured. Immobilized enzymes are widely used in industry. Ntre of science applictions Methods o production o lactose-ree milk and its advantages. Skills Experimental design: accurate quantitative measurements in enzyme experiments require replicates to ensure reliability. Design o experiments to test the efect o temperature, pH and substrate concentration on the activity o enzymes. Experimental investigation o a actor afecting enzyme activity. (Practical 3) active sites nd enzymes Enzymes have an active site to which specic substrates bind . Enzymes are globular proteins that work as catalysts they speed up chemical reactions without being altered themselves. Enzymes are oten called biological catalysts because they are made by living cells and speed up biochemical reactions. The substances that enzymes convert into products in these reactions are called substrates. A general equation or an enzyme- catalysed reaction is: e nzym e product substrate _______ Figure 1 Computer-generated image of the enzyme hexokinase, with a molecule of its substrate glucose bound to the active site. The enzyme bonds a second substrate, phosphate, to the glucose, to make glucose phosphate 96 Enzymes are ound in all living cells and are also secreted by some cells to work outside. Living organisms produce many dierent enzymes literally thousands o them. Many dierent enzymes are needed, as enzymes only catalyse one biochemical reaction and thousands o reactions take place in cells, nearly all o which need to be catalysed. This property is called enzymesubstrate sp ecifcity. It is a signifcant dierence between enzymes and non- biological catalysts such as the metals that are used in catalytic converters o vehicles. To be able to explain enzymesubstrate specifcity, we must look at the mechanism by which enzymes speed up reactions. This involves the 2 . 5 en z yM e s substrate, or substrates binding to a special region on the surace o the enzyme called the active site (see fgure 1 ) . The shape and chemical properties o the active site and the substrate match each other. This allows the substrate to bind, but not other substances. Substrates are converted into products while they are bound to the active site and the products are then released, reeing the active site to catalyse another reaction. data-ba qutio: Biosynthesis of glycogen The Nobel Prize or Medicine was won in 1 947 by Gerty C ori and her husband C arl. They isolated two enzymes that convert glucose phosphate into glycogen. Glycogen is a polysaccharide, composed o glucose molecules bonded together in two ways, called 1 , 4 and 1 , 6 bonds ( see fgure 2 ) . 4 4 bonding Figure 2 1 2 3 1 1 4 bonding plus a 6 bond forming a side-branch a) D escribe the shape o C urve B . [2 ] b) Explain the shape o C urve B . [2 ] % conversion 1 C urve B was obtained using enzymes that had not been heat- treated. Bonding in glycogen Explain why two dierent enzymes are needed or the synthesis o glycogen rom glucose phosphate. 80 60 [2 ] 40 The ormation o side-branches increases the rate at which glucose phosphate molecules can be linked on to a growing glycogen molecule. Explain the reason or this. [2 ] 20 C urve A was obtained using heat- treated enzymes. Explain the shape o curve A. [2 ] B A 10 20 30 40 50 min Figure 3 shows the percentage conversion of glucose phosphate to glycogen by the two enzymes, over a 50-minute period enzym activity Enzyme catalysis involves molecular motion and the collision of substrates with the active site. E nzyme activity is the catalysis o a reaction by an enzyme. There are three stages: The substrate binds to the active site o the enzyme. S ome enzymes have two substrates that bind to dierent parts o the active site. While the substrates are bound to the active site they change into dierent chemical substances, which are the products o the reaction. The products separate rom the active site, leaving it vacant or substrates to bind again. A substrate molecule can only bind to the active site i it moves very close to it. The coming together o a substrate molecule and an active site is known as a collision. This might suggest a high velocity impact between two vehicles on a road, but that would be a misleading image and we need to think about molecular motion in liquids to understand how substrateactive site collisions occur. With most reactions the substrates are dissolved in water around the enzyme. B ecause water is in a liquid state, its molecules and all 97 2 M O L E C U L AR B I O LO G Y toK Why hs he lck nd key mdel n been lly superseded by he induced-f mdel? The lock and key model and the induced-t model were both developed to help to explain enzyme activity. Models like these are simplied descriptions, which can be used to make predictions. Scientists test these predictions, usually by perorming experiments. I the results agree with the predictions, then the model is retained; i not then the model is modied or replaced. The German scientist Emil Fischer introduced the lock and key model in 1890. Daniel Koshland suggested the induced-t model in 1959 in the United States. The conormational changes predicted by Koshland's model were subsequently observed using high-resolution X-ray analysis o enzymes and other newly developed techniques. Although much experimental evidence has accumulated conrming predictions based on the induced-t model, it is still just viewed as a model o enzyme activity. the particles dissolved in it are in contact with each other and are in continual motion. E ach particle can move separately. The direction of movement repeatedly changes and is random, which is the basis of diffusion in liquids. B oth substrates and enzymes with active sites are able to move, though most substrate molecules are smaller than the enzyme so their movement is faster. S o, collisions between substrate molecules and the active site occur because of random movements of both substrate and enzyme. The substrate may be at any angle to the active site when the collision occurs. Successful collisions are ones in which the substrate and active site are correctly aligned to allow binding to take place. water molecules substrates active site part of enzyme Figure 4 Enzyme-substrate collisions. If random movements bring any of the substrate molecules close to the active site with the correct orientation, the substrate can bind to the active site Factors afecting enzyme activity aciviy Mking hyphesis Bacillus licheniformis lives in soil and on decomposing eathers. What is the reason or it producing a protease that works best at alkaline pH? Make a hypothesis to explain the observations. How could you test your hypothesis? 98 Temperature, pH and substrate concentration afect the rate o activity o enzymes. Enzyme activity is afected by temperature in two ways In liquids, the particles are in continual random motion. When a liquid is heated, the particles in it are given more kinetic energy. Both enzyme and substrate molecules therefore move around faster at higher temperatures and the chance of a substrate molecule colliding with the active site of the enzyme is increased. Enzyme activity therefore increases. When enzymes are heated, bonds in the enzyme vibrate more and the chance of the bonds breaking is increased. When bonds in the enzyme break, the structure of the enzyme changes, including the active site. This change is permanent and is called denaturation. When an enzyme molecule has been denatured, it is no longer able to catalyse reactions. As more and more enzyme molecules in a solution become denatured, enzyme activity falls. Eventually it stops altogether, when the enzyme has been completely denatured. So, as temperature rises there are reasons for both increases and decreases in enzyme activity. Figure 5 shows the effects of temperature on a typical enzyme. 2 . 5 en z yM e s Enzymes are sensitive to pH Most enzymes have an optimum pH at which their activity is highest. I the pH is increased or decreased rom the optimum, enzyme activity decreases and eventually stops altogether. When the hydrogen ion concentration is higher or lower than the level at which the enzyme naturally works, the structure o the enzyme is altered, including the active site. B eyond a certain pH the structure o the enzyme is irreversibly altered. This is another example o denaturation. E nzyme s do no t all have the same p H o p timu m in act, the re is a wide range . This re le cts the wide range o p H e nviro nme nts in which e nzyme s wo rk. Fo r e xamp le , the p ro te ase se cre te d b y Bacillus lichen iform is has a p H o p timum b e twe e n 9 and 1 0 . This b acte rium is cu lture d to p ro duce its alkaline - to le rant p ro te ase o r u se in b io lo gical lau ndry de te rge nts, which are alkaline . Figure 6 sho ws the p H range o so me o the p lace s whe re e nzyme s wo rk. Figu re 7 sho ws the e e cts o p H o n an e nzyme that is adap te d to wo rk at ne u tral p H. rate of reaction The pH scale is used to measure the acidity or alkalinity o a solution. The lower the pH, the more acid or the less alkaline a solution is. Acidity is due to the presence o hydrogen ions, so the lower the pH, the higher the hydrogen ion concentration. The pH scale is logarithmic. This means that reducing the pH by one unit makes a solution ten times more acidic. A solution at pH 7 is neutral. A solution at pH 6 is slightly acidic; pH 5 is ten times more acidic than pH 6, pH 4 is one hundred times more acidic than pH 6, and so on. rate at which reaction decreases owing to denaturation of enzyme molecules 0 20 optimum temperature actual rate of reaction 30 40 temperature/C 50 60 enzyme activity Key stomach acidic hot springs decaying plant matter large intestine small intestine alkaline lakes 1 2 3 4 5 6 E nzymes cannot catalyse reactions until the substrate binds to the active site. This happens because o the random movements o molecules in liquids that result in collisions between substrates and active sites. I the concentration o substrates is increased, substrateactive site collisions will take place more requently and the rate at which the enzyme catalyses its reaction increases. 7 8 9 10 Figure 6 Optimum pH at which enzyme activity is fastest (pH 7 is optimum for most enzymes) . As pH increases or decreases from the optimum, enzyme activity is reduced. This is because the shape of the active site is altered so the substrate does not t so well. Most enzymes are denatured by very high or low pH, so the enzyme no longer catalyses the reaction. enzyme activity I the relationship between substrate concentration and enzyme activity is plotted on a graph, a distinctive curve is seen ( fgure 8) , rising less and less steeply, but never quite reaching a maximum. 10 Figure 5 Temperature and Enzyme activity is afected by substrate concentration However, there is another trend that needs to be considered. Ater the binding o a substrate to an active site, the active site is occupied and unavailable to other substrate molecules until products have been ormed and released rom the active site. As the substrate concentration rises, more and more o the active sites are occupied at any moment. A greater and greater proportion o substrateactive site collisions are thereore blocked. For this reason, the increases in the rate at which enzymes catalyse reactions get smaller and smaller as substrate concentration rises. rate at which reaction increases owing to increased kinetic energy of substrate and enzyme molecules pH Figure 7 pH and enzyme activity 99 2 M O L E C U L AR B I O LO G Y Denaturation Enzymes can be denatured. enzyme activity Enzymes are proteins, and like other proteins their structure can be irreversibly altered by certain conditions. This process is denaturation and both high temperatures and either high or low pH can cause it. substrate concentration When an enzyme has been denatured, the active site is altered so the substrate can no longer bind, or i its binds, the reaction that the enzyme normally catalyses does not occur. In many cases denaturation causes enzymes that were dissolved in water to become insoluble and orm a precipitate. Figure 8 The efect o substrate concentration on enzyme activity Quantitative experiments Experimental design: accurate quantitative measurements in enzyme experiments require replicates to ensure reliability. O ur understanding o enzyme activity is based on evidence rom experiments. To obtain strong evidence these experiments must be careully designed and ollow some basic principles: measurements should be accurate, which in science means close to the true value; and the experiment should be repeated, so that the replicate results can be compared to assess how reliable they are. the results o the experiment should be quantitative, not j ust descriptive; data-base questions: Digesting jello cubes a) describing whether the solution around the cubes is colourless or a shade o pink or red Figure 9 shows apparatus that can be used to investigate protein digestion. tube b) taking a sample o the solution and measuring its absorbance in a colorimeter tight-tting lid c) nding the mass o the cubes using an electronic balance. [3 ] protease in a solution with known pH Figure 9 100 I method ( c) was chosen, discuss whether it would be better to nd the mass o all o the cubes o j ello together, or nd the mass o each one separately. [2 ] 3 I the j ello cubes have a mass o 0.5 grams, state whether it is accurate enough to measure their mass to: gelatine cubes Tube used to investigate the rate o digestion o gelatine I the cubes are made rom sugar- ree j ello ( j elly) , the colouring that they contain will gradually be released as the protein is digested by the protease. The questions below assume that strawberryfavoured j ello with red colouring has been used! 1 2 Explain whether these methods o assessing the rate o protein digestion are acceptable: a) the nearest gram ( g) b) the nearest milligram ( mg) c) the nearest microgram ( g) . [3 ] 2 . 5 en z yM e s 4 To obtain accurate mass measurements o the j ello cubes, it is necessary to remove them rom the tube and dry their surace to ensure that there are no drips o solution rom the tube adhering. Explain the reason or drying the surace o the blocks. [2 ] 7 D raw a graph o the results in the table. 8 D escribe the relationship between pH and papain activity. [3 ] 9 D iscuss the conclusions that can be drawn rom this data about the precise optimum pH o papain. [2 ] Table 1 gives the results that were obtained using sugar-ree jello cubes and a protease called papain, extracted rom the fesh o resh pineapples. 5 6 D iscuss whether the results in table 1 are reliable. [2 ] Most o the results were obtained using an extract o protease rom one pineapple, but ater this ran out, a second pineapple was used to obtain more protease or use in the experiment. a) Deduce which results were obtained using the second extract. [1 ] b) S uggest how the use o a second extract could have aected the results. [2 ] ph Ma dcra (mg) 2 80 87 77 3 122 127 131 4 163 166 164 5 171 182 177 6 215 210 213 7 167 163 84 8 157 157 77 9 142 146 73 [5 ] Table 1 Designing enzyme experiments Design o experiments to test the efect o temperature, pH and substrate concentration on the activity o enzymes. 1 2 The actor that you are going to investigate is the independent variable. You need to decide: clock could be used to measure the time taken or a colour change; how you are going to vary it, or example with substrate concentration you would obtain a solution with the highest concentration and dilute it to get lower concentrations; what units should be used or measuring the dependent variable, or example seconds rather than minutes or hours would be used or measuring a rapid colour change; what units should be used or measuring the independent variable, or example temperature is measured in degrees C elsius; how many repeats you need to get reliable enough results. what range you need or the independent variable, including the highest and lowest levels and the number o intermediate levels. The variable that you measure to nd out how ast the enzyme is catalysing the reaction is the dependent variable. You need to decide: how you are going to measure it, including the choice o meter or other measuring device, or example an electronic stop 3 Other actors that could aect the dependent are control variables. You need to decide: what all the control variables are; how each o them can be kept constant; what level they should be kept at, or example temperature should be kept at the optimum or the enzyme i pH is being investigated, but actors that might inhibit enzymes should be kept at a minimum level. 101 2 M O L E C U L AR B I O LO G Y enzym xprimnts Experimental investigation o a actor afecting enzyme activity. There are many worthwhile enzyme experiments. The method that ollows can be used to investigate the eect o substrate concentration on the activity o catalase. C atalase is one o the most widespread enzymes. It catalyses the conversion o hydrogen peroxide, a toxic by- product o metabolism, into water and oxygen. The apparatus shown in fgure 1 0 can be used to investigate the activity o catalase in yeast. The experiment could be repeated using the same concentration o yeast, but dierent hydrogen peroxide concentrations. Another possible investigation would be to assess the catalase concentrations in other cell types, such as liver, kidney or germinating seeds. These tissues would have to be macerated and then mixed with water at the same concentration as the yeast. 1 D escribe how the activity o the enzyme catalase could be measured using the apparatus shown in fgure 1 0. [2 ] 2 Explain why a yeast suspension must always be thoroughly stirred beore a sample o it is taken or use in an experiment. [2] 3 S tate two actors, apart rom enzyme concentration, that should be kept Figure 11 102 Enzyme experiment constant i investigating the eect o substrate concentration. [2 ] 4 Predict whether the enzyme activity will change more i substrate concentration is increased by 0. 2 mol dm - 3 or i it is decreased by the same amount. [2 ] 5 Explain why tissues such as liver must be macerated beore investigating catalase activity in them. [2 ] Safety goggles must be worn if this experiment is performed. Care should be taken not to get hydrogen peroxide on the skin. oxygen yeast three-way tap measuring cylinder water 0.8 mol dm 2 3 hydrogen peroxide Figure 10 Apparatus for measuring catalase activity water 2 . 5 en z yM e s data-ba qutio: Designing an experiment to fnd the eect o temperature on lipase. Lipase converts fats into fatty acids and glycerol. It therefore causes a decrease in pH. This pH change can be used to measure the activity of lipase. Figure 1 2 shows suitable apparatus. 2 tube contents mixed when both have reached target temperature thermometer 3 4 thermostatically controlled water bath Figure 12 lipase [2 ] b) S tate the units for measuring the dependent variable. [1 ] c) Explain the need for at least three replicate results for each temperature in this experiment. [2 ] a) List the control factors that must be kept constant in this experiment. [3 ] b) Explain how these control factors can be kept constant. [2 ] c) S uggest a suitable level for each control factor. [3 ] S uggest reasons for: a) milk being used to provide a source of lipids in this experiment rather than vegetable oil. [1 ] milk mixed with sodium carbonate (an alkali) and phenolphthalein (a pH indicator) b) the thermometer being placed in the tube containing the larger, rather than the smaller, volume of liquid [1 ] Apparatus for investigating the activity of lipase Phenolphthalein is pink in alkaline conditions, but becomes colourless when the pH drops to 7. The time taken for this colour change can be used to measure the activity of lipase at different temperatures. Alternatively, pH changes could be followed using a pH probe and data- logging software. 1 a) Explain how you would measure the dependent variable accurately. c) the substrate being added to the enzyme, rather than the enzyme to the substrate. 5 S ketch the shape of graph that you would expect from this experiment, with a temperature range from 0 C to 80 C on the x- axis and time taken for the indicator to change colour on the y- axis. [2 ] 6 Explain whether lipase from human pancreas or from germinating castor oil seeds would be expected to have the higher optimum temperature. [2 ] a) State the independent variable in this experiment and how you would vary it. [2 ] b) S tate the units for measuring the independent variable. [1 ] c) State an appropriate range for the independent variable. [2 ] [1 ] Immobilized enzymes Immobilized enzymes are widely used in industry. In 1 897 the B uchner brothers, Hans and E duard, showed that an extract of yeast, containing no yeast cells, would convert sucrose into alcohol. The door was opened to the use of enzymes to catalyse chemical processes outside living cells. Louis Pasteur had claimed that fermentation of sugars to alcohol could only occur if living cells were present. This was part of the theory of 103 2 M O L E C U L AR B I O LO G Y toK Wha is he diference beween dgma and hery? Ater the discovery in the 19th century o the conversion o sugar into alcohol by yeast, a dispute developed between two scientists, Justus von Liebig and Louis Pasteur. In 1860 Pasteur argued that this process, called ermentation, could not occur unless live yeast cells were present. Liebig claimed that the process was chemical and that living cells were not needed. Pasteurs view refected the vitalistic dogma that the substances in animals and plants could only be made under the infuence o a vital spirit or vital orce. These contrasting views were as much infuenced by political and religious actors as by scientic evidence. The dispute was only resolved ater the death o both men. In 1897 the Buchner brothers, Hans and Eduard, showed that an extract o yeast, containing no yeast cells, did indeed convert sucrose into alcohol. The vitalistic dogma was overthrown and the door was opened to the use o enzymes to catalyse chemical processes outside living cells. vitalism, which stated that substances in animals and plants can only be made under the infuence o a vital spirit or vital orce. The articial synthesis o urea, described in sub- topic 2 . 1 , had provided evidence against vitalism, but the B uchners research provided a clearer alsication o the theory. More than 5 00 enzymes now have commercial uses. Figure 1 3 shows a classication o commercially useul enzymes. Some enzymes are used in more than one type o industry. other industries 5% agriculture 11% miscellaneous 4% medical 21% biosensor 16% food & nutrition 23% biotechnology 46% environment 13% energy 3% Figure 13 The enzymes used in industry are usually immobilized. This is attachment o the enzymes to another material or into aggregations, so that movement o the enzyme is restricted. There are many ways o doing this, including attaching the enzymes to a glass surace, trapping them in an alginate gel, or bonding them together to orm enzyme aggregates o up to 0. 1 mm diameter. Enzyme immobilization has several advantages. 104 The enzyme can easily be separated rom the products o the reaction, stopping the reaction at the ideal time and preventing contamination o the products. Ater being retrieved rom the reaction mixture the enzyme may be recycled, giving useul cost savings, especially as many enzymes are very expensive. Immobilization increases the stability o enzymes to changes in temperature and pH, reducing the rate at which they are degraded and have to be replaced. S ubstrates can be exposed to higher enzyme concentrations than with dissolved enzymes, speeding up reaction rates. 2 . 6 s tru ctu r e o f d n a an d r n a lctose-free mik Methods o production o lactose-ree milk and its advantages. Lactose is the sugar that is naturally present in milk. It can be converted into glucose and galactose by the enzyme lactase: lactose glucose + galactose. Lactase is obtained rom Kluveromyces lactis, a type o yeast that grows naturally in milk. B iotechnology companies culture the yeast, extract the lactase rom the yeast and puriy it or sale to ood manuacturing companies. There are several reasons or using lactase in ood processing: S ome people are lactose-intolerant and cannot drink more than about 2 5 0 ml o milk per day, unless it is lactose- reduced ( see fgure 1 4) . Galactose and glucose are sweeter than lactose, so less sugar needs to be added to sweet oods containing milk, such as milk shakes or ruit yoghurt. Lactose tends to crystallize during the production o ice cream, giving a gritty texture. B ecause glucose and galactose are more soluble than lactose they remain dissolved, giving a smoother texture. B acteria erment glucose and galactose more quickly than lactose, so the production o yoghurt and cottage cheese is aster. Thailand South India Crete France Finland Sweden 0% 50% 100% lactose intolerance Figure 14 Rates of lactose intolerance 2.6 s dna rna understnding The nucleic acids DNA and RNA are polymers o nucleotides. DNA difers rom RNA in the number o strands normally present, the base composition and the type o pentose. DNA is a double helix made o two antiparallel strands o nucleotides linked by hydrogen bonding between complementary base pairs. Ntre of science Using models as representation o the real world: Crick and Watson used model-making to discover the structure o DNA. appictions Crick and Watsons elucidation o the structure o DNA using model-making. Skis Drawing simple diagrams o the structure o single nucleotides and o DNA and RNA, using circles, pentagons and rectangles to represent phosphates, pentoses and bases. 105 2 M O L E C U L AR B I O LO G Y Nucleic cids nd nucleotides The nucleic acids DNA and RNA are polymers o nucleotides. phosphate sugar base O O P O 5 CH 2 O O 1 C C N Nucleic acids were frst discovered in material extracted rom the nuclei o cells, hence their name. There are two types o nucleic acid: D NA and RNA. Nucleic acids are very large molecules that are constructed by linking together nucleotides to orm a polymer. Nucleotides consist o three parts: 4 C3 2 OH Figure 1 C a sugar, which has fve carbon atoms, so is a pentose sugar; OH a p hosp hate group, which is the acidic, negatively- charged part o nucleic acids; and a base that contains nitrogen and has either one or two rings o atoms in its structure. The parts of a nucleotide Figure 1 shows these parts and how they are linked together. The base and the phosphate are both linked by covalent bonds to the pentose sugar. Figure 2 shows a nucleotide in symbolic orm. To link nucleotides together into a chain or polymer, covalent bonds are ormed between the phosphate o one nucleotide and the pentose sugar o the next nucleotide. This creates a strong backbone or the molecule o alternating sugar and phosphate groups, with a base linked to each sugar. Figure 2 A simpler representation of a nucleotide There are our dierent bases in both D NA and RNA, so there are our dierent nucleotides. The our dierent nucleotides can be linked together in any sequence, because the phosphate and sugar used to link them are the same in every nucleotide. Any base sequence is thereore possible along a D NA or RNA molecule. This is the key to nucleic acids acting as a store o genetic inormation the base sequence is the store o inormation and the sugar phosphate backbone ensures that the store is stable and secure. Difeences between DNa nd rNa DNA difers rom RNA in the number o strands normally present, the base composition and the type o pentose. HOH 2 C OH O H H H H OH HOH 2 C H 2 There are usually two polymers o nucleotides in D NA but only one in RNA. The polymers are oten reerred to as strands, so D NA is double- stranded and RNA is single-stranded. 3 The our bases in D NA are adenine, cytosine, guanine and thymine. The our bases in RNA are adenine, cytosine, guanine and uracil, so the dierence is that uracil is present instead o thymine in RNA. H OH OH The sugar within DNA is deoxyribose (top) and the sugar in RNA is ribose (bottom) 106 The sugar within D NA is deoxyribose and the sugar in RNA is ribose. Figure 3 shows that deoxyribose has one ewer oxygen atom than ribose. The ull names o D NA and RNA are based on the type o sugar in them deoxyribonucleic acid and ribonucleic acid. OH H Figure 3 1 H O H There are three important dierences between the two types o nucleic acid: 2 . 6 s tru ctu r e o f d n a an d r n a d-b qi: Chargafs data D NA samples from a range of species were analysed in terms of their nucleotide composition by Edwin C hargaff, an Austrian biochemist, and by others. The data is presented in table 1 . 1 2 C ompare the base composition of Mycobacterium tuberculosis ( a prokaryote) with the base composition of the eukaryotes shown in the table. [2 ] C alculate the base ratio A+ G/T + C , for humans and for Mycobacterium tuberculosis. S how your working. [2 ] s dna Gp 3 4 5 E valuate the claim that in the D NA of eukaryotes and prokaryotes the amount of adenine and thymine are equal and the amounts of guanine and cytosine are equal. [2 ] E xplain the ratios between the amounts of bases in eukaryotes and prokaryotes in terms of the structure of D NA. [2 ] S uggest reasons for the difference in the base composition of bacteriophage T2 and the polio virus. [2 ] ai Gi cyi thymi Human Mammal 31.0 19.1 18.4 31.5 Cattle Mammal 28.7 22.2 22.0 27.2 Salmon Fish 29.7 20.8 20.4 29.1 Sea urchin Invertebrate 32.8 17.7 17.4 32.1 Wheat Plant 27.3 22.7 22.8 27.1 Yeast Fungus 31.3 18.7 17.1 32.9 Mycobacterium tuberculosis Bacteriophage T2 Polio virus Bacterium Virus Virus 15.1 32.6 30.4 34.9 18.2 25.4 35.4 16.6 19.5 14.6 32.6 0.0 Table 1 Dwing DNa nd rNa molecules Drawing simple diagrams of the structure of single nucleotides and of DNA and RNA, using circles, pentagons and rectangles to represent phosphates, pentoses and bases. The structure of D NA and RNA molecules can be shown in diagrams using simple symbols for the subunits: circles for phosphates; pentagons for pentose sugar; rectangles for bases. Figure 2 shows the structure of a nucleotide, using these symbols. The base and the phosphate are linked to the pentose sugar. The base is linked to C 1 the carbon atom on the right hand side of the pentose sugar. The phosphate is linked to C 5 the carbon atom on the side Figure 4 Simplifed diagram o RNA 107 2 M O L E C U L AR B I O LO G Y covalent bond P S A P chain on the upper let side o the pentose sugar. The positions o these carbon atoms are shown in fgure 1 . S T To show the structure o RNA, draw a polymer o nucleotides, with a line to show the covalent bond linking the phosphate group o each nucleotide to the pentose in the next nucleotide. The phosphate is linked to C 3 o the pentose the carbon atom that is on the lower let. P P S C S G P S P S G I you have drawn the structure o RNA correctly, the two ends o the polymer will be dierent. They are reerred to as the 3 and the 5 terminals. P The phosphate o another nucleotide could be linked to the C 3 atom o the 3 terminal. The pentose o another nucleotide could be linked to the phosphate o the 5 terminal. S A T P S C P P Hydrogen bonds are formed between two bases Key: S sugar A P phosphate C T nitrogenous bases G Figure 5 Simplifed diagram o DNA Structure of DNa 5 end 3 end complementary base pairs S P S P A T S G S C P hydrogen bonds S P P C S S G P A T P S S S P S P T S S P G P C S G Each strand consists o a chain o nucleotides linked by covalent bonds. P The two strands are parallel but run in opposite directions so they are said to be antiparallel. O ne strand is oriented in the direction 5 to 3 and the other is oriented in the direction 3 to 5 . The two strands are wound together to orm a double helix. The strands are held together by hydrogen bonds between the nitrogenous bases. Adenine ( A) is always paired with thymine ( T) and guanine ( G) with cytosine ( C ) . This is reerred to as comp lementary base p airing, meaning that A and T complement each other by orming base pairs and similarly G and C complement each other by orming base pairs. G S S P S P S sugarphosphate backbone S C S 3 end P 5 end Figure 6 The double helix 108 D rawings o the structure o D NA on paper cannot show all eatures o the three-dimensional structure o the molecule. Figure 6 represents some o these eatures. P S DNA is a double helix made of two antiparallel strands of nucleotides linked by hydrogen bonding between complementary base pairs. P A C A P P P C T G S S To show the structure o DNA, draw a strand o nucleotides, as with RNA, then a second strand alongside the frst. The second strand should be run in the opposite direction, so that at each end o the DNA molecule, one strand has a C 3 terminal and the other a C 5 terminal. The two strands are linked by hydrogen bonds between the bases. Add letters or names to indicate the bases. Adenine (A) only pairs with thymine (T) and cytosine (C ) only pairs with guanine (G) . 2 . 6 s tru ctu r e o f d n a an d r n a d-b qi: The bases in DNA Look at the molecular models in fgure 7 and answer the ollowing questions. 1 2 3 Identiy three similarities between adenine and guanine. [3 ] S tate one dierence between adenine and the other bases. [1 ] 4 C ompare the structure o cytosine and thymine. Each o the bases in D NA has a nitrogen atom bonded to a hydrogen atom in a similar position, which appears in the lower let in each case in fgure 7. D educe how this nitrogen is used when a nucleotide is being assembled rom its subunits. [2 ] 5 Guanine Adenine [4] Although the bases have some shared eatures, each one has a distinctive chemical structure and shape. Remembering the unction o D NA, explain the importance or the bases each to be distinctive. [5 ] Cytosine Thymine Figure 7 Molecular models Using models as representation of the real world: Crick and Watson used model-making to discover the structure of DNA. The word model in English is derived rom the Latin word modus, meaning manner or method. Models were originally architects plans, showing how a new building might be constructed. Threedimensional models were then developed to give a more realistic impression o what a proposed building would be like. Molecular models also show a possible structure in three dimensions, but whereas architects models are used to decide whether a building should become reality in the uture, molecular models help us to discover what the structure o a molecule actually is. Models in science are not always three- dimensional and do not always propose structures. They can be theoretical concepts and they can represent systems or processes. The common eature o models is that they are proposals, which are made to be tested. As with architecture, models in science are oten rej ected and replaced. Model- making played a critical part in C rick and Watsons discovery o the structure o D NA, but it took two attempts beore they were successul. 109 2 M O L E C U L AR B I O LO G Y toK crik nd Wtsons models of DNa struture Wha is he relaive rle cmpeiin and cperain in scienifc research? Crick and Watsons discovery o the structure o DNA using model-making. Three prominent research groups openly competed to elucidate the structure o DNA: Watson and Crick were working at Cambridge; Maurice Wilkins and Rosalind Franklin were working at Kings College o the University o London; and Linus Pauling's research group was operating out o Caltech in the United States. C rick and Watsons success in discovering the structure o D NA was based on using the evidence to develop possible structures or D NA and testing them by model- building. Their rst model consisted o a triple helix, with bases on the outside o the molecule and magnesium holding the two strands together with ionic bonds to the phosphate groups on each strand. The helical structure and the spacing between subunits in the helix tted the X- ray diraction pattern obtained by Rosalind Franklin. A stereotype o scientists is that they take a dispassionate approach to investigation. The truth is that science is a social endeavour involving a number o emotion-infuenced interactions between science. In addition to the joy o discovery, scientists seek the esteem o their community. Within research groups, collaboration is important, but outside o their research group competition oten restricts open communication that might accelerate the pace o scientic discovery. On the other hand, competition may motivate ambitious scientists to work tirelessly. It was dicult to get all parts o this model to t together satisactorily and it was rej ected when Franklin pointed out that there would not be enough magnesium available to orm the cross links between the strands. Another deciency o this rst model was that is that it did not take account o C hargas nding that the amount o adenine equals the thymine and the amount o cytosine equals the amount o guanine. To investigate the relationship between the bases in D NA pieces o cardboard were cut out to represent their shapes. These showed that A- T and C - G base pairs could be ormed, with hydrogen bonds linking the bases. The base pairs were equal in length so would t between two outer sugar-phosphate backbones. Another fash o insight was needed to make the parts o the molecule t together: the two strands in the helix had to run in opposite directions they must be antiparallel. C rick and Watson were then able to build their second model o the structure o D NA. They used metal rods and sheeting cut to shape and held together with small clamps. B ond lengths were all to scale and bond angles correct. Figure 8 shows C rick and Watson with the newly constructed model. The model convinced all those who saw it. A typical comment was It j ust looked right. The structure immediately suggested a mechanism or copying D NA. It also led quickly to the realization that the genetic code must consist o triplets o bases. In many ways the discovery o D NA structure started the great molecular biology revolution, with eects that are still reverberating in science and in society. Figure 8 Crick and 110 Watson and their DNA model 2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n 2.7 dna p, p understnding The replication o DNA is semi-conservative and depends on complementary base pairing. Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds. DNA polymerase links nucleotides together to orm a new strand, using the pre-existing strand as a template. Transcription is the synthesis o mRNA copied rom the DNA base sequences by RNA polymerase. Translation is synthesis o polypeptides on ribosomes. The amino acid sequence o polypeptides is determined by mRNA according to the genetic code. Codons o three bases on mRNA correspond to one amino acid in a polypeptide. Translation depends on complementary base pairing between codons on mRNA and anticodons on tRNA. applictions Use o Taq DNA polymerase to produce multiple copies o DNA rapidly by the polymerase chain reaction (PCR) . Production o human insulin in bacteria as an example o the universality o the genetic code allowing gene transer between species. Skills Use a table o the genetic code to deduce which codon(s) corresponds to which amino acid. Analysis o Meselson and Stahls results to obtain support or the theory o semiconservative replication o DNA. Use a table o mRNA codons and their corresponding amino acids to deduce the sequence o amino acids coded by a short mRNA strand o known base sequence. Deducing the DNA base sequence or the mRNA strand. Ntre of science Obtaining evidence or scientifc theories: Meselson and Stahl obtained evidence or the semi-conservative replication o DNA. Semi-conservtive repliction of DNa The replication o DNA is semi-conservative and depends on complementary base pairing. When a cell prepares to divide, the two strands o the double helix separate ( see fgure 2 ) . Each o these original strands serves as a guide, or template, or the creation o a new strand. The new strands are ormed by adding nucleotides, one by one, and linking them together. The result is two D NA molecules, both composed o an original strand and a newly synthesized strand. For this reason, D NA replication is reerred to as being semi-conservative. 111 2 M O L E C U L AR B I O LO G Y adenine thymine cytosine guanine guanine cytosine thymine The base sequence on the template strand determines the base sequence on the new strand. Only a nucleotide carrying a base that is complementary to the next base on the template strand can successully be added to the new strand (fgure 1 ) . This is because complementary bases orm hydrogen bonds with each other, stabilizing the structure. I a nucleotide with the wrong base started to be inserted, hydrogen bonding between bases would not occur and the nucleotide would not be added to the chain. The rule that one base always pairs with another is called complementary base pairing. It ensures that the two D NA molecules that result rom DNA replication are identical in their base sequences to the parent molecule that was replicated. obtaining evidence fr the thery f semicnservative replicatin adenine Obtaining evidence or scientifc theories: Meselson and Stahl obtained evidence or the semi-conservative replication o DNA. Figure 1 S emi- conservative replication is an example o a scientifc theory that seemed intuitively right, but nonetheless needed to be backed up with evidence. Laboratories around the world attempted to confrm experimentally that replication o D NA is semi- conservative and soon convincing evidence had been obtained. Parental DNA G C C G C G A T G C T A T A C G Replication fork A T G C A T G C T A C T A T A C G T A C C G A A T A T C G T A A T A T G C A T T A G Parental strand Figure 2 G C A T T A G C New strand New Parental strand strand Semi-conservative replication In 1 95 8 Matthew Meselson and Franklin S tahl published the results o exceedingly elegant experiments that provided very strong evidence or semi- conservative replication. They used 1 5 N, a rare isotope o nitrogen that has one more neutron than the normal 14 N isotope, so is denser. In the 1 93 0s Harold Urey had developed methods o puriying stable isotopes that could be used as tracers in biochemical pathways. 1 5 N was one o these. Meselson and S tahl devised a new method o separating D NA containing 1 5 N in its bases rom D NA with 1 4N. The technique is called caesium chloride density gradient centriugation. A solution o caesium chloride is spun in an ultracentriuge at nearly 45 , 000 revolutions per minute or 2 0 hours. The dense caesium ions tend to move towards the bottom o the tube but do not sediment ully because o diusion. A gradient is established, with the greatest caesium concentration, and thereore density, at the bottom and the lowest at the top o the tube. Any substance centriuged with the caesium chloride solution becomes concentrated at a level corresponding with its density. Meselson and S tahl cultured the bacterium E. coli or ourteen generations in a medium where the only nitrogen source was 1 5 N. Almost all nitrogen atoms in the bases o the D NA in the bacteria were thereore 1 5 N. They then transerred the bacteria abruptly to a medium in which all the nitrogen was 1 4 N. At the temperature used to culture them, the generation time was 5 0 minutes the bacteria divided and thereore replicated their D NA once every 5 0 minutes. 112 2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n Meselson and S tahl collected samples o D NA rom the bacterial culture or several hours rom the time when it was transerred to the 1 4 N medium. They extracted the D NA and measured its density by caesium chloride density gradient centriugation. The D NA could be detected because it absorbs ultraviolet light, and so created a dark band when the tubes were illuminated with ultraviolet. Figure 3 shows the results. In the next part o this sub- topic there is guidance in how to analyse the changes in position o the dark bands. avy nw xpm hqu Meselson and Stahl used three techniques in their experiments that that were relatively new. Identiy a technique used by them that was developed: a) by Urey in the 1930s b) by Pickels in the 1 940s c) by M eselson and Stahl them selves in the 1 950s. avy 0 0.3 0.7 1.0 1.5 2.0 2.5 3.0 4.0 generations Figure 3 Meselson nd Sthls DNa repliction experiments Analysis o Meselson and Stahls results to obtain support or the theory o semi-conservative replication o DNA. The data- based question below will guide you through the analysis o Meselson and S tahls results and help to build your skills in this aspect o science. Mg h vy To model helicase activity you could use some two-stranded rope or string and a split key ring. The strands in the rope are helical and represent the two strands in DNA. Open the key ring and put one strand o the rope inside it. Close the ring so that the other strand is outside. Slide the ring along the string to separate the strands. What problems are revealed by this model o the activity o helicase? Use the internet to fnd the solution used by living organisms. d-b qu: The Meselson and Stahl experiment In order or cell division to occur, DNA must be duplicated to ensure that progeny cells have the same genetic inormation as the parent cells. The process o duplicating DNA is termed replication. The MeselsonStahl experiment sought to understand the mechanism o replication. Did it occur in a conservative ashion, a semi-conservative ashion or in a dispersive ashion (see fgure 4) ? Meselson and Stahl grew E. coli in a medium containing heavy nitrogen ( 1 5 N) or a number o generations. They then transerred the bacteria to a 1 4N medium. S amples o the bacteria were taken over a period o time and separated by density gradient centriugation, a method in which heavier molecules settle urther down in acentriuge tube than lighter ones. 1 The single band o D NA at the start ( 0 generations) had a density o 1 . 72 4 g cm -3 . The main band o D NA ater our generations had a density o 1 . 71 0 g cm -3 . Explain how D NA with a lower density had been produced by the bacteria. [2 ] 113 2 M O L E C U L AR B I O LO G Y 2 a) Estimate the density o the D NA ater one generation. [2 ] b) Explain whether the density o D NA ater one generation alsifes any o the three possible mechanisms or D NA replication shown in fgure 4. [3 ] 3 4 5 6 Predict the results o centriuging a mixture o D NA rom 0 generations and 2 generations. [2 ] a) D escribe the results ater two generations, including the density o the D NA. [3 ] b) E xplain whether the results ater two generations alsiy any o the three possible mechanisms or D NA replication. [3 ] Explain the results ater three and our generations. [2 ] Figure 4 shows D NA rom E. coli at the start ( 0 generations) and ater one generation, with strands o D NA containing 1 5 N shown red and strands containing 1 4N shown green. Redraw either ( a) , ( b) or ( c) , choosing the mechanism that is supported by Meselson and S tahls experiment. Each D NA molecule can be shown as two parallel lines rather than a helix and the colours do not have to be red and green. D raw the D NA or two more generations o replication in a medium containing 1 4N. [3 ] Dispersive Conservative Semi-conservative Newly synthesized strand Original template strand Figure 4 Three possible mechanisms for DNA replication Helicase Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds. B eore D NA replication can occur, the two strands o the molecule must separate so that they can each act as a template or the ormation o a new strand. The separation is carried out by helicases, a group o enzymes that use energy rom ATP. The energy is required or breaking hydrogen bonds between complementary bases. One well-studied helicase consists o six globular polypeptides arranged in a donut shape. The polypeptides assemble with one strand o the D NA molecule passing through the centre o the donut and the other outside it. Energy rom ATP is used to move the helicase along the DNA molecule, breaking the hydrogen bonds between bases and parting the two stands. D ouble- stranded D NA cannot be split into two strands while it is still helical. Helicase thereore causes the unwinding o the helix at the same time as it separates the strands. 114 2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n DNa polymese DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template. O nce helicase has unwound the double helix and split the D NA into two strands, replication can begin. Each o the two strands acts as a template or the ormation o a new strand. The assembly o the new strands is carried out by the enzyme D NA polymerase. D NA polymerase always moves along the template strand in the same direction, adding one nucleotide at a time. Free nucleotides with each o the our possible bases are available in the area where D NA is being replicated. Each time a nucleotide is added to the new strand, only one o the our types o nucleotide has the base that can pair with the base at the position reached on the template strand. D NA polymerase brings nucleotides into the position where hydrogen bonds could orm, but unless this happens and a complementary base pair is ormed, the nucleotide breaks away again. O nce a nucleotide with the correct base has been brought into position and hydrogen bonds have been ormed between the two bases, D NA polymerase links it to the end o the new strand. This is done by making a covalent bond between the phosphate group o the ree nucleotide and the sugar o the nucleotide at the existing end o the new strand. The pentose sugar is the 3 terminal and the phosphate group is the 5 terminal, so D NA polymerase adds on the 5 terminal o the ree nucleotide to the 3 terminal o the existing strand. D NA polymerase gradually moves along the template strand, assembling the new strand with a base sequence complementary to the template strand. It does this with a very high degree o fdelity very ew mistakes are made during D NA replication. Pcr the polymese hin etion Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR) . The polymerase chain reaction ( PC R) is a technique used to make many copies o a selected D NA sequence. O nly a very small quantity o the D NA is needed at the start. The D NA is loaded into a PC R machine in which a cycle o steps repeatedly doubles the quantity o the selected D NA. This involves double- stranded D NA being separated into two single strands at one stage o the cycle and single strands combining to orm double-stranded D NA at another stage. The two strands in D NA are held together by hydrogen bonds. These are weak interactions, but in a D NA molecule there are large numbers o them so they hold the two strands together successully at the temperatures normally encountered by most cells. I D NA is heated to a high temperature, the hydrogen bonds eventually break and the two strands separate. I the D NA is then cooled hydrogen bonds can orm, so the strands pair up again. This is called re- annealing. The PC R machine separates DNA strands by heating them to 95 C or fteen seconds. It then cools the DNA quickly to 5 4 C . This would allow reannealing o parent strands to orm double-stranded DNA. However, a large excess o short sections o single-stranded DNA called primers is present. The 115 2 M O L E C U L AR B I O LO G Y primers bind rapidly to target sequences and as a large excess o primers is present, they prevent the re-annealing o the parent strands. C opying o the single parent strands then starts rom the primers. The next stage in PCR is synthesis o doublestranded DNA, using the single strands with primers as templates. The enzyme Taq DNA polymerase is used to do this. It was obtained rom a bacterium, Thermus aquaticus, ound in hot springs, including those o Yellowstone National Park. The temperatures o these springs range rom 50 C to 80 C. Enzymes in most organisms would rapidly denature at such high temperatures, but those o Thermus aquaticus, including its DNA polymerase, are adapted to be very heat-stable to resist denaturation. Taq DNA polymerase is used because it can resist the brie period at 95 C used to separate the DNA strands. It would work at the lower temperature o 5 4 C that is used to attach the primers, but its optimum temperature is 72 C . The reaction mixture is thereore heated to this temperature or the period when Taq DNA polymerase is working. At this temperature it adds about 1 ,000 nucleotides per minute, a very rapid rate o DNA replication. When enough time has elapsed or replication o the selected base sequence to be complete, the next cycle is started by heating to 95 C . A cycle o PC R can be completed in less than two minutes. Thirty cycles, which ampliy the D NA by a actor o a billion, take less than an hour. With the help o Taq D NA polymerase, PC R allows the production o huge numbers o copies o a selected base sequence in a very short time. Select the DNA sequence to be copied Twice as many DNA molecules can be copied in the next cycle Raise temperature 15 seconds to 95C to separate the two strands 80 seconds Raise temperature to 72C to allow rapid DNA replication by Taq DNA polymerase Figure 5 Lower temperature abruptly to 54C to allow binding of primers to DNA 25 seconds Figure 6 Transcription Transcription is the synthesis of mRNA copied from the DNA base sequences by RNA polymerase. This sequence o bases in a gene does not, in itsel, give any observable characteristic in an organism. The unction o most genes is to speciy the sequence o amino acids in a particular polypeptide. It is proteins that oten directly or indirectly determine the observable characteristics o an individual. Two processes are needed to produce a specifc polypeptide, using the base sequence o a gene. The frst o these is transcrip tion. Transcription is the synthesis o RNA, using D NA as a template. B ecause RNA is single- stranded, transcription only occurs along one o the two strands o D NA. What ollows is an outline o transcription: 116 The enzyme RNA polymerase binds to a site on the D NA at the start o a gene. 2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n RNA polymerase moves along the gene separating D NA into single strands and pairing up RNA nucleotides with complementary bases on one strand o the D NA. There is no thymine in RNA, so uracil pairs in a complementary ashion with adenine. RNA polymerase orms covalent bonds between the RNA nucleotides. The RNA separates rom the D NA and the double helix reorms. Transcription stops at the end o the gene and the completed RNA molecule is released. The product o transcription is a molecule o RNA with a base sequence that is complementary to the template strand o D NA. This RNA has a base sequence that is identical to the other strand, with one exception there is uracil in place o thymine. So, to make an RNA copy o the base sequence o one strand o a D NA molecule, the other strand is transcribed. The D NA strand with the same base sequence as the RNA is called the sense strand. The other strand that acts as the template and has a complementary base sequence to both the RNA and the sense strand is called the antisense strand. RNA polymerase free RNA nucleotides direction of transcription antisense strand of DNA 3 5 5 3 sense strand of DNA RNA molecule Figure 7 The second o the two processes needed to produce a specifc polypeptide is translation. Translation is the synthesis o a polypeptide, with an amino acid sequence determined by the base sequence o a molecule o RNA. The production o RNA by transcription and how its base sequence is determined by a gene was described in the previous part o this sub- topic. Translation takes place on cell structures in the cytoplasm known as ribosomes. Ribosomes are complex structures that consist o a small and a large subunit, with binding sites or each o the molecules that take part in the translation. Figure 9 shows the two subunits o a ribosome. Each is composed o RNA molecules (pink and yellow) and proteins (purple) . Part o the large subunit (green) is the site that makes peptide bonds between amino acids, to link them together into a polypeptide. TRANSCRIPTION Translation is synthesis of polypeptides on ribosomes. DNA RNA TRANSLATION Translation POLYPEPTIDE Figure 8 117 2 M O L E C U L AR B I O LO G Y Figure 9 Large and small subunits of the ribosome with proteins shown in purple, ribosomal RNA in pink and yellow and the site that catalyses the formation of peptide bonds green Messenge rNa nd the genetic code The amino acid sequence of polypeptides is determined by mRNA according to the genetic code. RNA that carries the inormation needed to synthesize a polypeptide is called messenger RNA, usually abbreviated to mRNA. The length o mRNA molecules varies depending on the number o amino acids in the polypeptide but an average length or mammals is about 2,000 nucleotides. In the genome there are many dierent genes that carry the inormation needed to make a polypeptide with a specifc amino acid sequence. At any time a cell will only need to make some o these polypeptides. O nly certain genes are thereore transcribed and only certain types o mRNA will be available or translation in the cytoplasm. C ells that need or secrete large amounts o a particular polypeptide make many copies o the mRNA or that polypeptide. For example, insulin- secreting cells in the pancreas make many copies o the mRNA needed to make insulin. Although most RNA is mRNA, there are other types; or example, transer RNA is involved in decoding the base sequence o mRNA into an amino acid sequence during translation and ribosomal RNA is part o the structure o the ribosome. They are usually reerred to as tRNA and rRNA. data-base questions: Interpreting electron micrographs The electron micrographs in fgure 1 0 show transcription, translation and D NA replication. show up more clearly. Identiy each o these structures: 1 a) the red structure in the central micrograph 2 118 D educe, with reasons, which process is occurring in each electron micrograph. The colour in the electron micrographs has been added to make the dierent structures [5 ] b) the thin blue molecule near the lower edge o the right- hand micrograph 2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n c) the blue molecules o variable length attached to this thin blue molecule e) the green molecules in the let- hand micrograph. [5] d) the red molecule in the let-hand micrograph Figure 10 codons Codons of three bases on mRNA correspond to one amino acid in a polypeptide. The translation dictionary that enables the cellular machinery to convert the base sequence on the mRNA into an amino acid sequence is called the genetic code. There are our dierent bases and twenty amino acids, so one base cannot code or one amino acid. There are sixteen combinations o two bases, which is still too ew to code or all o the twenty amino acids. Living organisms thereore use a triplet code, with groups o three bases coding or an amino acid. A sequence o three bases on the mRNA is called a codon. E ach codon codes or a specifc amino acid to be added to the polypeptide. Table 1 lists all o the 64 possible codons. The three bases o an mRNA codon are designated in the table as frst, second and third positions. Note that dierent codons can code or the same amino acid. For example the codons GUU and GUC both code or the amino acid valine. For this reason, the code is said to be degenerate. Note also that three codons are stop codons that code or the end o translation. Amino acids are carried on another kind o RNA, called tRNA. Each amino acid is carried by a specifc tRNA, which has a three- base anticodon complementary to the mRNA codon or that particular amino acid. f p (5 ) U C A G s p u Phe Phe Leu Leu Leu Leu Leu Leu IIe IIe IIe Met Val Val Val Val c Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala a Tyr Tyr Stop Stop His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu G Cys Cys Stop Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly th p (3 ) U C A G U C A G U C A G U C A G Table 1 119 2 M O L E C U L AR B I O LO G Y Deoding base sequenes Use of a table of the genetic code to deduce which codon(s) corresponds to which amino acid; use of a table of mRNA codons and their corresponding amino acids to deduce the sequence of amino acids coded by a short mRNA strand of known base sequence; deducing the DNA base sequence for the mRNA strand. There is no need to try to memorize the genetic code, but i a table showing it is available, you should be able to make various deductions. 1 Which codons correspond to an amino acid? Three letters are used to indicate each amino acid in the table o the genetic code. Each o the 20 amino acids has between one and six codons. Read o the three letters o each codon or the amino acid. For example, the amino acid methionine, shown as Met on the table, has one codon which is AUG. 2 Questions 1 What base sequence in D NA would be transcribed to give the base sequence of a strand of mRNA? A strand o mRNA is produced by transcribing the anti- sense strand o the D NA. This thereore has a D educe the codons or a) Tryptophan ( Trp) What amino acid sequence would be translated from a sequence of codons in a strand of mRNA? The frst three bases in the mRNA sequence are the codon or the frst amino acid, the next three bases are the codon or the second base and so on. Look down the let hand side o the table to fnd the frst base o a codon, across the top o the table to fnd the second base and down the right hand side to fnd the third base. For example, GCA codes or the amino acid alanine, which is abbreviated to Ala in the table. 3 base sequence complementary to the mRNA. For example, the codon AUG in mRNA is transcribed rom the base sequence TAC on the antisense strand o the D NA. A longer example is that the base sequence GUAC GUAC G is transcribed rom C ATGC ATGC . Note that adenine pairs with thymine in D NA but with uracil in RNA. b) Tyrosine ( Tyr) 2 c) Arginine ( Arg) [3 ] D educe the amino acid sequences that correspond to these mRNA sequences: [3 ] a) AC G 3 b) C AC GGG c) C GC GC GAGG [3 ] I mRNA contains the base sequence C UC AUC GAAUAAC C C a) deduce the amino acid sequence o the polypeptide translated rom the mRNA [2 ] b) deduce the base sequence o the antisense strand transcribed to produce the mRNA. [2 ] codons and antiodons Translation depends on complementary base pairing between codons on mRNA and anticodons on tRNA. Three components work together to synthesize polypeptides by translation: 120 mRNA has a sequence o codons that specifes the amino acid sequence o the polypeptide; tRNA molecules have an anticodon o three bases that binds to a complementary codon on mRNA and they carry the amino acid corresponding to that codon; ribosomes act as the binding site or mRNA and tRNAs and also catalyse the assembly o the polypeptide. 2 . 7 d n a r e P l i c at i o n , t r a n s c r i P t i o n a n d t r a n s l at i o n A summary o the main events o translation ollows: 1 An mRNA binds to the small subunit o the ribosome. 2 A molecule o tRNA with an anticodon complementary to the frst codon to be translated on the mRNA binds to the ribosome. 3 A second tRNA with an anticodon complementary to the second codon on the mRNA then binds. A maximum o two tRNAs can be bound at the same time. 4 The ribosome transers the amino acid carried by the frst tRNA to the amino acid on the second tRNA, by making a new peptide bond. The second tRNA is then carrying a chain o two amino acids a dipeptide. 5 The ribosome moves along the mRNA so the frst tRNA is released, the second becomes the frst. 6 Another tRNA binds with an anticodon complementary to the next codon on the mRNA. 7 The ribosome transers the chain o amino acids carried by the frst tRNA to the amino acid on the second tRNA, by making a new peptide bond. S tages 4, 5 and 6 are repeated again and again, with one amino acid added to the chain each time the cycle is repeated. The process continues along the mRNA until a stop codon is reached, when the completed polypeptide is released. The accuracy o translation depends on complementary base pairing between the anticodon on each tRNA and the codon on mRNA. Mistakes are very rare, so polypeptides with a sequence o hundreds o amino acids are regularly made with every amino acid correct. amino acid growing polypeptide chain large sub unit of ribosome tRNA tRNA mRNA anticodon Figure 11 Production of human insulin in bacteria Production of human insulin in bacteria as an example of the universality of the genetic code allowing gene transfer between species. D iabetes in some individuals is due to destruction o cells in the pancreas that secrete the hormone insulin. It can be treated by inj ecting insulin into the blood. Porcine and bovine insulin, extracted rom the pancreases o pigs and cattle, have both been widely used. Porcine insulin has only one dierence in amino acid sequence rom human insulin and bovine insulin has three dierences. S hark insulin, which has been used or treating diabetics in Japan, has seventeen dierences. 121 2 M O L E C U L AR B I O LO G Y D espite the dierences in the amino acid sequence between animal and human insulin, they all bind to the human insulin receptor and cause lowering o blood glucose concentration. However, some diabetics develop an allergy to animal insulins, so it is preerable to use human insulin. In 1 982 human insulin became commercially available or the rst time. It was produced using genetically modied E. coli bacteria. S ince then methods o production have been developed using yeast cells and more recently safower plants. Each o these species has been genetically modied by transerring the gene or making human insulin to it. This is done in such a way that the gene is transcribed to produce mRNA and the mRNA is translated to produce harvestable quantities o insulin. The insulin produced has exactly the same amino acid sequence as i the gene was being transcribed and translated in human cells. This may seem obvious, but it depends on each tRNA with a particular anticodon having the same amino acid attached to it as in humans. In other words, E. coli, yeast and safower ( a prokaryote, a ungus and a plant) all use the same genetic code as humans ( an animal) . It is ortunate or Figure 12 genetic engineers that all organisms, with very ew exceptions, use the same genetic code as it makes gene transer possible between widely diering species. 2.8 cell respiration Figure 12 Text to be added. understnding Cell respiration is the controlled release o energy rom organic compounds to produce ATP. ATP rom cell respiration is immediately available as a source o energy in the cell. Anaerobic cell respiration gives a small yield o ATP rom glucose. Aerobic cell respiration requires oxygen and gives a large yield o ATP rom glucose. Ntre of science Assessing the ethics o scientifc research: the use o invertebrates in respirometer experiments has ethical implications. 122 applictions Use o anaerobic cell respiration in yeasts to produce ethanol and carbon dioxide in baking. Lactate production in humans when anaerobic respiration is used to maximize the power o muscle contractions. Skills Analysis o results rom experiments involving measurement o respiration rates in germinating seeds or invertebrates using a respirometer. 2 . 8 c e l l r e s P i r at i o n relese of enegy by cell espition Cell respiration is the controlled release of energy from organic compounds to produce ATP. C ell respiration is one o the unctions o lie that all living cells perorm. O rganic compounds are broken down to release energy, which can then be used in the cell. For example, energy is released in muscle fbres by breaking down glucose into carbon dioxide and water. The energy can then be used or muscle contraction. In humans the source o the organic compounds broken down in cell respiration is the ood that we eat. C arbohydrates and lipids are oten used, but amino acids rom proteins may be used i we eat more protein than needed. Plants use carbohydrates or lipids previously made by photosynthesis. Figure 1 Breaking down 8 grams of glucose in cell respiration provides enough energy to sprint 100 metres C ell respiration is carried out using enzymes in a careul and controlled way, so that as much as possible o the energy released is retained in a usable orm. This orm is a chemical substance called adenosine triphosphate, almost always abbreviated to ATP. To make ATP, a phosphate group is linked to adenosine diphosphate, or AD P. E nergy is required to carry out this reaction. The energy comes rom the breakdown o organic compounds. ATP is not transerred rom cell to cell and all cells require a continuous supply. This is the reason or cell respiration being an essential unction o lie in all cells. aTP is souce of enegy ATP from cell respiration is immediately available as a source of energy in the cell. C ells require energy or three main types o activity. S ynthesizing large molecules like D NA, RNA and proteins. Pumping molecules or ions across membranes by active transport. Moving things around inside the cell, such as chromosomes, vesicles, or in muscle cells the protein fbres that cause muscle contraction. cell respiration ADP 1 phosphate ATP active cell processes Figure 2 The energy or all o these processes is supplied by ATP. The advantage o ATP as an energy supply is that the energy is immediately available. It is released simply by splitting ATP into AD P and phosphate. The AD P and phosphate can then be reconverted to ATP by cell respiration. When energy rom ATP is used in cells, it is ultimately all converted to heat. Although heat energy may be useul to keep an organism warm, it cannot be reused or cell activities and is eventually lost to the environment. This is the reason or cells requiring a continual source o ATP or cell activities. Figure 3 Infra red photo of toucan showing that it is warmer than its surroundings due to heat generated by respiration. Excess heat is dissipated by sending warm blood to the beak 123 2 M O L E C U L AR B I O LO G Y anerobic respirtion Anaerobic cell respiration gives a small yield of ATP from glucose. Glucose is broken down in anaerobic cell respiration without using any oxygen. The yield o ATP is relatively small, but the ATP can be produced quickly. Anaerobic cell respiration is thereore useul in three situations: Figure 4 The mud in mangrove swamps is defcient in oxygen. Mangrove trees have evolved vertical roots called pneumatophores which they use to obtain oxygen rom the air when a short but rapid burst o ATP production is needed; when oxygen supplies run out in respiring cells; in environments that are decient in oxygen, or example waterlogged soils. The products o anaerobic respiration are not the same in all organisms. In humans, glucose is converted to lactic acid, which is usually in a dissolved orm known as lactate. In yeast and plants glucose is converted to ethanol and carbon dioxide. B oth lactate and ethanol are toxic in excess, so must be removed rom the cells that produce them, or be produced in strictly limited quantities. activity S ummary equations does bioethnol solve or mke more problems? glucose There has been much debate about bioethanol production. A renewable fuel that cuts down on carbon emissions is obviously desirable. What are the arguments against bioethanol production? lactate AD P ATP This occurs in animals including humans. glucose ethanol + carbon dioxide AD P ATP This occurs in yeasts and plants. Yest nd its uses Use of anaerobic cell respiration in yeasts to produce ethanol and carbon dioxide in baking. Yeast is a unicellular ungus that occurs naturally in habitats where glucose or other sugars are available, such as the surace o ruits. It can respire either aerobically or anaerobically. Anaerobic cell respiration in yeast is the basis or production o oods, drinks and renewable energy. B read is made by adding water to four, kneading the mixture to make dough and then baking it. Usually an ingredient is added to the dough to create bubbles o gas, so that the baked bread has a lighter texture. Yeast is oten this ingredient. Ater kneading, the dough is kept warm to encourage the yeast to respire. Any oxygen in the dough is soon used up so the yeast carries out anaerobic cell respiration. The carbon dioxide produced by anaerobic cell respiration cannot escape rom the dough and orms bubbles. The swelling o the dough due to Figure 5 124 2 . 8 c e l l r e s P i r at i o n the production o bubbles o carbon dioxide is called rising. Ethanol is also produced by anaerobic cell respiration, but it evaporates during baking. B ioethanol is ethanol produced by living organisms, or use as a renewable energy source. Although any plant matter can be utilized as a eed stock and various living organisms can be used to convert the plant matter into ethanol, most bioethanol is produced rom sugar cane and corn ( maize) , using yeast. Yeast converts sugars into ethanol in large ermenters by anaerobic respiration. O nly sugars can be converted, so starch and cellulose must rst be broken down into sugars. This is done using enzymes. The ethanol produced by the yeasts is puried by distillation and various methods are then used to remove water rom it to improve its combustion. Most bioethanol is used as a uel in vehicles, sometimes in a pure state and sometimes mixed with gasoline ( petrol) . Figure 6 d-b qu: Monitoring anaerobic cell respiration in yeast The apparatus in gure 7 was used to monitor mass changes during the brewing o wine. The fask was placed on an electronic balance, which was connected to a computer or data-logging. The results are shown in gure 8. C alculate the total loss o mass during the experiment and the mean daily loss. airlock to prevent entry of oxygen electronic balance connected to a datalogging computer yeast in a solution of sugar and nutrients E xplain the loss o mass. 3 S uggest two reasons or the increasing rate o mass loss rom the start o the experiment until day 6. [2 ] 4 S uggest two reasons or the mass remaining constant rom day 1 1 onwards. [2 ] [3 ] 555 550 545 555.00 Figure 7 [3 ] 560 mass / g 1 2 Yeast data-logging apparatus 0 1 2 3 4 5 6 7 8 9 time / days Figure 8 Monitoring anaerobic cell 10 11 12 13 respiration in yeast anerobic respirtion in humns Lactate production in humans when anaerobic respiration is used to maximize the power of muscle contractions. The lungs and blood system supply oxygen to most organs o the body rapidly enough or aerobic respiration to be used, but sometimes we resort to anaerobic cell respiration in muscles. The reason is that anaerobic respiration can supply ATP very rapidly or a short period o time. It is 125 2 M O L E C U L AR B I O LO G Y thereore used when we need to maximize the power o muscle contractions. In our ancestors maximally powerul muscle contractions will have been needed or survival by allowing escape rom a predator or catching o prey during times o ood shortage. These events rarely occur in our lives today. Instead anaerobic respiration is more likely to be used during training or sport. These are examples: weight liters during the lit; short- distance runners in races up to 400 metres; long- distance runners, cyclists and rowers during a sprint fnish. Anaerobic cell respiration involves the production o lactate, so when it is being used to supply ATP, the concentration o lactate in a muscle increases. There is a limit to the concentration that the body can tolerate and this limits how much anaerobic respiration can be done. This is the reason or the short timescale over which the power o muscle contractions can be maximized. We can only sprint or a short distance not more than 400 metres. Ater vigorous muscle contractions, the lactate must be broken down. This involves the use o oxygen. It can take several minutes or enough oxygen to be absorbed or all lactate to be broken down. The demand or oxygen that builds up during a period o anaerobic respiration is called the oxygen debt. Figure 9 Short bursts of intense exercise are fuelled by ATP from anaerobic cell respiration aerobic respirtion Aerobic cell respiration requires oxygen and gives a large yield of ATP from glucose. I oxygen is available to a cell, glucose can be more ully broken down to release a greater quantity o energy than in anaerobic cell respiration. Whereas the yield o ATP is only two molecules per glucose with anaerobic cell respiration, it is more than thirty per glucose with aerobic cell respiration. Aerobic cell respiration involves a series o chemical reactions. C arbon dioxide and water are produced. In most organisms carbon dioxide is a waste product that has to be excreted, but the water is oten useul. In humans about hal a litre is produced per day. glucose + oxygen carbon dioxide + water AD P to ATP Figure 10 The desert rat never needs to drink despite only eating dry foods, because aerobic cell respiration supplies its water needs 126 In eukaryotic cells most o the reactions o aerobic cell respiration, including all o the reactions that produce carbon dioxide, happen inside the mitochondrion. 2 . 8 c e l l r e s P i r at i o n respiometes Analysis of results from experiments involving measurement of respiration rates in germinating seeds or invertebrates using a respirometer. A respirometer is any device that is used to measure respiration rate. There are many possible designs. Most involve these parts: in volume. I possible the temperature inside the respirometer should be controlled using a thermostatically controlled water bath. A sealed glass or plastic container in which the organism or tissue is placed. Respirometers can be used to perorm various experiments: An alkali, such as potassium hydroxide, to absorb carbon dioxide. the respiration rate o dierent organisms could be compared; A capillary tube containing fuid, connected to the container. the eect o temperature on respiration rate could be investigated; respiration rates could be compared in active and inactive organisms. O ne possible design o respirometer is shown in gure 1 1 , but it is possible to design simpler versions that require only a syringe with a capillary tube attached to it. I the respirometer is working correctly and the organisms inside are carrying out aerobic cell respiration, the volume o air inside the respirometer will reduce and the fuid in the capillary tube will move towards the container with the organisms. This is because oxygen is used up and carbon dioxide produced by aerobic cell respiration is absorbed by the alkali. The position o the fuid should be recorded several times. I the rate o movement o the fuid is relatively even, the results are reliable. I the temperature inside the respirometer fuctuates, the results will not be reliable because an increase in air temperature causes an increase graduated 1 cm 3 syringe wire basket containing animal tissue lter paper rolled to form a wick potassium hydroxide solution capillary tube Figure 11 Diagram of a respirometer The table below shows the results o an experiment in which the eect o temperature on respiration in germinating pea seeds was investigated. To analyse these results you should rst check to see i the repeats at each temperature are close enough or you to decide that the results are reliable. You should then calculate mean results or each temperature. The next stage is to plot a graph o the mean results, with temperature on the horizontal x-axis and the rate o movement o fuid on the vertical y-axis. Range bars can be added to the graph by plotting the lowest and highest result at each temperature and joining them with a ruled line. The graph will allow you to conclude what the relationship is between the temperature and the respiration rate o the germinating peas. tmpu (c) Mvm fud pm (mm m - 1 ) 1 dg 2d dg 3d dg 5 2.0 1.5 2.0 10 2.5 2.5 3.0 15 3.5 4.0 4.0 20 5.5 5.0 6.0 25 6.5 8.0 7.5 30 11.5 11.0 9.5 127 2 M O L E C U L AR B I O LO G Y data-bas qustions: Oxygen consumption in tobacco hornworms 1 a) Predict, using the data in the graphs, how the respiration rate o a larva will change as it grows rom moulting until it reaches the critical weight. [1 ] b) Explain the change in respiration rate that you have described. [2 ] 2 a) D iscuss the trends in respiration rate in larvae above the critical weight. [2 ] 3 S uggest a reason or earlier moulting in larvae reared in air with reduced oxygen content. [2 ] before critical weight 5th instar 0.12 0.10 0.08 0.06 0.04 0.02 after critical weight 0.16 0.14 0.12 0.10 0.08 1 0.025 2 3 4 5 6 4th instar 0.020 0.015 0.010 0.005 7 8 9 10 11 12 13 0.032 0.030 0.028 0.026 0.024 0.022 0.020 0.018 0.20.30.40.50.60.70.80.9 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.000 3rd instar 4 6 0.0 0.0 weight (g) 1.0 1.1 1.2 1.3 1.4 0.009 0.008 0.007 0.006 0.005 0.004 0.003 6 18 0.1 0 . weight (g) 0.2 0 0.2 2 0.2 4 0.2 6 Each data point on the graphs shows the body mass and respiration rate o one larva. For each instar the results have been divided into younger larvae with low to intermediate body mass and older larvae with intermediate to high body mass. The results are plotted on separate graphs. The intermediate body mass is reerred to as the critical weight. The researchers reared some tobacco hornworms in air with reduced oxygen content. They ound that the instar larvae moulted at a lower body mass than larvae reared in normal air with 2 0% oxygen. 0.0 8 0.1 0 0.1 2 0.1 4 The graphs below (fgure 1 2 ) show measurements made using a simple respirometer o the respiration rate o 3rd, 4th and 5 th instar larvae. D etails o the methods are given in the paper published by the biologists who carried out the research. The reerence to the research is C allier V and Nijhout H F (2 01 1 ) C ontrol o body size by oxygen supply reveals size-dependent and size-independent mechanisms o molting and metamorphosis. PNAS;1 08:1 46641 4669. This paper is reely available on the internet at http://www.pnas.org/ content/1 08/35 /1 4664.ull.pd+ html. b) S uggest reasons or the dierence in the trends between the periods below and above the critical weight. [2 ] respiration rate (ml O 2 /min) Tobacco hornworms are the larvae o Manduca sexta. Adults o this species are moths. Larvae emerge rom the eggs laid by the adult emale moths. There are a series o larval stages called instars. Each instar grows and then changes into the next one by shedding its exoskeleton and developing a new larger one. The exoskeleton includes the tracheal tubes that supply oxygen to the tissues. Figure 12 Respiration rates of tobacco hornworms (after Callier and Nijhout, 2011) ethics of animal us in rspiromtrs Assessing the ethics o scientifc research: the use o invertebrates in respirometer experiments has ethical implications. It is important or all scientists to assess the ethics o their research. There has been intense debate about the ethics o using animals in experiments. When discussing ethical issues, do 128 we consider the consequences such as benefts to students who are learning science? D o we consider intentions? For example, i the animals are harmed unintentionally does that change 2 . 9 Ph o to s yn th e s i s whether the experiment was ethical or not? Are there absolute principles o right and wrong: or example, can we say that animals should never be subj ect to conditions that are outside what they would encounter in their natural habitat? B eore carrying out respirometer experiments involving animals these questions should be answered to help to decide whether the experiments are ethically acceptable: 1 Is it acceptable to remove animals rom their natural habitat or use in an experiment and can they be saely returned to their habitat? 2 Will the animals suer pain or any other harm during the experiment? 3 C an the risk o accidents that cause pain or suering to the animals be minimized during the experiment? In particular, can contact with the alkali be prevented? 4 Is the use o animals in the experiment essential or is there an alternative method that avoids using animals? It is particularly important to consider the ethics o animal use in respirometer experiments because the International B accalaureate Organization has issued a directive that laboratory or eld experiments and investigations need to be undertaken in an ethical way. An important aspect o this is that experiments should not be undertaken in schools that infict pain or harm on humans or other living animals. 2.9 P understnding Photosynthesis is the production o carbon compounds in cells using light energy. Visible light has a range o wavelengths with violet the shortest wavelength and red the longest. Chlorophyll absorbs red and blue light most eectively and refects green light more than other colours. Oxygen is produced in photosynthesis rom photolysis o water. Energy is needed to produce carbohydrates and other carbon compounds rom carbon dioxide. Temperature, light intensity and carbon dioxide concentration are possible limiting actors on the rate o photosynthesis. applictions Changes to the Earths atmosphere, oceans and rock deposition due to photosynthesis. Skills Design o experiments to investigate limiting actors on photosynthesis. Separation o photosynthetic pigments by chromatography. Drawing an absorption spectrum or chlorophyll and an action spectrum or photosynthesis. Ntre of science Experimental design: controlling relevant variables in photosynthesis experiments is essential. 129 2 M O L E C U L AR B I O LO G Y What is photosynthesis? Photosynthesis is the production of carbon compounds in cells using light energy. Living organisms require complex carbon compounds to build the structure of their cells and to carry out life processes. S ome organisms are able to make all the carbon compounds that they need using only light energy and simple inorganic substances such as carbon dioxide and water. The process that does this is called photosynthesis. Photosynthesis is an example of energy conversion, as light energy is converted into chemical energy in carbon compounds. The carbon compounds produced include carbohydrates, proteins and lipids. Figure 2 The trees in one hectare of redwood forest in California can have a biomass of more than 4,000 tonnes, mostly carbon compounds produced by photosynthesis Figure 1 Leaves absorb carbon dioxide and light and use them in photosynthesis Separating photosynthetic pigments by chromatography Separation of photosynthetic pigments by chromatography. (Practical 4) C hloroplasts contain several types of chlorophyll and other pigments called accessory pigments. B ecause these pigments absorb different ranges of wavelength of light, they look a different colour to us. Pigments can be separated by chromatography. You may be familiar with paper chromatography but thin layer chromatography gives better results. This is done with a plastic strip that has been coated with a thin layer of a porous material. A spot containing pigments extracted from leaf tissue is placed near one end of the strip. A solvent is allowed to run up the strip, to separate the different types of pigment. 130 1 Tear up a leaf into small pieces and put them in a mortar. 2 Add a small amount of sand for grinding. Figure 3 Thin layer chromatography 2 . 9 Ph o to s yn th e s i s 3 Add a small volume o propanone ( acetone) . 4 Use the pestle to grind the lea tissue and dissolve out the pigments. Carotene orange 0.98 5 I the propanone all evaporates, add a little more. Chlorophyll a blue green 0.59 6 When the propanone has turned dark green, allow the sand and other solids to settle, then pour the propanone o into a watch glass. Chlorophyll b yellow green 0.42 Phaeophytin olive green 0.81 Xanthophyll 1 yellow 0.28 Xanthophyll 2 yellow 0.15 7 8 9 Use a hair drier to evaporate o all the propanone and water rom the cells cytoplasm. When you have just a smear o dry pigments in the watch glass, add 34 drops o propanone and use a paint brush to dissolve the pigments. Use the paint brush to transer a very small amount o the pigment solution to the TLC strip. Your aim is to make a very small spot o pigment in the middle o the strip, 1 0 millimetres rom one end. It should be very dark. This is achieved by repeatedly putting a small drop onto the strip and then allowing it to dry beore adding another amount. You can speed up drying by blowing on the spot or by using the hair drier. 1 0 When the spot is dark enough, slide the other end o the strip into the slot in a cork or bung that fts into a tube that is wider than the TLC strip. The slot should hold the strip frmly. 1 1 Insert the cork and strip into a specimen tube. The TLC strip should extend nearly to the bottom o the tube, but not quite touch. sp umb clu da mv (mm) 1 2 3 4 5 rf nam f pgm Pgm clu f pgm rf 1 2 Mark the outside o the tube j ust below the level o the spot on the TLC strip. 1 3 Take the strip and cork out o the tube. 1 4 Pour running solvent into the specimen tube up to the level that you marked. 1 5 Place the specimen tube on a lab bench where it will not be disturbed. C areully lower the TLC strip and cork into the tube, so that the tube is sealed and the TLC strip is j ust dipping into the running solvent. The solvent must NO T touch the pigment spot. 1 6 Leave the tube completely alone or about fve minutes, to allow the solvent to run up through the TLC strip. You can watch the pigments separate, but D O NO T TO UC H THE TUB E . 1 7 When the solvent has nearly reached the top o the strip, remove it rom the tube and separate it rom the cork. 1 8 Rule two pencil lines across the strip, one at the level reached by the solvent and one at the level o the initial pigment spot. 1 9 D raw a circle around each o the separated pigment spots and a cross in the centre o the circle. 6 7 8 Figure 4 Chromatogram of leaf pigments Table o standard R values 131 2 M O L E C U L AR B I O LO G Y 2 0 Using a ruler with millimetre markings, measure the distance moved by the running solvent ( the distance between the two lines) and the distance moved by each pigment ( the distance between the lower line and the cross in the centre o the circle) . 2 1 C alculate the R or each pigment, where R is the distance run by the pigment divided by the distance run by the solvent. 22 Show all your results in the table above, starting with the pigment that had moved least ar. Waveengths of ight Visible light has a range o wavelengths with violet the shortest wavelength and red the longest. Sunlight or simply light is made up o all the wavelengths o electromagnetic radiation that our eyes can detect. It is thereore visible to us and other wavelengths are invisible. There is a spectrum o electromagnetic radiation rom very short to very long wavelengths. Shorter wavelengths such as X-rays and ultraviolet radiation have high energy; longer wavelengths such as inrared radiation and radio waves have lower energy. Visible light has wavelengths longer than ultraviolet and shorter than inrared. The range o wavelengths o visible light is 400 to 700 nanometres. When droplets o water in the sky split sunlight up and a rainbow is ormed, dierent colours o light are visible. This is because sunlight is a mixture o dierent wavelengths, which we see as dierent colours, including violet, blue, green and red. Violet and blue are the shorter wavelengths and red is the longest wavelength. The wavelengths o light that are detected by the eye are also those used by plants in photosynthesis. A reason or this is that they are emitted by the sun and penetrate the E arths atmosphere in larger quantities than other wavelengths, so are particularly abundant. Figure 5 In a rainbow the wavelengths of visible light are separated solar radiation reaching the Earths surface/W m 2 2 1.5 blue 5 4502 500 nm green 5 5252 575 nm red 5 6502 700 nm 1.0 0.5 0 500 1000 1500 2000 2500 3000 wavelength /nm Figure 6 The spectrum of electromagnetic radiation reaching the Earths surface light absorption by chorophy Chlorophyll absorbs red and blue light most eectively and refects green light more than other colours. 132 The frst stage in photosynthesis is the absorption o sunlight. This involves chemical substances called pigments. A white or transparent substance does not absorb visible light. Pigments are substances that do 2 . 9 Ph o to s yn th e s i s absorb light and thereore appear coloured to us. Pigments that absorb all o the colours appear black, because they emit no light. There are pigments that absorb some wavelengths o visible light but not others. For example, the pigment in a gentian fower absorbs all colours except blue. It appears blue to us, because this part o the sunlight is refected and can pass into our eye, to be detected by cells in the retina. Photosynthesizing organisms use a range o pigments, but the main photosynthetic pigment is chlorophyll. There are various orms o chlorophyll but they all appear green to us. This is because they absorb red and blue light very eectively, but the intermediate green light much less eectively. Wavelengths o green light thereore are refected. This is the reason or the main colour in ecosystems dominated by plants being green. Figure 7 Gentian fowers contain the pigment delphinidin, which refects blue light and absorbs all other wavelengths. absorption nd ction spectr Drawing an absorption spectrum for chlorophyll and an action spectrum for photosynthesis. When drawing both action and absorption spectra, the horizontal x-axis should have the legend wavelength, with nanometres shown as the units. The scale should extend rom 400 to 700 nanometres. O n an action spectrum the y-axis should be used or a measure o the relative amount o photosynthesis. This is oten given as a percentage o the maximum rate, with a scale rom 0 to 1 00% . It is not dicult to explain why action and absorption spectra are very similar: photosynthesis can only occur in wavelengths o light that chlorophyll or the other photosynthetic pigments can absorb. 100 chlorophyll a chlorophyll b carotenoids % absorption An action spectrum is a graph showing the rate o photosynthesis at each wavelength o light. An absorption spectrum is a graph showing the percentage o light absorbed at each wavelength by a pigment or a group o pigments. 400 Figure 8 500 600 wavelength (nm) 700 Absorption spectra o plant pigments O n an absorption spectrum the y- axis should have the legend % absorption, with a scale rom 0 to 1 00% . Ideally data points or specic wavelengths should be plotted and then a smooth curve be drawn through them. I this is not possible, the curve rom a published spectrum could be copied. photosynthesis (% of max rate) 100 400 Figure 9 500 600 wavelength (nm) 700 Action spectrum o a plant pigment 133 2 M O L E C U L AR B I O LO G Y data-bas qustions: Growth of tomato seedlings in red, green and blue light Tomato seeds were germinated and grown or 3 0 days in light produced by red, orange, green and blue light emitting diodes. Four dierent colours o LE D were tested and two combinations o colours. In every treatment the tomato plants received the same intensity o photons o light. The peak wavelength o light emitted by each wavelength is shown in the table below, together with the mean lea area and height o the seedlings. Plants oten grow tall, with weak stems and small leaves when they are receiving insufcient light or photosynthesis. 1 Plot a graph to show the relationship between wavelength, lea area and height. Hint: i you need two dierent scales on the y-axis you can put one on the let hand side o the graph and the other on the right hand side. D o not attempt to plot the results or combinations o LE D s. [6] 2 Using your graph, deduce the relationship between the lea area o the seedlings and their height. [1 ] 3 E valuate the data in the table or a grower o tomato crops in greenhouses who is considering using LED s to provide light. [3 ] Pak wavngt o igt mitt by led (nm) la ara o sings (m 2 ) higt o sings (mm) Red 630 5.26 192 Orange 600 4.87 172 Green 510 5.13 161 Blue 450 7.26 128 Red and Blue 5.62 99 Red, Green and Blue 5.92 85 coours o leds Source: Xiaoying, Shirong, Taotao, Zhigang and Tezuka (2012) . Regulation o the growth and photosynthesis o cherry tomato seedlings by diferent light irradiations o light emitting diodes (LED) . African Journal of Biotechnology Vol. 11(22) , pp. 6169-6177 oxygen prductin in phtsynthesis Oxygen is produced in photosynthesis from photolysis of water. O ne o the essential steps in photosynthesis is the splitting o molecules o water to release electrons needed in other stages. H 2 O 4e + 4H + + O 2 This reaction is called photolysis because it only happens in the light and the word lysis means disintegration. All o the oxygen generated in photosynthesis comes rom photolysis o water. O xygen is a waste product and diuses away. Figure 10 Photosynthesizing organisms seem insignicant in relation to the size o the Earth but over billions o years they have changed it signicantly 134 efts o potosyntsis on t eart Changes to the Earths atmosphere, oceans and rock deposition due to photosynthesis. Prokaryotes were the frst organisms to perorm photosynthesis, starting about 3,500 million years ago. They were joined millions o years later by algae and plants, which have been carrying out photosynthesis ever since. 2 . 9 Ph o to s yn th e s i s One consequence o photosynthesis is the rise in the oxygen concentration o the atmosphere. This began about 2,400 million years ago (mya) , rising to 2% by volume by 2,200 mya. This is known as the Great Oxidation Event. At the same time the E arth experienced its frst glaciation, presumably due to a reduction in the greenhouse eect. This could have been due to the rise in oxygenation causing a decrease in the concentration o methane in the atmosphere and photosynthesis causing a decrease in carbon dioxide concentration. B oth methane and carbon dioxide are potent greenhouse gases. The increase in oxygen concentrations in the oceans between 2 , 400 and 2 , 2 00 mya caused the oxidation o dissolved iron in the water, causing it to precipitate onto the sea bed. A distinctive rock ormation was produced called the banded iron ormation, with layers o iron oxide alternating with other minerals. The reasons or the banding are not yet ully understood. The banded iron ormations are the most important iron ores, so it is thanks to photosynthesis in bacteria billions o years ago that we have abundant supplies o steel today. av dfr mpr Pl cmp mpr (%) CO 2 N2 Ar O2 H 2O 98 1 1 0 0.04 78 1 21 0.1 Venus Earth Mars 0 96 2.5 1.5 2.5 0.1 What are the main diferences between the composition o the Earth's atmospheres and the atmosphere o the other planets. What is the cause o these diferences? The oxygen concentration o the atmosphere remained at about 2 % rom 2 , 2 00 mya until about 75 0- 63 5 mya. There was then a signifcant rise to 2 0% or more. This corresponds with the period when many groups o multicellular organisms were evolving. 40 av 30 lg 20 1500 10 CO 2 uptake/mol h 2 1 oxygen/% of atmosphere 50 1000 0 4.0 3.0 2.0 Millions of years ago ( 1,000) 1.0 0 Figure 11 Production of carbohydrates 75 150 225 300 light intensity /J dm 2 2 s 2 1 of an experiment in which the rate of photosynthesis was found by measuring the uptake of carbon dioxide 1 What is the reason or a CO 2 uptake rate o 200 in darkness? 2 What can you predict about cell respiration and photosynthesis at the point where the net rate o CO 2 uptake is zero? carbon dioxide + water carbohydrate + oxygen To carry out this process, energy is required. A chemical reaction that involves putting in energy is described as endothermic. Reactions involving the production o oxygen are usually endothermic in living systems. Reactions involving combining smaller molecules to make larger ones are also oten endothermic and molecules o carbohydrate such as glucose are much larger than carbon dioxide or water. 0 200 Figure 12 The graph shows the results Energy is needed to produce carbohydrates and other carbon compounds rom carbon dioxide. Plants convert carbon dioxide and water into carbohydrates by photosynthesis. The simple equation below summarizes the process: 500 135 2 M O L E C U L AR B I O LO G Y ativity increase in biomass of grass /kg ha - 1 h - 1 co 2 nentrtin 40 30 limiting fators 20 Temperature, light intensity and carbon dioxide concentration are possible limiting factors on the rate of photosynthesis. 10 0 210 100 200 300 400 CO 2 /cm 3 m - 3 air Figure 13 In this graph the rate of photosynthesis was measured indirectly by measuring the change in plant biomass. 1 2 The energy for the conversion of carbon dioxide into carbohydrate is obtained by absorbing light. This is the reason for photosynthesis only occurring in the light. The energy absorbed from light does not disappear it is converted to chemical energy in the carbohydrates. The maximum carbon dioxide concentration of the atmosphere is 380 cm 3 m 3 air. Why is the concentration often lower near leaves? In what weather conditions is carbon dioxide concentration likely to be the limiting factor for photosynthesis? The rate of photosynthesis in a plant can be affected by three external factors: temperature; light intensity; carbon dioxide concentration. E ach of these factors can limit the rate if they are below the optimal level. These three factors are therefore called limiting factors. According to the concept of limiting factors, under any combination of light intensity, temperature and carbon dioxide concentration, only one of the factors is actually limiting the rate of photosynthesis. This is the factor that is furthest from its optimum. If the factor is changed to make it closer to the optimum, the rate of photosynthesis increases, but changing the other factors will have no effect, as they are not the limiting factor. O f course, as the limiting factor is moved closer to its optimum, while keeping the other factors constant, a point will be reached where this factor is no longer the one that is furthest from its optimum and another factor becomes the limiting factor. For example, at night, light intensity is presumably the limiting factor for photosynthesis. When the sun rises and light intensity increases, temperature will usually take over as the limiting factor. As the temperature increases during the morning, carbon dioxide concentration might well become the limiting factor. controed variabes in imiting fator experiments Experimental design: controlling relevant variables in photosynthesis experiments is essential. In any experiment, it is important to control all variables other than the independent and dependent variable that you are investigating. The independent variable is the one that you deliberately vary in the experiment with a range of levels that you choose. The dependent variable is what you measure during the experiment, to see if it is affected by the independent variable. 136 2 . 9 Ph o to s yn th e s i s It is essential during this type o experiment to be sure that the independent variable is the only actor that could be aecting the dependent variable. All other variables that might aect the independent variable must thereore be controlled. These are questions that you need to answer when you are designing an experiment to investigate a limiting actor on photosynthesis: Which limiting actor will you investigate? This will be your independent variable. How will you measure the rate o photosynthesis? This will be your dependent variable. How will you keep the other limiting actors at a constant and optimal level? These will be your controlled variables. Investigating limiting factors Design of experiments to investigate limiting factors on photosynthesis. There are many possible experimental designs. A method that can be used to investigate the eect o carbon dioxide concentration is given below. You could either modiy this to investigate a dierent limiting actor or you could develop an entirely dierent design. Investigating the efect o carbon dioxide on photosynthesis acv tmprur 100 % of maximum rate I a stem o pondweed such as Elodea, Cabomba or Myriophyllum is placed upside- down in water and the end o the stem is cut, bubbles o gas may be seen to escape. I these are collected and tested, they are ound to be mostly oxygen, produced by photosynthesis. The rate o oxygen production can be measured by counting the bubbles. Factors that might aect the rate o photosynthesis can be varied to fnd out what eect this has. In the method below carbon dioxide concentration is varied. 50 0 1 Enough water to fll a large beaker is boiled and allowed to cool. This removes carbon dioxide and other dissolved gases. 2 The water is poured repeatedly rom one beaker to another, to oxygenate the water. Very little carbon dioxide will dissolve. 3 A stem o pondweed is placed upside-down in the water and the end o its stem is cut. No bubbles are expected to emerge, as the water contains almost no carbon dioxide. The temperature o the water should be about 25 C and the water should be very brightly illuminated. Suitable apparatus is shown in fgure 1 6. 1 Enough sodium hydrogen carbonate is added to the beaker to raise the carbon dioxide concentration by 0.01 mol dm - 3 . I bubbles emerge, they are counted or 3 0 seconds, repeating the counts until two or three consistent results are obtained. What was the optimum temperature for photosynthesis in this plant? 2 What was the maximum temperature for photosynthesis? 4 0 10 20 30 40 50 temperature/C Figure 14 In this graph the rate of photosynthesis was measured indirectly by measuring the change in plant biomass 137 2 M O L E C U L AR B I O LO G Y sodium hydrogen carbonate 5 Enough sodium hydrogen carbonate is added to raise the concentration by another 0.01 mol dm 3 . B ubble counts are done in the same way. 6 The procedure above is repeated again and again until further increases in carbon dioxide do not affect the rate of bubble production. Questions 1 pondweed Why are the following procedures necessary? a) B oiling and then cooling the water before the experiment. b) Keeping the water at 2 5 C and brightly illuminating it. c) Repeating bubble counts until several consistent counts have been obtained. water at 25 C 2 What other factor could be investigated using bubble counts with pondweed and how would you design the experiment? 3 How could you make the measurement of the rate of oxygen production more accurate? light source Figure 15 Apparatus or measuring photosynthesis rates in diferent concentrations o carbon dioxide 138 Question s Questions 1 2 Lipase is a digestive enzyme that accelerates the breakdown o triglycerides in the small intestine. In the laboratory, the rate o activity o lipase can be detected by a decline in pH. Explain what causes the pH to decline. [4] a) ( i) ( ii) S tate the mass units that are shown in the equation. [2 ] b) ( i) % of protien digested c) Explain how it is possible to synthesize such large masses o ATP during races. [3 ] d) D uring a 1 00 m race, 80 g o ATP is needed but only 0.5 dm 3 o oxygen is consumed. D educe how ATP is being produced. [3 ] lgh f Vm f xyg cmd c rac/m rpra drg h rac/dm 3 immobilized papain 80 dissolved papain 60 C alculate the mass o ATP produced per [2 ] dm 3 o oxygen. ( ii) C alculate the mass o ATP produced per race in table 1 . [4] Papain is a protease that can be extracted rom pineapple ruits. Figure 1 7 shows the eect o temperature on the activity o papain. The experiment was perormed using papain dissolved in water and then repeated with the same quantity o papain that had been immobilized by attaching it to a solid surace. The results show the percentage o the protein in the reaction mixture that was digested in a fxed time. 100 S tate the volume units that are shown in the equation. [1 ] 40 20 1500 36 10,000 150 42,300 700 Table 1 0 Figure 17 a) ( i) O utline the eects o temperature on the activity o dissolved papain. [2 ] ( ii) E xplain the eects o temperature on the activity o dissolved papain. [2 ] b) ( i) C ompare the eect o temperature on the activity o immobilized papain with the eect on dissolved papain. [2 ] ( ii) S uggest a reason or the dierence that you have described. [2 ] (iii) In some parts o the human body, enzymes are immobilized in membranes. Suggest one enzyme and a part o the body where it would be useul or it to be immobilized in a membrane. [2] 3 The equation below summarizes the results o metabolic pathways used to produce ATP, using energy rom the oxidation o glucose. glucose + oxygen + (ADP + Pi) 1 80 g 1 34.4 dm 3 1 8.25 kg carbon dioxide + water + ATP 1 34.4 dm3 1 08 g 1 8.25 kg 4 Figure 1 8 shows the eects o varying light intensity on the carbon dioxide absorption by leaves, at dierent, fxed carbon dioxide concentrations and temperatures. a) D educe the limiting actor or photosynthesis at: ( i) W ( ii) X ( iii) Y ( iv) Z. [4] b) Explain why curves I and II are the same between 1 and 7 units o light intensity. [3 ] c) Explain the negative values or carbon dioxide absorption when the leaves were in low light intensities. [3 ] 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 rate of CO 2 absorption / arbitrary units 20 30 40 50 60 70 80 temperature /C Figure 18 Z IV 0.4%CO 2 at 30C III 0.4%CO 2 at 20C X Y II 0.13%CO 2 at 30C I 0.13%CO 2 at 20C W 1 2 3 4 5 6 7 light intensity / arbitrary units 139 2 M O L E C U L AR B I O LO G Y 5 Figure 1 9 shows the results o an experiment in which Chlorella cells were given light o wavelengths rom 660 nm ( red) up to 700 nm ( ar red) . The rate o oxygen production by photosynthesis was measured and the yield o oxygen per photon o light was calculated. This gives a measure o the efciency o photosynthesis at each wavelength. The experiment was then repeated with supplementary light with a wavelength o 65 0 nm at the same time as each o the wavelengths rom 660 to 700 nm, but with the same overall intensity o light as in the frst experiment. yeild of oxygen molecules per photon of light with supplementary light without supplementary light 0.10 0.05 680 700 wavelength (nm) Figure 19 Photon yield o photosynthesis in diferent light intensities 140 b) D escribe the eect o the supplementary light. [2 ] c) E xplain how the error bars help in drawing conclusions rom this experiment. [2 ] d) The probable maximum yield o oxygen was 0. 1 2 5 molecules per photon o light. C alculate how many photons are needed to produce one oxygen molecule in photosynthesis. [2 ] e) O xygen production by photolysis involves this reaction: 4H 2 O O 2 + 2 H 2 O + 4H + + 4e E ach photon o light is used to excite an electron ( raise it to a higher energy level) . C alculate how many times each electron produced by photolysis must be excited during the reactions o photosynthesis. [2 ] 0.15 0 660 a) D escribe the relationship between wavelength o light and oxygen yield, when there was no supplementary light. [2 ] 3C E LGLE Bn IEOtLI CO sG Y Iroducio E very living organism inherits a blueprint or lie rom its parents. The inheritance o genes ollows patterns. C hromosomes carry genes in a linear sequence that is shared by members o a species. Alleles segregate during meiosis allowing new combinations to be ormed by the usion o gametes. B iologists have developed techniques or artifcial manipulation o D NA, cells and organisms. 3.1 Genes Uderadig A gene is a heritable actor that consists o a length o DNA and inuences a specic characteristic. A gene occupies a specic position on one type o chromosome. The various specic orms o a gene are alleles. Alleles difer rom each other by one or a ew bases only. New alleles are ormed by mutation. The genome is the whole o the genetic inormation o an organism. The entire base sequence o human genes was sequenced in the Human Genome Project. Applicaio The causes o sickle cell anemia, including a base substitution mutation, a change to the base sequence o mRNA transcribed rom it and a change to the sequence o a polypeptide in hemoglobin. Comparison o the number o genes in humans with other species. skill Use o a database to determine diferences in the base sequence o a gene in two species. naure of ciece Developments in scientic research ollow improvements in technology: gene sequencers, essentially lasers and optical detectors, are used or the sequencing o genes. 141 3 G e n e ti cs What is a gene? A gene is a heritable actor that consists o a length o DNA and infuences a specic characteristic. Genetics is the branch o biology concerned with the storage o inormation in living organisms and how this inormation can be passed rom parents to progeny. The word genetics was used by biologists long beore the method o inormation storage was understood. It came rom the word genesis, meaning origins. B iologists were interested in the origins o eatures such as baldness, blue eyes and much more. S omething must be the cause o these eatures and be passed on to ospring where the eatures would again develop. Experiments in the 1 9th century showed that there were indeed actors in living organisms that infuenced specic characteristics and that these actors were heritable. They could be passed on to ospring by pea plants, ruit fies and all other organisms. There was intense research into genetics rom the early 2 0th century onwards and the word gene was invented or the heritable actors. O ne obvious question was the chemical composition o genes. B y the middle o the 2 0th century there was strong evidence that genes were made o D NA. There are relatively ew D NA molecules in a cell j ust 46 in a typical human cell or example yet there are thousands o genes. We can thereore deduce that each gene consists o a much shorter length o D NA than a chromosome and that each chromosome carries many genes. Comparing numbers of genes Comparison o the number o genes in humans with other species. How many genes does it take to make a bacterium, a banana plant or a bat, and how many are needed to make a human? We see ourselves as more complex in structure, physiology and behaviour so we might expect to Group Prokaryotes Brief description Numbers of genes Haemophilus infuenzae Pathogenic bacterium 1,700 Escherichia coli Gut bacterium 3,200 Protoctista Trichomonas vaginalis Unicellular parasite 60,000 Fungi Saccharomyces cerevisiae (Yeast) Unicellular ungus 6,000 Plants Oryza sativa (Rice) Crop grown or ood 41,000 Arabidopsis thaliana (Thale cress) Small annual weed 26,000 Populus trichocarpa (Black cottonwood) Large tree 46,000 Drosophila melanogaster (Fruit fy) Larvae consume ripe ruit 14,000 Caenorhabditis elegans Small soil roundworm 19,000 Homo sapiens (Humans) Large omnivorous biped 23,000 Daphnia pulex (Water fea) Small pond crustacean 31,000 Animals 142 Name of species have more genes. The table shows whether this is true. It gives a range o predicted gene numbers. They are based on evidence rom the D NA o these species but are not precise counts o gene numbers as these are not yet known. 3 .1 GEN Es Where are genes located? Activity A gene occupies a specifc position on one type o chromosome. Etimating the number of human gene E xperiments in which dierent varieties o plant or animals are crossed show that genes are linked in groups and each group corresponds to one o the types o chromosome in a species. For example, there are our groups o linked genes in ruit fies and our types o chromosome. Maize has ten groups o linked genes and ten types o chromosome and in humans the number o both is 2 3 . 7q22.2 7q15.2 7q21.3 7q21.1 7q14.3 7q14.1 7q12.1 7q12.3 7q11.22 7q21.3 7q21.13 7q21.11 7q31.33 7q31.31 7q31.1 7q22.2 7q33 7q35 7q32.2 7q36.2 Each gene occupies a specic position on the type o chromosome where it is located. This position is called the locus o the gene. Maps showing the sequence o genes along chromosomes in ruit fies and other organisms were produced by crossing experiments, but much more detailed maps can now be produced when the genome o a species is sequenced. In October 1970 Scientifc American published an estimate that the human genome might consist o as many as 10 million genes. How many times greater than the current predicted number is this? What reasons can you give or such a huge overestimate in 1970? Figure 1 Chromosome 7: an example o a human chromosome. It consists o a single DNA molecule with approximately 170 million base pairs about 5% o the human genome. The pattern o banding, obtained by staining the chromosome, is diferent rom other human chromosomes. Several thousand genes are located on chromosome 7, mostly in the light bands, each o which has a unique identiying code. The locus o a ew o the genes on chromosome 7 is shown What are alleles? The various specifc orms o a gene are alleles. Gregor Mendel is usually regarded as the ather o genetics. He crossed varieties o pea plants, or example tall pea plants with dwar peas and white- fowered pea plants with purple-fowered. Mendel deduced that the dierences between the varieties that he crossed together were due to dierent heritable actors. We now know that these pairs o heritable actors are alternative orms o the same gene. For example there are two orms o the gene that infuences height, one making pea plants tall and the other making the plants dwar. These dierent orms are called alleles. There can be more than two alleles o a gene. O ne o the rst examples o multiple alleles to be discovered is in mice. A gene that infuences coat colour has three alleles, making the mice yellow, grey and black. There are three alleles o the gene in humans that determines AB O blood groups. In some cases there are large numbers o dierent alleles o a gene, or example the gene that infuences eye colour in ruit fies. As alleles are alternative orms o the same gene, they occupy the same position on one type o chromosome they have the same locus. O nly one allele can occupy the locus o the gene on a chromosome. Most animal and plant cells have two copies o each type o chromosome, so Figure 2 Diferent coat colours in mice 143 3 G e n e ti cs we can expect two copies o a gene to be present. These could be two o the same allele o the gene or two dierent alleles. Diferences between alleles Alleles difer rom each other by one or a ew bases only. A gene consists o a length o D NA, with a base sequence that can be hundreds or thousands o bases long. The dierent alleles o a gene have slight variations in the base sequence. Usually only one or a very small number o bases are dierent, or example adenine might be present at a particular position in the sequence in one allele and cytosine at that position in another allele. Positions in a gene where more than one base may be present are called single nucleotide polymorphisms, abbreviated to S NPs and pronounced snips. Several snips can be present in a gene, but even then the alleles o the gene dier by only a ew bases. Comparing genes Use o a database to determine diferences in the base sequence o a gene in two species One outcome o the Human Genome Project is that the techniques that were developed have enabled the sequencing o other genomes. This allows gene sequences to be compared. The results o this comparison can be used to determine evolutionary relationships. Also, the identifcation o conserved sequences allows species to be chosen or exploring the unction o that sequence. C hoose Fast A and the sequence should appear. C opy the sequence and paste it into a .txt fle or notepad fle. Repeat with a number o dierent species that you want to compare and save the fles. To have the computer align the sequence or you, download the sotware called C lustalX and run it. In the File menu, choose Load S equences. C hoose gene rom the search menu. Enter the name o a gene plus the organism, such as cytochrome oxidase 1 ( C O X1 ) or pan ( chimpanzee) . S elect your fle. Your sequences should show up in the C lustalX window. Under the Alignment menu choose D o C omplete Alignment. The example below shows the sequence alignment o 9 dierent organisms. Move your mouse over the section Genomic regions, transcripts, and products until Nucleotide Links appears. 144 Go to the website called GenB ank ( http://www.ncbi. nlm.nih.gov/pubmed/) Figure 3 3 .1 GEN Es Data-baed quetion: COX-2, smoking and stomach cancer C O X- 2 is a gene that codes or the enzyme cyclooxygenase. The gene consists o over 6 , 000 nucleotides. Three single nucleotide polymorphisms have been discovered that are associated with gastric adenocarcinoma, a cancer o the stomach. O ne o these S NPs occurs at nucleotide 1 1 9 5 . The base at this nucleotide can be either adenine or guanine. A large survey in C hina involved sequencing both copies o the C O X- 2 gene in 3 5 7 patients who had developed gastric adenocarcinoma and in 9 85 people who did not have the disease. All o these people were asked whether they had ever smoked cigarettes. Table 1 shows the 3 5 7 patients with gastric adenocarcinoma categorized according to whether they were smokers or non- smokers and whether they had two copies o C O X-2 with G at nucleotide 1 1 95 ( GG) or at least one copy o the gene with A at this position ( AG or AA) . The results are shown as percentages. Table 2 shows the same categorization or the 985 people who did not have this cancer. 1 Predict, using the data, which o bases G or A is more common at nucleotide 1 1 95 in the controls. [2 ] 2 a) Calculate the total percentage o the patients that were smokers and the total percentage o controls that were smokers. [2 ] b) Explain the conclusion that can be drawn rom the dierence in the percentages. [2 ] 3 4 D educe, with a reason, whether G or A at nucleotide 1 1 95 is associated with an increased risk o gastric adenocarcinoma. [2 ] D iscuss, using the data, whether the risk o gastric adenocarcinoma is increased equally in all smokers. [2 ] GG AG or AA Smokers 9.8% 43.7% Non-smokers 9.5% 40.0% Table 1 Patients with cancer GG AG or AA Smokers 9.4% 35.6% Non-smokers 12.6% 42.4% Table 2 Patients without cancer Mutation Activity New alleles are ormed by mutation. New allele New alleles are ormed rom other alleles by gene mutation. Mutations are random changes there is no mechanism or a particular mutation being carried out. The most signifcant type o mutation is a base substitution. One base in the sequence o a gene is replaced by a dierent base. For example, i adenine was present at a particular point in the base sequence it could be substituted by cytosine, guanine or thymine. Recent research into mutation involved nding the base sequence o all genes in parents and their ofspring. It showed that there was one base mutation per 1.2 10 8 bases. Calculate how many new alleles a child is likely to have as a result o mutations in their parents. Assume that there are 25,000 human genes and these genes are 2,000 bases long on average. A random change to an allele that has developed by evolution over perhaps millions o years is unlikely to be benefcial. Almost all mutations are thereore either neutral or harmul. S ome mutations are lethal they cause the death o the cell in which the mutation occurs. Mutations in body cells are eliminated when the individual dies, but mutations in cells that develop into gametes can be passed on to ospring and cause genetic disease. Source: Campbell, CD, et al. (2012) Estimating the human mutation rate using autozygosity in a founder population. Nature Genetics, 44: 1277-1281. doi: 10.1038/ng.2418 145 3 G e n e ti cs TOK sickle cell anemia What criteria can be used to distinguish between correlation and cause and efect? There is a correlation between high requencies o the sickle-cell allele in human populations and high rates o inection with Falciparum malaria. Where a correlation exists, it may or may not be due to a causal link. Consider the inormation in fgure 4 to decide whether sickle-cell anemia causes inection with malaria. b) a) Key Frequency of Hb s allele (%) 1520 1015 510 The causes o sickle cell anemia, including a base substitution mutation, a change to the base sequence o mRNA transcribed rom it and a change to the sequence o a polypeptide in hemoglobin. S ickle- cell anemia is the commonest genetic disease in the world. It is due to a mutation o the gene that codes or the alpha- globin polypeptide in hemoglobin. The symbol or this gene is Hb. Most humans have the allele Hb A . I a base substitution mutation converts the sixth codon o the gene rom GAG to GTG, a new allele is ormed, called Hb S. The mutation is only inherited by ospring i it occurs in a cell o the ovary or testis that develops into an egg or sperm. When the Hb S allele is transcribed, the mRNA produced has GUG as its sixth codon instead o GAG, and when this mRNA is transcribed, the sixth amino acid in the polypeptide is valine instead o glutamic acid. This change causes hemoglobin molecules to stick together in tissues with low oxygen concentrations. The bundles o hemoglobin molecules that are ormed are rigid enough to distort the red blood cells into a sickle shape. 05 Figure 4 Map ( a) shows the requency o the sickle cell allele and map (b) shows malaria afected areas in Arica and Western Asia These sickle cells cause damage to tissues by becoming trapped in blood capillaries, blocking them and reducing blood fow. When sickle cells return to high oxygen conditions in the lung, the hemoglobin bundles break up and the cells return to their normal shape. These changes occur time ater time, as the red blood cells circulate. Both the hemoglobin and the plasma membrane are damaged and the lie o a red blood cell can be shortened to as little as 4 days. The body cannot replace red blood cells at a rapid enough rate and anemia thereore develops. So, a small change to a gene can have very harmul consequences or individuals that inherit the gene. It is not known how oten this mutation has occurred but in some parts o the world the Hb S allele is remarkably common. In parts o East Arica up to 5 % o newborn babies have two copies o the allele and develop severe anemia. Another 3 5 % have one copy so make both normal hemoglobin and the mutant orm. These individuals only suer mild anemia. Figure 5 Micrographs o sickle cells and normal red blood cells 146 3 .1 GEN Es Wha is a genome? The genome is the whole of the genetic information of an organism. Among biologists today the word genome means the whole o the genetic inormation o an organism. Genetic inormation is contained in D NA, so a living organisms genome is the entire base sequence o each o its D NA molecules. In humans the genome consists o the 46 molecules that orm the chromosomes in the nucleus plus the D NA molecule in the mitochondrion. This is the pattern in other animals, though the number o chromosomes is usually dierent. In plant species the genome is the D NA molecules o chromosomes in the nucleus plus the D NA molecules in the mitochondrion and the chloroplast. The genome o prokaryotes is much smaller and consists o the D NA in the circular chromosome, plus any plasmids that are present. the Human Genome Projec The entire base sequence of human genes was sequenced in the Human Genome Project. The Human Genome Proj ect began in 1 990. Its aim was to fnd the base sequence o the entire human genome. This proj ect drove rapid improvements in base sequencing techniques, which allowed a drat sequence to be published much sooner than expected in 2 000 and a complete sequence in 2 003 . Although knowledge o the entire base sequence has not given us an immediate and total understanding o human genetics, it has given us what can be regarded as a rich mine o data, which will be worked by researchers or many years to come. For example, it is possible to predict which base sequences are protein- coding genes. There are approximately 2 3 , 000 o these in the human genome. O riginally, estimates or the number o genes were much higher. Another discovery was that most o the genome is not transcribed. O riginally called j unk D NA, it is being increasingly recognized that within these j unk regions, there are elements that aect gene expression as well as highly repetitive sequences, called satellite D NA. The genome that was sequenced consists o one set o chromosomes it is a human genome rather than the human genome. Work continues to fnd variations in sequence between dierent individuals. The vast maj ority o base sequences are shared by all humans giving us genetic unity, but there are also many single nucleotide polymorphisms which contribute to human diversity. S ince the publication o the human genome, the base sequence o many other species has been determined. C omparisons between these genomes reveal aspects o the evolutionary history o living organisms that were previously unknown. Research into genomes will be a developing theme o biology in the 2 1 st century. Activity Ethic of genome reearch Ethical questions about genome research are worth discussing. Is it ethical to take a DNA sample from ethnic groups around the world and sequence it without their permission? Is it ethical for a biotech company to patent the base sequence of a gene to prevent other companies from using it to conduct research freely? Who should have access to this genetic information? Should employers, insurance companies and law enforcement agencies know our genetic makeup? 147 3 G e n e ti cs techniques used for genome sequencing Developments in scientifc research ollow improvements in technology: gene sequencers, essentially lasers and optical detectors, are used or the sequencing o genes. The idea o sequencing the entire human genome seemed impossibly dicult at one time but improvements in technology towards the end o the 20th century made it possible, though still very ambitious. These improvements continued once the project was underway and drat sequences were thereore completed much sooner than expected. Further advances are allowing the genomes o other species to be sequenced at an ever increasing rate. To sequence a genome, it is rst broken up into small lengths o D NA. Each o these is sequenced separately. To nd the base sequence o a ragment o D NA, single- stranded copies o it are made using D NA polymerase, but the process is stopped beore the whole base sequence has been copied by putting small quantities o a non- standard nucleotide into the reaction mixture. This is done separately with non-standard nucleotides carrying each o the our possible D NA bases. Four samples o D NA copy o varying length are produced, each with one o our D NA bases at the end o each copy. These our samples are separated according to length by gel electrophoresis. For each number o nucleotides in the copy there is a band in j ust one o the our tracks in the gel, rom which the sequence o bases in the D NA can be deduced. fuorescent marker is used or the copies ending in each o the our bases. The samples are mixed together and all the D NA copies are separated in one lane o a gel according to the number o nucleotides. A laser scans along the lane to make the fuorescent markers fuoresce. An optical detector is used to detect the colours o fuorescence along the lane. There is a series o peaks o fuorescence, corresponding to each number o nucleotides A computer deduces the base sequence rom the sequence o colours o fuorescence detected. The maj or advance in technology that speeded up base sequencing by automating it is this: 148 C oloured fuorescent markers are used to mark the D NA copies. A dierent colour o Figure 6 Sequencing read from the DNA of Pinor Noir variety of grape 3 .2 Ch rOmOsOm Es 3.2 Coooe Udertadig Prokaryotes have one chromosome consisting o a circular DNA molecule. Some prokaryotes also have plasmids but eukaryotes do not. Eukaryote chromosomes are linear DNA molecules associated with histone proteins. In a eukaryote species there are diferent chromosomes that carry diferent genes. Homologous chromosomes carry the same sequence o genes but not necessarily the same alleles o those genes. Diploid nuclei have pairs o homologous chromosomes. Haploid nuclei have one chromosome o each pair. The number o chromosomes is a characteristic eature o members o a species. A karyogram shows the chromosomes o an organism in homologous pairs o decreasing length. Sex is determined by sex chromosomes and autosomes are chromosomes that do not determine sex. Applicatio Cairnss technique or measuring the length o DNA molecules by autoradiography. Comparison o genome size in T2 phage, Escherichia coli, Drosophila melanogaster, Homo sapiens and Paris japonica. Comparison o diploid chromosome numbers o Homo sapiens, Pan troglodytes, Canis familiaris, Oryza sativa, Parascaris equorum. Use o karyotypes to deduce sex and diagnose Down syndrome in humans. skill Use o online databases to identiy the locus o a human gene and its protein product. nature of ciece Developments in scientic research ollow improvements in techniques: autoradiography was used to establish the length o DNA molecules in chromosomes. Bacterial chromoome Prokaryotes have one chromosome consisting o a circular DNA molecule. The structure of prokaryotic cells was described in sub- topic 1 . 2 . In most prokaryotes there is one chromosome, consisting of a circular D NA molecule containing all the genes needed for the basic life processes of the cell. The D NA in bacteria is not associated with proteins, so is sometimes described as naked. 149 3 G e n e ti cs B ecause only one chromosome is present in a prokaryotic cell, there is usually only a single copy o each gene. Two identical copies are present briefy ater the chromosome has been replicated, but this is a preparation or cell division. The two genetically identical chromosomes are moved to opposite poles and the cell then splits in two. Plasmids Some prokaryotes also have plasmids but eukaryotes do not. Plasmids are small extra DNA molecules that are commonly ound in prokaryotes but are very unusual in eukaryotes. They are usually small, circular and naked, containing a ew genes that may be useul to the cell but not those needed or its basic lie processes. For example, genes or antibiotic resistance are oten located in plasmids. These genes are benecial when an antibiotic is present in the environment but are not at other times. Plasmids are not always replicated at the same time as the chromosome o a prokaryotic cell or at the same rate. Hence there may be multiple copies o plasmids in a cell and a plasmid may not be passed to both cells ormed by cell division. C opies o plasmids can be transerred rom one cell to another, allowing spread through a population. It is even possible or plasmids to cross the species barrier. This happens i a plasmid that is released when a prokaryotic cell dies is absorbed by a cell o a dierent species. It is a natural method o gene transer between species. Plasmids are also used by biologists to transer genes between species articially. Figure 1 (a) Circular DNA molecule from a bacterium (b) Bacterium preparing to divide trimethoprim resistance genes to help the plasmid spread penicillin family resistance disinfectant resistance streptomycin family resistance vancomycin resistance Figure 2 The pLW1043 plasmid Usig autoradiography to measure DnA molecules Developments in scientifc research ollow improvements in techniques: autoradiography was used to establish the length o DNA molecules in chromosomes. Quantitative data is usually considered to be the strongest type o evidence or or against a hypothesis, but in biology it is sometimes images that provide the most convincing evidence. 150 D evelopments in microscopy have allowed images to be produced o structures that were previously invisible. These sometimes conrm existing ideas but sometimes also change our understanding. 3 .2 Ch rOmOsOm Es Autoradiography was used by biologists rom the 1 940s onwards to discover where specic substances were located in cells or tissues. John C airns used the technique in a dierent way in the 1 96 0s. He obtained images o whole D NA molecules rom E. coli bacteria. At the time it was not clear whether the bacterial chromosome was a single D NA molecule or more than one, but the images produced by C airns answered this question. They also revealed replication orks in D NA or the rst time. C airnss technique was used by others to investigate the structure o eukaryote chromosomes. Measurig the legth of DnA molecules Cairnss technique for measuring the length of DNA molecules by autoradiography. John C airns produced images o D NA molecules rom E.coli using this technique: C ells were grown or two generations in a culture medium containing tritiated thymidine. Thymidine consists o the base thymine linked to deoxyribose and is used by E. coli to make nucleotides that it uses in D NA replication. Tritiated thymidine contains tritium, a radioactive isotope o hydrogen, so radioactively labelled D NA was produced by replication in the E. coli cells. The cells were then placed onto a dialysis membrane and their cell walls were digested using the enzyme lysozyme. The cells were gently burst to release their D NA onto the surace o the dialysis membrane. A thin lm o photographic emulsion was applied to the surace o the membrane and let in darkness or two months. D uring that time some o the atoms o tritium in the D NA decayed and emitted high energy electrons, which react with the lm. At the end o the two-month period the lm was developed and examined with a microscope. At each point where a tritium atom decayed there is a dark grain. These indicate the position o the D NA. The images produced by C airns showed that the chromosome in E. coli is a single circular D NA molecule with a length o 1 , 1 00 m. This is remarkably long given that the length o the E coli cells is only 2 m. Autoradiography was then used by other researchers to produce images o eukaryotic chromosomes. An image o a chromosome rom the ruit fy Drosophila melanogaster was produced that was 1 2 , 00 0 m long. This corresponded with the total amount o D NA known to be in a D. melanogaster chromosome, so or this species at least a chromosome contains one very long D NA molecule. In contrast to prokaryotes, the molecule was linear rather than circular. Figure 3 Eukaryote chromosomes Eukaryote chromosomes are linear DNA molecules associated with histone proteins. C hromosomes in eukaryotes are composed o D NA and protein. The D NA is a single immensely long linear D NA molecule. It is associated with histone proteins. Histones are globular in shape and are wider 151 3 G e n e ti cs than the D NA. There are many histone molecules in a chromosome, with the D NA molecule wound around them. Adj acent histones in the chromosome are separated by short stretches o the D NA molecule that are not in contact with histones. This gives a eukaryotic chromosome the appearance o a string o beads during interphase. Diferences between chromosomes In a eukaryote species there are diferent chromosomes that carry diferent genes. Eukaryote chromosomes are too narrow to be visible with a light microscope during interphase. During mitosis and meiosis the chromosomes become much shorter and atter by supercoiling, so are visible i stains that bind either D NA or proteins are used. In the frst stage o mitosis the chromosomes can be seen to be double. There are two chromatids, with identical DNA molecules produced by replication. Figure 4 In an electron micrograph the histones give a eukaryotic chromosome the appearance of a string of beads during interphase 7S DNA thr OH PH phe 16S cyt b pro There are at least two dierent types in every eukaryote but in most species there are more than that. In humans or example there are 2 3 types o chromosome. val 23S leu PL glu N6 gln ala control loop asn ribosomal RNA cys transfer RNAs protein coding gene tyr N5 leu ser his ser N4 a rg When the chromosomes are examined during mitosis, dierent types can be seen. They dier both in length and in the position o the centromere where the two chromatids are held together. The centromere can be positioned anywhere rom close to an end to the centre o the chromosome. lys N 3gly OX3 ATPase N1 ile f-met N2 trp OL OX1 asp OX2 Figure 5 Gene map of the human mitochondrial chromosome. There are genes on both of the two DNA strands. The chromosomes in the nucleus are much longer, carry far more genes and are linear rather than circular Every gene in eukaryotes occupies a specifc position on one type o chromosome, called the locus o the gene. Each chromosome type thereore carries a specifc sequence o genes arranged along the linear D NA molecule. In many chromosomes this sequence contains over a thousand genes. C rossing experiments were done in the past to discover the sequence o genes on chromosome types in Drosophila melanogaster and other species. The base sequence o whole chromosomes can now be ound, allowing more accurate and complete gene sequences to be deduced. Having the genes arranged in a standard sequence along a type o chromosome allows parts o chromosomes to be swapped during meiosis. Homologous chromosomes Homologous chromosomes carry the same sequence o genes but not necessarily the same alleles o those genes. I two chromosomes have the same sequence o genes they are homologous. Homologous chromosomes are not usually identical to each other because, or at least some o the genes on them, the alleles are dierent. I two eukaryotes are members o the same species, we can expect each o the chromosomes in one o them to be homologous with at least one chromosome in the other. This allows members o a species to interbreed. 152 3 .2 Ch rOmOsOm Es Data-baed quetion: Comparing the chromosomes of mice and humans Activity Figure 6 shows all of the types of chromosome in mice and in humans. Numbers and colours are used to indicate sections of mouse chromosomes that are homologous to sections of human chromosomes. Mouse and human genetic similarities Mouse chromosomes 1 2 3 10 9 2 11 15 6 2 18 1 10 10 22 21 19 12 6 Human chromosomes 7 7 2 3 10 12 13 13 2 7 14 15 7 6 3 10 14 8 5 13 8 22 14 17 19 X 16 5 12 21 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 11 15 6 3 18 6 16 21 6 19 18 2 10 18 5 water at 25 C 18 19 20 21 22 X Y Y X 10 Figure 6 Chromosomes 1 2 D educe the number of types of chromosomes in mice and in humans. [2 ] Identify the two human chromosome types that are most similar to mouse chromosomes. [2 ] 3 Identify mouse chromosomes which contain sections that are not homologous to human chromosomes. [2 ] 4 S uggest reasons for the many similarities between the mouse and human genomes. 5 polystyrene disc with hole cut through beaker garlic bulb 1 Y 11 9 1 Garlic has large chromosomes so is an ideal choice for looking at chromosomes. Cells in mitosis are needed. Garlic bulbs grow roots if they are kept for 3 or 4 days with their bases in water, at about 25C. Root tips with cells in mitosis are yellow in colour, not white. 16 17 16 22 3 2 11 19 19 4 19 11 16 10 11 7 1 1 8 11 15 4 4 9 19 19 9 4 3 1 8 7 3 12 22 7 2 16 5 5 8 8 20 11 6 4 micocope invetigation of galic coooe [2 ] D educe how chromosomes have mutated during the evolution of animals such as mice and humans. [2 ] 2 Root tips are put in a mixture of a stain that binds to the chromosomes and acid, which loosens the connections between the cell walls. A length of about 5 mm is suitable. Ten parts of acetoorcein to one part of 1.0 mol dm -3 hydrochloric acid gives good results. stainacid mixture 5 mm long garlic root tip watch glass 3 The roots are heated in the stainacid mixture on a hot plate, to 80C for 5 minutes. One of the root tips is put on a microscope slide, cut in half and the 2.5 mm length furthest from the end of the root is discarded. root tip watch glass Comparing the genome sizes 8 7 1 5 3 6 2 4 Comparison of genome size in T2 phage, Escherichia coli, Drosophila melanogaster, Homo sapiens and Paris japonica. The genomes of living organisms vary by a huge amount. The smallest genomes are those of viruses, though they are not usually regarded as living organisms. The table on the next page gives the genome size of one virus and four living organisms. O ne of the four living organisms is a prokaryote. It has much the smallest genome. The genome size of eukaryotes depends on the size and number of chromosomes. It is correlated with the complexity of the organism, but is not directly proportional. There are several reasons for this. The proportion of the D NA that acts as functional genes is very variable and also the amount of gene duplication varies. hot plate set at 80 C 4 A drop of stain and a cover slip is added and the root tip is squashed to spread out the cells to form a layer one cell thick. The chromosomes can then be examined and counted and the various phases of mitosis should also be visible. thumb pressing down to squash root tip cover slip microscope slide folded lter paper 153 3 G e n e ti cs Organism Genome size (million base pairs) T2 phage 0.18 5 Escherichia coli Description Virus that attacks Escherichia coli Gut bacterium 140 Fruit fy Homo sapiens 3,000 Humans Paris japonica 150,000 Drosophila melanogaster Woodland plant Finding the loci of human genes Use o online databases to identiy the locus o a human gene and its protein product. The locus o a gene is its particular position on homologous chromosomes. Online databases can be used to fnd the locus o human genes. There is an example o such a database in the Online Mendelian Inheritance in Man website, maintained by Johns Hopkins University. S earch or the abbreviation O MIM to open the home page. C hoose Search Gene Map. Enter the name o a gene into the S earch Gene Map box. This should bring up a table with inormation about the gene, including its locus, starting with the chromosome on which the gene is located. S uggestions o human genes are shown on the right. An alternative to entering the name o a gene is to select a chromosome rom 1 2 2 or one o the sex chromosomes X or Y. A complete sequence o gene loci will be displayed, together with the total number o gene loci on that chromosome. Gene name Description of gene DRD4 A gene that codes or a dopamine receptor that is implicated in a variety o neurological and psychiatric conditions. CFTR A gene that codes or a chloride channel protein. An allele o this gene causes cystic brosis. HBB The gene that codes or the beta-globin subunit o hemoglobin. An allele o this gene causes sickle cell anemia. F8 The gene that codes or Factor VIII, one o the proteins needed or the clotting o blood. The classic orm o hemophilia is caused by an allele o this gene. TDF Testis determining actor the gene that causes a etus to develop as a male. Haploid nuclei Haploid nuclei have one chromosome o each pair. A haploid nucleus has one chromosome o each type. It has one ull set o the chromosomes that are ound in its species. Haploid nuclei in humans contain 2 3 chromosomes or example. Gametes are the sex cells that use together during sexual reproduction. Gametes have haploid nuclei, so in humans both egg and sperm cells contain 2 3 chromosomes. 154 3 .2 Ch rOmOsOm Es Diploid nuclei Diploid nuclei have pairs of homologous chromosomes. A diploid nucleus has two chromosomes of each type. It has two full sets of the chromosomes that are found in its species. D iploid nuclei in humans contain 46 chromosomes for example. When haploid gametes fuse together during sexual reproduction, a zygote with a diploid nucleus is produced. When this divides by mitosis, more cells with diploid nuclei are produced. Many animals and plants consist entirely of diploid cells, apart from the cells that they are using to produce gametes for sexual reproduction. D iploid nuclei have two copies of every gene, apart from genes on the sex chromosomes. An advantage of this is that the effects of harmful recessive mutations can be avoided if a dominant allele is also present. Also, organisms are often more vigorous if they have two different alleles of genes instead of j ust one. This is known as hybrid vigour and is the reason for strong growth of F 1 hybrid crop plants. Figure 7 Mosses coat the trunks of the laurel trees in this forest in the Canary Islands. Mosses are unusual because their cells are haploid. In most eukaryotes the gametes are haploid but not the parent that produces them Chromosome numbers The number of chromosomes is a characteristic feature of members of a species. O ne of the most fundamental characteristics of a species is the number of chromosomes. O rganisms with a different number of chromosomes are unlikely to be able to interbreed so all the interbreeding members of a species need to have the same number of chromosomes. The number of chromosomes can change during the evolution of a species. It can decrease if chromosomes become fused together or increase if splits occur. There are also mechanisms that can cause the chromosome number to double. However, these are rare events and chromosome numbers tend to remain unchanged over millions of years of evolution. Figure 8 Trillium luteum cell with a diploid number of 12 chromosomes. Two of each type of chromosome are present Comparing chromosome numbers Comparison of diploid chromosome numbers of Homo sapiens, Pan troglodytes, Canis familiaris, Oryza sativa, Parascaris equorum. The Oxford English D ictionary consists of twenty large volumes, each containing a large amount of information about the origins and meanings of words. This information could have been published in a smaller number of larger volumes or in a larger number of smaller volumes. There is a parallel with the numbers and sizes of chromosomes in eukaryotes. Some have a few large chromosomes and others have many small ones. All eukaryotes have at least two different types of chromosome, so the diploid chromosome number is at least four. In some cases it is over a hundred. The table on the next page shows the diploid chromosome number of selected species. 155 3 G e n e ti cs scientifc name o pecie Figure 9 Who has more chromosomes a dog or its owner? Englih name Diploid chromoome number Parascaris equorum horse threadworm 4 Oryza sativa rice 24 Homo sapiens humans 46 Pan troglodytes chimpanzee 48 Canis amiliaris dog 78 Data-baed quetion: Diferences in chromosome number Plant Haplopappus gracilis Luzula purpurea (woodrush) Crepis capillaris Vicia aba (eld bean) Brassica oleracea (cabbage) Citrullus vulgaris (water melon) Lilium regale (royal lily) Bromus texensis Camellia sinesis (Chinese tea) Magnolia virginiana (sweet bay) Arachis hypogaea (peanut) Cofea arabica (cofee) Stipa spartea (porcupine grass) Chrysoplenum alterniolium (saxirage) Aster laevis (Michaelmas daisy) Glyceria canadensis (manna grass) Carya tomentosa (hickory) Magnolia cordata Rhododendron keysii Chromoome number 4 6 8 12 18 22 24 28 30 38 40 44 46 48 54 60 64 76 78 Animal Parascaris equorum (horse threadworm) Aedes aegypti (yellow ever mosquito) Drosophila melanogaster (ruity) Musca domestica (house y) Chorthippus parallelus (grasshopper) Cricetulus griseus (Chinese hamster) Schistocerca gregaria (desert locust) Desmodus rotundus (vampire bat) Mustela vison (mink) Felis catus (domestic cat) Mus musculus (mouse) Mesocricetus auratus (golden hamster) Homo sapiens (modern humans) Pan troglodytes (chimpanzee) Ovis aries (domestic sheep) Capra hircus (goat) Dasypus novemcinctus (armadillo) Ursus americanus (American black bear) Canis amiliaris (dog) Table 1 156 1 There are many different chromosome numbers in the table, but some numbers are missing, for example, 5 , 7, 1 1 , 1 3 . Explain why none of the species has 1 3 chromosomes. [3 ] 2 D iscuss, using the data in the table, the hypothesis that the more complex an organism is, the more chromosomes it has. [4] 3 E xplain why the size of the genome of a species cannot be deduced from the number of chromosomes. [1 ] 4 S uggest, using the data in table 1 , a change in chromosome structure that may have occurred during human evolution. [2 ] 3 .2 Ch rOmOsOm Es sex determination female male XX XY Sex is determined by sex chromosomes and autosomes are chromosomes that do not determine sex. There are two chromosomes in humans that determine sex: X X the Y chromosome is much smaller and has its centromere near the end. B ecause the X and Y chromosomes determine sex they are called the sex chromosomes. All the other chromosomes are autosomes and do not affect whether a fetus develops as a male or female. The X chromosome has many genes that are essential in both males and females. All humans must therefore have at least one X chromosome. The Y chromosome only has a small number of genes. A small part of the Y chromosome has the same sequence of genes as a small part of the X chromosome, but the genes on the remainder of the Y chromosome are not found on the X chromosome and are not needed for female development. XX X the X chromosome is relatively large and has its centromere near the middle. Y XX XY XY 1 female : 1 male Figure 10 Determination of gender O ne Y chromosome gene in particular causes a fetus to develop as a male. This is called either S RY or TD F. It initiates the development of male features, including testes and testosterone production. B ecause of this gene a fetus with one X and one Y chromosome develops as a male. A fetus that has two X chromosomes and no Y chromosome does not have the TD F gene so ovaries develop instead of testes and female sex hormones are produced, not testosterone. Females have two X chromosomes. Females pass on one of their two X chromosomes in each egg cell, so all offspring inherit an X chromosome from their mother. The gender of a human is determined at the moment of fertilization by one chromosome carried in the sperm. This can either be an X or a Y chromosome. When sperm are formed, half contain the X chromosome and half the Y chromosome. D aughters inherit their fathers X chromosome and sons inherit his Y chromosome. Karyogram A karyogram shows the chromosomes of an organism in homologous pairs of decreasing length. The chromosomes of an organism are visible in cells that are in mitosis, with cells in metaphase giving the clearest view. S tains have to be used to make the chromosomes show up. S ome stains give each chromosome type a distinctive banding pattern. If dividing cells are stained and placed on a microscope slide and are then burst by pressing on the cover slip, the chromosomes become spread. O ften they overlap each other, but with careful searching a cell can usually be found with no overlapping chromosomes. A micrograph can be taken of the stained chromosomes. 157 3 G e n e ti cs TOK To what extent is determining gender or sporting competition a scientifc question? Gender testing was introduced at the 1968 Olympic games to address concerns that women with ambiguous physiological genders would have an unair advantage. This has proven to be problematic or a number o reasons. The chromosomal standard is problematic as non-disjunction can lead to situations where an individual might technically be male, but might not defne hersel in that way. People with two X chromosomes can develop hormonally as a male and people with an X and a Y can develop hormonally as a emale. Originally analysis involved cutting out all the chromosomes and arranging them manually but this process can now be done digitally. The chromosomes are arranged according to their size and structure. The position of the centromere and the pattern of banding allow chromosomes that are of a different type but similar size to be distinguished. As most cells are diploid, the chromosomes are usually in homologous pairs. They are arranged by size, starting with the longest pair and ending with the smallest. The practice o gender testing was discontinued in 1996 in part because o human rights issues including the right to sel-expression and the right to identiy one's own gender. Rather than being a scientifc question, it is more airly a social question. Figure 11 Karyogram o a human emale, with fuorescent staining Karyotypes and Down syndrome Use o karyotypes to deduce sex and diagnose Down syndrome in humans. A karyogram is an image of the chromosomes of an organism, arranged in homologous pairs of decreasing length. A karyotype is a property of an organism it is the number and type of chromosomes that the organism has in its nuclei. Karyotypes are studied by looking at karyograms. They can be used in two ways: 1 To deduce whether an individual is male or female. If two XX chromosomes are present the individual is female whereas one X and one Y indicate a male. 2 To diagnose Down syndrome and other chromosome abnormalities. This is usually done using fetal cells taken from the uterus during pregnancy. If there are three copies of chromosome 2 1 in the karyotype instead of two, the child has Down syndrome. This is sometimes called trisomy 21 . While individuals vary, some of the component features of the syndrome are hearing loss, heart and vision disorders. Mental and growth retardation are also common. Figure 12 Child with trisomy 21 or Down syndrome 158 3.3 mEiOsis Data-based questions: A human karyotype The karyogram shows the karyotype of a fetus. 1 S tate which chromosome type is a) longest b) shortest. 2 [2 ] D istinguish between the structure of a) human chromosome 2 and chromosome 1 2 b) the human X and Y chromosome. [4] 3 D educe with a reason the sex of the fetus. [2 ] 4 E xplain whether the karyotype shows any abnormalities. [2 ] Figure 13 3.3 meo Udertadig One diploid nucleus divides by meiosis to produce our haploid nuclei. The halving o the chromosome number allows a sexual lie cycle with usion o gametes. DNA is replicated beore meiosis so that all chromosomes consist o two sister chromatids. The early stages o meiosis involve pairing o homologous chromosomes and crossing over ollowed by condensation. Orientation o pairs o homologous chromosomes prior to separation is random. Separation o pairs o homologous chromosomes in the rst division o meiosis halves the chromosome number. Crossing over and random orientation promotes genetic variation. Fusion o gametes rom diferent parents promotes genetic variation. Applicatio Non-disjunction can cause Down syndrome and other chromosome abnormalities. Studies showing age o parents inuences chances o non-disjunction. Methods used to obtain cells or karyotype analysis e.g. chorionic villus sampling and amniocentesis and the associated risks. skill Drawing diagrams to show the stages o meiosis resulting in the ormation o our haploid cells. nature of ciece Making careul observations: meiosis was discovered by microscope examination o dividing germ-line cells. 159 3 G e n e ti cs the discovery of meiosis Making careful observations: meiosis was discovered by microscope examination of dividing germ-line cells. When improved microscopes had been developed in the 1 9th century that gave detailed images o cell structures, it was discovered that some dyes specifcally stained the nucleus o the cell. These dyes revealed thread-like structures in dividing nuclei that were named chromosomes. From the 1 880s onwards a group o German biologists carried out careul and detailed observations o dividing nuclei that gradually revealed how mitosis and meiosis occur. We can appreciate the considerable achievements o these biologists i we try to repeat the observations that they made. The preparation o microscope slides showing meiosis is challenging. Suitable tissue can be obtained rom the developing anthers inside a lily bud or rom the testis o a dissected locust. The tissue must be fxed, stained and then squashed on a microscope slide. Oten no cells in meiosis are visible or the images are not clear enough to show details o the process. Even with prepared slides made by experts it is difcult to understand the images as chromosomes orm a variety o bizarre shapes during the stages o meiosis. A key observation was that in the horse threadworm (Parascaris equorum) there are two chromosomes in the nuclei o egg and sperm cells, whereas the ertilized egg contains our. This indicated that the one diploid cell Nuclear divisions unlike mitosis had already been observed during gamete development in both animals and plants. These divisions were identifed as the method used to halve the chromosome number and they were named meiosis. The sequence o events in meiosis was eventually worked out by careul observation o cells taken rom the ovaries o rabbits ( Oryctolagus cuniculus) between 0 and 2 8 days old. The advantage o this species is that in emales meiosis begins at birth and occurs slowly over many days. Figure 1 Meiosis in ouline 2n One diploid nucleus divides by meiosis to produce four haploid nuclei. meiosis I two haploid cells n n meiosis II four haploid cells chromosome number is doubled by ertilization. The observation led to the hypothesis that there must be a special nuclear division in every generation that halves the chromosome number. n n Figure 2 Overview of meiosis n n Meiosis is one o the two ways in which the nucleus o a eukaryotic cell can divide. The other method is mitosis, which was described in sub- topic 1 . 6. In meiosis the nucleus divides twice. The frst division produces two nuclei, each o which divides again to give a total o our nuclei. The two divisions are known as meiosis I and meiosis II. The nucleus that undergoes the frst division o meiosis is diploid it has two chromosomes o each type. C hromosomes o the same type are known as homologous chromosomes. Each o the our nuclei produced by meiosis has j ust one chromosome o each type they are haploid. Meiosis involves a halving o the chromosome number. It is thereore known as a reduction division. The cells produced by meiosis I have one chromosome o each type, so the halving o the chromosome number happens in the frst division, 160 3.3 mEiOsis not the second division. The two nuclei produced by meiosis I have the haploid number o chromosomes, but each chromosome still consists o two chromatids. These chromatids separate during meiosis II, producing our nuclei that have the haploid number o chromosomes, with each chromosome consisting o a single chromatid. Meiosis and sexual life cycles The halving of the chromosome number allows a sexual life cycle with fusion of gametes. The lie cycles o living organisms can be sexual or asexual. In an asexual lie cycle the ospring have the same chromosomes as the parent so are genetically identical. In a sexual lie cycle there are dierences between the chromosomes o the ospring and the parents, so there is genetic diversity. In eukaryotic organisms, sexual reproduction involves the process o ertilization. Fertilization is the union o sex cells, or gametes, usually rom two dierent parents. Fertilization doubles the number o chromosomes each time it occurs. It would thereore cause a doubling o chromosome number every generation, i the number was not also halved at some stage in the lie cycle. This halving o chromosome number happens during meiosis. Meiosis can happen at any stage during a sexual lie cycle, but in animals it happens during the process o creating the gametes. B ody cells are thereore diploid and have two copies o most genes. Meiosis is a complex process and it is not at the moment clear how it developed. What is clear is that its evolution was a critical step in the origin o eukaryotes. Without meiosis there cannot be usion o gametes and the sexual lie cycle o eukaryotes could not occur. Figure 4 Fledgling owls (bottom) produced by a sexual life cycle have diploid body cells but mosses ( top) have haploid cells Data-baed queton: Life cycles Figure 3 shows the lie cycle o humans and mosses, with n being used to represent the haploid number o chromosomes and 2 n to represent the diploid number. Sporophytes o mosses grow on the main moss plant and consist o a stalk and a capsule in which spores are produced. 1 2 O utline fve similarities between the lie cycle o a moss and o a human. [5 ] D istinguish between the lie cycles o a moss and a human by giving fve dierences. [5 ] egg n sperm n human male 2n sperm n egg n zygote 2n human female 2n moss plant n Key Figure 3 mitosis meiosis fertilization zygote 2n spore n sporophyte 2n 161 3 G e n e ti cs Replicatio of DnA before meiosis 2n 2n 2n n n n DNA is replicated before meiosis so that all chromosomes consist of two sister chromatids. interphase homologous chromosomes D uring the early stages o meiosis the chromosomes gradually shorten by supercoiling. As soon as they become visible it is clear that each chromosome consists o two chromatids. This is because all D NA in the nucleus is replicated during the interphase beore meiosis, so each chromosome consists o two sister chromatids. Initially the two chromatids that make up each chromosome are genetically identical. This is because D NA replication is very accurate and the number o mistakes in the copying o the D NA is extremely small. meiosis I n meiosis II n n We might expect the D NA to be replicated again between the frst and the second division o meiosis, but it does not happen. This explains how the chromosome number is halved during meiosis. O ne diploid nucleus, in which each chromosome consists o two chromatids, divides twice to produce our haploid nuclei in which each chromosome consists o one chromatid. Figure 5 Outline of meiosis Bivalets formatio ad crossig over The early stages of meiosis involve pairing of homologous chromosomes and crossing over followed by condensation. Some o the most important events o meiosis happen at the start o meiosis I while the chromosomes are still very elongated and cannot be seen with a microscope. Firstly homologous chromosomes pair up with each other. Because DNA replication has already occurred, each chromosome consists o two chromatids and so there are our DNA molecules associated in each pair o homologous chromosomes. A pair o homologous chromosomes is bivalent and the pairing process is sometimes called synapsis. Soon ater synapsis, a process called crossing over takes place. The molecular details o this need not concern us here, but the outcome is very important. A junction is created where one chromatid in each o the homologous chromosomes breaks and rejoins with the other chromatid. Crossing over occurs at random positions anywhere along the chromosomes. At least one crossover occurs in each bivalent and there can be several. Figure 6 A pair of homologous chromosomes contains four chromatids and is sometimes called a tetrad. Five chiasmata are visible in this tetrad, showing that crossing over can occur more than once B ecause a crossover occurs at precisely the same position on the two chromatids involved, there is a mutual exchange o genes between the chromatids. As the chromatids are homologous but not identical, some alleles o the exchanged genes are likely to be dierent. C hromatids with new combinations o alleles are thereore produced. Radom orietatio of bivalets Orientation of pairs of homologous chromosomes prior to separation is random. While pairs o homologous chromosomes are condensing inside the nucleus o a cell in the early stages o meiosis, spindle microtubules are growing rom the poles o the cell. Ater the nuclear membrane has 162 3.3 mEiOsis broken down, these spindle microtubules attach to the centromeres o the chromosomes. The attachment o the spindle microtubules is not the same as in mitosis. The principles are these: E ach chromosome is attached to one pole only, not to both. The two homologous chromosomes in a bivalent are attached to dierent poles. The pole to which each chromosome is attached depends on which way the pair o chromosomes is acing. This is called the orientation. The orientation o bivalents is random, so each chromosome has an equal chance o attaching to each pole, and eventually o being pulled to it. The orientation o one bivalent does not aect other bivalents. The consequences o the random orientation o bivalents are discussed in the section on genetic diversity later in this topic. MITOSIS Halving the chromosome number Separation o pairs o homologous chromosomes in the frst division o meiosis halves the chromosome number. The movement o chromosomes is not the same in the frst division o meiosis as in mitosis. Whereas in mitosis the centromere divides and the two chromatids that make up a chromosome move to opposite poles, in meiosis the centromere does not divide and whole chromosomes move to the poles. either or MEIOSIS Figure 7 Comparison of attachment of chromosomes to spindle microtubules in mitosis and meiosis Initially the two chromosomes in each bivalent are held together by chiasmata, but these slide to the end o the chromosomes and then the chromosomes can separate. This separation o homologous chromosomes is called disj unction. O ne chromosome rom each bivalent moves to one o the poles and the other chromosome to the other pole. The separation o pairs o homologous chromosomes to opposite poles o the cell halves the chromosome number o the cell. It is thereore the frst division o meiosis that is the reduction division. B ecause one chromosome o each type moves to each pole, both o the two nuclei ormed in the frst division o meiosis contain one o each type o chromosome, so they are both haploid. Obtaining cells from a fetus Methods used to obtain cells or karyotype analysis e.g. chorionic villus sampling and amniocentesis and the associated risks. Two procedures are used or obtaining cells containing the etal chromosomes needed or producing a karyotype. Amniocentesis involves passing a nee dle through the mothe r' s ab domen wall, using ultrasound to guide the needle. The needle is used to withdraw a sample o amniotic luid containing etal cells rom the amniotic sac. The second procedure is chorionic villus sampling. A sampling tool that enters through the vagina is used to obtain cells rom the chorion, one o the membranes rom which the placenta develops. This can be done earlier in the pregnancy than amniocentesis, but whereas the risk o miscarriage with amniocentesis is 1 % , with chorionic villus sampling it is 2 % . 163 3 G e n e ti cs Diagrams of the stages of meiosis Drawing diagrams to show the stages of meiosis resulting in the formation of four haploid cells. In mitosis our stages are usually recognized: prophase, metaphase, anaphase and telophase. Meiosis can also be divided into these stages, but each stage happens twice: in meiosis I and then a second time in meiosis II. The main events o each stage in mitosis also happen in meiosis: prophase: condensation o chromosomes; metaphase: attachment o spindle microtubules; anaphase: movement o chromosomes to the poles; telophase: decondensation o chromosomes. Usually we draw biological structures rom actual specimens, oten looking at them down a microscope. Preparation o microscope slides showing meiosis is worth attempting but it is challenging. Permanent slides usually have more cells visible in meiosis than temporary mounts, but even then it is difcult to interpret the structure o bivalents rom their appearance. This is why we usually construct diagrams o meiosis rather than draw stages rom specimens on microscope slides! The frst division o meiosis Prophase i Cell has 2n chromosomes (double chromatid) : n is haploid number of chromosomes. Homologous chromosomes pair (synapsis) . Crossing over occurs. nuclear membrane spindle microtubules and centriole Prophase I metaphase i Spindle microtubules move homologous pairs to equator of cell. Orientation of paternal and maternal chromosomes on either side of equator is random and independent of other homologous pairs. bivalents aligned on the equator Metaphase I Anaphase i Homologous pairs are separated. One chromosome of each pair moves to each pole. homologous chromosomes being pulled to opposite poles Anaphase I Telophase i 164 Chromosomes uncoil. During interphase that follows, no replication occurs. Reduction of chromosome number from diploid to haploid completed. Cytokinesis occurs. cell has divided across the equator Telophase I 3.3 mEiOsis The second division of meiosis Prophae ii Chromosomes, which still consist of two chromatids, condense and become visible. Prophase II metaphae ii Metaphase II Anaphae ii Centromeres separate and chromatids are moved to opposite poles. Anaphase II Telophae ii Chromatids reach opposite poles. Nuclear envelope forms. Cytokinesis occurs. Telophase II Meiosis and genetic variation Crossing over and random orientation promotes genetic variation. When two parents have a child, they know that it will inherit an unpredictable mixture of characteristics from each of them. Much of the unpredictability is due to meiosis. E very gamete produced by a parent has a new combination of alleles meiosis is a source of endless genetic variation. Apart from the genes on the X and Y chromosomes, humans have two copies of each gene. In some cases the two copies are the same allele and there will be one copy of that allele in every gamete produced by the parent. There are likely to be thousands of genes in the parents genome 165 3 G e n e ti cs Activity I g is the number o genes in a genome with diferent alleles, 2 g is the number o combinations o these alleles that can be generated by meiosis. I there were just 69 genes with diferent alleles (3 in each o the 23 chromosome types in humans) there would be 590,295,810,358,705, 700,000 combinations. Assuming that all humans are genetically diferent, and that there are 7,000,000 humans, calculate the percentage o all possible genomes that currently exist. where the two alleles are dierent. Each o the two alleles has an equal chance o being passed on in a gamete. Let us suppose that there is a gene with the alleles A and a. Hal o the gametes produced by the parent will contain A and hal will contain a. Let us now suppose that there is another gene with the alleles B and b. Again hal o the gametes will contain B and hal b. However, meiosis can result in gametes with dierent combinations o these genes: AB , Ab, aB and ab. There are two processes in meiosis that generate this diversity. 50% probability a B A b B a b A B b telophase I A a prophase I 50% probability a b A B b a B A metaphase I Figure 8 Random orientation in metaphase I 1. Random orientation o bivalents In metaphase I the orientation o bivalents is random and the orientation o one bivalent does not infuence the orientation o any o the others. Random orientation o bivalents is the process that generates genetic variation among genes that are on dierent chromosome types. For every additional bivalent, the number o possible chromosome combinations in a cell produced by meiosis doubles. For a haploid number o n, the number o possible combinations is 2 n. For humans with a haploid number o 2 3 this amounts to 2 23 or over 8 million combinations. 2. Crossing over Without crossing over in prophase I, combinations o alleles on chromosomes would be orever linked together. For example, i one chromosome carried the combination C D and another carried cd, only these combinations could occur in gametes. C rossing over allows linked genes to be reshufed, to produce new combinations such as C d and cD . It increases the number o allele combinations that can be generated by meiosis so much that it is eectively innite. Fertilization and genetic variation Fusion o gametes rom diferent parents promotes genetic variation. The usion o gametes to produce a zygote is a highly signicant event both or individuals and or species. Figure 9 166 It is the start o the lie o a new individual. It allows alleles rom two dierent individuals to be combined in one new individual. 3.3 mEiOsis The combination o alleles is unlikely ever to have existed beore. Fusion o gametes thereore promotes genetic variation in a species. Genetic variation is essential or evolution. no-disjuctio ad Dow sydrome Non-disjunction can cause Down syndrome and other chromosome abnormalities. Meiosis is sometimes subj ect to errors. O ne example o this is when homologous chromosomes ail to separate at anaphase. This is termed non- disj unction. This can happen with any o the pairs o homologous chromosomes. B oth o the chromosomes move to one pole and neither to the other pole. The result will be a gamete that either has an extra chromosome or is defcient in a chromosome. I the gamete is involved in human ertilization, the result will be an individual with either 45 or 47 chromosomes. An abnormal number o chromosomes will oten lead to a person possessing a syndrome, i.e. a collection o physical signs or symptoms. For example trisomy 2 1 , also known as D own syndrome, is due to a non- disj unction event that leaves the individual with three o chromosome number 2 1 instead o two. While individuals vary, some o the component eatures o the syndrome include hearing loss, heart and vision disorders. Mental and growth retardation are also common. Most other trisomies in humans are so serious that the ospring do not survive. B abies are sometimes born with trisomy 1 8 and trisomy 1 3 . Non-disj unction can also result in the birth o babies with abnormal numbers o sex chromosomes. Klineelters syndrome is caused by having the sex chromosomes XXY. Turners syndrome is caused by having only one sex chromosome, an X. diploid parent cell with two chromosome 21 non-disjunction during meiosis gamete with no chromosome 21 gamete with two chromosome 21 cell dies fusion of gametes normal haploid gamete trisomy: zygote with three chromosome 21 Figure 10 How non-disjunction can give rise to Down syndrome trisomy 21 all chromosomal abnormalities Studies showing age o parents infuences chances o non-disjunction The data presented in fgure 1 1 shows the relationship between maternal age and the incidence o trisomy 2 1 and o other chromosomal abnormalities. 1 O utline the relationship between maternal age and the incidence o chromosomal abnormalities in live births. [2 ] 2 a) For mothers 40 years o age, determine the probability that they will give birth to a child with trisomy 2 1 . [1 ] b) Using the data in fgure 1 1 , calculate the probability that a mother o 40 years o age will give birth to a child with a chromosomal abnormality other than trisomy 2 1 . incidence (% of all live births) Paretal age ad o-disjuctio 14 12 10 8 6 4 2 0 20 40 60 maternal age (years) Figure 11 The incidence of trisomy 21 [2 ] and other chromosomal abnormalities as a function of maternal age 167 3 G e n e ti cs 3 4 O nly a small number of possible chromosomal abnormalities are ever found among live births, and trisomy 2 1 is much the commonest. S uggest reasons for these trends. [3 ] D iscuss the risks parents face when choosing to postpone having children. [2 ] 3.4 inhertance Udertadig Mendel discovered the principles o inheritance 168 with experiments in which large numbers o pea plants were crossed. Gametes are haploid so contain one allele o each gene. The two alleles o each gene separate into diferent haploid daughter nuclei during meiosis. Fusion o gametes results in diploid zygotes with two alleles o each gene that may be the same allele or diferent alleles. Dominant alleles mask the efects o recessive alleles but co-dominant alleles have joint efects. Many genetic diseases in humans are due to recessive alleles o autosomal genes. Some genetic diseases are sex-linked and some are due to dominant or co-dominant alleles. The pattern o inheritance is diferent with sex-linked genes due to their location on sex chromosomes. Many genetic diseases have been identied in humans but most are very rare. Radiation and mutagenic chemicals increase the mutation rate and can cause genetic disease and cancer. Applicatio Inheritance o ABO blood groups. Red-green colour-blindness and hemophilia as examples o sex-linked inheritance. Inheritance o cystic brosis and Huntingtons disease. Consequences o radiation ater nuclear bombing o Hiroshima and Nagasaki and the nuclear accidents at Chernobyl. skill Construction o Punnett grids or predicting the outcomes o monohybrid genetic crosses. Comparison o predicted and actual outcomes o genetic crosses using real data. Analysis o pedigree charts to deduce the pattern o inheritance o genetic diseases. nature of ciece Making quantitative measurements with replicates to ensure reliability: Mendels genetic crosses with pea plants generated numerical data. 3 . 4 i N h E r i TAN CE Mendel and the principles of inheritance Mendel discovered the principles of inheritance with experiments in which large numbers of pea plants were crossed. When living organisms reproduce, they pass on characteristics to their ospring. For example, when blue whales reproduce, the young are also blue whales they are members o the same species. More than this, variations, such as the markings on the skin o a blue whale, can be passed on. We say that the ospring inherit the parents characteristics. However, some characteristics cannot be inherited. S cars seen on the tails o some blue whales caused by killer whale attacks and cosmetic surgery in humans are examples o this. According to current theories, acquired characteristics such as these cannot be inherited. Inheritance has been discussed since the time o Hippocrates and earlier. For example, Aristotle observed that children sometimes resemble their grandparents more than their parents. Many o the early theories involved blending inheritance, in which ospring inherit characters rom both parents and so have characters intermediate between those o their parents. S ome o the observations that biologists made in the rst hal o the 1 9th century could not be explained by blending inheritance, but it was not until Mendel published his paper E xperiments in Plant Hybridization that an alternative theory was available. Figure 1 Hair styles are acquired characteristics and are ortunately not inherited by ofspring Mendels experiments were done using varieties o pea plant, each o which reliably had the same characters when grown on its own. Mendel careully crossed varieties o pea together by transerring the male pollen rom one variety to the emale parts in fowers o another variety. He collected the pea seeds that were ormed as a result and grew them to nd out what their characters were. Mendel repeated each cross with many pea plants. He also did this experiment with seven dierent pairs o characters and so his results reliably demonstrated the principles o inheritance in peas, not j ust an isolated eect. In 1 866 Mendel published his research. For over thirty years his ndings were largely ignored. Various reasons have been suggested or this. One actor was that his experiments used pea plants and there was not great interest in the pattern o inheritance in that species. In 1 900 several biologists rediscovered Mendels work. They quickly did cross-breeding experiments with other plants and with animals. These conrmed that Mendels theory explained the basis o inheritance in all plants and animals. Replicates and reliability in Mendels experiments Making quantitative measurements with replicates to ensure reliability: Mendel's genetic crosses with pea plants generated numerical data. Gregor Mendel is regarded by most biologists as the ather o genetics. His success is sometimes attributed to being the rst to use pea plants or research into inheritance. Peas have clear characteristics such as red or white fower colour that can easily be ollowed rom one generation to the next. They can also be crossed to produce hybrids or they can be allowed to sel- pollinate. 169 3 G e n e ti cs In act Mendel was not the rst to use pea plants. Thomas Andrew Knight, an English horticulturalist, had conducted research at D ownton C astle in Hereordshire in the late 1 8th century and published his results in the Philosophical Transactions o the Royal S ociety. Knight made some important discoveries: pollen is collected from the anthers male and emale parents contribute equally to the ospring; characters such as white fower colour that apparently disappear in ospring can reappear in the next generation, showing that inheritance is discrete rather than blending; lower petal called the keel one character such as red fower colour can show a stronger tendency than the alternative character. self pollinating peas: if the ower is left untouched, the anthers inside the keel pollinate the stigma Although Mendel was not as pioneering in his experiments as sometimes thought, he deserves credit or another aspect o his research. Mendel was a pioneer in obtaining quantitative results and in having large numbers o replicates. He also did seven dierent cross experiments, not just one. Table 1 shows the results o his monohybrid crosses. It is now standard practice in science to include repeats in experiments to demonstrate the reliability o results. Repeats can be compared to see how close they are. Anomalous results can be identied and excluded rom analysis. S tatistical tests can be done to assess the signicance o dierences between treatments. It is also standard practice to repeat whole experiments, using a dierent organism or dierent treatments, to test a hypothesis in dierent ways. Mendel should thereore be regarded as one o the athers o genetics, but even more we should think o him as a pioneer o research methods in biology. Paental plants Figure 2 Cross and sel pollination (a) Prediction based on blending inheritance tall plants 3 dwarf plants pea plants with an intermediate height (b) Actual results tall plants 3 dwarf plants pea plants as tall as the tall parent Figure 3 Example o a monohybrid cross experiment. All the hybrid plants produced by crossing two varieties together had the same character as one o the parents and the character o the other parent was not seen. This is a clear alsifcation o the theory o blending inheritance hybid plants Ofsping om sel-pollinating te ybids ratio Tall stem dwar stem All tall 787 tall : 277 dwar 2.84 : 1 Round seed wrinkled seed All round 5474 round : 1850 wrinkled 2.96 : 1 Yellow cotyledons green cotyledons All yellow 6022 yellow : 2001 green 3.01 : 1 Purple fowers white fowers All purple 705 purple : 224 white 3.15 : 1 Full pods constricted pods All ull 882 ull : 299 constricted 2.95 : 1 Green unripe pods yellow unripe pods All green 428 green : 152 yellow 2.82 : 1 Flowers along stem fowers at stem tip All along stem 651 along stem : 207 at tip Table 1 170 cross pollinating peas: pollen from another plant is dusted on to the stigma here 3.14 : 1 3 . 4 i N h E r i TAN CE Gamete Gametes are haploid so contain one allele o each gene. Gametes are cells that fuse together to produce the single cell that is the start of a new life. They are sometimes called sex cells, and the single cell produced when male and female gametes fuse is a zygote. Male and female gametes are different in size and motility. The male gamete is generally smaller than the female one. It is usually able to move whereas the female gamete moves less or not at all. In humans, for example, the sperm has a much smaller volume than the egg cell and uses its tail to swim to the egg. Parents pass genes on to their offspring in gametes. Gametes contain one chromosome of each type so are haploid. The nucleus of a gamete therefore only has one allele of each gene. This is true of both male and female gametes, so male and female parents make an equal genetic contribution to their offspring, despite being very different in overall size. Figure 4 Pollen on the anthers o a fower contains the male gamete o the plant. The male gametes contain one allele o each o the plants Zygote Fusion o gametes results in diploid zygotes with two alleles o each gene that may be the same allele or diferent alleles. When male and female gametes fuse, their nuclei j oin together, doubling the chromosome number. The nucleus of the zygote contains two chromosomes of each type so is diploid. It contains also two alleles of each gene. If there were two alleles of a gene, A and a, the zygote could contain two copies of either allele or one of each. The three possible combinations are AA, Aa and aa. S ome genes have more than two alleles. For example, the gene for AB O blood groups in humans has three alleles: I A, I B and i. This gives six possible combinations of alleles: three with two of the same allele, IAIA, IB I B and ii three with two different alleles, IAIB , I Ai and I B i. segregation of allele The two alleles o each gene separate into diferent haploid daughter nuclei during meiosis. D uring meiosis a diploid nucleus divides twice to produce four haploid nuclei. The diploid nucleus contains two copies of each gene, but the haploid nuclei contain only one. If two copies of one allele of a gene were present, each of the haploid nuclei will receive one copy of this allele. For example, if the two alleles were PP, every gamete will receive one copy of P. If two different alleles were present, each haploid nucleus will receive either one of the alleles or the other allele, not both. For example, if the two alleles were Pp, 5 0% of the haploid nuclei would receive P and 5 0% would receive p. Figure 5 Most crop plants are pure-bred strains with two o the same allele o each gene 171 3 G e n e ti cs TOK Did mendel alter his results for publication? In 1936, the English statistician R.A. Fisher published an analysis o Mendels data. His conclusion was that the data o most, i not all, o the experiments have been alsied so as to agree closely with Mendels expectations. Doubts still persist about Mendel's data a recent estimate put the chance o getting seven ratios as close to 3:1 as Mendels at 1 in 33,000. 1 2 To get ratios as close to 3:1 as Mendel's would have required a miracle o chance. What are the possible explanations apart rom a miracle o chance? Many distinguished scientists, including Louis Pasteur, are known to have discarded results when they did not t a theory. Is it acceptable to do this? How can we distinguish between results that are due to an error and results that alsiy a theory? What standard do you use as a student in rejecting anomalous data? The separation o alleles into dierent nuclei is called segregation. It breaks up existing combinations o alleles in a parent and allows new combinations to orm in the ospring. Dominant, recessive and co-dominant alleles Dominant alleles mask the efects o recessive alleles but co-dominant alleles have joint efects. In each o Mendels seven crosses between dierent varieties o pea plant, all o the ospring showed the character o one o the parents, not the other. For example, in a cross between a tall pea plant and a dwar pea plant, all the ospring were tall. The dierence in height between the parents is due to one gene with two alleles: the tall parents have two copies o an allele that makes them tall, TT the dwar parents have two copies o an allele that makes them dwar, tt they each pass on one allele to the ospring, which thereore has one o each allele, Tt when the two alleles are combined in one individual, it is the allele or tallness that determines the height because the allele or tallness is dominant the other allele, that does not have an eect i the dominant allele is present, is recessive. In each o Mendels crosses one o the alleles was dominant and the other was recessive. However, some genes have pairs o alleles where both have an eect when they are present together. They are called co-dominant alleles. A well-known example is the fower colour o Mirabilis jalapa. I a red-fowered plant is crossed with a white-fowered plant, the ospring have pink fowers. there is an allele or red fowers, C R there is an allele or white fowers, C W these alleles are co-dominant so C RC W gives pink fowers. The usual reason or dominance o one allele is that this allele codes or a protein that is active and carries out a unction, whereas the recessive allele codes or a non- unctional protein. Figure 6 There are co-dominant alleles of the gene for coat colour in Icelandic horses. 172 3 . 4 i N h E r i TAN CE parents: Monohybrid crosses only involve one character, or example the height o a pea plant, so they involve only one gene. Most crosses start with two pure- breeding parents. This means that the parents have two o the same allele, not two dierent alleles. E ach parent thereore produces j ust one type o gamete, containing one copy o the allele. Their ospring are also identical, although they have two dierent alleles. The ospring obtained by crossing the parents are called F 1 hybrids or the F 1 generation. eggs or pollen T lle po t tt dwarf Figure 7 Explanation of Mendels 3:1 ratio parents: genotype phenotype C WC W CRCR white owers red owers D educe the colour o coat that is due to a recessive allele, with two reasons or your answer. [3 ] 3 C hoose suitable symbols or the alleles or grey and albino coat and list the possible combinations o alleles o mice using your symbols, together with the coat colours associated with each combination o alleles. [3 ] CW CRCW pink owers CW po l le n F1 hybrids genotype phenotype C WC R pink CRCR red C WC W white gs CW 2 CR R C alculate the ratio between grey and albino ospring, showing your working. [2 ] Tt tall eg 1 t tT tall C In the early years o the 2 0th century, many crossing experiments were done in a similar way to those o Mendel. The French geneticist Lucien C unot used the house mouse, Mus musculus, to see whether the principles that Mendel had discovered also operated in animals. He crossed normal grey- coloured mice with albino mice. The hybrid mice that were produced were all grey. These grey hybrids were crossed together and produced 1 98 grey and 72 albino ospring. gs TT tall Figure 8 shows the results o a cross between red and white fowered plants o Mirabilis jalapa. It explains the F 2 ratio o one red to two pink to one white fowered plant. Data-based questons: Coat colour in the house mouse T n Tt tall stem eg Figure 7 shows Mendels cross between tall and dwar plants. It explains the F 2 ratio o three tall to one dwar plant. t F1 hybrids genotype phenotype The F 1 hybrids have two dierent alleles o the gene, so they can each produce two types o gamete. I two F 1 hybrids are crossed together, or i an F 1 plant is allowed to sel-pollinate, there are our possible outcomes. This can be shown using a 2 2 table, called a Punnett grid ater the geneticist who rst used this type o table. The ospring o a cross between two F 1 plants are called the F 2 generation. To make a Punnett grid as clear as possible the gametes should be labeled and both the alleles and the character o the our possible outcomes should be shown on the grid. It is also useul to give an overall ratio below the Punnett grid. tt dwarf stem TT tall stem CR Construction of Punnett grids for predicting the outcomes of monohybrid genetic crosses. genotype phenotype T Punnett grids C WC W pink Figure 8 A cross involving co-dominance 173 3 G e n e ti cs 4 t ica annulata 5 Figure 9 Using a Punnett grid, explain how the observed ratio o grey and albino mice was produced. [5 ] The albino mice had red eyes in addition to white coats. S uggest how one gene can determine whether the mice had grey ur and black eyes or white ur and red eyes. [2 ] Data-based questions: The two-spot ladybird Adalia bipunctata is a species o ladybird. In North America ladybirds are called ladybugs. The commonest orm o this species is known as typica. There is a rarer orm called annulata. B oth orms are shown in fgure 9. Figure 10 F 1 Figure 11 hybrid ofspring 1 C ompare the typica and annulata orms o Adalia bipunctata. [2 ] 2 The dierences between the two orms are due to a single gene. I male and emale typica are mated together, all the ospring are typica. S imilarly, the ospring produced when annulata orms are mated are all annulata. Explain the conclusions that can be drawn. [2 ] 3 When typica is mated with annulata, the F 1 hybrid ospring are not identical to either parent. Examples o these F 1 hybrid ospring are shown in fgure 1 0. D istinguish between the F 1 hybrid ospring and the typica and annulata parents. [3 ] 4 I F 1 hybrid ospring are mated with each other, the ospring include both typica and annulata orms, and also ospring with the same wing case markings as the F 1 hybrid ospring. F2 ofspring Activity ABO blood groups It is possible for two parents to have an equal chance of having a child with blood group A, B, AB or O. What would be the genotypes of the parents? a) Use a genetic diagram to explain this pattern o inheritance. [6] b) Predict the expected ratio o phenotypes. [2 ] ABO blood groups Inheritance of ABO blood groups. The AB O blood group system in humans is an example o co- dominance. It is o great medical importance: beore blood is transused, it is vital to fnd out the blood group o a patient and ensure that it is matched. Unless this is done, there may be complications due to coagulation o red blood cells. O ne gene determines the AB O blood group o a person. The genotype IA IA gives blood group A and the genotype IBIB gives group B . Neither IA nor IB is dominant over the other allele and a person with the genotype IA IB has a dierent blood group, called AB . There is a third allele o the AB O blood group gene, usually called i. A person with the genotype ii is in blood group O . The genotypes IA i and IBi give blood groups A and B respectively, showing that i is 174 recessive to both IA and IB. The reasons or two alleles being co- dominant and the other allele being recessive are as ollows: All o the three alleles cause the production o a glycoprotein in the membrane o red blood cells. IA alters the glycoprotein by addition o acetylgalactosamine. This altered glycoprotein is absent rom people who do not have the allele IA so i exposed to it they make anti-A antibodies. IB alters the glycoprotein by addition o galactose. This altered glycoprotein is not present in people who do not have the allele IB so i exposed to it they make anti-A antibodies. 3 . 4 i N h E r i TAN CE The genotype IAIB causes the glycoprotein to be altered by addition o acetyl-galactosamine and galactose. As a consequence neither anti-A nor anti- B antibodies are produced. This genotype thereore gives a dierent phenotype to IA IA and IBIB so the alleles IA and IB are co- dominant. either o the IA or IB alleles is also present the glycoprotein is altered by addition o acetyl-galactosamine or galactose. IAIA and IA i thereore give the same phenotype, as do IBIB and IBi. The allele i is recessive because it causes production o the basic glycoprotein: i The allele i is recessive because it does not cause the production o a glycoprotein. IA IA and IA i thereore give the same phenotype and so do IBIB and IBi. Group O Group A anti-A anti-B anti-A Group B anti-A Figure 12 anti-B Group AB anti-B anti-A anti-B Blood group can easily be determined using test cards tesing predicions in cross-breeding experimens Comparison of predicted and actual outcomes of genetic crosses using real data. It is in the nature o science to try to fnd general principles that explain natural phenomena and not j ust to describe individual examples o a phenomenon. Mendel discovered principles o inheritance that have great predictive power. We can still use them to predict the outcomes o genetic crosses. Table 2 lists possible predictions in monohybrid crosses. The actual outcomes o genetic crosses do not usually correspond exactly with the predicted outcomes. This is because there is an element o chance involved in the inheritance o genes. The tossing o a coin is a simple analogy. We expect the coin to land 5 0% o times with each o its two aces uppermost, but i we toss it 1 , 000 times we do not expect it to land precisely 5 00 times with one ace showing and 5 00 times with the other ace showing. An important skill in biology is deciding whether the results o an experiment are close enough to the predictions or us to accept that they ft, or whether the dierences are too great and either the results or the predictions must be alse. An obvious trend is that the greater the dierence between observed and expected results, the less likely that the dierence is due to chance and the more likely that the predictions do not ft the results. To assess obj ectively whether results ft predictions, statistical tests are used. For genetic crosses the chi-squared test can be used. This test is described later in the book in sub- topic 4.1 . 175 3 G e n e ti cs Cross Predicted outcome Example Pure-breeding parents one with dominant alleles and one with recessive alleles are crossed. All o the ofspring will have the same character as the parent with dominant alleles. All ofspring o a cross between purebreeding tall and dwar pea plants will be tall. Pure-breeding parents that have diferent co-dominant alleles are crossed. All o the ofspring will have the same character and the character will be diferent rom either parent. All ofspring o a cross between red and white owered Mirabilis jalapa plants will have pink owers. Two parents each with one dominant and one recessive allele are crossed. Three times as many ofspring have the character o the parent with dominant alleles as have the character o the parent with the recessive alleles. 3:1 ratio o tall to dwar pea plants rom a cross between two parents that each have one allele or tall height and one allele or dwar height. A parent with one dominant and one recessive allele is crossed with a parent with two recessive alleles. Equal proportions o ofspring with the character o an individual with a dominant allele and the character o an individual with recessive alleles. 1:1 ratio rom a cross between a dwar pea plant and a tall plant with one allele or tall height and one or dwar height . Table 2 Data-based questions: Analysing genetic crosses 1 Figure 13 Antirrhinum fowers (a) wild type, (b) peloric C harles D arwin crossed pure breeding wild- type Antirrhinum majus plants, which have bilaterally symmetric fowers, with pure breeding plants with peloric fowers that are radially symmetric. All the F 1 ospring produced bilaterally symmetric fowers. D arwin then crossed the F 1 plants together. In the F 2 generation there were 8 8 plants with bilaterally symmetric fowers and 3 7 with peloric fowers. a) C onstruct a Punnett grid to predict the outcome o the cross between the F 1 plants. [3 ] b) D iscuss whether the actual results o the cross are close enough to support the predicted outcome. [2 ] c) Peloric Antirrhinum majus plants are extremely rare in wild populations o this species. Suggest reasons or this. [1 ] 2 176 There are three varieties o pheasant with eather coloration called light, ring and bu. When light pheasants were bred together, only light ospring were produced. S imilarly, when ring were crossed with ring, all the ospring were ring. When bu pheasants were crossed with bu there were 75 light ospring, 68 ring and 1 41 bu. a) C onstruct a Punnett grid to predict the outcome o breeding together bu pheasants. [3 ] b) D iscuss whether the actual results o the cross are close enough to support the predicted outcome. [2 ] 3 . 4 i N h E r i TAN CE 3 Mary and Herschel Mitchell investigated the inheritance o a character called poky in the ungus Neurospora crassa. Poky strains o the ungus grow more slowly than the wild- type. The results are shown in table 3 . male paent Feale paent Nube o wld type ofspng Nube o poky ofspng Wild type Wild type 9,691 90 Poky Poky 0 10,591 Wild type Poky 0 7,905 Poky Wild type 4,816 43 Table 3 a) D iscuss whether the data fts any o the Mendelian ratios in table 1 ( page 1 70) . [2 ] b) S uggest a reason or all the ospring being poky in a cross between wild type and poky strains when a wild type is the male parent. [2 ] c) S uggest a reason or a small number o poky ospring in a cross between wild type and poky strains when a wild type is the emale parent. [1 ] Figure 14 Feather coloration rom a buf pheasant Genetic diseases due to recessive alleles Many genetic diseases in humans are due to recessive alleles of autosomal genes. A genetic disease is an illness that is caused by a gene. Most genetic diseases are caused by a recessive allele o a gene. The disease thereore only develops in individuals that do not have the dominant allele o the gene, usually because they have two copies o the recessive allele. I a person has one allele or the genetic disease and one dominant allele, they will not show symptoms o the disease, but they can pass on the recessive allele to their ospring. These individuals are called carriers. Genetic diseases caused by a recessive allele usually appear unexpectedly. B oth parents o a child with the disease must be carriers, but as they do not show symptoms o the disease, they are unaware o this. The probability o these parents having a child with the disease is 2 5 per cent ( see fgure 1 5 ) . C ystic fbrosis is an example o a genetic disease caused by a recessive allele. It is described later in this sub- topic. Other causes of genetic diseases Some genetic diseases are sex-linked and some are due to dominant or co-dominant alleles. A small proportion o genetic diseases are caused by a dominant allele. It is not possible to be a carrier o these diseases. I a person has one dominant allele then they themselves will develop the disease. I one Aa Aa A a AA not carrier Aa A a aA aa carrier do not develop the disease develops the genetic disease Figure 15 Genetic diseases caused by a recessive allele 177 3 G e n e ti cs bb Bb B b b Bb develops the disease b bb does not develop the disease Figure 16 Genetic diseases caused by a dominant allele parent has the allele or the disease, the chance o a child inheriting it is 5 0 per cent ( see fgure 1 6) . Huntingtons disease is an example o a genetic disease caused by a dominant allele. It is described later in this sub- topic. A very small proportion o genetic diseases are caused by co- dominant alleles. An example is sickle- cell anemia. The molecular basis o this disease was described in sub- topic 3 . 1 . The normal allele or hemoglobin is Hb A and the sickle cell allele is Hb S . Figure 1 7 shows the three possible combinations o alleles and the characteristics that result. Individuals that have one Hb A and one Hb S allele do not have the same characteristics as those who have two copies o either allele, so the alleles are co- dominant. Most genetic diseases aect males and emales in the same way but some show a dierent pattern o inheritance in males and emales. This is called sex linkage. The causes o sex linkage and two examples, red- green colour- blindness and hemophilia, are described later in this sub- topic. alleles : Hb A Hb A alleles : Hb A Hb s characteristics : - susceptible to malaria - not anemic characteristics : - increased resistance to malaria - mild anemia alleles : Hb S Hb S characteristics : - susceptible to malaria - severe anemia normal red blood cell shape Figure 17 sickle-cell shape Efects o Hb A and Hb S alleles Cystic fbrosis and Huntingtons disease Inheritance o cystic fbrosis and Huntingtons disease. C ystic fbrosis is the commonest genetic disease in parts o E urope. It is due to a recessive allele o the C FTR gene. This gene is located on chromosome 7 and the gene product is a chloride ion channel that is involved in secretion o sweat, mucus and digestive j uices. The recessive alleles o this gene result in chloride channels being produced that do not unction properly. S weat containing excessive amounts o sodium chloride is produced, but digestive j uices and mucus are secreted with insufcient sodium chloride. As a result not enough water moves by osmosis into the 178 secretions, making them very viscous. S ticky mucus builds up in the lungs causing inections and the pancreatic duct is usually blocked so digestive enzymes secreted by the pancreas do not reach the small intestine. In some parts o E urope one in twenty people have an allele or cystic fbrosis. As the allele is recessive, a single copy o the allele does not have any eects. The chance o two parents 1 1 both being a carrier o the allele is __ __ , 20 20 1 which is ___ . The chance o such parents having 40 0 a child with cystic fbrosis can be ound using a Punnett grid. 3 . 4 i N h E r i TAN CE father Cc C c C CC normal Cc normal (carrier) c cC normal (carrier) cc cystic brosis mother Cc B ecause of the late onset, many people diagnosed with Huntingtons disease have already had children. A genetic test can show before symptoms would develop whether a young person has the dominant allele, but most people at risk choose not to have the test. About one in 1 0, 000 people have a copy of the Huntingtons allele, so it is very unlikely for two parents both to have a copy. A person can nonetheless develop the disease if only one of their parents has the allele because it is dominant. ratio 3 normal : 1 cystic brosis father Hh Huntingtons disease is due to a dominant allele of the HTT gene. This gene is located on chromosome 4 and the gene product is a protein named huntingtin. The function of huntingtin is still being researched. The dominant allele of HTT causes degenerative changes in the brain. S ymptoms usually start when a person is between 3 0 and 5 0 years old. C hanges to behaviour, thinking and emotions become increasingly severe. Life expectancy after the start of symptoms is about 2 0 years. A person with the disease eventually needs full nursing care and usually succumbs to heart failure, pneumonia or some other infectious disease. H h h Hh Huntingtons disease hh normal h Hh Huntingtons disease hh normal mother hh ratio 1 normal : 1 Huntingtons disease sex-linked gene The pattern o inheritance is diferent with sex-linked genes due to their location on sex chromosomes. Plants such as peas are hermaphrodite they can produce both male and female gametes. When Thomas Andrew Knight did crossing experiments between pea plants in the late 1 8th century, he discovered that the results were the same whichever character was in the male gamete and which in the female gamete. For example, these two crosses gave the same results: pollen from a plant with green stems placed onto on the stigma of a plant with purple stems; pollen from a plant with purple stems placed onto on the stigma of a plant with green stems. 179 3 G e n e ti cs red eye XRY X rX R red XrY white XrXR red Y Xr R X Xr white eye XrXr XrY white XR XR XR Xr red Y red XR Y red XR Y red Key XR X chromosome with allele for red eye (dominant) Xr X chromosome with allele for white eye (recessive) Y Y chromosome Figure 18 Reciprocal crosses normal-winged males vestigial- winged emales; vestigial-winged males normal- winged emales. These crosses gave dierent results: red-eyed males white- eyed emales gave only red- eyed ospring; white- eyed males red- eyed emales gave red-eyed emales and white- eyed males. r XR white eye X rY O ne o the rst examples o sex linkage was discovered by Thomas Morgan in the ruit fy, Drosophila. This small insect is about 4 mm long and completes its lie cycle in two weeks, allowing crossing experiments to be done quickly with large numbers o fies. Most crosses in Drosophila do not show sex linkage. For example, these reciprocal crosses give the same results: X XR red eye XRXR Plants always give the same results when reciprocal crosses such as these are carried out, but in animals the results are sometimes dierent. An inheritance pattern where the ratios are dierent in males and emales is called sex linkage. sex-linkage Geneticists had observed that the inheritance o genes and o chromosomes showed clear parallels and so genes were likely to be located on chromosomes. It was also known that emale Drosophila have two copies o a chromosome called X and males only have one copy. Morgan deduced that sex linkage o eye colour could thereore be due to the eye colour gene being located on the X chromosome. Male Drosophila also have a Y chromosome, but this does not carry the eye- colour gene. Figure 1 8 explains the inheritance o eye colour in Drosophila. In crosses involving sex linkage, the alleles should always be shown as a superscript letter on a letter X to represent the X chromosome. The Y chromosome should also be shown though it does not carry an allele o the gene. Red-green colour-blindness and hemophilia Red-green colour-blindness and hemophilia as examples of sex-linked inheritance. Many examples o sex linkage have been discovered in humans. They are almost all due to genes located on the X chromosome, as there are very ew genes on the Y chromosome. Two examples o sex-linked conditions due to genes on the X chromosomes are described here: red- green colour- blindness and hemophilia. Red- green colour- blindness is caused by a recessive allele o a gene or one o the photoreceptor proteins. These proteins are made by cone cells in the retina o the eye and detect specic wavelength ranges o visible light. Figure 19 A person with red-green colour-blindness cannot clearly distinguish between the colours o the fowers and the leaves 180 3 . 4 i N h E r i TAN CE proteins involved in the clotting o blood. Lie expectancy is only about ten years i hemophilia is untreated. Treatment is by inusing Factor VIII, purifed rom the blood o donors. XH Xh Xh XH Y XH Y H XH XH normal Y Whereas red- green colour-blindness is a mild disability, hemophilia is a lie- threatening genetic disease. Although there are some rarer orms o the disease, most cases o hemophilia are due to an inability to make Factor VIII, one o the XH KEY XH X chromosome carrying the allele for normal blood clotting Xh X chromosome carrying the allele for hemophilia. X Males have only one X chromosome, which they inherit rom their mother. I that X chromosome carries the red- green colour- blindness allele then the son will be red- green colour- blind. In parts o northern E urope the percentage o males with this disability is as high as 8% . Girls are red- green colour- blind i their ather is red-green colourblind and they also inherit an X chromosome carrying the recessive gene rom their mother. We can predict that the percentage o girls with colour- blindness in the same parts o E urope to be 8% 8% = 0.64% . The actual percentage is about 0.5 % , ftting this prediction well. XH Blood should stop quickly owing rom a pricked fnger but in hemophiliacs bleeding continues or much longer as blood does not clot properly Xh Figure 20 The gene or Factor VIII is located on the X chromosome. The allele that causes hemophilia is recessive. The requency o the hemophilia allele is about 1 in 1 0, 000. This is thereore the requency o the disease in boys. Females can be carriers o the recessive hemophilia allele but they only develop the disease i both o their X chromosomes carry the allele. The requency in 1 2 = 1 in 1 00, 000, 000. girls theoretically is ( _____ 1 0,000 ) In practice, there have been even ewer cases o girls with hemophilia due to lack o Factor VIII than this. O ne reason is that the ather would have to be hemophiliac and decide to risk passing on the condition to his children. XH Xh carrier XH Y normal Xh Y hemophiliac Pedigree charts Analysis of pedigree charts to deduce the pattern of inheritance of genetic diseases. It isnt possible to investigate the inheritance o genetic diseases in humans by carrying out cross experiments. Pedigree charts can be used instead to deduce the pattern o inheritance. These are the usual conventions or constructing pedigree charts: males are shown as squares; emales are shown as circles; squares and circles are shaded or crosshatched to indicate whether an individual is aected by the disease; parents and children are linked using a T, with the top bar o the T between the parents; Roman numerals indicate generations; 181 3 G e n e ti cs Arabic numbers are used or individuals in each generation. their children will be albino, we could only expect to see that ratio i the parents had very large numbers o children. The actual ratio o 1 in 2 is not unexpected and does not show that our deductions about the inheritance o albinism are incorrect. Example 1 Albinism in humans generation I 1 2 Example 2 Vitamin D-resistant rickets D eductions: generation II 1 2 3 Two unaected parents only have unaected children but two aected parents have children with vitamin D - resistant rickets, suggesting that this disease is caused by a dominant allele. The ospring o the parents in generation I are all aected daughters and unaected sons. This suggests sex linkage although the number o ospring is too small to be sure o the inheritance pattern. I vitamin D - resistant rickets is caused by a dominant X- linked allele, daughters o the ather in generation I would inherit his X chromosome carrying the dominant allele, so all o his daughters would have the disease. This data in the pedigree shows that this and so supports the theory. S imilarly i vitamin D -resistant rickets is caused by a dominant X- linked allele, the mother with the disease in generation II would have one X chromosome carrying the dominant allele or the disease and one with the recessive allele. All o her ospring would have a 5 0% chance o inheriting this X chromosome and o having the disease. The data in the pedigree fts this and so supports the theory. 4 Key: normal pigmentation albino D eductions: Two o the children are albino and yet the parents both have normal pigmentation. This suggests that albinism is caused by a recessive allele ( m) and normal pigmentation by a dominant allele ( M) . There are both daughters and sons with albinism suggesting that the condition is not sex- linked. B oth males and emales are albino only i they have two copies o the recessive albinism allele ( mm) . The albino children must have inherited an allele or albinism rom both parents. B oth parents must also have one allele or normal pigmentation as they are not albino. The parents thereore have the alleles Mm. The chance o a child o these parents having albinism is 1 . Although on average 1 in 4 o 4 Key: vitamin D-resistant rickets not aected Figure 21 182 Pedigree of a family with cases of vitamin D-resistant rickets 3 . 4 i N h E r i TAN CE Data-based questons: Deducing genotypes from pedigree charts The pedigree chart in fgure 2 2 shows fve generations o a amily aected by a genetic disease. 1 2 Explain, using evidence rom the pedigree, whether the condition is due to a recessive or a dominant allele. [3 ] Explain what the probability is o the individuals in generation V having: I 1 2 3 4 II 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 III 1 2 3 4 IV 1 2 3 4 5 6 7 8 V ? ? ? ? 1 2 3 4 unaected male unaected female aected male aected female Figure 22 Example of a pedigree chart a) two copies o a recessive allele; 3 b) one recessive and one dominant allele; c) two copies o the dominant allele. D educe, with reasons, the possible alleles o: a) 1 in generation III; b) 1 3 in generation II. [3 ] 4 [2 ] S uggest two examples o genetic diseases that would ft this inheritance pattern. [2 ] Genetic diseases in humans Many genetic diseases have been identifed in humans but most are very rare. S everal genetic diseases have already been described in this sub- topic, including sickle- cell anemia, cystic fbrosis, hemophilia and Huntingtons disease. There are other well- known examples, such as phenylketonuria ( PKU) , Tay-S achs disease and Marans syndrome. Medical research has already identifed more than 4, 000 genetic diseases and more no doubt remain to be ound. Given this large number o genetic diseases, it might seem surprising that most o us do not suer rom any o them. The reason or this is that most genetic diseases are caused by very rare recessive alleles which ollow Mendelian patterns o inheritance. The chance o inheriting one allele or any specifc disease is small but to develop the disease two alleles must be inherited and the chance o this is extremely small. It is now possible to sequence the genome o an individual human relatively cheaply and quickly and large numbers o humans are being sequenced to allow comparisons. This research is revealing the number o rare recessive alleles that a typical individual is carrying that could cause a genetic disease. C urrent estimates are that the number is between 75 and 2 00 alleles among the 2 5 , 000 or so genes in the human genome. An individual can only produce a child with a genetic disease due to one o these recessive alleles i the other parent o the child has the same rare allele. Figure 23 Alleles from two parents come together when they have a child. There is a small chance that two recessive alleles will come together and cause a genetic disease 183 3 G e n e ti cs Causes of mutation Radiation and mutagenic chemicals increase the mutation rate and can cause genetic disease and cancer. Figure 24 Abraham Lincolns eatures resemble Marans syndrome but a more recent theory is that he sufered rom MEN2B, another genetic disease A gene consists o a length o D NA, with a base sequence that can be hundreds or thousands o bases long. The dierent alleles o a gene have slight variations in the base sequence. Usually only one or a very small number o bases are dierent. New alleles are ormed rom other alleles by gene mutation. A mutation is a random change to the base sequence o a gene. Two types o actor can increase the mutation rate. Radiation increases the mutation rate i it has enough energy to cause chemical changes in D NA. Gamma rays and alpha particles rom radioactive isotopes, short- wave ultraviolet radiation and X- rays are all mutagenic. S ome chemical substances cause chemical changes in D NA and so are mutagenic. Examples are benzo[a] pyrene and nitrosamines ound in tobacco smoke and mustard gas used as a chemical weapon in the First World War. Mutations are random changes there is no mechanism or a particular mutation being carried out. A random change to an allele that has developed by evolution over perhaps millions o years is unlikely to be benefcial. Almost all mutations are thereore either neutral or harmul. Mutations o the genes that control cell division can cause a cell to divide endlessly and develop into a tumour. Mutations are thereore a cause o cancer. Figure 25 The risk o mutations due to radiation rom nuclear waste is minimized by careul storage Mutations in body cells, including those that cause cancer, are eliminated when the individual dies, but mutations in cells that develop into gametes can be passed on to ospring. This is the origin o genetic diseases. It is thereore particularly important to minimize the number o mutations in gamete- producing cells in the ovaries and testes. C urrent estimates are that one or two new mutations occur each generation in humans, adding to the risk o genetic diseases in children. Consequences of nuclear bombing and accidents at nuclear power stations Consequences of radiation after nuclear bombing of Hiroshima and Nagasaki and the nuclear accidents at Chernobyl. The common eature o the nuclear bombing o Hiroshima and Nagasaki and the nuclear accidents at Three Mile Island and C hernobyl is that radioactive isotopes were released into the environment and as a result people were exposed to potentially dangerous levels o radiation. When the atomic bombs were detonated over Hiroshima and Nagasaki 1 5 0 , 0 0 0 2 5 0 , 00 0 184 people either died directly or within a ew months. The health o nearly 1 0 0 , 0 0 0 survivors has been ollowed since then by the Radiation E ects Research Foundation in Japan. Another 2 6 , 0 0 0 people who were not exposed to radiation have been used as a control group. B y 2 0 1 1 the survivors had developed 1 7 , 448 tumours, but only 8 5 3 o these could be 3 . 4 i N h E r i TAN CE attributed to the eects o radiation rom the atomic bombs. into the atmosphere in total. The eects were widespread and severe: Apart rom cancer the other main eect o the radiation that was predicted was mutations, leading to stillbirths, malormation or death. The health o 1 0, 000 children that were etuses when the atomic bombs were detonated and 77, 000 children that were born later in Hiroshima and Nagasaki has been monitored. No evidence has been ound o mutations caused by the radiation. There are likely to have been some mutations, but the number is too small or it to be statistically signifcant even with the large numbers o children in the study. 4 km 2 o pine orest downwind o the reactor turned ginger brown and died. Horses and cattle near the plant died rom damage to their thyroid glands. Lynx, eagle owl, wild boar and other wildlie subsequently started to thrive in a zone around C hernobyl rom which humans were excluded. B ioaccumulation caused high levels o radioactive caesium in fsh as ar away as Scandinavia and Germany and consumption o lamb contaminated with radioactive caesium was banned or some time as ar away as Wales. C oncentrations o radioactive iodine in the environment rose and resulted in drinking water and milk with unacceptably high levels. More than 6, 000 cases o thyroid cancer have been reported that can be attributed to radioactive iodine released during the accident. According to the report C hernobyls Legacy Health, Environmental and S ocio- Economic Impacts, produced by The C hernobyl Forum, there is no clearly demonstrated increase in solid cancers or leukemia due to radiation in the most aected populations. D espite the lack o evidence o mutations due to the atomic bombs, survivors have sometimes elt that they were stigmatized. S ome ound that potential wives or husbands were reluctant to marry them or ear that their children might have genetic diseases. The accident at C hernobyl, Ukraine, in 1 986 involved explosions and a fre in the core o a nuclear reactor. Workers at the plant quickly received atal doses o radiation. Radioactive isotopes o xenon, krypton, iodine, caesium and tellurium were released and spread over large parts o E urope. About six tonnes o uranium and other radioactive metals in uel rom the reactor was broken up into small particles by the explosions and escaped. An estimated 5 , 2 00 million GB q o radioactive material was released Incidence per 100,000 in Belarus 12 Actvty adults (1934) 10 Cangng ates of tyod cance adolescents (1518) When would you expect the cases o thyroid cancer in young adults to start to drop, based on the data in fgure 26? Cases per 100,000 children (014) 8 6 4 2 0 1984 1986 1988 1990 Figure 26 Incidence of thyroid 1992 1994 1996 1998 2000 2002 2004v cancer in Belarus after the Chernobyl accident 185 3 G e n e ti cs Data-baed quetion: The aftermath of Chernobyl Mutations can cause a cell to become a tumour cell. The release of 6 . 7 tonnes of radioactive material from the nuclear power station at C hernobyl in 1 9 8 6 was therefore the cause of large numbers of deaths due to cancer. The UN C hernob yl Forum stated that up to 4, 0 0 0 people may ultimately die as a result of the disaster, but Green Party members of the E uropean Parliament commissioned a report from a radiation scientist, which gave an estimate of 3 0 , 0 0 0 to 6 0 , 0 0 0 extra deaths. O ne way of obtaining an estimate is to use data from previous radiation exposures, such as the detonation of nuclear warheads at Hiroshima and Nagasaki in 1 9 45 . The data below is an analysis of deaths due to leukemia and cancer b etween 1 9 5 0 and 1 9 9 0 among those exposed to radiation from these warheads. It was published by the Radiation E ffects Research Foundation. Figure 27 Humans have been excluded from a large zone near the Chernobyl reactor. Some plants and animals have shown deformities that may be due to mutations radiation Numbe of death Etimate of exce Pecentage of death doe ange in people expoed death ove contol attibutable to (sv) to adiation goup adiation expoue Leukemia 0.0050.2 70 10 0.20.5 27 13 48 0.51 23 17 74 56 47 >1 Cancer 0.0050.2 3391 63 2 0.20.5 646 76 12 0.51 342 79 23 308 121 39 >1 1 C alculate the percentage of excess deaths over control groups due to leukemia in people exposed to ( a) 0. 005 - 0. 02 Sv ( sieverts) of radiation ( b) >1 Sv of radiation. [4] 2 C onstruct a suitable type of graph or chart to represent the data in the right- hand column of the table, including the two percentages that you have calculated. There should be two y- axes, for the leukemia deaths and the cancer deaths. [4] 3 C ompare the effect of radiation on deaths due to leukemia and deaths due to cancer. [3 ] D iscuss, with reasons, what level of radiation might be acceptable in the environment. [4] 4 186 3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y 3.5 Genetc odfcaton and botecnoog Udertadig Gel electrophoresis is used to separate proteins or ragments o DNA according to size. PCR can be used to ampliy small amounts o DNA. DNA proling involves comparison o DNA. Genetic modication is carried out by gene transer between species. Clones are groups o genetically identical organisms, derived rom a single original parent cell. Many plant species and some animal species have natural methods o cloning. Animals can be cloned at the embryo stage by breaking up the embryo into more than one group o cells. Methods have been developed or cloning adult animals using diferentiated cells. Applicatio Use o DNA proling in paternity and orensic investigations. Gene transer to bacteria with plasmids using restriction endonucleases and DNA ligase. Assessment o the potential risks and benets associated with genetic modication o crops. Production o cloned embryos by somatic-cell nuclear transer. skill Design o an experiment to assess one actor afecting the rooting o stem-cuttings. Analysis o examples o DNA proles. Analysis o data on risks to monarch butteries o Bt crops. nature of ciece Assessing risks associated with scientic research: scientists attempt to assess the risks associated with genetically modied crops or livestock. Gel electrophorei Gel electrophoresis is used to separate proteins or ragments o DNA according to size. Gel electrophoresis involves separating charged molecules in an electric eld, according to their size and charge. Samples are placed in wells cast in a gel. The gel is immersed in a conducting fuid and an electric eld is applied. Molecules in the sample that are charged will move through the gel. Molecules with negative and positive charges move in opposite directions. Proteins may be positively or negatively charged so can be separated according to their charge. The gel used in gel electrophoresis consists o a mesh o laments that resists the movement o molecules in a sample. D NA molecules rom eukaryotes are too long to move through the gel, so they must be broken up into smaller ragments. All D NA molecules carry negative charges so move in the same direction during gel electrophoresis, but not DNA samples negative electrode 2 sample well gel 1 positive electrode large fragments 2 direction of migration small fragments 1 Figure 1 Procedure for gel electrophoresis 187 3 G e n e ti cs at the same rate. S mall ragments move aster than large ones so they move urther in a given time. Gel electrophoresis can thereore be used to separate ragments o D NA according to size. DnA amplifcatio by PCR PCR can be used to amplify small amounts of DNA. Figure 2 Small samples o DNA being extracted rom ossil bones o a Neanderthal or amplifcation by PCR The polymerase chain reaction is used to make large numbers o copies o D NA. It is almost always simply called PC R. The details o this technique are described in sub- topic 2 . 7 . O nly a very small amount o D NA is needed at the start o the process in theory j ust a single molecule. Within an hour or two, millions o copies can be made. This makes it possible to study the D NA urther without the risk o using up a limited sample. For example, D NA extracted rom ossils can be amplifed using PC R. Very small amounts o D NA rom blood, semen or hairs can also be amplifed or use in orensic investigations. PC R is not used to copy the entire set o D NA molecules in a sample such as blood or semen. White blood cells contain all chromosomes o the person rom whom the blood came, or example, and together the sperm cells in a sample o semen contain a mans entire genome. Instead PC R is used to copy specifc D NA sequences. A sequence is selected or copying by using a primer that binds to the start o the desired sequence. The primer binds by complementary base pairing. The selectivity o PC R allows particular desired sequences to be copied rom a whole genome or even greater mixture o DNA. One test or the presence o genetically modifed ingredients in oods involves the use o a primer that binds to the genetically modifed D NA. Any such DNA present is amplifed by the PC R, but i there is none present the PC R has no eect. Data-based questions: PCR and Neanderthals Samples o D NA were recently obtained rom ossil bones o a Neanderthal ( Homo neanderthalensis) . They were amplifed using PC R. A section o the Neanderthal mitochondrial D NA was sequenced and compared with sequences rom 994 humans and 1 6 chimpanzees. The bar chart in fgure 3 shows how many basesequence dierences were ound within the sample o humans, between the humans and the 188 Neanderthal and between the humans and the chimpanzees. frequency of number of dierences / % The evolution o groups o living organisms can be studied by comparing the base sequences o their D NA. I a species separates into two groups, dierences in base sequence between the two species accumulate gradually over long periods o time. The number o dierences can be used as an evolutionary clock. 25 humanNeanderthal 20 15 humanhuman humanchimp 10 5 0 0 Figure3 5 10 15 20 25 30 35 40 45 50 55 60 65 number of dierences in base sequence Number o dierences in base sequences between humans, chimps and Neanderthals 3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y 1 S tate the most common number o dierences in base sequence between pairs o humans. [1 ] 2 Humans and Neanderthals are both classifed in the genus Homo and chimpanzees are classifed in the genus Pan. D iscuss whether this classifcation is supported by the data in the bar chart. [3 ] 3 Suggest a limitation to drawing any conclusion rom the humanNeanderthal comparison. [1 ] DnA proflig DNA profling involves comparison o DNA. D NA profling involves these stages: A sample o D NA is obtained, either rom a known individual or rom another source such as a ossil or a crime scene. Sequences in the D NA that vary considerably between individuals are selected and are copied by PC R. The copied D NA is split into ragments using restriction endonucleases. The ragments are separated using gel electrophoresis. This produces a pattern o bands that is always the same with D NA taken rom one individual. This is the individual' s D NA profle. The profles o dierent individuals can be compared to see which bands are the same and which are dierent. Figure 4 DNA profles are oten reerred to as DNA fngerprints as they are used in a similar way to real fngerprints to distinguish one individual rom all others Paterity ad oresic ivestigatios Use o DNA profling in paternity and orensic investigations. D NA profling is used in orensic investigations. B lood stains on a suspects clothing could be shown to come rom the victim. B lood stains at the crime scene that are not rom the victim could be shown to come rom the suspect. A single hair at the crime scene could be shown to come rom the suspect. S emen rom a sexual crime could be shown to come rom the suspect. In each example the DNA profle o material rom the crime scene is compared with the DNA profle o a sample o DNA taken rom the suspect or the victim. I the pattern o bands matches exactly it is highly likely that the two samples o DNA are rom the same person. This can provide very strong evidence o who committed the crime. Some countries now have databases o DNA profles, which have allowed many criminal cases to be solved. D NA profling is also used in paternity investigations. These are done to fnd out whether a man is the ather o a child. There are various reasons or paternity investigations being requested. Men sometimes claim that they are not the ather o a child to avoid having to pay the mother to raise the child. Women who have had multiple partners may wish to identiy the biological ather o a child. A child may wish to prove that a deceased man was their ather in order to show that they are their heir. D NA profles o the mother, the child and the man are needed. D NA profles o each o the samples are prepared and the patterns o bands are compared. I any bands in the childs profle do not occur in the profle o the mother or man, another person must be the ather. 189 3 G e n e ti cs Aalysis o DnA profles Analysis o examples o DNA profles. Analysis o D NA profles in orensic investigations is straightorward: two D NA samples are very likely to have come rom the same person i the pattern o bands on the profle is the same. victim specimen 1 2 suspects 3 Figure 5 Which o the three suspects DNA fngerprints matches the specimen recovered rom the crime scene? Analysis o DNA profles in paternity investigations is more complicated. Each o the bands in the childs DNA profle must be the same as a band in the biological mother or athers profle. Every band in the childs profle must be checked to make sure that it occurs either in the mothers profle or in the profle o the man presumed to be the ather. I one or more bands do not, another man must have been the biological ather. Geetic modifcatio Genetic modifcation is carried out by gene transer between species. Molecular biologists have developed techniques that allow genes to be transerred between species. The transer o genes rom one species to another is known as genetic modifcation. It is possible because the genetic code is universal, so when genes are transerred between species, the amino acid sequence translated rom them is unchanged the same polypeptide is produced. Genes have been transerred rom eukaryotes to bacteria. O ne o the early examples was the transer o the gene or making human insulin to a bacterium. This was done so that large quantities o this hormone can be produced or treating diabetics. Genetic modifcation has been used to introduce new characteristics to animal species. For example, goats have been produced that secrete milk containing spider silk protein. S pider silk is immensely strong, but spiders could not be used to produce it commercially. Figure 6 Genes have been transerred rom daodil plants to rice, to make the rice produce a yellow pigment in its seeds 190 Genetic modifcation has also been used to produce many new varieties o crop plant. These are known as genetically modifed or GM crops. For example genes rom snapdragons have been transerred to tomatoes to produce ruits that are purple rather than red. The production o golden rice involved the transer o three genes, two rom daodil plants and 3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y one rom a bacterium, so that the yellow pigment - carotene is produced in the rice grains. Actvt Scientists have an obligation to consider the ethical implications o their research. Discuss the ethics o the development o golden rice. -carotene is a precursor to vitamin A. The development o golden rice was intended as a solution to the problem o vitamin A defciency, which is a signifcant cause o blindness among children globally. techniques for gene ransfer o baceria Gene transer to bacteria with plasmids using restriction endonucleases and DNA ligase. Genes can be transerred rom one species to another by a variety o techniques. Together these techniques are known as genetic engineering. Gene transer to bacteria usually involves plasmids, restriction enzymes and D NA ligase. A plasmid is a small extra circle o D NA. The smallest plasmids have about 1 , 000 base pairs ( 1 kbp) , but they can have over 1 , 000 kbp. They occur commonly in bacteria. The most abundant plasmids are those with genes that encourage their replication in the cytoplasm and transer rom one bacterium to another. There are thereore some parallels with viruses but plasmids are not pathogenic and natural selection avours plasmids that coner an advantage on a bacterium rather than a disadvantage. B acteria use plasmids to exchange genes, so naturally absorb them and incorporate them into their main circular D NA molecule. Plasmids are very useul in genetic engineering. Bacterial cell Plasmid mRNA extracted from human pancreatic cells Plasmid obtained from bacteria mRNA cDNA mRNA treated with reverse transcriptase to make complementary DNA (cDNA) Restriction enzymes, also known as endonucleases, are enzymes that cut D NA molecules at specifc base sequences. They can be used to cut open plasmids and also to cut out desired genes rom larger D NA molecules. S ome restriction enzymes have the useul property o cutting the two strands o a D NA molecule at dierent points. This leaves single- stranded sections called sticky ends. The sticky ends created by any one particular restriction enzyme have complementary base sequences so can be used to link together pieces o D NA, by hydrogen bonding between the bases. Plasmid and cDNA fused using DNA ligase Recombinant plasmid introduced into host cells Bacteria multiply in a fermenter and produce insulin Separation and purication of human insulin D NA ligase is an enzyme that j oins D NA molecules together frmly by making sugarphosphate bonds between nucleotides. When the desired gene has been inserted into a plasmid using sticky ends there are still nicks in each sugarphosphate backbone o the D NA but D NA ligase can be used to seal these nicks. An obvious requirement or gene transer is a copy o the gene being transerred. It is usually easier to obtain messenger RNA transcripts o genes than the genes themselves. Reverse transcriptase is an enzyme that makes D NA copies o RNA molecules called cD NA. It can be used to make the D NA needed or gene transer rom messenger RNA. Plasmid cut with restriction enzyme Human insulin can be used by diabetic patients Figure 7 shows the steps involved in one example o gene transer. It has been used to create genetically modifed E. coli bacteria that are able to manuacture human insulin, or use in treating diabetes 191 3 G e n e ti cs Assessing the risks o genetic modifcation Assessing risks associated with scientifc research: scientists attempt to assess the risks associated with genetically modifed crops or livestock. Figure 8 The biohazard symbol indicates any organism or material that poses a threat to the health of living organisms especially humans There have been many ears expressed about the possible dangers o genetic modifcation. These ears can be traced back to the 1 970s when the frst experiments in gene transer were being conducted. Paul B erg planned an experiment in which D NA rom the monkey virus S V40 was going to be inserted into the bacterium E. coli. O ther biologists expressed serious concerns because SV40 was known to cause cancer in mice and E. coli lives naturally in the intestines o humans. There was thereore a risk o the genetically engineered bacterium causing cancer in humans. S ince then many other risks associated with genetic modifcation have been identifed. There has been ferce debate both among scientists and between scientists and non- scientists about the saety o the research and the saety o using genetically modifed organisms. This has led to bans being imposed in some countries, with potentially useul applications o GM crops or livestock let undeveloped. Almost everything that we do carries risks and it is not possible to eliminate risk entirely, either in science or in other aspects o our lives. It is natural or humans to assess the risk o an action and decide whether or not go ahead with it. This is what scientists must do assess the risks associated with their research beore carrying it out. The risks can be assessed in two ways: GM corn (maize) is widely grown in North America What is the chance o an accident or other harmul consequence? How harmul would the consequence be? Figure 9 I there is a high chance o harmul consequences or a signifcant chance o very harmul consequences then research should not be done. Risks and benefts o GM crops 192 Assessment o the potential risks and benefts associated with genetic modifcation o crops. is disagreement, because gene transer to crop plants is a relatively recent procedure, the issues involved are very complex and in science it oten takes decades or disputes to be resolved. GM crops have many potential benefts. These have been publicized widely by the corporations that produce GM seed, but they are questioned by opponents o the technology. Even basic issues such as whether GM crops increase yields and reduce pesticide and herbicide use have been contested. It is not surprising that there Potential benefts can be grouped into environmental benefts, health benefts and agricultural benefts. Economic benefts o GM crops are not included here, because they cannot be assessed on a scientifc basis using experimental evidence. It would be impossible in the time available or IB students to assess all claimed 3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y benefts or all GM crops. Instead it is better to select one claim rom the list given here and assess it or one crop. Much o the evidence relating to potential benefts and also to risks is reely available. C laims about environmental benefts o GM crop s: Pest- resistant crop varieties can be produced by transerring a gene or making a toxin to the plants. Less insecticide then has to be sprayed on to the crop so ewer bees and other benefcial insects are harmed. Use o GM crop varieties reduces the need or plowing and spraying crops, so less uel is needed or arm machinery. The shel- lie o ruit and vegetables can be improved, reducing wastage and reducing the area o crops that have to be grown. C laims about the health benefts o GM crop s: The nutritional value o crops can be improved, or example by increasing the vitamin content. Varieties o crops could be produced lacking allergens or toxins that are naturally present in them. GM crops could be engineered that produce edible vaccines so by eating the crop a person would be vaccinated against a disease. C laims about agricultural benefts o GM crop s: Varieties resistant to drought, cold and salinity can be produced by gene transer, expending the range over which crops can be produced and increasing total yields. A gene or herbicide resistance can be transerred to crop plants allowing all other plants to be killed in the growing crop by spraying with herbicide. With less weed competition crop yields are higher. Herbicides that kill all plants can be used to create weed- ree conditions or sowing non- GM crops but they cannot be used once the crop is growing. C rop varieties can be produced that are resistant to diseases caused by viruses. Figure 10 Wild plants growing next to a crop of GM maize These diseases currently reduce crop yields signifcantly and the only current method o control is to reduce transmission by killing insect vectors o the viruses with insecticides. A wide variety o concerns about GM crops have been raised. S ome o these, such as the eect on armers incomes, cannot be assessed on scientifc grounds so are not relevant here. The remaining concerns can be grouped into health risks, environmental risks and agricultural risks. To make overall j udgments about the saety o GM crops, each risk needs to be assessed careully, using all the available experimental evidence. This needs to be done on a case by case basis as it is not possible to assess the risks and benefts o one GM crop rom experiments perormed on another one. There is no consensus among all scientists or non- scientists yet about GM crops and it is thereore important or as many o us as possible to look at the evidence or the claims and counter- claims, rather than the publicity. Any o the risks that are included here could be selected or detailed scrutiny. C laims made about health risks o GM crop s: Proteins produced by transcription and translation o transerred genes could be 193 3 G e n e ti cs plants, plant-eating insects and organisms that eed on them where GM rather than non-GM crops are being grown. toxic or cause allergic reactions in humans or livestock that eat GM crops. Antibiotic resistance genes used as markers during gene transer could spread to pathogenic bacteria. Transerred genes could mutate and cause unexpected problems that were not riskassessed during development o GM crops. C laims made about agricultural risks of GM crop s: Some seed rom a crop is always spilt and germinates to become unwanted volunteer plants that must be controlled, but this could become very dicult i the crop contains herbicide resistance genes. Widespread use o GM crops containing a toxin that kills insect pests will lead to the spread o resistance to the toxin in the pests that were the initial problem and also to the spread o secondary pests that are resistant to the toxin but were previously scarce. Farmers are not permitted by patent law to save and re-sow GM seed rom crops they have grown, so strains adapted to local conditions cannot be developed. C laims made about environmental risks of GM crop s: Non- target organisms could be aected by toxins that are intended to control pests in GM crop plants. Genes transerred to crop plants to make them herbicide resistant could spread to wild plants, turning them into uncontrollable super- weeds. Biodiversity could be reduced i a lower proportion o sunlight energy passes to weed Analysing risks to monarch butterfies o Bt corn Analysis o data on risks to monarch butterfies o Bt crops. Insect pests o crops can be controlled by spraying with insecticides but varieties have been recently been produced by genetic engineering that produce a toxin that kills insects. A gene was transerred rom the bacterium Bacillus thuringiensis that codes or Bt toxin. The toxin is a protein. It kills members o insect orders that contain butterfies, moths, fies, beetles, bees and ants. The genetically engineered corn varieties produce Bt toxin in all parts o the plant including pollen. Bt varieties o many crops have been produced, including Zea mays. In North America this crop is called corn, while in B ritain it is known as maize, or corn on the cob. The crop is attacked by various insect pests including corn borers, which are the larvae o the moth Ostrinia nubilalis. C oncerns have been expressed about the eects o Bt corn on non-target species o insect. O ne particular species o concern is the monarch butterfy, Danaus plexippus. The larvae o the monarch butterfy eed on leaves o milkweed, Asclepias curassavica. This plant sometimes grows close enough to corn crops to become dusted with the wind- dispersed corn pollen. There is thereore a risk that monarch larvae might be poisoned by Bt toxin in pollen rom GM corn crops. This risk has been investigated experimentally. D ata rom these experiments is available or analysis. 194 To investigate the eect o pollen rom Bt corn on the larvae o monarch butterfies the ollowing procedure was used. Leaves were collected rom milkweed plants and were lightly misted with water. A spatula o pollen was gently tapped over the leaves to deposit a ne dusting. The leaves were placed in water- lled tubes. Five three-dayold monarch butterfy larvae were placed on each lea. The area o lea eaten by the larvae was monitored over our days. The mass o the larvae was measured ater our days. The survival o the larvae was monitored over our days. Three treatments were included in the experiment, with ve repeats o each treatment: leaves not dusted with pollen ( blue) leaves dusted with non- GM pollen ( yellow) leaves dusted with pollen rom Bt corn ( red) 100 75 50 25 0 2 3 4 5 6 7 2 3 Time (days) 1 2 3 Time (days) 4 1.5 1 0.5 0 The results are shown in the table, bar chart and graph on the right. 1 1 2 Cumulative leaf consumption per larva Data-based questons: Transgenic pollen and monarch larvae Survival of monarch larvae (%) 3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y a) List the variables that were kept constant in the experiment. [3 ] b) Explain the need to keep these variables constant. [2 ] a) C alculate the total number o larvae used in the experiment. [2 ] b) Explain the need or replicates in experiments. [2 ] The bar chart and the graph show mean results and error bars. Explain how error bars help in the analysis and evaluation o data. [2 ] Explain the conclusions that can be drawn rom the percentage survival o larvae in the three treatments. [2 ] Suggest reasons or the dierences in lea consumption between the three treatments. [3 ] Predict the mean mass o larvae that ed on leaves dusted with non- GM pollen. [2 ] O utline any dierences between the procedures used in this experiment and processes that occur in nature, which might aect whether monarch larvae are actually harmed by Bt pollen. [2 ] 4 Source: Losey JE, Rayor LS, Carter ME (May 1999) . Transgenic pollen harms monarch larvae. Nature 399 (6733) : 214. Treatment Mean mass of surviving larvae (g) Leaves not dusted with pollen 0.38 Leaves dusted with Not available non-GM pollen Leaves dusted with 0.16 pollen from Bt corn Actvt Estatng te sze of a cone A total of 130,000 hectares of Russet Burbank potatoes were planted in Idaho in 2011. The mean density of planting of potato tubers was 50,000 per hectare. Estimate the size of the clone at the time of planting and at the time of harvest. Clones Clones are groups of genetically identical organisms, derived from a single original parent cell. A zygote, produced by the usion o a male and emale gamete, is the rst cell o a new organism. B ecause zygotes are produced by sexual reproduction, they are all genetically dierent. A zygote grows and develops into an adult organism. I it reproduces sexually, its 195 3 G e n e ti cs Activity ospring will be genetically dierent. In some species organisms can also reproduce asexually. When they do this, they produce genetically identical organisms. The production o genetically identical organisms is called cloning and a group o genetically identical organisms is called a clone. How many potato clones are there in this photo? Although we do not usually think o them in this way, a pair o identical twins is the smallest clone that can exist. They are either the result o a human zygote dividing into two cells, which each develop into separate embryos, or an embryo splitting into two parts which each develop into a separate individual. Identical twins are not identical in all their characteristics and have, or example, dierent fngerprints. A better term or them is monozygotic. More rarely identical triplets, quadruplets and even quintuplets have been produced. S ometimes a clone can consist o very large numbers o organisms. For example, commercially grown potato varieties are huge clones. Large clones are ormed by cloning happening again and again, but even so all the organisms may be traced back to one original parent cell. natural methods of cloig Many plant species and some animal species have natural methods of cloning. Figure 11 Identical twins are an example of cloning Although the word clone is now used or any group o genetically identical organisms, it was frst used in the early 2 0th century or plants produced by asexual reproduction. It comes rom the Greek word or twig. Many plants have a natural method o cloning. The methods used by plants are very varied and can involve stems, roots, leaves or bulbs. Two examples are given here: A single garlic bulb, when planted, uses its ood stores to grow leaves. These leaves produce enough ood by photosynthesis to grow a group o bulbs. All the bulbs in the group are genetically identical so they are a clone. A strawberry plant grows long horizontal stems with plantlets at the end. These plantlets grow roots into the soil and photosynthesize using their leaves, so can become independent o the parent plant. A healthy strawberry plant can produce ten or more genetically identical new plants in this way during a growing season. Natural methods o cloning are less common in animals but some species are able to do it. Figure 12 One bulb of garlic clones itself to produce a group of bulbs by the end of the growing season 196 Hydra clones itsel by a process called budding ( sub- topic 1 .6, fgure 1 , page 5 1 ) . Female aphids can give birth to ospring that have been produced entirely rom diploid egg cells that were produced by mitosis rather than meiosis. The ospring are thereore clones o their mother. 3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y Investigating actors afecting the rooting o stem-cuttings Design o an experiment to assess one actor afecting the rooting o stem-cuttings. S tem- cuttings are short lengths o stem that are used to clone plants artifcially. I roots develop rom the stem, the cutting can become an independent new plant. 1 2 3 Many plants can be cloned rom cuttings. Ocimum basilicum roots particularly easily. Nodes are positions on the stem where leaves are attached. With most species the stem is cut below a node. Leaves are removed rom the lower hal o the stem. I there are many large leaves in the upper hal they can also be reduced. 4 The lowest third o the cutting is inserted into compost or water. C ompost should be sterile and contain plenty o both air and water. 5 A clear plastic bag with a ew holes cut in it prevents excessive water loss rom cuttings inserted in compost. 6 Rooting normally takes a ew weeks. Growth o new leaves usually indicates that the cutting has developed roots. whether the cutting is placed in water or compost what type o compost is used how warm the cuttings are kept whether a plastic bag is placed over the cuttings whether holes are cut in the plastic bag. You should think about these questions when you design your experiment: 1 What is your independent variable? 2 How will you measure the amount o root ormation, which is your dependent variable? 3 Which variables should you keep constant? 4 How many dierent types o plant should you use? 5 How many cuttings should you use or each treatment? Not all gardeners have success when trying to clone plants using root cuttings. S uccessul gardeners are sometimes said to have green fngers but a biologist would rej ect this as the reason or their success. E xperiments can give evidence about the actors that determine whether cuttings root or not. You can design and carry out an experiment to investigate one o the actors on the list below, or another actor o your own. Possible actors to investigate: whether the stem is cut above or below a node how long the cutting is whether the end o the stem is let in the air to callus over how many leaves are let on the cutting whether a hormone rooting powder is used 197 3 G e n e ti cs Cloning animal embryos Animals can be cloned at the embryo stage by breaking up the embryo into more than one group o cells. At an early stage o development all cells in an animal embryo are pluripotent ( capable o developing into all types o tissue) . It is thereore theoretically possible or the embryo to divide into two or more parts and each part to develop into a separate individual with all body parts. This process is called splitting or ragmentation. C oral embryos have been observed to clone themselves by breaking up into smaller groups o cells or even single cells, presumably because this increases the chance o one embryo surviving. Formation o identical twins could be regarded as cloning by splitting, but most animal species do not appear to do this naturally. However, it is possible to break up animal embryos artifcially and in some cases the separated parts develop into multiple embryos. In livestock, an egg can be ertilized in vitro and allowed to develop into a multicellular embryo. Individual cells can be separated rom the embryo while they are still pluripotent and transplanted into surrogate mothers. Only a limited number o clones can be obtained this way, because ater a certain number o divisions the embryo cells are no longer pluripotent. Splitting o embryos is usually most successul at the eight-cell stage. Figure 13 Sea urchin embryo (a) 4-cell stage (b) blastula stage consisting of a hollow ball of cells There has been little interest in this method o artifcial cloning because at the embryo stage it is not possible to assess whether a new individual produced by sexual reproduction has desirable characteristics. Cloning adult animals using diferentiated cells Methods have been developed or cloning adult animals using diferentiated cells. It is relatively easy to clone animal embryos, but at that stage it is impossible to know whether the embryos will have desirable characteristics. O nce the embryos have grown into adults it is easy to assess their characteristics, but it is much more difcult to clone them. This is because the cells that make up the body o an adult animal are dierentiated. To produce all the tissues in a new animal body undierentiated pluripotent cells are needed. The biologist John Gurdon carried out experiments on cloning in the rog Xenopus as a postgraduate student in Oxord during the 1 950s. He removed nuclei rom body cells o Xenopus tadpoles and transplanted them into egg cells rom which the nucleus had been removed. The egg cells into which the nuclei were transplanted developed as though they were zygotes. They carried out cell division, cell growth and dierentiation to orm all the tissues o a normal Xenopus rog. In 201 2 Gurdon was awarded the Nobel Prize or Physiology or Medicine or his pioneering research. Figure 14 Xenopus tadpoles 198 C loning using dierentiated cells prove d to b e much more diicult in mammals. The irst cloned mammal was D olly the shee p in 1 9 9 6 . Apart rom the ob vious reproductive use s o this type o cloning, there is also interest in it or therape utic reasons. I this procedure 3 . 5 G E N E T i C m O D i F i C AT i O N A N D B i O T E C h N O l O G y was done with humans, the embryo would consist of pluripotent stem cells, which could be used to regenerate tissues for the adult. B e cause the cells would be genetically ide ntical to those of the adult from whom the nucleus was obtaine d they would not cause rej ection problems. Methods used to produce Dolly Production of cloned embryos by somatic-cell nuclear transfer. The production of D olly was a pioneering development in animal cloning. The method that was used is called somatic-cell nuclear transfer. A somatic cell is a normal body cell with a diploid nucleus. The method has these stages: Adult cells were taken from the udder of a Finn D orset ewe and were grown in the laboratory, using a medium containing a low concentration of nutrients. This made genes in the cells inactive so that the pattern of differentiation was lost. Unfertilized eggs were taken from the ovaries of a S cottish B lackface ewe. The nuclei were removed from these eggs. O ne of the cultured cells from the Finn D orset was placed next to each egg cell, inside the zona pellucida around the egg, which is a protective coating of gel. A small electric pulse was used to cause the two cells to fuse together. About 1 0% of the fused cells developed like a zygote into an embryo. Figure 15 Dolly with Dr Ian Wilmut, the embryologist who led the team that produced her The embryos were then inj ected when about seven days old into the uteri of other ewes that could act as surrogate mothers. This was done in the same way as in IVF. O nly one of the 2 9 embryos implanted successfully and developed through a normal gestation. This was D olly. egg without a nucleus fused with donor cell using a pulse of electricity cell taken from udder of donor adult and cultured in laboratory for six days unfertilized egg taken from another sheep. Nucleus removed from the egg Figure 16 A method embryo resulting from fusion of udder cell and egg transfered to the uterus of a third sheep which acts as the surrogate mother surrogate mother gives birth to lamb. Dolly is genetically identical with the sheep that donated the udder cell (the donor) or cloning an adult sheep using diferentiated cells 199 3 G e n e ti cs Questions 1 Human somatic cells have 46 chromosomes, while our closest primate relatives, the chimpanzee, the gorilla and the orangutan all have 48 chromosomes. One hypothesis is that the human chromosome number 2 was ormed rom the usion o two chromosomes in a primate ancestor. The image below shows human chromosome 2 compared to chromosome 1 2 and 1 3 rom the chimpanzee. a) C ompare the human chromosome 2 with the two chimpanzee chromosomes ( fgure 1 7) . [3 ] The cheetah ( Acinonyx jubatus) is an endangered species o large cat ound in S outh and East Arica. A study o the level o variation o the cheetah gene pool was carried out. In one part o this study, blood samples were taken rom 1 9 cheetahs and analysed or the protein transerrin using gel electrophoresis. The results were compared with the electrophoresis patterns or blood samples rom 1 9 domestic cats ( Felis sylvestris) . Gel electrophoresis can be used to separate proteins using the same principles as in D NA profling. The bands on the gel which represent orms o the protein transerrin are indicated. transferrin H C b) The ends o chromosomes, called telomeres, have many repeats o the same short D NA sequence. I the usion hypothesis were true, predict what would be ound in the region o the chromosome where the usion is hypothesized to have occurred. [2 ] 3 Figure 17 origin 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 cheetahs The pedigree in fgure 1 8 shows the AB O groups o three generations o a amily. I II III AB B O B 1 2 3 4 B A B O O 1 2 3 4 5 O A B O ? 1 2 3 4 5 Figure 18 transferrin origin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a) D educe the genotype o each person in the amily. [4] b) D educe the possible blood groups o individual III 5 , with the percentage chance o each. [2 ] c) D educe the possible blood groups and the percentage chance o each blood group: 200 domestic cats Figure 19 Using fgure 1 9, deduce with reasons: a) the number o domestic cats and the number o cheetahs that were heterozygous or the transerrin gene; [2 ] ( i) o children o individual III 1 and his partner who is also in blood group O [2 ] b) the number o alleles o the transerrin gene in the gene pool o domestic cats; [2 ] ( ii) o children o III 2 and her partner who is in blood group AB . [2 ] c) the number o alleles o the transerrin gene in the gene pool o cheetahs. [1 ] 4 E co lo gy Intrdutin E cosystems require a continuous supply o energy to uel lie processes and to replace energy lost as heat. C ontinued availability o carbon and other chemical elements in ecosystems depends on cycles. The uture survival o living organisms including humans depends on sustainable ecological communities. C oncentrations o gases in the atmosphere have signifcant eects on climates experienced at the Earths surace. 4.1 Species, communities and ecosystems Understandin Species are groups o organisms that can potentially interbreed to produce ertile ofspring. Members o a species may be reproductively isolated in separate populations. Species have either an autotrophic or heterotrophic method o nutrition (a ew species have both methods) . Consumers are heterotrophs that eed on living organisms by ingestion. Detritivores are heterotrophs that obtain organic nutrients rom detritus by internal digestion. Saprotrophs are heterotrophs that obtain organic nutrients rom dead organic matter by external digestion. A community is ormed by populations o diferent species living together and interacting with each other. A community orms an ecosystem by its interactions with the abiotic environment. Autotrophs and heterotrophs obtain inorganic nutrients rom the abiotic environment. The supply o inorganic nutrients is maintained by nutrient cycling. Ecosystems have the potential to be sustainable over long periods o time. Skis Classiying species as autotrophs, consumers, detritivores or saprotrophs rom a knowledge o their mode o nutrition. Testing or association between two species using the chi-squared test with data obtained by quadrat sampling. Recognizing and interpreting statistical signicance. Setting up sealed mesocosms to try to establish sustainability. (Practical 5) Nature f siene Looking or patterns, trends and discrepancies: plants and algae are mostly autotrophic but some are not. 201 41 E c o lo g y Species Species are groups o organisms that can potentially interbreed to produce ertile ofspring. B irds o paradise inhabit Papua New Guinea and other Australasian islands. In the breeding season the males do elaborate and distinctive courtship dances, repeatedly carrying out a series o movements to display their exotic plumage. O ne reason or this is to show to a emale that they are ft and would be a suitable partner. Another reason is to show that they are the same type o bird o paradise as the emale. Figure 1 A bird of paradise in Papua New Guinea There are orty- one dierent types o bird o paradise. E ach o these usually only reproduces with others o its type and hybrids between the dierent types are rarely produced. For this reason each o the orty- one types o bird o paradise remains distinct, with characters that are dierent to those o other types. B iologists call types o organism such as these sp ecies. Although ew species have as elaborate courtship rituals as birds o paradise, most species have some method o trying to ensure that they reproduce with other members o their species. When two members o the same species mate and produce ospring they are interbreeding. O ccasionally members o dierent species breed together. This is called cross- breeding. It happens occasionally with birds o paradise. However, the ospring produced by cross- breeding between species are almost always inertile, which prevents the genes o two species becoming mixed. The reproductive separation between species is the reason or each species being a recognizable type o organism with characters that distinguish it rom even the most closely related other species. In summary, a species is a group o organisms that interbreed to produce ertile ospring. Populations Members o a species may be reproductively isolated in separate populations. A population is a group o organisms o the same species who live in the same area at the same time. I two populations live in dierent areas they are unlikely to interbreed with each other. This does not mean that they are dierent species. I they potentially could interbreed, they are still members o the same species. I two populations o a species never interbreed then they may gradually develop dierences in their characters. Even i there are recognizable dierences, they are considered to be the same species until they cannot interbreed and produce ertile ospring. In practice it can be very difcult to decide whether two populations have reached this point and biologists sometimes disagree about whether populations are the same or dierent species. 202 4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S arph hrrph r Species have either an autotrophic or heterotrophic method o nutrition (a ew species have both methods) . All organisms need a supply o organic nutrients, such as glucose and amino acids. They are needed or growth and reproduction. Methods o obtaining these carbon compounds can be divided into two types: some organisms make their own carbon compounds rom carbon dioxide and other simple substances they are autotrophic, which means sel-eeding; some organisms obtain their carbon compounds rom other organisms they are heterotrophic, which means eeding on others. S ome unicellular organisms use both methods o nutrition. Euglena gracilis or example has chloroplasts and carries out photosynthesis when there is sufcient light, but can also eed on detritus or smaller organisms by endocytosis. O rganisms that are not exclusively autotrophic or heterotrophic are mixotrophic. Figure 3 Arabidopsis thaliana the autotroph that molecular biologists use as a model plant Figure 4 Humming birds are heterotrophic; the plants from which they obtain nectar are autotrophic Figure 5 Euglena an unusual organism as it can feed both autotrophically and heterotrophically trs pl lgl r Looking or patterns, trends and discrepancies: plants and algae are mostly autotrophic but some are not. Almost all plants and algae are autotrophic they make their own complex organic compounds using carbon dioxide and other simple substances. A supply o energy is needed to do this, which plants and algae obtain by absorbing light. Their method o autotrophic nutrition is thereore photosynthesis and they carry it out in chloroplasts. av Glpgs rss The tortoises that live on the Galpagos islands are the largest in the world. They have sometimes been grouped together into one species, Chelinoidis nigra, but more recently have been split into separate species. Discuss whether each o these observations indicates that populations on the various islands are separate species: The Galpagos tortoises are poor swimmers and cannot travel rom one island to another so they do not naturally interbreed. Tortoises rom diferent islands have recognizable diferences in their characters, including shell size and shape. Tortoises rom diferent islands have been mated in zoos and hybrid ofspring have been produced but they have lower ertility and higher mortality than the ofspring o tortoises rom the same island. Figure 2 Galpagos tortoise This trend or plants and algae to make their own carbon compounds by photosynthesis in chloroplasts is ollowed by the majority o species. However there are small numbers o both plants and algae that do not ft the trend, because although they are recognizably plants or algae, they 203 41 E c o lo g y do not contain chloroplasts and they do not carry out photosynthesis. These species grow on other plants, obtain carbon compounds rom them and cause them harm. They are thereore parasitic. To decide whether parasitic plants alsiy the theory that plants and algae are groups o autotrophic species or whether they are j ust minor and insignifcant discrepancies we need to consider how many species there are and how they evolved. The number o parasitic plants and algae is relatively small only about 1 % o all plant and algal species. It is almost certain that the original ancestral species o plant and alga were autotrophic and that the parasitic species evolved rom them. C hloroplasts can quite easily be lost rom cells, but cannot easily be developed. Also, parasitic species are diverse and occur in many dierent amilies. This pattern suggests that parasitic plants have evolved repeatedly rom photosynthetic species. B ecause o this evidence, ecologists regard plants and algae as groups o autotrophs, with a small number o exceptional species that are parasitic. data-base questions: Unexpected diets Although we usually expect plants to be autotrophs and animals to be consumers, living organisms are very varied and do not always conorm to our expectations. Figures 6 to 9 show our organisms with diets that are unexpected. 1 Which o the organisms is autotrophic? [4] 2 Which o the organisms is heterotrophic? [4] 3 O the organisms that are heterotrophic, deduce which is a consumer, which a detritivore and which a saprotroph. [4] Figure 7 Ghost orchid: grows underground in woodland, eeding of dead organic matter, occasionally growing a stem with owers above ground 204 Figure 8 Euglena: unicell that lives in ponds, using its chloroplasts or photosynthesis, but also ingesting dead organic matter by endocytosis Figure 6 Venus y trap: grows in swamps, with green leaves that carry out photosynthesis and also catch and digest insects, to provide a supply o nitrogen Figure 9 Dodder: grows parasitically on gorse bushes, using small root-like structures to obtain sugars, amino acids and other substances it requires, rom the gorse 4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S csrs Consumers are heterotrophs that feed on living organisms by ingestion. Heterotrophs are divided into groups by ecologists according to the source o organic molecules that they use and the method o taking them in. O ne group o heterotrophs is called consumers. C onsumers eed o other organisms. These other organisms are either still alive or have only been dead or a relatively short time. A mosquito sucking blood rom a larger animal is a consumer that eeds on an organism that is still alive. A lion eeding o a gazelle that it has killed is a consumer. Figure 10 Red kite (Milvus milvus) is a consumer that feeds on live prey but also on dead animal remains (carrion) C onsumers ingest their ood. This means that they take in undigested material rom other organisms. They digest it and absorb the products o digestion. Unicellular consumers such as Paramecium take the ood in by endocytosis and digest it inside vacuoles. Multicellular consumers such as lions take ood into their digestive system by swallowing it. C onsumers are sometimes divided up into trophic groups according to what other organisms they consume. Primary consumers eed on autotrophs; secondary consumers eed on primary consumers and so on. In practice, most consumers do not ft neatly into any one o these groups because their diet includes material rom a variety o trophic groups. Figure 11 Yellow-necked mouse (Apodemus favicollis) is a consumer that feeds mostly on living plant matter, especially seeds, but also on living invertebrates drvrs Sprrphs Detritivores are heterotrophs that obtain organic nutrients from detritus by internal digestion. Saprotrophs are heterotrophs that obtain organic nutrients from dead organic matter by external digestion. O rganisms discard large quantities o organic matter, or example: Saprotrophs secrete digestive enzymes into the dead organic matter and digest it externally. They then absorb the products o digestion. Many types o bacteria and ungi are saprotrophic. They are also known as decomposers because they break down carbon compounds in dead organic matter and release elements such as nitrogen into the ecosystem so that they can be used again by other organisms. dead leaves and other parts o plants eathers, hairs and other dead parts o animal bodies eces rom animals. This dead organic matter rarely accumulates in ecosystems and instead is used as a source o nutrition by two groups o heterotroph detritivores and saprotrophs. D etritivores ingest dead organic matter and then digest it internally and absorb the products o digestion. Large multicellular detritivores such as earthworms ingest the dead matter into their gut. Unicellular organisms ingest it into ood vacuoles. The larvae o dung beetles eed by ingestion o eces rolled into a ball by their parent. Figure 12 Saprotrophic fungi growing over the surfaces of dead leaves and decomposing them by secreting digestive enzymes 205 41 E c o lo g y TOK Identifying modes of nutrition to wh exen do he lssifion sysems (lbels nd egories) we use se limis o wh we pereive? Classiying species as autotrophs, consumers, detritivores or saprotrophs rom a knowledge o their mode o nutrition. There are innite ways to divide up our observations. Organisms can be organized in a number o ways by scientists: by morphology (physical similarity to other organisms) , phylogeny (evolutionary history) and niche (ecological role) . In everyday language, we classiy organisms such as domesticated or wild; dangerous or harmless; edible or toxic. By answering a series o simple questions about an organisms mode o nutrition it is usually possible to deduce what trophic group it is in. These questions are presented here as a dichotomous key, which consists o a series o pairs o choices. The key works or unicellular and multicellular organisms but does not work or parasites such as tapeworms or ungi that cause diseases in plants. All multicellular autotrophs are photosynthetic and have chloroplasts containing chlorophyll. Feeds on living or recently killed organisms = CONSUMERS Feeds on dead organic matter = DETRITIVORES Either ingests organic matter by endocytosis (no cell walls) or by taking it into its gut. aiviy START HERE cleruing Cell walls present. No ingestion of organic matter. No gut. Figure 14 Secretes enzymes into its environment to digest dead organic matter = SAPROTROPHS Enzymes not secreted. Only requires simple ions and compounds such as CO 2 = AUTOTROPHS In a classic essay written in 1972, the physicist Philip Anderson stated this: The ability to reduce everything to simple fundamental laws does not imply the ability to start from those laws and reconstruct the universe. At each level of complexity entirely new properties appear. Clearcutting is the most common and economically protable orm o logging. It involves clearing every tree in an area so that no canopy remains. With reerence to the concept o emergent properties, suggest why the ecological community oten ails to recover ater clearcutting. 206 communiies A community is ormed by populations o diferent species living together and interacting with each other. An important part o ecology is research into relationships between organisms. These relationships are complex and varied. In some cases the interaction between two species is o benet to one species and harms the other, or example the relationship between a parasite and its host. In other cases both species benet, as when a hummingbird eeds on nectar rom a fower and helps the plant by pollinating it. All species are dependent on relationships with other species or their long- term survival. For this reason a population o one species can never live in isolation. Groups o populations live together. A group 4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S o populations living together in an area and interacting with each other is known in ecology as a community. Typical communities consist o hundreds or even thousands o species living together in an area. Figure 13 A coral reef is a complex community with many interactions between the populations. Most corals have photosynthetic unicellular algae called zooxanthellae living inside their cells Field work associations between species Testing for association between two species using the chi-squared test with data obtained by quadrat sampling. Quadrats are square sample areas, usually marked out using a quadrat rame. Quadrat sampling involves repeatedly placing a quadrat rame at random positions in a habitat and recording the numbers o organisms present each time. The quadrat is placed precisely at the distances determined by the two random numbers. I this procedure is ollowed correctly, with a large enough number o replicates, reliable estimates o The usual procedure or randomly positioning quadrats is this: A base line is marked out along the edge o the habitat using a measuring tape. It must extend all the way along the edge o the habitat. Random numbers are obtained using either a table or a random number generator on a calculator. A frst random number is used to determine a distance along the measuring tape. All distances along the tape must be equally likely. A second random number is used to determine a distance out across the habitat at right angles to the tape. All distances across the habitat must be equally likely. Figure 15 Quadrat sampling of seaweed populations on a rocky shore 207 41 E c o lo g y population sizes are obtained. The method is only suitable or plants and other organisms that are not motile. Quadrat sampling is not suitable or populations o most animals, or obvious reasons. I the presence or absence o more than one species is recorded in every quadrat during sampling o a habitat, it is possible to test or an association between species. Populations are oten unevenly distributed because some parts o the habitat are more suitable or a species than others. I two species occur in the same parts o a habitat, they will tend to be ound in the same quadrats. This is known as a positive association. There can also be negative associations, or the distribution o two species can be independent. There are two possible hypotheses: 2 C alculate the expe cted requencies, assuming indepe ndent distribution, or each o the our species combinations. E ach e xpe cted requency is calculated rom value s on the contingency table using this equation: row total column total expected = ___ grand total requency 3 C alculate the number o degrees o reedom using this equation. degrees o reedom = ( m 1 ) ( n 1 ) where m and n are the numbe r o rows and number o columns in the contingency table. 4 Find the critical region or chi- squared rom a table o chi- squared values, using the degrees o reedom that you have calculated and a signifcance level ( p) o 0.05 ( 5 % ) . The critical region is any value o chi-squared larger than the value in the table. 5 C alculate chi-squared using this equation: H 0 : two species are distributed independently ( the null hypothesis) . H 1 : two species are associated ( either positively so they tend to occur together or negatively so they tend to occur apart) . We can test these hypotheses using a statistical procedure the chi- squared test. ( fo - fe) 2 X2 = _ fe The chi- squared test is only valid i all the expected requencies are 5 or larger and the sample was taken at random rom the population. where fo is the observed requency fe is the expected requency and Method for chi-squared test 1 is the sum o. Draw up a contingency table o observed requencies, which are the numbers o quadrats containing or not containing the two species. Species A present Species A absent 6 C ompare the calculated value o chi- squared with the critical region. I the calculated value is in the critical region, there is evidence at the 5 % level or an association between the two species. We can rej ect the hypothesis H 0 . I the calculated value is not in the critical region, because it is equal or below the value obtained rom the table o chisquared values, H 0 is not rej ected. There is no evidence at the 5 % level or an association between the two species. Row totals Species B present Species B absent Column totals C alculate the row and column totals. Adding the row totals or the column totals should give the same grand total in the lower right cell. 208 4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S d-bs qss: Chi-squared testing Figure 1 6 shows an area on the summit o C aer C aradoc, a hill in S hropshire, E ngland. The area is grazed by sheep in summer and hill walkers cross it on grassy paths. There are raised hummocks with heather (Calluna vulgaris) growing in them. A visual survey o this site suggested that Rhytidiadelphus squarrosus, a species o moss growing in this area, was associated with these heather hummocks. The presence or absence o the heather and the moss was recorded in a sample o 1 00 quadrats, positioned randomly. Results Sps Frq Heather only 9 Moss only 7 Both species 57 Neither species 27 3 Calculate the number o degrees o reedom. [2 ] 4 Find the critical region or chi- squared at a signifcance level o 5 % . [2 ] 5 C alculate chi- squared. 6 S tate the two alternative hypotheses, H 0 and H 1 , and evaluate them using the calculated value or chi- squared. [4] 7 Suggest ecological reasons or an association between the heather and the moss. [4] 8 Explain the methods that should have been used to position quadrats randomly in the area o study. [3 ] [4] Questions 1 C onstruct a contingency table o observed values. [4] 2 C alculate the expected values, assuming no association between the species. [4] Figure 16 Caer Caradoc, Shropshire Statistical signifcance Recognizing and interpreting statistical signifcance. B iologists oten use the phrase statistically signifcant when discussing results o an experiment. This reers to the outcome o a statistical hypothesis test. There are two alternative types o hypothesis: H 0 is the null hypothesis and is the belie that there is no relationship, or example that two means are equal or that there is no association or correlation between two variables. H 1 is the alternative hypothesis and is the belie that there is a relationship, or example that two means are dierent or that there is an association between two variables. The usual procedure is to test the null hypothesis, with the expectation o showing that it is alse. A statistic is calculated using the results o the research and is compared with a range o possible values called the critical region. I the calculated statistic exceeds the critical region, the null hypothesis is considered to be alse and is thereore rej ected, though we cannot say that this has been proved with certainty. When a biologist states that results were statistically signifcant it means that i the null hypothesis ( H 0 ) was true, the probability o getting results as extreme as the observed results would be very small. A decision has to be made about how small this probability needs to be. This is known as the signifcance level. It is the cut- o point or the probability o rej ecting the null 209 41 E c o lo g y hypothesis when in act it was true. A level o 5 % is oten chosen, so the probability is less than one in twenty. That is the minimum acceptable signicance level in published research. I there is a dierence between the mean results or the two treatments in an experiment, a statistical test will show whether the dierence is signicant at the 5 % level. I it is, there is a less than 5 % probability o such a large dierence between the sample means arising by chance, even when the population means are equal. We say that there is statistically signicant evidence that the population means dier. In the example o testing or an association between two species, described on previous pages, the chi-squared test shows whether there is a less than 5 % probability o the dierence between the observed and the expected results being as large as it is without the species being either positively or negatively associated. When results o biological research are displayed on a bar chart, letters are oten used to indicate statistical signicance. Two dierent letters, usually a and b, indicate mean results with a statistically signicant dierence. Two o the same letter such as a and a indicates that any dierence is not statistically signicant. Ecosystems A community forms an ecosystem by its interactions with the abiotic environment. A community is composed o all organisms living in an area. These organisms could not live in isolation they depend on their nonliving surroundings o air, water, soil or rock. Ecologists reer to these surroundings as the abiotic environment. In some cases the abiotic environment exerts a powerul infuence over the organisms. For example the wave action on a rocky shore creates a very specialized habitat and only organisms adapted to it can survive. On clis, the rock type determines whether there are ledges on which birds can nest. There are also many cases where living organisms infuence the abiotic environment. Sand dunes are an example o this. They develop along coasts where sand is blown up the shore and specialized plants grow in the loose wind-blown sand. The roots o these plants stabilize the sand and their leaves break the wind and encourage more sand to be deposited. So, not only are there complex interactions within communities, there are also many interactions between organisms and the abiotic environment. The community o organisms in an area and their non-living environment can thereore be considered to be a single highly complex interacting system, known as an ecosystem. Ecologists study both the components o ecosystems and the interactions between them. inorganc nutrents Autotrophs and heterotrophs obtain inorganic nutrients from the abiotic environment. Living organisms need a supply o chemical elements: Figure 17 Grasses in an area of developing sand dunes 210 C arbon, hydrogen and oxygen are needed to make carbohydrates, lipids and other carbon compounds on which lie is based. 4 . 1 S P e c i e S , c o m m u n i t i e S a n d e c o S yS t e m S Nitrogen and phosphorus are also needed to make many o these compounds. Approximately fteen other elements are needed by living organisms. S ome o them are used in minute traces only, but they are nonetheless essential. Autotrophs obtain all o the elements that they need as inorganic nutrients rom the abiotic environment, including carbon and nitrogen. Heterotrophs on the other hand obtain these two elements and several others as part o the carbon compounds in their ood. They do however obtain other elements as inorganic nutrients rom the abiotic environment, including sodium, potassium and calcium. nr ls The supply of inorganic nutrients is maintained by nutrient cycling. There are limited supplies on Earth o chemical elements. Although living organisms have been using the supplies or three billion years, they have not run out. This is because chemical elements can be endlessly recycled. O rganisms absorb the elements that they require as inorganic nutrients rom the abiotic environment, use them and then return them to the environment with the atoms unchanged. Recycling o chemical elements is rarely as simple as shown in this diagram and oten an element is passed rom organism to organism beore it is released back into the abiotic environment. The details vary rom element to element. The carbon cycle is dierent rom the nitrogen cycle or example. E cologists reer to these schemes collectively as nutrient cycles. The word nutrient is oten ambiguous in biology but in this context it simply means an element that an organism needs. The carbon cycle is described as an example o a nutrient cycle in subtopic 4.2 and the nitrogen cycle in O ption C . Reserves of an element in the abiotic environment Element forming part of a living organism Ssbl f sss Ecosystems have the potential to be sustainable over long periods of time. The concept o sustainability has risen to prominence recently because it is clear that some current human uses o resources are unsustainable. S omething is sustainable i it can continue indefnitely. Human use o ossil uels is an example o an unsustainable activity. Supplies o ossil uels are fnite, are not currently being renewed and cannot thereore carry on indefnitely. Natural ecosystems can teach us how to live in a sustainable way, so that our children and grandchildren can live as we do. There are three requirements or sustainability in ecosystems: nutrient availability detoxifcation o waste products energy availability. Figure 18 Living organisms have been recycling for billions of years 211 41 E c o lo g y Nutrients can be recycled indefnitely and i this is done there should not be a lack o the chemical elements on which lie is based. The waste products o one species are usually exploited as a resource by another species. For example, ammonium ions released by decomposers are absorbed and used or an energy source by Nitrosomonas bacteria in the soil. Ammonium is potentially toxic but because o the action o these bacteria it does not accumulate. Figure 19 Sunlight supplies energy to a forest ecosystem and nutrients are recycled ativity E nergy cannot be recycled, so sustainability depends on continued energy supply to ecosystems. Most energy is supplied to ecosystems as light rom the sun. The importance o this supply can be illustrated by the consequences o the eruption o Mount Tambora in 1 81 5 . D ust in the atmosphere reduced the intensity o sunlight or some months aterwards, causing crop ailures globally and deaths due to starvation. This was only a temporary phenomenon, however, and energy supplies to ecosystems in the orm o sunlight will continue or billions o years. cve eosystems Organisms have been ound living in total darkness in caves, including eyeless fsh. Discuss whether ecosystems in dark caves are sustainable. Figure 20 shows a small ecosystem with photosynthesizing plants near artifcial lighting in a cave that is open to visitors in Cheddar Gorge. Discuss whether this is more or less sustainable than ecosystems in dark caves. Mesocosms Setting up sealed mesocosms to try to establish sustainability. (Practical 5) Mesocosms are small experimental areas that are set up as ecological experiments. Fenced- o enclosures in grassland or orest could be used as terrestrial mesocosms; tanks set up in the laboratory can be used as aquatic mesocosms. E cological experiments can be done in replicate mesocosms, to fnd out the eects o varying one or more conditions. For example, tanks could be set up with and without fsh, to investigate the eects o fsh on aquatic ecosystems. Another possible use o mesocosms is to test what types o ecosystems are sustainable. This involves sealing up a community o organisms together with air and soil or water inside a container. You should consider these questions beore setting up either aquatic or terrestrial mesocosms: Figure 20 212 Large glass j ars are ideal but transparent plastic containers could also be used. S hould the sides o the container be transparent or opaque? Which o these groups o organisms must be included to make up a sustainable community: autotrophs, consumers, saprotrophs and detritivores? How can we ensure that the oxygen supply is sufcient or all the organisms in the mesocosm as once it is sealed, no more oxygen will be able to enter. How can we prevent any organisms suering as a result o being placed in the mesocosm? 4. 2 e n erG y Flo w 4.2 eg f Understanding Most ecosystems rely on a supply o energy rom sunlight. Light energy is converted to chemical energy in carbon compounds by photosynthesis. Chemical energy in carbon compounds fows through ood chains by means o eeding. Energy released by respiration is used in living organisms and converted to heat. Living organisms cannot convert heat to other Nature of science orms o energy. Experimental design: accurate quantitative Heat is lost rom ecosystems. measurements in osmosis experiments Energy losses between trophic levels restrict are essential. the length o ood chains and the biomass o higher trophic levels. Skills Quantitative representations o energy fow using pyramids o energy. Nature of science Use theories to explain natural phenomena: the concept o energy fow explains the limited length o ood chains. Sunlight and ecosystems Most ecosystems rely on a supply o energy rom sunlight. For most biological communities, the initial source of energy is sunlight. Living organisms can harvest this energy by photosynthesis. Three groups of autotroph carry out photosynthesis: plants, eukaryotic algae including seaweeds that grow on rocky shores, and cyanobacteria. These organisms are often referred to by ecologists as producers. Heterotrophs do not use light energy directly, but they are indirectly dependent on it. There are several groups of heterotroph in ecosystems: consumers, saprotrophs and detritivores. All of them use carbon compounds in their food as a source of energy. In most ecosystems all or almost all energy in the carbon compounds will originally have been harvested by photosynthesis in producers. The amount of energy supplied to ecosystems in sunlight varies around the world. The percentage of this energy that is harvested by producers and therefore available to other organisms also varies. In the S ahara D esert, for example, the intensity of sunlight is very high but little of it becomes available to organisms because there are very few producers. In the redwood forests of C alifornia the intensity of sunlight is less than in the S ahara but much more energy becomes available to organisms because producers are abundant. 213 41 E c o lo g y ativity cynobteri in ves Cyanobacteria are photosynthetic bacteria that are oten very abundant in marine and reshwater ecosystems. Figure 1 shows an area o green cyanobacteria on an area o wall in a cave that is illuminated by artifcial light. The surrounding areas are normally dark. I the artifcial light was not present, what other energy sources could be used by bacteria in caves? dt-bse questions: Insolation Insolation is a measure o solar radiation The two maps in fgure 2 show annual mean insolation at the top o the Earths atmosphere (upper map) and at the Earths surace (lower map) . Questions 1 State the relationship between distance rom the equator and insolation at the top o the Earths atmosphere. [1 ] 2 S tate the mean annual insolation in Watts per square metre or the most northerly part o Australia 3 4 a) at the top o the atmosphere [1 ] b) at the Earths surace. [1 ] S uggest reasons or dierences in insolation at the Earths surace between places that are at the same distance rom the equator. [2 ] Tropical rainorests are ound in equatorial regions o all continents. They have very high rates o photosynthesis. Evaluate the hypothesis that this is due to very high insolation. Include named parts o the world in your answer. [5 ] Figure 1 0 40 Figure 2 214 80 120 160 200 240 280 320 360 400 w/m 2 4. 2 e n erG y Flo w Energy conversion Light energy is converted to chemical energy in carbon compounds by photosynthesis. activit Bush d st fs Producers absorb sunlight using chlorophyll and other photosynthetic pigments. This converts the light energy to chemical energy, which is used to make carbohydrates, lipids and all the other carbon compounds in producers. Producers can release energy rom their carbon compounds by cell respiration and then use it or cell activities. Energy released in this way is eventually lost to the environment as waste heat. However, only some o the carbon compounds in producers are used in this way and the largest part remains in the cells and tissues o producers. The energy in these carbon compounds is available to heterotrophs. Energy in food chains Chemical energy in carbon compounds fows through ood chains by means o eeding. A ood chain is a sequence o organisms, each o which eeds on the previous one. There are usually between two and ve organisms in a ood chain. It is rare or there to be more organisms in the chain. As they do not obtain ood rom other organisms, producers are always the rst organisms in a ood chain. The subsequent organisms are consumers. Primary consumers eed on producers; secondary consumers eed on primary consumers; tertiary consumers eed on secondary consumers, and so on. No consumers eed on the last organism in a ood chain. Consumers obtain energy rom the carbon compounds in the organisms on which they eed. The arrows in a ood chain thereore indicate the direction o energy fow. Figure 3 Figure 3 shows a bush re in Australia. What energy conversion is happening in a bush re? Bush and orest res occur naturally in some ecosystems. Suggest two reasons or this hypothesis: There are ewer heterotrophs in ecosystems where res are common compared to ecosystems where res are not common. Figure 4 is an example o a ood chain rom the orests around Iguazu alls in northern Argentina. Figure 4 Respiration and energy release Energy released by respiration is used in living organisms and converted to heat. Living organisms need energy or cell activities such as these: Synthesizing large molecules like D NA, RNA and proteins. Pumping molecules or ions across membranes by active transport. Moving things around inside the cell, such as chromosomes or vesicles, or in muscle cells the protein bres that cause muscle contraction. ATP supplies energy or these activities. Every cell produces its own ATP supply. 215 41 E c o lo g y All cells can produce ATP by cell respiration. In this process carbon compounds such as carbohydrates and lipids are oxidized. These oxidation reactions are exothermic and the energy released is used in endothermic reactions to make ATP. So cell respiration transers chemical energy rom glucose and other carbon compounds to ATP. The reason or doing this is that the chemical energy in carbon compounds such as glucose is not immediately usable by the cell, but the chemical energy in ATP can be used directly or many dierent activities. The second law o thermodynamics states that energy transormations are never 1 00% efcient. Not all o the energy rom the oxidation o carbon compounds in cell respiration is transerred to ATP. The remainder is converted to heat. S ome heat is also produced when ATP is used in cell activities. Muscles warm up when they contract or example. Energy rom ATP may reside or a time in large molecules when they have been synthesized, such as D NA and proteins, but when these molecules are eventually digested the energy is released as heat. data-base questions a) D escribe the relationship between external temperature and respiration rate in yellowbilled magpies. [3 ] b) Explain the change in respiration rate as temperature drops rom + 1 0 C to 1 0 C . c) S uggest a reason or the change in respiration rate as temperature increased rom 3 0 C to 40 C . respiration rate (mW g1 ) 20 Figure 5 shows the results o an experiment in which yellow- billed magpies (Pica nuttalli) were put in a cage in which the temperature could be controlled. The birds rate o respiration was measured at seven dierent temperatures, rom 1 0 C to + 40 C . B etween 1 0 C and 3 0 C the magpies maintained constant body temperature, but above 3 0 C body temperature increased. 15 10 5 0 -10 0 10 20 30 temperature (C) 40 50 Figure 5 Cell [3 ] [2 ] respiration rates at diferent temperatures in yellow-billed magpies d) S uggest two reasons or the variation in respiration rate between the birds at each temperature. [2 ] Heat energy in ecosystems Living organisms cannot convert heat to other forms of energy. Living organisms can perorm various energy conversions: Light energy to chemical energy in photosynthesis. C hemical energy to kinetic energy in muscle contraction. C hemical energy to electrical energy in nerve cells. C hemical energy to heat energy in heat-generating adipose tissue. They cannot convert heat energy into any other orm o energy. 216 4. 2 e n erG y Flo w Heat losses from ecosystems Heat is lost rom ecosystems. Heat resulting rom cell respiration makes living organisms warmer. This heat can be useul in making cold-blooded animals more active. B irds and mammals increase their rate o heat generation i necessary to maintain their constant body temperatures. According to the laws o thermodynamics in physics, heat passes rom hotter to cooler bodies, so heat produced in living organisms is all eventually lost to the abiotic environment. The heat may remain in the ecosystem or a while, but ultimately is lost, or example when heat is radiated into the atmosphere. Ecologists assume that all energy released by respiration or use in cell activities will ultimately be lost rom an ecosystem. acivi thikig bu g chgs What energy conversions are required to shoot a basketball? What is the nal orm o the energy? expiig h gh f fd chis Use theories to explain natural phenomena: the concept o energy fow explains the limited length o ood chains. I we consider the diet o a top carnivore that is at the end o a ood chain, we can work out how many stages there are in the ood chain leading up to it. For example, i an osprey eeds on sh such as salmon that ed on shrimps, which ed on phytoplankton, there are our stages in the ood chain. There are rarely more than our or ve stages in a ood chain. We might expect ood chains to be limitless, with one species being eaten by another ad innitum. This does not happen. In ecology, as in all branches o science, we try to explain natural phenomena such as the restricted length o ood chains using scientic theories. In this case it is the concept o energy fow along ood chains and the energy losses that occur between trophic levels that can provide an explanation. Energy losses and ecosystems Energy losses between trophic levels restrict the length o ood chains and the biomass o higher trophic levels. Figure 6 An inrared camera image o an Arican grey parrot (Psittacus erithacus) shows how much heat is being released to the environment by dierent parts o its body Biomass is the total mass o a group o organisms. It consists o the cells and tissues o those organisms, including the carbohydrates and other carbon compounds that they contain. Because carbon compounds have chemical energy, biomass has energy. Ecologists can measure how much energy is added per year by groups o organisms to their biomass. The results are calculated per square metre o the ecosystem so that dierent trophic levels can be compared. When this is done, the same trend is always ound: the energy added to biomass by each successive trophic level is less. In secondary consumers, or example, the amount o energy is always less per year per square metre o ecosystem than in primary consumers. The reason or this trend is loss o energy between trophic levels. Most o the energy in ood that is digested and absorbed by organisms in a trophic level is released by them in respiration or Figure 7 The osprey (Pandion halietus) is a fsh-eating top carnivore 217 41 E c o lo g y use in cell activities. It is thereore lost as heat. The only energy available to organisms in the next trophic level is chemical energy in carbohydrates and other carbon compounds that have not been used up in cell respiration. activity Slmon nd soy Most salmon eaten by humans is produced in sh arms. The salmon have traditionally been ed on sh meal, mostly based on anchovies harvested o the coast o South America. These have become scarce and expensive. Feeds based on plant products such as soy beans are increasingly being used. In terms o energy ow, which o these human diets is most and least efcient? 1 Salmon ed on sh meal 2 Salmon ed on soy beans 3 Soy beans. The organisms in a trophic level are not usually entirely consumed by organisms in the next trophic level. For example, locusts sometimes consume all the plants in an area but more usually only parts o some plants are eaten. Predators may not eat material rom the bodies o their prey such as bones or hair. E nergy in uneaten material passes to saprotrophs or detritivores rather than passing to organisms in the next trophic level. Not all parts o ood ingested by the organisms in a trophic level are digested and absorbed. Some material is indigestible and is egested in eces. Energy in eces does not pass on along the ood chain and instead passes to saprotrophs or detritivores. B ecause o these losses, only a small proportion o the energy in the biomass o organisms in one trophic level will ever become part o the biomass o organisms in the next trophic level. The fgure o 1 0% is oten quoted, but the level o energy loss between trophic levels is variable. As the losses occur at each stage in a ood chain, there is less and less energy available to each successive trophic level. Ater only a ew stages in a ood chain the amount o energy remaining would not be enough to support another trophic level. For this reason the number o trophic levels in ood chains is restricted. B iomass, measured in grams, also diminishes along ood chains, due to loss o carbon dioxide and water rom respiration and loss rom the ood chain o uneaten or undigested parts o organisms. The biomass o higher trophic levels is thereore usually smaller than that o lower levels. There is generally a higher biomass o producers, the lowest trophic level o all, than o any other trophic level. decomposers (16,000 kJ m 2 yr1 ) secondary consumer (200 kJ m 2 yr1 ) primary consumer (2,500 kJ m 2 yr1 ) plankton (150,000 kJ m 2 yr1 ) Figure 8 An energy pyramid for an aquatic ecosystem (not to scale) secondary consumer (3,000 MJ m 2 yr1 ) primary consumer (7,000 MJ m 2 yr1 ) producers (50,000 MJ m 2 yr1 ) Figure 9 218 Pyramid of energy for grassland Pyramids of energy Quantitative representations o energy ow using pyramids o energy. The amount o energy converted to new biomass by each trophic level in an ecological community can be represented with a pyramid o energy. This is a type o bar chart with a horizontal bar or each trophic level. The amounts o energy should be per unit area per year. Oten the units are kilojoules per metre squared per year (kJ m -2 yr-1 ) . The pyramid should be stepped, not triangular, starting with the producers in the lowest bar. The bars should be labelled producer, frst consumer, second consumer and so on. I a suitable scale is chosen, the length o each bar can be proportional to the amount o energy that it shows. Figure 8 shows an example o a pyramid o energy or an aquatic ecosystem. To be more accurate, the bars should be drawn with relative widths that match the relative energy content at each trophic level. Figure 9 shows a pyramid o energy or grassland, with the bars correctly to scale. 4. 2 e n erG y Flo w dt-bs qustis: a simple food web A sinkhole is a surace eature which orms when an underground cavern collapses. Montezuma Well in the Sonoran desert in Arizona is a sinkhole flled with water. It is an aquatic ecosystem that lacks fsh, due in part to the extremely high concentrations o dissolved C O 2 . The dominant top predator is Belostoma bakeri, a giant water insect that can grow to 70 mm in length. Figure 1 0 shows a ood web or Montezuma Well. 1 2 3 4 5 6 7 C ompare the roles o Belostoma bakeri and Ranatra montezuma within the ood web. [2 ] D educe, with a reason, which organism occupies more than one trophic level. [2 ] D educe using P values: a) what would be the most common ood chain in this web [2 ] b) what is the preerred prey o B. bakeri? [1 ] C onstruct a pyramid o energy or the frst and second trophic levels. [3 ] C alculate the percentage o energy lost between the frst and second trophic levels. [2 ] D iscuss the difculties o classiying organisms into trophic levels. [2 ] Outline the additional inormation that would be required to complete the pyramid o energy or the third and ourth trophic level. [1 ] Ranatra montezuma 235,000 kJ ha 1 yr1 P = 1.0 gm 2 yr1 Belostoma bakeri 588,000 kJ ha 1 yr1 P = 2.8 gm 2 yr1 Telebasis salva 1,587,900 kJ ha 1 yr1 P = 7.9 gm 2 yr1 Hyalella montezuma 30,960,000 kJ ha 1 yr1 P = 215 gm 2 yr1 phytoplankton - Metaphyton 234,342,702 kJ ha 1 yr1 P = 602 g C m 2 yr1 piphyton 427,078,320 kJ ha 1 yr1 P = 1,096 g C m 2 yr1 Figure 10 A food web for Montezuma Well. P values represent the biomass stored in the population of that organism each year. Energy values represent the energy equivalent of that biomass. Arrows indicate trophic linkages and arrow thickness indicates the relative amount of energy transferred between trophic levels 219 41 E c o lo g y 4.3 carbon yling Understanding Appliations Autotrophs convert carbon dioxide into carbohydrates and other carbon compounds. In aquatic habitats carbon dioxide is present as a dissolved gas and hydrogen carbonate ions. Carbon dioxide diuses rom the atmosphere or water into autotrophs. Carbon dioxide is produced by respiration and diuses out o organisms into water or the atmosphere. Methane is produced rom organic matter in anaerobic conditions by methanogenic archaeans and some diuses into the atmosphere. Methane is oxidized to carbon dioxide and water in the atmosphere. Peat orms when organic matter is not ully decomposed because o anaerobic conditions in waterlogged soils. Partially decomposed organic matter rom past geological eras was converted into oil and gas in porous rocks or into coal. Carbon dioxide is produced by the combustion o biomass and ossilized organic matter. Animals such as ree-building corals and molluscs have hard parts that are composed o calcium carbonate and can become ossilized in limestone. Estimation o carbon fuxes due to processes in the carbon cycle. Analysis o data rom atmosphere monitoring stations showing annual fuctuations. Skills Construct a diagram o the carbon cycle. Nature o siene Making accurate, quantitative measurements: it is important to obtain reliable data on the concentration o carbon dioxide and methane in the atmosphere. carbon fxation Autotrophs convert carbon dioxide into carbohydrates and other carbon compounds. Autotrophs absorb carbon dioxide from the atmosphere and convert it into carbohydrates, lipids and all the other carbon compounds that they require. This has the effect of reducing the carbon dioxide concentration of the atmosphere. The mean C O 2 concentration of the atmosphere is currently approximately 0.03 9% or 3 90 micromoles per mole ( mol/mol) but it is lower above parts of the Earths surface where photosynthesis rates have been high. 220 4 . 3 c ar B o n c ycli n G dt-bse quests: Carbon dioxide concentration The two maps in fgure 1 were produced by NAS A. They show the carbon dioxide concentration o the atmosphere eight kilometres above the surace o the E arth, in May and O ctober 2 01 1 . 1 S tate whether O ctober is in the spring or all( autumn) in the southern hemisphere. [1 ] 2 a) D istinguish between carbon dioxide concentrations in May and O ctober in the northern hemisphere. [1 ] b) Suggest reasons or the dierence. [2 ] a) Distinguish between the carbon dioxide concentrations in May between the northern and the southern hemisphere. [1 ] b) S uggest reasons or the dierence. [2 ] 3 4 a) D educe the part o the Earth that had the lowest mean carbon dioxide concentration between May and O ctober 2 01 1 . [1 ] b) S uggest reasons or the carbon dioxide concentration being lowest in this area. [2 ] Figure 1 carbon dioxide in solution In aquatic habitats carbon dioxide is present as a dissolved gas and hydrogen carbonate ions. C arbon dioxide is soluble in water. It can either remain in water as a dissolved gas or it can combine with water to orm carbonic acid ( H 2 C O 3 ) . C arbonic acid can dissociate to orm hydrogen and hydrogen carbonate ions ( H + and HC O -3 ) . This explains how carbon dioxide can reduce the pH o water. B oth dissolved carbon dioxide and hydrogen carbonate ions are absorbed by aquatic plants and other autotrophs that live in water. They use them to make carbohydrates and other carbon compounds. Absorption of arbon dioxide Carbon dioxide difuses rom the atmosphere or water into autotrophs. Autotrophs use carbon dioxide in the production o carbon compounds by photosynthesis or other processes. This reduces the concentration o carbon dioxide inside autotrophs and sets up a concentration gradient between cells in autotrophs and the air or water around. C arbon dioxide thereore diuses rom the atmosphere or water into autotrophs. In land plants with leaves this diusion usually happens through stomata in the underside o the leaves. In aquatic plants the entire surace o the leaves and stems is usually permeable to carbon dioxide, so diusion can be through any part o these parts o the plant. atvt pH hges k ps Ecologists have monitored pH in rock pools on sea shores that contain animals and also photosynthesizing algae. The pH o the water rises and alls in a 24-hour cycle, due to changes in carbon dioxide concentration in the water. The lowest values o about pH 7 have been ound during the night, and the highest values o about pH 10 have been ound when there was bright sunlight during the day. What are the reasons or these maxima and minima? The pH in natural pools or articial aquatic mesocosms could be monitored using data loggers. 221 41 E c o lo g y Release of carbon dioxide from cell respiration Carbon dioxide is produced by respiration and difuses out o organisms into water or the atmosphere. C arbon dioxide is a waste product o aerobic cell respiration. It is produced in all cells that carry out aerobic cell respiration. These can be grouped according to trophic level o the organism: non-photosynthetic cells in producers or example root cells in plants animal cells saprotrophs such as ungi that decompose dead organic matter. C arbon dioxide produced by respiration diuses out o cells and passes into the atmosphere or water that surrounds these organisms. data-base questions: Data-logging pH in an aquarium 1 E xplain the changes in light intensity during the experiment. [2 ] 2 D etermine how many days the data logging covers. [2 ] a) D educe the trend in pH in the light. [1 ] b) Explain this trend. [2 ] 3 pH sensor (pH) 7.50 100 light intensity pH 90 7.45 80 70 7.40 60 50 7.35 40 30 7.30 20 light intensity /arbitrary units Figure 2 shows the pH and light intensity in an aquarium containing a varied community o organisms including pondweeds, newts and other animals. The data was obtained by data logging using a pH electrode and a light meter. The aquarium was illuminated articially to give a 24-hour cycle o light and dark using a lamp controlled by a timer. 10 7.25 0.14:02:31 0.23:13:11 3.08:23:50 4.17:34:30 06 February 2013 14:02:31 absolute time (d.hh:mm:ss) 0 6.02:45:09 Figure 2 4 a) D educe the trend in pH in darkness. [1 ] b) Explain this trend. [2 ] Methanogenesis Methane is produced rom organic matter in anaerobic conditions by methanogenic archaeans and some difuses into the atmosphere. In 1 776 Alessandro Volta collected bubbles o gas emerging rom mud in a reed bed on the margins o Lake Maggiore in Italy, and ound that it was infammable. He had discovered methane, though Volta did not give it this name. Methane is produced widely in anaerobic environments, as it is a waste product o a type o anaerobic respiration. Three dierent groups o anaerobic prokaryotes are involved. 1 222 B acteria that convert organic matter into a mixture o organic acids, alcohol, hydrogen and carbon dioxide. 4 . 3 c ar B o n c ycli n G 2 B acteria that use the organic acids and alcohol to produce acetate, carbon dioxide and hydrogen. 3 Archaeans that produce methane rom carbon dioxide, hydrogen and acetate. They do this by two chemical reactions: C O 2 + 4H 2 C H 4 + 2 H 2 O C H3C O O H C H4 + C O 2 The archaeans in this third group are thereore methanogenic. They carry out methanogenesis in many anaerobic environments: Mud along the shores and in the bed o lakes. Swamps, mires, mangrove orests and other wetlands where the soil or peat deposits are waterlogged. Guts o termites and o ruminant mammals such as cattle and sheep. Landfll sites where organic matter is in wastes that have been buried. S ome o the methane produced by archaeans in these anaerobic environments diuses into the atmosphere. C urrently the concentration in the atmosphere is between 1 .7 and 1 .85 micromoles per mole. Methane produced rom organic waste in anaerobic digesters is not allowed to escape and instead is burned as a uel. Figure 3 Waterlogged woodlanda typical habitat for methanogenic prokaryotes oxidatin f methane Methane is oxidized to carbon dioxide and water in the atmosphere. Molecules o methane released into the atmosphere persist there on average or only 1 2 years, because it is naturally oxidized in the stratosphere. Monatomic oxygen ( O ) and highly reactive hydroxyl radicals ( O H ) are involved in methane oxidation. This explains why atmospheric concentrations are not high, despite large amounts o production o methane by both natural processes and human activities. Peat frmatin Peat forms when organic matter is not fully decomposed because of anaerobic conditions in waterlogged soils. In many soils all o rganic matte r such as de ad le ave s rom plants is e ve ntually dige ste d by saprotrophic b acte ria and ungi. S apro trop hs o btain the oxygen that they ne ed or re spiration rom air spaces in the so il. In some e nvironments water is unable to drain o ut o so ils so they be co me wate rlogged and anae rob ic. S ap rotrophs cannot thrive in these co nditions so de ad organic matter is not ully deco mposed. Acidic conditions te nd to de ve lo p, urthe r inhib iting sapro trop hs and also me thanogens that might b re ak down the o rganic matte r. Figure 4 Peat deposits form a blanket on a boggy hill top at Bwlch Groes in North Wales 223 41 E c o lo g y data-base questions: Release of carbon from tundra soils Soils in tundra ecosystems typically contain large amounts o carbon in the orm o peat. This accumulates because o low rates o decomposition o dead plant organic matter by saprotrophs. To investigate this, ecologists collected samples o soil rom areas o tussock vegetation near Toolik Lake in Alaska. Some o the areas had been ertilized with nitrogen and phosphorus every year or the previous eight years (TF) and some had not (TC ) . The soils were incubated or 1 00-day periods at either 7 or 1 5C. Some samples were kept moist (M) and others were saturated with water (W) . The initial carbon content o the soils was measured and the amount o carbon dioxide given o during the experiment was monitored. The bar chart in fgure 5 shows the results. 1 S tate the eect o increasing the temperature o the soils on the rate o release o carbon. b) Explain the reasons or this eect. 40 TC percentage of initial C a) 30 2 TF a) 7M 7W 15M treatment group 15W [2 ] 3 O utline the eects o ertilizers on rates o release o carbon rom the soils. [2 ] 4 D iscuss whether dierences in temperature, amount o water in the soil or amount o ertilizer have the greatest impact on the release o carbon. [2 ] 10 0 [2 ] C ompare the rates o release o carbon in moist soils with those in soils saturated with water. [2 ] b) S uggest reasons or the dierences. 20 [2 ] Figure 5 Large quantities o partially decomposed organic matter have accumulated in some ecosystems and become compressed to orm a dark brown acidic material called peat. About 3 % o the Earths land surace is covered by peat and as the depth is ten metres or more in some places, the total quantities o this material are immense. Fossilized organic matter Partially decomposed organic matter from past geological eras was converted into oil and gas in porous rocks or into coal. C arbon and some compounds o carbon are chemically very stable and can remain unchanged in rocks or hundreds o millions o years. There are large deposits o carbon rom past geological eras. These deposits are the result o incomplete decomposition o organic matter and its burial in sediments that became rock. Figure 6 Coal at a power station 224 C oal is ormed when deposits o peat are buried under other sediments. The peat is compressed and heated, gradually turning into coal. Large coal deposits were ormed during the Pennsylvanian subperiod o the C arbonierous. There was a cycle o sea level rises and alls; coastal swamps ormed as the level ell and were destroyed and buried when the level rose and the sea spread inland. Each cycle has let a seam o coal. 4 . 3 c ar B o n c ycli n G Oil and natural gas are ormed in the mud at the bottom o seas and lakes. C onditions are usually anaerobic and so decomposition is oten incomplete. As more mud or other sediments are deposited the partially decomposed matter is compressed and heated. C hemical changes occur, which produce complex mixtures o liquid carbon compounds or gases. We call these mixtures crude oil and natural gas. Methane orms the largest part o natural gas. Deposits are ound where there are porous rocks that can hold them such as shales and also impervious rocks above and below the porous rocks that prevent the deposits escape. combustion Carbon dioxide is produced by the combustion of biomass and fossilized organic matter. I organic matter is heated to its ignition temperature in the presence o oxygen it will set light and burn. The oxidation reactions that occur are called combustion. The products o complete combustion are carbon dioxide and water. Figure 7 Carbon dioxide is released by combustion of the leaves of sugar cane In some parts o the world it is natural or there to be periodic fres in orests or grassland. C arbon dioxide is released rom the combustion o the biomass in the orest or grassland. In these areas the trees and other organisms are oten well adapted to fres and communities regenerate rapidly aterwards. In other areas fres due to natural causes are very unusual, but humans sometimes cause them to occur. Fire is used to clear areas o tropical rainorest or planting oil palms or or cattle ranching. C rops o sugar cane are traditionally burned shortly beore they are harvested. The dry leaves burn o, leaving the harvestable stems. C oal, oil and natural gas are dierent orms o ossilized organic matter. They are all burned as uels. The carbon atoms in the carbon dioxide released may have been removed rom the atmosphere by photosynthesizing plants hundreds o millions o years ago. limestone Figure 8 Kodonophylluma Silurian coral, in limestone from Wenlock Edge. The calcium carbonate skeletons of the coral are clearly visible embedded in more calcium carbonate that precipitated 420 million years ago in shallow tropical seas Animals such as reef-building corals and molluscs have hard parts that are composed of calcium carbonate and can become fossilized in limestone. S ome animals have hard body parts composed o calcium carbonate ( C aC O 3 ) : mollusc shells contain calcium carbonate; hard corals that build rees produce their exoskeletons by secreting calcium carbonate. When these animals die, their sot parts are usually decomposed quickly. In acid conditions the calcium carbonate dissolves away but in neutral or alkaline conditions it is stable and deposits o it rom hard animal parts can orm on the sea bed. In shallow tropical seas calcium Figure 9 Chalk cliffs on the south coast of England. Chalk is a form of limestone that consists almost entirely of 90-million-yearold shells of tiny unicellular animals called foraminifera 225 41 E c o lo g y carbonate is also deposited by precipitation in the water. The result is limestone rock, where the deposited hard parts o animals are oten visible as ossils. Approximately 1 0% o all sedimentary rock on Earth is limestone. About 1 2 % o the mass o the calcium carbonate is carbon, so huge amounts o carbon are locked up in limestone rock on Earth. carbon yle diagrams Construct a diagram of the carbon cycle. Ecologists studying the carbon cycle and the recycling o other elements use the terms pool and fux. A pool is a reserve o the element. It can be organic or inorganic. For example the carbon dioxide in the atmosphere is an inorganic pool o carbon. The biomass o producers in an ecosystem is an organic pool. A fux is the transer o the element rom one pool to another. An example o carbon fux is the absorption o carbon dioxide rom the atmosphere and its conversion by photosynthesis to plant biomass. D iagrams can be used to represent the carbon cycle. Text boxes can be used or pools and labeled arrows or fuxes. Figure 1 0 shows an illustrated diagram which can be converted to a diagram o text boxes and arrows. Figure 1 0 only shows the carbon cycle or terrestrial ecosystems. A separate diagram could be constructed or marine or aquatic ecosystems, or a combined diagram or all ecosystems. In marine and aquatic ecosystems, the inorganic reserve o carbon is dissolved carbon dioxide and hydrogen carbonate, which is absorbed by producers and by various means is released back into the water. CO 2 in atmosphere fu e l s cell respiration in saprotrophs and detritivores ce in pr ll r od es uc pi r er at tos ynt carbon in organic compounds in producers co m b feeding egestion incomplete decomposition and fossilization of organic matter coal Figure 10 Carbon cycle 226 oil and gas is ion death carbon in dead organic matter hes s u stio n of f ossil cell respiration in consumers pho 4 . 3 c ar B o n c ycli n G carbon fuxes Estimation o carbon fuxes due to processes in the carbon cycle. The carbon cycle diagram in gure 1 0 shows processes that transer carbon rom one pool to another but it does not show the quantities o these fuxes. It is not possible to measure global carbon fuxes precisely but as these quantities are o great interest, scientists have produced estimates or them. Estimates are based on many measurements in individual natural ecosystems or in mesocosms. Fux/ggtes e- 1 120 119.6 92.8 90.0 1.6 Pess Photosynthesis Cell respiration Ocean uptake Ocean loss Deorestation and land use changes Burial in marine sediments Combustion o ossil uels Global carbon fuxes are extremely large so estimates are in gigatonnes (petagrams) . One gigatonne is 1 ,01 5 grams. Table 1 shows estimates based on Ocean Biogeochemical Dynamics, Sarmiento and Gruber, 2006, Princeton University Press. 0.2 6.4 Table 1 dt-bse quests: Oak woodland and carbon dioxide concentrations C arbon fuxes have been measured since 1 998 in deciduous woodland at Alice Holt Research Forest in E ngland. The trees are mainly oaks, Quercus robur and Quercus petraea, with some ash, Fraxinus excelsior. They were planted in 1 93 5 and are now nearly 2 0 metres tall. C arbon dioxide concentrations are measured 2 0 times a second. From these measurements the net ecosystem production can be deduced. This is the net fux o carbon dioxide between the orest and the atmosphere. Positive values indicate an increase in the carbon pool o the orest and negative values indicate a decrease due to net loss o carbon dioxide. The graph shows the daily average net ecosystem production or several years and also the cumulative net ecosystem production. 1 C alculate whether the carbon pool in the biomass o the orest increases or decreases on more days in the year. [1 ] 2 D educe the months in which the carbon pool o biomass in the orest was highest and lowest. [2 ] 3 Explain the reasons or increases in the carbon pool o biomass in the orest during part o the year and decreases in other parts. 4 State the annual carbon fux to or rom the orest. [2 ] 5 Suggest a reason based on the data or encouraging the planting o more oak orests. [1 ] 25 20 15 15 10 10 5 5 0 0 50 100 150 200 250 300 530 5 cumulative NEP (t CO 2 ha 1 ) daily average NEP (kg CO 2 ha 1 h 1 ) 20 0 [4] 5 10 10 day of year 15 227 41 E c o lo g y Environmental monitoring Making accurate, quantitative measurements: it is important to obtain reliable data on the concentration o carbon dioxide and methane in the atmosphere. C arbon dioxide and methane concentrations in the atmosphere have very important eects. C arbon dioxide concentrations aect photosynthesis rates and the pH o seawater. B oth gases infuence global temperatures and as a result the extent o ice sheets at the poles. Indirectly they thereore aect sea levels and the position o coast lines. Through their eects on the amount o heat energy in the oceans and the atmosphere they aect ocean currents, the distribution o rainall and also the requency and severity o extreme weather events such as hurricanes. C onsider these hypotheses and predictions: The carbon dioxide concentration o the atmosphere is currently higher than at any time in the past twenty million years. Human activities have increased the carbon dioxide and methane concentrations in the Earths atmosphere. Human activity will cause atmospheric carbon dioxide concentrations to rise rom 3 97 micromoles per mole in 2 01 4 to a level above 600 by the end o the century. Reliable data are an essential prerequisite or evaluating hypotheses and predictions such as these. Reliable measurements o atmospheric carbon dioxide and methane concentration are needed over as long a period as possible beore we can evaluate the past and possible uture consequences o human activity. D ata on concentrations o gases in the atmosphere is collected by the Global Atmosphere Watch programme o the World Meteorological O rganization, an agency o the United Nations. Research stations in various parts o the world now monitor the atmosphere, but Mauna Loa O bservatory on Hawaii has records rom the longest period. C arbon dioxide concentrations have been measured rom 1 95 9 onwards and methane rom 1 984. These and other reliable records are o immense value to scientists. Trends in atmospheric carbon dioxide Analysis o data rom atmosphere monitoring stations showing annual fuctuations. D ata rom atmosphere monitoring stations is reely available allowing any person to analyse it. There are both long- term trends and annual fuctuations in the data. The Mauna Loa O bservatory in Hawaii produces vast amounts o data and data rom this and other monitoring stations are available or analysis. Figure 11 Hawaii from space. Mauna Loa is near the centre of the largest island 228 4 . 4 c l i m at e c H a n G e 4.4 c hg Understandin Carbon dioxide and water vapour are the most signicant greenhouse gases. Other gases including methane and nitrogen oxides have less impact. The impact o a gas depends on its ability to absorb long-wave radiation as well as on its concentration in the atmosphere. The warmed Earth emits longer-wave radiation (heat) . Longer-wave radiation is reabsorbed by greenhouse gases which retains the heat in the atmosphere. Global temperatures and climate patterns are infuenced by concentrations o greenhouse gases. There is a correlation between rising atmospheric concentrations o carbon dioxide since the start o the industrial revolution two hundred years ago and average global temperatures. Recent increases in atmospheric carbon dioxide are largely due to increases in the combustion o ossilized organic matter. Applications Correlations between global temperatures and carbon dioxide concentrations on Earth. Evaluating claims that human activities are not causing climate change. Threats to coral rees rom increasing concentrations o dissolved carbon dioxide. Nature of science Assessing claims: assessment o the claims that human activities are not causing climate change. greenhouse ases Carbon dioxide and water vapour are the most signicant greenhouse gases. The Earth is kept much warmer than it otherwise would be by gases in the atmosphere that retain heat. The effect of these gases has been likened to that of the glass that retains heat in a greenhouse and they are therefore known as greenhouse gases, though the mechanism of heat retention is not the same. The greenhouse gases that have the largest warming effect on the Earth are carbon dioxide and water vapour. C arbon dioxide is released into the atmosphere by cell respiration in living organisms and also by combustion of biomass and fossil 229 41 E c o lo g y uels. It is removed rom the atmosphere by photosynthesis and by dissolving in the oceans. Water vapour is ormed by evaporation rom the oceans and also transpiration in plants. It is removed rom the atmosphere by rainall and snow. Water continues to retain heat ater it condenses to orm droplets o liquid water in clouds. The water absorbs heat energy and radiates it back to the Earths surace and also refects the heat energy back. This explains why the temperature drops so much more quickly at night in areas with clear skies than in areas with cloud cover. other greenhuse gases Other gases including methane and nitrogen oxides have less impact. Figure 1 Satellite image of Hurricane Andrew in the Gulf of Mexico. Hurricanes are increasing in frequency and intensity as a result of increases in heat retention by greenhouse gases Although carbon dioxide and water vapour are the most signicant greenhouse gases there are others that have a smaller but nonetheless signicant eect. Methane is the third most signicant greenhouse gas. It is emitted rom marshes and other waterlogged habitats and rom landll sites where organic wastes have been dumped. It is released during extraction o ossil uels and rom melting ice in polar regions. Nitrous oxide is another signicant greenhouse gas. It is released naturally by bacteria in some habitats and also by agriculture and vehicle exhausts. The two most abundant gases in the Earths atmosphere, oxygen and nitrogen, are not greenhouse gases as they do not absorb longer- wave radiation. All o the greenhouse gases together thereore make up less than 1 % o the atmosphere. Assessing the impact f greenhuse gases The impact of a gas depends on its ability to absorb long-wave radiation as well as on its concentration in the atmosphere. Two actors together determine the warming impact o a greenhouse gas: how readily the gas absorbs long- wave radiation; and the concentration o the gas in the atmosphere. For example, methane causes much more warming per molecule than carbon dioxide, but as it is at a much lower concentration in the atmosphere its impact on global warming is less. The concentration o a gas depends on the rate at which it is released into the atmosphere and how long on average it remains there. The rate at which water vapour enters the atmosphere is immensely rapid, but it remains there only nine days on average, whereas methane remains in the atmosphere or twelve years and carbon dioxide or even longer. 230 4 . 4 c l i m at e c H a n G e lon-waveenth emissions from Earth TOK The warmed Earth emits longer-wave radiation. Qusos xs bou h ry o sf phoo. wh osqus gh hs hv or h pub prpo d udrsdg o s? The warmed surface of the Earth absorbs short- wave energy from the sun and then re- emits it, but at much longer wavelengths. Most of the re- emitted radiation is infrared, with a peak wavelength of 1 0, 000 nm. The peak wavelength of solar radiation is 400 nm. spectral intensity Figure 2 shows the range of wavelengths of solar radiation that pass through the atmosphere to reach the Earths surface and warm it ( red) and the range of much longer wavelengths emitted by the Earth that pass out through the atmosphere ( blue) . The smooth red and blue curves show the range of wavelengths expected to be emitted by bodies of the temperature of the Earth and the sun. UV 0.2 Visible Much o what science investigates involves entities and concepts beyond everyday experience o the world, such as the nature and behaviour o electromagnetic radiation or the build-up o invisible gases in the atmosphere. This makes it difcult or scientists to convince the general public that such phenomenon actually exist particularly when the consequences o accepting their existance might run counter to value systems or entrenched belies. Infrared 1 10 70 wavelength (m) Figure 2 greenhouse ases Longer-wave radiation is reabsorbed by greenhouse gases which retains the heat in the atmosphere. 2 5 3 0% of the short-wavelength radiation from the sun that is passing through the atmosphere is absorbed before it reaches the Earths surface. Most of the solar radiation absorbed is ultraviolet light, which is absorbed by ozone. 7075 % of solar radiation therefore reaches the Earths surface and much of this is converted to heat. A far higher percentage of the longer- wavelength radiation re-emitted by the surface of the Earth is absorbed before it has passed out to space. B etween 70% and 85 % is captured by greenhouse gases in the atmosphere. This energy is re- emitted, some towards the E arth. The effect is global warming. Without it the mean temperature at the Earths surface would be about 1 8C . Key short-wave radiation from the sun long-wave radiation from earth Figure 3 The greenhouse efect 231 41 E c o lo g y Greenhouse gases in the Earths atmosphere only absorb energy in specifc wavebands. Figure 4 below shows total percentage absorption o radiation by the atmosphere. The graph also shows the bands o wavelengths absorbed by individual gases. The wavelengths re-emitted by the E arth are between 5 and 70nm. Water vapour, carbon dioxide, methane and nitrous oxide all absorb some o these wavelengths, so each o them is a greenhouse gas. percent 100 75 Total absorption and scattering 50 25 0 0.2 1 10 70 major components Water vapour Carbon dioxide Oxygen and ozone Methane Nitrous oxide 0.2 1 10 70 wavelength (m) Figure 4 global temperatures and carbon dioxide concentrations Correlations between global temperatures and carbon dioxide concentrations on Earth. I the concentration o any o the greenhouse gases in the atmosphere changes, we can expect the size o its contribution to the greenhouse eect to change and global temperatures to rise or all. We can test this hypothesis using the carbon dioxide concentration o the atmosphere, because it has changed considerably. To deduce carbon dioxide concentrations and temperatures in the past, columns o ice have been drilled in the Antarctic. The ice has built up over thousands o years, so ice rom deeper down is older than ice near the surace. B ubbles o air trapped in the ice can be extracted and analysed to fnd the carbon dioxide concentration. Global temperatures can be deduced rom ratios o hydrogen isotopes in the water molecules. Figure 5 shows results or an 800, 000 year period beore the present. They were obtained rom an ice core drilled in D ome C on the Antarctic plateau by the European Proj ect or Ice C oring in 232 Antarctica. D uring this part o the current Ice Age there has been a repeating pattern o rapid periods o warming ollowed by much longer periods o gradual cooling. There is a very striking correlation between carbon dioxide concentration and global temperatures the periods o higher carbon dioxide concentration repeatedly coincide with periods when the Earth was warmer. The same trend has been ound in other ice cores. D ata o this type are consistent with the hypothesis that rises in carbon dioxide concentration increase the greenhouse eect. It is important always to remember that correlation does not prove causation, but in this case we know rom other research that carbon dioxide is a greenhouse gas. At least some o the temperature variation over the past 800,000 years must thereore have been due to rises and alls in atmospheric carbon dioxide concentrations. 4 . 4 c l i m at e c H a n G e CO 2 /ppmv 300 250 D/% (temperature proxy) 200 -380 warm 9C -410 -440 cold 800,000 600,000 400,000 age (years before present) 200,000 0 Figure 5 Data from the European Project for Ice Coring in the Antarctic Dome C ice core d-bs qusos: CO 2 concentrations and global temperatures 0.6 temperature anomaly (C) Figure 6 shows atmospheric carbon dioxide concentrations. The red line shows direct measurements at Mauna Loa O bservatory. The points show carbon dioxide concentrations measured rom trapped air in polar ice cores. parts per million by volume 380 360 Annual average Five year average 0.2 0 -0.2 Direct measurments Ice core measurments -0.4 340 1880 320 1900 1920 1940 1960 1980 2000 Figure 7 300 2 280 260 1750 1800 1850 1900 1950 2000 3 Figure 6 Figure 7 shows a record o global average temperatures compiled by the NAS A Goddard Institute or S pace S tudies. The green points are annual averages and the red curve is a rolling ve-year average. The values are given as the deviation rom the mean temperature between 1 961 and 1 990. 1 0.4 D iscuss whether the measurements o carbon dioxide concentration rom ice cores are consistent with direct measurements at Mauna Loa. [2 ] 4 C ompare the trends in carbon dioxide concentration and global temperatures between 1 880 and 2 008. [2 ] Estimate the change in global average temperature between a) 1 900 and 2 000 [1 ] b) 1 905 and 2 005 [1 ] a) S uggest reasons or global average temperatures alling or a ew years during a period with an overall trend o rising temperatures. [2 ] b) D iscuss whether these alls indicate that carbon dioxide concentration does not infuence global temperatures. [2 ] 233 41 E c o lo g y greenhouse ases and climate patterns Global temperatures and climate patterns are infuenced by concentrations o greenhouse gases. The surace o the Earth is warmer than it would be with no greenhouse gases in the atmosphere. Mean temperatures are estimated to be 3 2 C higher. I the concentration o any o the greenhouse gases rises, more heat will be retained and we should expect an increase in global average temperatures. This does not mean that global average temperatures are directly proportional to greenhouse gas concentrations. O ther actors have an infuence, including Milankovitch cycles in the E arths orbit and variation in sunspot activity. Even so, increases in greenhouse gas concentrations will tend to cause higher global average temperatures and also more requent and intense heat waves. Global temperatures infuence other aspects o climate. Higher temperatures increase the evaporation o water rom the oceans and thereore periods o rain are likely to be more requent and protracted. The amount o rain delivered during thunderstorms and other intense bursts is likely to increase very signicantly. In addition, higher ocean temperatures cause tropical storms and hurricanes to be more requent and more powerul, with aster wind speeds. The consequences o any rise in global average temperature are unlikely to be evenly spread. Not all areas would become warmer. The west coast o Ireland and Scotland might become colder i the North Atlantic C urrent brought less warm water rom the Gul Stream to north-west Europe. The distribution o rainall would also be likely to change, with some areas becoming more prone to droughts and other areas to intense periods o rainall and fooding. Predictions about changes to weather patterns are very uncertain, but it is clear that just a ew degrees o warming would cause very proound changes to the Earths climate patterns. data-base questions: Phenology Phenologists are biologists who study the timing o seasonal activities in animals and plants, such as the opening o tree leaves and the laying o eggs by birds. Data such as these can provide evidence o climate changes, including global warming. 2 Identiy the year in which: a) the leaves opened earliest [1 ] b) mean temperatures in March and April were at their lowest. [1 ] Use the data in the graph to deduce the ollowing: a) the relationship between temperatures in March and April and the date o opening o leaves on horse chestnut trees. [1 ] b) whether there is evidence o global warming towards the end o the 2 0th century. -15 4 3 2 1 0 -1 -2 -3 -4 -10 -5 0 5 10 1970 1980 1990 year 234 1 15 2000 dierence in date of leaf opening / days dierence in mean temperature / C The date in the spring when new leaves open on horse chestnut trees ( Aesculus hippocastaneum) has been recorded in Germany every year since 1 95 1 . Figure 8 shows the dierence between each years date o lea opening and the mean date o lea opening between 1 970 and 2 000. Negative values indicate that the date o lea opening was earlier than the mean. The graph also shows the dierence between each years mean temperature during March and April and the overall mean temperature or these two months. The data or temperature was obtained rom the records o 3 5 German climate stations. [2 ] Figure 8 The relationship between temperature and horse chestnut leaf opening in Germany since 1951 Key: temperature leaf opening 4 . 4 c l i m at e c H a n G e Industrialization and climate change There is a correlation between rising atmospheric concentrations o carbon dioxide since the start o the industrial revolution two hundred years ago and average global temperatures. The graph o atmospheric carbon dioxide concentrations over the past 800, 000 years shown in gure 5 indicates that there have been large fuctuations. D uring glaciations the concentration dropped to as low as 1 80 parts per million by volume. D uring warm interglacial periods they rose as high as 3 00 ppm. The rise during recent times to concentrations nearing 400 ppm is thereore unprecedented in this period. Atmospheric carbon dioxide concentrations were between 2 60 and 2 80 ppm until the late 1 8th century. This is when concentrations probably started to rise above the natural levels, but as the rise was initially very slight, it is impossible to say exactly when an unnatural rise in concentrations began. Much o the rise has happened since 1 95 0. Figure 9 During the industrial revolution renewable sources of power including wind were replaced with power generated by burning fossil fuels In the late 1 8th century the industrial revolution was starting in some countries but the main impact o industrialization globally was in the second hal o the 2 0th century. More countries became industrialized, and combustion o coal, oil and natural gas increased ever more rapidly, with consequent increases in atmospheric carbon dioxide concentration. There is strong evidence or a correlation between atmospheric carbon dioxide concentration and global temperatures, but as already explained, other actors have an eect so temperatures are not directly proportional to carbon dioxide concentration. Nevertheless, since the start o the industrial revolution the correlation between rising atmospheric carbon dioxide concentration and average global temperatures is very marked. Burning fossil fuels Recent increases in atmospheric carbon dioxide are largely due to increases in the combustion o ossilized organic matter. As the industrial revolution spread rom the late 1 8th century onwards, increasing quantities o coal were being mined and burned, causing carbon dioxide emissions. E nergy rom combustion o the coal provided a source o heat and power. D uring the 1 9 th century the combustion o oil and natural gas became increasingly widespread in addition to coal. Increases in the burning o ossil uels were most rapid rom the 1 95 0s onwards and this coincides with the period o steepest rises in atmospheric carbon dioxide. It seems hard to doubt the conclusion that the burning o ossil uels has been a maj or contributory actor in the rise o atmospheric carbon dioxide concentrations to higher levels than experienced on Earth or more than 800, 000 years. TOK wh osus upb v of rsk? In situations where the public is at risk, scientists are called upon to advise governments on the setting o policies or restrictions to oset the risk. Because scientic claims are based largely on inductive observation, absolute certainty is difcult to establish. The precautionary principle argues that action to protect the public must precede certainty o risk when the potential consequences or humanity are catastrophic. Principle 15 o the 1992 Rio Declaration on the Environment and Development stated the principle in this way: Where there are threats o serious or irreversible damage, lack o ull scientic certainty shall not be used as a reason or postponing cost-efective measures to prevent environmental degradation. 235 41 E c o lo g y data-base questions: Comparing CO 2 emissions The bar chart in gure 1 0 shows the cumulative CO 2 emissions rom ossil uels o the European Union and ve individual countries between 1 950 and 2000. It also shows the total CO 2 emissions including orest clearance and other land use changes. 1 D iscuss reasons or higher cumulative C O 2 emissions rom combustion o ossil uels in the United States than in B razil. [3 ] 2 Although cumulative emissions between 1 95 0 and 2 000 were higher in the United S tates than any other country, there were our countries in which emissions per capita were higher in the year 2 000: Qatar, United Arab Emirates, Kuwait and B ahrain. Suggest reasons or the dierence. [3 ] 3 Although cumulative C O 2 emissions rom combustion o ossil uels in Indonesia and B razil between 1 95 0 and 2000 were relatively low, total C O 2 emissions were signicantly higher. S uggest reasons or this. [3 ] 4 Australia ranked seventh in the world or emissions o C O 2 in 2 000, but ourth when all greenhouse gases are included. S uggest a reason or the dierence. [1 ] 30% Figure 10 CO 2 from fossil fuels CO 2 from fossil fuels & land-use change percent of world total 25% 20% 15% 10% 5% 0% U.S. EU-25 Russia China Indonesia Brazil Assessing claims and counter-claims Assessing claims: assessment of the claims that human activities are not causing climate change. C limate change has been more hotly debated than almost any other area o science. A search o the internet will quickly reveal diametrically opposed views, expressed very vocierously. The author Michael C richton portrayed climate change scientists as eco- terrorists who were prepared to use mass murder to promote their work in his novel S tate o Fear. What reasons could there be or such erce opposition to climate change science and or what reason do climate change scientists deend their ndings so vigorously? These questions are worth discussing. There are many actors that could be having an infuence: 236 Scientists are trained to be cautious about their claims and to base their ideas on evidence. They are expected to admit when there are uncertainties and this can give the impression that evidence is weaker than it actually is. Global climate patterns are very complex and it is dicult to make predictions about the consequences o urther increases in greenhouse gas concentrations. There can be tipping points in climate patterns where sudden massive changes occur. This makes prediction even more dicult. The consequences o changes in global climate patterns could be very severe or humans and or other species so many eel that there is a need or immediate action even i uncertainties remain in climate change science. C ompanies make huge prots rom coal, oil and natural gas and it is in their interests or ossil uel combustion to continue to grow. It would not be surprising i they paid or reports to be written that minimized the risks o climate change. 4 . 4 c l i m at e c H a n G e oppsitin t the climate change science Evaluating claims that human activities are not causing climate change. Many claims that human activities are not causing climate change have been made in newspapers, on television and on the internet. One example o this is: Global warming stopped in 1 998, yet carbon dioxide concentrations have continued to rise, so human carbon dioxide emissions cannot be causing global warming. This claim ignores the act that temperatures on Earth are infuenced by many actors, not j ust greenhouse gas concentrations. Volcanic activity and cycles in ocean currents can cause signicant variations rom year to year. B ecause o such actors, 1 998 was an unusually warm year and also because o them some recent years have been cooler than they otherwise would have been. Global warming is continuing but not with equal increases each year. Humans are emitting carbon dioxide by burning ossil uels and there is strong evidence that carbon dioxide causes warming, so the claim is not supported by the evidence. C laims that human activities are not causing climate change will continue and these claims need to be evaluated. As always in science, we should base our evaluations on reliable evidence. There is now considerable evidence about emissions o greenhouse gases by humans, about the eects o these gases and about changing climate patterns. Not all sources on the internet are trustworthy and we need to be careul to distinguish between websites with objective assessments based on reliable evidence and others that show bias. d-bs qusos: Uncertainty in temperature rise projections Figure 1 1 shows computer-generated orecasts or average global temperatures, based on eight dierent scenarios or the changes in the emissions o greenhouse gases. The light green band includes the ull range o orecasts rom research centres around the world, and the dark green band shows the range o most o the orecasts. Figure 1 2 shows orecasts or arctic temperatures, based on two o the emissions scenarios. 1 2 3 4 5 6 Identiy the code or the least optimistic emissions scenario. 6 5 4 3 2 AIB AIT AIFI A2 B1 B2 IS92a 1 0 0 0 0 0 0 0 0 0 0 0 0 0 199 200 201 202 203 204 205 206 207 208 209 210 [1 ] S tate the minimum and maximum orecasts or average global temperature change. [2 ] C alculate the dierence between the A2 and B 2 orecasts o global average temperature rise. [2 ] C ompare the orecasts or arctic temperatures with those or global average temperatures. [2 ] S uggest uncertainties, apart rom greenhouse gas emissions, which aect orecasts or average global temperatures over the next 1 00 years. [2 ] Discuss how much more condent we can be in orecasts based on data rom a number o dierent research centres, rather than one. [3 ] Figure 11 Forecast global average temperatures 7 Discuss whether the uncertainty in temperature orecasts justies action or inaction. [4] 8 D iscuss whether it is possible to balance environmental risks with socio- economic and livelihood risks or whether priorities need to be established. [4] 7 6 A2 B2 5 4 3 2 1 0 2000 2020 2040 2060 2080 2100 Figure 12 Forecast arctic temperature 237 41 E c o lo g y coral reefs and arbon dioxide Threats to coral rees rom increasing concentrations o dissolved carbon dioxide. In addition to its contribution to global warming, emissions o carbon dioxide are having eects on the oceans. Over 500 billion tonnes o carbon dioxide released by humans since the start o the industrial revolution have dissolved in the oceans. The pH o surace layers o the Earths oceans is estimated to have been 8.1 79 in the late 1 8th century when there had been little industrialization. Measurements in the mid-1 990s showed that it had allen to 8.1 04 and current levels are approximately 8.069. This seemingly small change represents a 30% acidication. Ocean acidication will become more severe i the carbon dioxide concentration o the atmosphere continues to rise. Marine animals such as ree- building corals that deposit calcium carbonate in their skeletons need to absorb carbonate ions rom seawater. The concentration o carbonate ions in seawater is low, because they are not very soluble. D issolved carbon dioxide makes the carbonate concentration even lower as a result o some interrelated chemical reactions. C arbon dioxide reacts with water to orm carbonic acid, which dissociates into hydrogen and hydrogen carbonate ions. Hydrogen ions react with dissolved carbonate ions, reducing their concentration. make their skeletons. Also, i seawater ceases to be a saturated solution o carbonate ions, existing calcium carbonate tends to dissolve, so existing skeletons o ree-building corals are threatened. In 2 01 2 oceanographers rom more than 2 0 countries met in S eattle and agreed to set up a global scheme or monitoring ocean acidication. There is already evidence or concerns about corals and coral rees. Volcanic vents near the island o Ischia in the Gul o Naples have been releasing carbon dioxide into the water or thousands o years, reducing the pH o the seawater. In the area o acidied water there are no corals, sea urchins or other animals that make their skeletons rom calcium carbonate. In their place other organisms fourish such as sea grasses and invasive algae. This could be the uture o coral rees around the world i carbon dioxide continues to be emitted rom burning ossil uels. C O 2 + H 2 O H 2 C O 3 H + + HC O -3 H + + C O 23 HC O 3 I carbonate ion concentrations drop it is more dicult or ree- building corals to absorb them to activity Draw a graph o oceanic pH rom the 18th century onwards, using the gures given in the text above, and extrapolate the curve to obtain an estimate o when the pH might drop below 7. 238 Figure 13 Skeleton of calcium carbonate from a reef-building coral TOK wht re the potentil impcts of funding bis? The costs o scientic research is oten met by grant agencies. Scientists submit research proposals to agencies, the application is reviewed and i successul, the research can proceed. Questions arise when the grant agency has a stake in the study's outcome. Further, grant applications might ask scientists to project outcomes or suggest applications o the research beore it has even begun. The sponsor may und several diferent research groups, suppressing results that run counter to their interests and publishing those that support their industry. For example, a 2006 review o studies examining the health efects o cell phone use revealed that studies unded by the telecommunications industry were statistically least likely to report a signicant efect. Pharmaceutical research, nutrition research and climate change research are all areas where claims o unding bias have been prominent in the media. QueStion S Questions a) C alculate the energy lost by plant respiration. [2 ] b) C onstruct a pyramid o energy or this grassland. [3 ] Drought Index The total solar energy received by a grassland is 5 l0 5 kJ m - 2 yr - 1 . The net production o the grassland is 5 1 0 2 kJ m - 2 yr - 1 and its gross production is 6 1 0 2 kJ m - 2 yr - 1 . The total energy passed on to primary consumers is 60 kJ m - 2 yr - 1 . O nly 1 0 per cent o this energy is passed on to the secondary consumers. Area of tree mortality/km 2 1 4 Warm/dry 3 long-term average 2 1 0 1 2 3 Cool/moist 2000 1500 1000 500 0 1930 1940 1950 1960 1970 1980 1990 2000 Figure 15 Tree mortality and drought index 2 a) Figure 1 4 shows the energy fow through a temperate orest. The energy fow is shown per square metre per year ( kJ m - 2 yr - 1 ) . lost 5,223,120 b) ( i) C ompare the beetle outbreaks in the 1 970s and 1 990s. [2 ] respiration 24,024 green plants 172 14,448 decomposers c) consumers storage (e.g. wood) 5,036 Figure 14 a) The chart shows that 99.1 7 per cent o the sunlight energy in the temperate orest is lost. Predict with a reason whether a greater or lesser percentage o sunlight energy would be lost in desert. [2 ] b) O nly a small part o the net production o plants in the temperate orest passes to herbivores. Explain the reasons or this. [2 ] 3 Warmer temperatures avour some species o pest, or example the spruce beetle. Since the rst maj or outbreak in 1 992 , it has killed approximately 400, 000 hectares o trees in Alaska and the C anadian Yukon. The beetle normally needs two years to complete its lie cycle, but it has recently been able to do it in one year. The graphs in gure 1 5 show the drought index, a combination o temperatures and precipitation, and the area o spruce trees destroyed annually. 4 CO 2 concentration/ppm sunlight energy 5,266,800 Identiy the two periods when the drought index remained high or three or more years. [2 ] ( ii) S uggest reasons or the dierences between the outbreaks. [2 ] Predict rates o destruction o spruce trees in the uture, with reasons or your answer. [4] Figure 1 6 shows monthly average carbon dioxide concentrations or B aring Head, New Zealand and Alert, C anada. 390 385 380 375 370 365 360 355 350 345 340 335 330 Key Alert station, Canada Baring Head, New Zealand 76 78 80 82 84 86 88 90 92 94 96 98 00 02 04 year Figure 16 a) S uggest why scientists have chosen such areas as Mauna Loa, B aring Head and Alert as the locations or monitoring stations. [1 ] b) C ompare the trends illustrated in both graphs. c) [2 ] Explain why the graphs show dierent patterns. [3 ] 239 41 e c o lo G y 5 Figure 1 7 shows the concentration o CO 2 in the atmosphere, measured in parts per million (ppm) . In a orest, concentrations o CO 2 change over the course o the day and change with height. The top o the orest is reerred to as the canopy. tundra above ground taiga root above ground height/m soil 320 330 320 310 30 Top forest canopy 320 grasslands 340 350 340350 0 0 360 6 12 soil 18 24 time of day / hours soil equatorial forest above ground ( i) S tate the highest concentration o C O 2 reached in the canopy. [1 ] soil ( ii) D etermine the range o concentration ound in the canopy. [2 ] b) ( i) State the time o day ( or night) when the highest levels o C O 2 are detected. root root savannah Figure 17 above ground soil root root Figure 18 The distribution of nitrogen in the three organic matters compartments for each of six major biomes [1 ] ( ii) The highest levels o C O 2 are detected j ust above the ground. D educe two reasons why this is the case. [2 ] 240 above ground 330 10 6 deciduous forest above ground 305 c) soil 310 ppm 20 a) root Give an example o an hour when C O 2 concentrations are reasonably uniorm over the ull range o heights. [1 ] Within an ecosystem, nitrogen can be stored in one o three organic matter compartments: above ground, in roots and in the soil. Figure 1 8 shows the distribution o nitrogen in the three organic matter compartments or each o six maj or biomes. a) Deduce what the above ground compartment consists o in an ecosystem. [1 ] b) S tate which biome has the largest above ground compartment. [1 ] c) Explain why it is difcult to grow crops in an area where equatorial orest has been cleared o its vegetation. [2 ] d) S tate the name o the process carried out by decomposers and detritus eeders that releases C O 2 into the atmosphere. [1 ] e) f) Suggest why most o the nitrogen in a tundra ecosystem is in the soil. [1 ] Explain why warming due to climate change might cause a release o C O 2 rom tundra soil. [2 ] 5C E LELvOB Lu t I O n an d B I O d I vE r s I t Y I O LO GY Iocio There is overwhelming evidence or the theory that the diversity o lie has evolved, and continues to evolve by natural selection. The ancestry o groups o species can be deduced by comparing their base or amino acid sequences. S pecies are named and classifed using an internationally agreed system. 5.1 Evidence for evolution ueig Evolution occurs when heritable characteristics o a species change. The ossil record provides evidence or evolution. Selective breeding o domesticated animals shows that artifcial selection can cause evolution. Evolution o homologous structures by adaptive radiation explains similarities in structure when there are dierences in unction. Populations o a species can gradually diverge into separate species by evolution. Continuous variation across the geographical range o related populations matches the concept o gradual divergence. applicio Comparison o the pentadactyl limb o mammals, birds, amphibians and reptiles with dierent methods o locomotion. Development o melanistic insects in polluted areas. ne of ciece Looking or patterns, trends and discrepancies: there are common eatures in the bone structure o vertebrate limbs despite their varied use. 241 5 E vo l u t i o n an d b i o d i vE r s i t y Evolution in summary Evolution occurs when heritable characteristics of a species change. There is strong evidence or characteristics o species changing over time. B iologists call this process evolution. It lies at the heart o a scientifc understanding o the natural world. An important distinction should be drawn between acquired characteristics that develop during the lietime o an individual and heritable characteristics that are passed rom parent to ospring. E volution only concerns heritable characteristics. Figure 1 Fossils o dinosaurs show there were animals on Earth in the past that had diferent characteristics rom those alive today The mechanism o evolution is now well understood it is natural selection. D espite the robustness o evidence or evolution by natural selection, there is still widespread disbelie among some religious groups. There are stronger obj ections to the concept that species can evolve than to the logic o the mechanism that inevitably causes evolution. It is thereore important to look at the evidence or evolution. Evidence from fossils The fossil record provides evidence for evolution. In the frst hal o the 1 9 th century, the sequence in which layers or strata o rock were deposited was worked out and the geological eras were named. It became obvious that the ossils ound in the various layers were dierent there was a sequence o ossils. In the 2 0th century, reliable methods o radioisotope dating revealed the ages o the rock strata and o the ossils in them. There has been a huge amount o research into ossils, which is the branch o science called palaeontology. It has given us strong evidence that evolution has occurred. Figure 2 Many trilobite species evolved over hundreds o millions o years but the group is now totally extinct 242 The sequence in which ossils appear matches the sequence in which they would be expected to evolve, with bacteria and simple algae appearing frst, ungi and worms later and land vertebrates later still. Among the vertebrates, bony fsh appeared about 42 0 million years ago ( mya) , amphibians 3 40 mya, reptiles 3 2 0 mya, birds 2 5 0 mya and placental mammals 1 1 0 mya. The sequence also fts in with the ecology o the groups, with plant ossils appearing beore animal, plants on land beore animals on land, and plants suitable or insect pollination beore insect pollinators. Many sequences o ossils are known, which link together existing organisms with their likely ancestors. For example, horses, asses and zebras, members o the genus Equus, are most closely related to rhinoceroses and tapirs. An extensive sequence o ossils, extending back over 60 million years, links them to Hyracotherium, an animal very similar to a rhinoceros. 5 .1 E vi D E n cE fo r E vo lu ti o n Daa-based qess: Missing links An obj ection to ossil evidence or evolution has been gaps in the record, called missing links, or example a link between reptiles and birds. (a) (b) (d) (g) (c) The discovery o ossils that ll in these gaps is particularly exciting or biologists. 1 2 (i) (h) 100 mm Drawings o ossils recently ound in Western China. They show Dilong paradoxus, a 130-million-year-old tyrannosauroid dinosaur with protoeathers. ad: bones o skull; e: teeth; g: tail vertebrae with protoeathers; hj: limb bones [2 ] D educe three similarities between Dilong paradoxus and reptiles that live on Earth today. [3 ] 3 Suggest a unction or the protoeathers o Dilong paradoxus. [1 ] 4 Suggest two eatures which Dilong paradoxus would have had to evolve to become capable o fight. [2 ] 5 Explain why it is not possible to be certain whether the protoeathers o Dilong paradoxus are homologous with the eathers o birds. [2 ] (j) (e) (f) C alculate the length o Dilong paradoxus, rom its head to the tip o its tail. Figure 3 Evidence from selective breeding Selective breeding o domesticated animals shows that artifcial selection can cause evolution. Humans have deliberately bred and used particular animal species or thousands o years. I modern breeds o livestock are compared with the wild species that they most resemble, the dierences are oten huge. Consider the dierences between modern egg-laying hens and the jungleowl o Southern Asia, or between Belgian Blue cattle and the aurochs o Western Asia. There are also many dierent breeds o sheep, cattle and other domesticated livestock, with much variation between breeds. It is clear that domesticated breeds have not always existed in their current orm. The only credible explanation is that the change has been achieved simply by repeatedly selecting or and breeding the individuals most suited to human uses. This process is called articial selection. The eectiveness o articial selection is shown by the considerable changes that have occurred in domesticated animals over periods o time that are very short, in comparison to geological time. It shows that selection can cause evolution, but it does not prove that evolution o species has actually occurred naturally, or that the mechanism or evolution is natural selection. Figure 4 Over the last 15,000 years many breeds o dog have been developed by artifcial selection rom domesticated wolves 243 5 E vo l u t i o n an d b i o d i vE r s i t y Homology and evolution Looking or patterns, trends and disrepanies: there are ommon eatures in the one struture o verterate lims despite their varied use. Vertebrate limbs are used in many dierent ways, such as walking, running, j umping, fying, swimming, grasping and digging. These varied uses require j oints that articulate in dierent ways, dierent velocities o movement and also dierent amounts o orce. It would be reasonable to expect them to have very dierent bone structure, but there are in act common eatures o bone structure that are ound in all vertebrate limbs. Patterns like this require explanation. The only reasonable explanation so ar proposed in this case is evolution rom a common ancestor. As a consequence, the common bone structure o vertebrate limbs has become a classic piece o evidence or evolution. Data-based questions: Domestication of corn A wild grass called teosinte that grows in C entral America was probably the ancestor o cultivated corn, Zea mays. When teosinte is grown as a crop, it gives yields o about 1 5 0 kg per hectare. This compares with a world average yield o corn o 4, 1 00 kg per hectare at the start o the 2 1 st century. Table 1 gives the lengths o some cobs. C orn was domesticated at least 7, 000 years ago. 1 C alculate the percentage dierence in length between teosinte and S ilver Queen. [2 ] 2 C alculate the percentage dierence in yield between teosinte and world average yields o corn. [2 ] 3 Suggest actors apart rom cob length, selected or by armers. [3 ] 4 Explain why improvement slows down over generations o selection. corn variety and origin Teosinte wild relative o orn Early primitive orn rom Colomia Peruvian anient orn rom 500 bc Imriado primitive orn rom Colomia Silver Queen modern sweetorn [3 ] length of ob (mm) 14 45 65 90 170 Table 1 Figure 5 Corn cobs Evidence from homologous structures Evolution o homologous strutures y adaptive radiation explains similarities in struture when there are diferenes in untion. D arwin pointed out in The Origin of Species that some similarities in structure between organisms are supercial, or example between a dugong and a whale, or between a whale and a sh. S imilarities like those between the tail ns o whales and shes are known as analogous structures. When we study them closely we nd that these structures are very dierent. An evolutionary interpretation is that they have had 244 5 .1 E vi D E n cE fo r E vo lu ti o n dierent origins and have become similar because they perorm the same or a similar unction. This is called convergent evolution. Homologous structures are the converse o this. They are structures that may look supercially dierent and perorm a dierent unction, but which have what D arwin called a unity o type. He gave the example o the orelimbs o a human, mole, horse, porpoise and bat and asked what could be more curious than to nd that they include the same bones, in the same relative positions, despite on the surace appearing completely dierent. The evolutionary explanation is that they have had the same origin, rom an ancestor that had a pentadactyl or vedigit limb, and that they have become dierent because they perorm dierent unctions. This is called adaptive radiation. There are many examples o homologous structures. They do not prove that organisms have evolved or had common ancestry and do not reveal anything about the mechanism o evolution, but they are dicult to explain without evolution. Particularly interesting are the structures that D arwin called rudimentary organs reduced structures that serve no unction. They are now called vestigial organs and examples o them are the beginnings o teeth ound in embryo baleen whales, despite adults being toothless, the small pelvis and thigh bone ound in the body wall o whales and some snakes, and o course the appendix in humans. These structures are easily explained by evolution as structures that no longer have a unction and so are being gradually lost. Pentadactyl limbs Comparison o the pentadactyl limb o mammals, birds, amphibians and reptiles with dierent methods o locomotion. The pentadactyl limb consists o these structures: Be se single bone in the proximal part femb humerus Hdmb emur two bones in the distal part radius and ulna group o wrist/ ankle bones carpals series o bones in each o fve digits metacarpals and metatarsals phalanges and phalanges classes that have limbs: amphibians, reptiles, birds and mammals. E ach o them has pentadactyl limbs: crocodiles walk or crawl on land and use their webbed hind limbs or swimming penguins use their hind limbs or walking and their orelimbs as fippers or swimming echidnas use all our limbs or walking and also use their orelimbs or digging rogs use all our limbs or walking and their hindlimbs or j umping. tibia and fbula tarsals The pattern o bones or a modication o it is present in all amphibians, reptiles, birds and mammals, whatever the unction o their limbs. The photos in gure 6 show the skeletons o one example o each o the our vertebrates D ierences can be seen in the relative lengths and thicknesses o the bones. Some metacarpals and phalanges have been lost during the evolution o the penguins orelimb. 245 5 E vo l u t i o n an d b i o d i vE r s i t y Activity Pentadactyl limbs in mammals mole horse Figure 6 porpoise speciation Populations o a species can gradually diverge into separate species by evolution. bat human Figure 7 Pentadactyl limbs (not to scale) Choose a colour code or the types o bone in a pentadactyl limb and colour the diagrams in fgure 7 to show the type o each bone. How is each limb used? What eatures o the bones in each limb make them well adapted to the use? 246 If two populations of a species become separated so that they do not interbreed and natural selection then acts differently on the two populations, they will evolve in different ways. The characteristics of the two populations will gradually diverge. After a time they will be recognizably different. If the populations subsequently merge and have the chance of interbreeding, but do not actually interbreed, it would be clear that they have evolved into separate species. This process is called speciation. S peciation often occurs after a population of a species extends its range by migrating to an island. This explains the large numbers of endemic species on islands. An endemic species is one that is found only in a certain geographical area. The lava lizards of the Galpagos Islands are an example of this. O ne species is present on all the main islands of the archipelago. O n six smaller islands there is a closely related but different species, formed by migration to the island and by subsequent divergence. 5 .1 E vi D E n cE fo r E vo lu ti o n Evidence from patterns of variation Pinta Continuous variation across the geographical range o related populations matches the concept o gradual divergence. Genovesa Marchena Santiago I populations gradually diverge over time to become separate species, then at any one moment we would expect to be able to nd examples o all stages o divergence. This is indeed what we nd in nature, as C harles D arwin describes in C hapter II o The Origin of Species. He wrote: Santa Cruz Fernandina Santa Fe Isabel a Espaola Santa Maria Many years ago, when comparing, and seeing others compare, the birds from the separate islands of the Galpagos Archipelago, both one with another, and with those from the American mainland, I was much struck how entirely vague and arbitrary is the distinction between species and varieties. San Cristbal key T. albemarlensis T. duncanensis T. delanonis T. habelii T. pacicus T. bivittatus T. grayii Figure 8 Distribution of lava lizards in the Galpagos Islands D arwin gave examples o populations that are recognizably dierent, but not to the extent that they are clearly separate species. O ne o his examples is the red grouse o B ritain and the willow ptarmigan o Norway. They have sometimes been classied as separate species and sometimes as varieties o the species Lagopus lagopus. This is a common problem or biologists who name and classiy living organisms. B ecause species can gradually diverge over long periods o time and there is no sudden switch rom being two populations o one species to being two separate species, the decision to lump populations together or split them into separate species remains rather arbitrary. The continuous range in variation between populations does not match either the belie that species were created as distinct types o organism and thereore should be constant across their geographic range or that species are unchanging. Instead it provides evidence or the evolution o species and the origin o new species by evolution. Industrial melanism Development o melanistic insects in polluted areas. D ark varieties o typically light- coloured insects are called melanistic. The most amous example o an insect with a melanistic variety is Biston betularia, the peppered moth. It has been widely used as an example o natural selection, as the melanistic variety became commoner in polluted industrial areas where it is better camoufaged than the pale peppered variety. A simple explanation o industrial melanism is this: Adult Biston betularia moths fy at night to try to nd a mate and reproduce. D uring the day they roost on the branches o trees. B irds and other animals that hunt in daylight predate moths i they nd them. TOK t wha exe a mpe mdes be sed es hees? The useulness o a theory is the degree to which it explains phenomenon and the degree to which it allows predictions to be made. One way to test the theory o evolution by natural selection is through the use o computer models. The Blind Watchmaker computer model is used to demonstrate how complexity can evolve rom simple orms through artifcial selection. The Weasel computer model is used to demonstrate how artifcial selection can increase the pace o evolution over random events. What eatures would a computer model have to include or it to simulate evolution by natural selection realistically? 247 5 E vo l u t i o n an d b i o d i vE r s i t y In unpolluted areas tree branches are covered in pale- coloured lichens and peppered moths are well camoufaged against them. Sulphur dioxide pollution kills lichens. S oot rom coal burning blackens tree branches. Melanic moths are well camoufaged against dark tree branches in polluted areas. In polluted areas the melanic variety o Biston betularia replaced the peppered variety over a relatively short time, but not in nonpolluted areas. Figure 9 Museum specimen of the peppered form of Biston betularia mounted on tree bark with lichens from an unpolluted area Figure 10 The ladybug Adalia bipunctata has a melanic form which has become common in polluted areas. A melanic male is mating with a normal female here B iologists have used industrial melanism as a classic example o evolution by natural selection. Perhaps because o this, research ndings have been repeatedly attacked. The design o some early experiments into camoufage and predation o the moths has been criticized and this has been used to cast doubt over whether natural selection ever actually occurs. Michael Majerus gives a careul evaluation o evidence about the development o melanism in Biston betularia and other species o moth in his book in the New Naturalist series (Moths, Michael Majerus, HarperCollins 2002) . His nding is that the evidence or industrial pollution causing melanism in Biston betularia and other species o moth is strong, though actors other than camoufage can also infuence survival rates o pale and melanic varieties. Data-based questions: Predation rates in Biston betularia One o the criticisms o the original experiments into predation o Biston betularia was that the moths were placed in exposed positions on tree trunks and that this is not normally where they roost. The moths were able to move to more suitable positions but even so the criticisms have persisted on some websites. Experiments done in the 1 980s tested the eect o the position in which the moths were placed. Peppered and melanic 248 orms ( ty o each) o Biston betularia were placed in exposed positions on tree trunks and 5 0 millimetres below a joint between a maj or branch and the tree trunk. This procedure was carried out at two oak woods, one in an unpolluted area o the New Forest in southern England and another in a polluted area near Stoke-on-Trent in the Midlands. The box plots in gure 1 1 show the percentage o moths eaten and moths surviving. 5 . 2 n At u r A l s E l E c t i o n 1 a) D educe, with a reason from the data, whether the moths were more likely to be eaten if they were placed on the exposed trunk or below the j unction of a main branch and the trunk. [2 ] b) Suggest a reason for the difference. 2 a) C ompare and contrast the survival rates of peppered and melanic moths in the New Forest. b) Explain the difference in survival rate between the two varieties in the New Forest. [1 ] [3 ] peppered New Forest/melanic/BJ New Forest/melanic/ET 4 D istinguish between the S toke- on- Trent and New Forest woodlands in relative survival rates of peppered and melanic moths. [2 ] Pollution due to industry has decreased greatly near S toke- on- Trent since the 1 980s. Predict the consequences of this change for Biston betularia. [4] 38 40 62 74 26 New Forest/peppered/ET 68 32 Stoke/melanic/BJ 72 28 Stoke/melanic/ET Stoke/peppered/BJ [3 ] 60 New Forest/peppered/BJ Stoke/peppered/ET melanic 3 Stoke on Trent and New Forest key not eaten ET = exposed trunk 0% 60 50 42 40 50 58 20% 40% 60% 80% 100% eaten BJ = branch junction Figure 11 Source: Howlett and Majerus (1987) The Understanding of industrial melanism in the peppered moth (Biston betularia) Biol. J.Linn.Soc. 30, 3144 5.2 naa ee uderstdig Natural selection can only occur i there is variation amongst members o the same species. Mutation, meiosis and sexual reproduction cause variation between individuals in a species. Adaptations are characteristics that make an individual suited to its environment and way o lie. Species tend to produce more ospring than the environment can support. Individuals that are better adapted tend to survive and produce more ospring while the less well adapted tend to die or produce ewer ospring. Individuals that reproduce pass on characteristics to their ospring. Natural selection increases the requency o characteristics that make individuals better adapted and decreases the requency o other characteristics leading to changes within the species. applictios Changes in beaks o fnches on Daphne Major. Evolution o antibiotic resistance in bacteria. ntre of sciece Use theories to explain natural phenomena: the theory o evolution by natural selection can explain the development o antibiotic resistance in bacteria. 249 5 E vo l u t i o n an d b i o d i vE r s i t y vrition Natural selection can only occur if there is variation amongst members of the same species. Figure 1 Populations o bluebells (Hyacinthoides non-scripta) mostly have blue fowers but white-fowered plants sometimes occur C harles D arwin developed his understanding of the mechanism that causes evolution over many years, after returning to England from his voyage around the world on HMS B eagle. He probably developed the theory of natural selection in the late 1 83 0s, but then worked to accumulate evidence for it. D arwin published his great work, The Origin of Species, in 1 85 9. In this book of nearly 5 00 pages, he explains his theory and presents the evidence for it that he had found over the previous 2 0 to 3 0 years. O ne of the observations on which D arwin based the theory of evolution by natural selection is variation. Typical populations vary in many respects. Variation in human populations is obvious height, skin colour, blood group and many other features. With other species the variation may not be so immediately obvious but careful observation shows that it is there. Natural selection depends on variation within populations if all individuals in a population were identical, there would be no way of some individuals being favoured more than others. source of rition Mutation, meiosis and sexual reproduction cause variation between individuals in a species. The causes of variation in populations are now well understood: Figure 2 Dandelions (Taraxacum ofcinale) appear to be reproducing sexually when they disperse their seed but the embryos in the seeds have been produced asexually so are genetically identical 1 Mutation is the original source of variation. New alleles are produced by gene mutation, which enlarges the gene pool of a population. 2 Meiosis produces new combinations of alleles by breaking up the existing combination in a diploid cell. Every cell produced by meiosis in an individual is likely to carry a different combination of alleles, because of crossing over and the independent orientation of bivalents. 3 S exual reproduction involves the fusion of male and female gametes. The gametes usually come from different parents, so the offspring has a combination of alleles from two individuals. This allows mutations that occurred in different individuals to be brought together. In species that do not carry out sexual reproduction the only source of variation is mutation. It is generally assumed that such species will not generate enough variation to be able to evolve quickly enough for survival during times of environmental change. adpttion Adaptations are characteristics that make an individual suited to its environment and way of life. O ne of the recurring themes in biology is the close relationship between structure and function. For example, the structure of a birds beak is correlated with its diet and method of feeding. The thick coat of a musk 250 5 . 2 n At u r A l s E l E c t i o n ox is obviously correlated with the low temperatures in its northerly habitats. The water storage tissue in the stem o a cactus is related to inrequent rainall in desert habitats. In biology characteristics such as these that make an individual suited to its environment or way o lie are called adaptations. The term adaptation implies that characteristics develop over time and thus that species evolve. It is important not to imply purpose in this process. According to evolutionary theory adaptations develop by natural selection, not with the direct purpose o making an individual suited to its environment. They do not develop during the lietime o one individual. C haracteristics that do develop during a lietime are known as acquired characteristics and a widely accepted theory is that acquired characteristics cannot be inherited. Avy Adapa f bd beak The our photographs o birds show the beaks o a heron, macaw, hawk and woodpecker. To what diet and method o eeding is each adapted? Overproduction o ofspring Species tend to produce more ofspring than the environment can support. Living organisms vary in the number o ospring they produce. An example o a species with a relatively slow breeding rate is the southern ground hornbill, Bucorvus leadbeateri. It raises one fedgling every three years on average and needs the cooperation o at least two other adults to do this. However they can live or as long as 7 0 years so in their lietime a pair could theoretically raise twenty ospring. Most species have a aster breeding rate. For example, the coconut palm, Cocos nucifera usually produces between 2 0 and 60 coconuts per year. Apart rom bacteria, the astest breeding rate o all may be in the ungus Calvatia gigantea. It produces a huge ruiting body called a giant puball in which there can be as many as 7 trillion spores ( 7, 000, 000, 000, 000) . Figure 3 D espite the huge variation in breeding rate, there is an overall trend in living organisms or more ospring to be produced than the environment can support. D arwin pointed out that this will tend to lead to a struggle or existence within a population. There will be competition or resources and not every individual will obtain enough to allow them to survive and reproduce. Figure 4 The breeding rate of pairs of southern ground hornbills, Bucorvus leadbeateri, is as low as 0.3 young per year 251 5 E vo l u t i o n an d b i o d i vE r s i t y Activity simulation of natural election Make ten or more artifcial fsh using modelling clay, or some other malleable material. Drop each o them into a measuring cylinder o water and time how long each takes to reach the bottom. Discard the hal o the models that were slowest. Pair up the astest models and make intermediate shapes, to represent their ospring. Random new shapes can also be introduced to simulate mutation. Test the new generation and repeat the elimination o the slowest and the breeding o the astest. Does one shape gradually emerge? Describe its eatures. diferential survival an reprouction Individuals that are better adapted tend to survive and produce more ospring while the less well adapted tend to die or produce ewer ospring. C hance plays a part in deciding which individuals survive and reproduce and which do not, but the characteristics o an individual also have an infuence. In the struggle or existence the less well- adapted individuals tend to die or ail to reproduce and the best adapted tend to survive and produce many ospring. This is natural selection. An example that is oten quoted is that o the girae. It can graze on grass and herbs but is more adapted to browse on tree leaves. In the wet season its ood is abundant but in the dry season there can be periods o ood shortage when the only remaining tree leaves are on high branches. Giraes with longer necks are better adapted to reaching these leaves and surviving periods o ood shortage than those with shorter necks. Inheritance Individuals that reproduce pass on characteristics to their ospring. Much o the variation between individuals can be passed on to ospring it is heritable. Maasai children inherit the dark skin colour o their parents or example and children o light- skinned north European parents inherit a light skin colour. Variation in behaviour can be heritable. The direction o migration to overwintering sites in the blackcap Sylvia atricapilla is an example. D ue to dierences in their genes, some birds o this species migrate southwestwards rom Germany to Spain or the winter and others northwestwards to B ritain. Not all eatures are passed on to ospring. Those acquired during the lietime o an individual are not usually inherited. An elephant with a broken tusk does not have calves with broken tusks or example. I a person develops darker skin colour through exposure to sunlight, the darker skin is not inherited. Acquired characteristics are thereore not signicant in the evolution o a species. Progressive change Natural selection increases the requency o characteristics that make individuals better adapted and decreases the requency o other characteristics leading to changes within the species. B ecause better- adapted individuals survive, they can reproduce and pass on characteristics to their ospring. Individuals that are less well adapted have lower survival rates and less reproductive success. This leads to an increase in the proportion o individuals in a population with 252 5 . 2 n At u r A l s E l E c t i o n characteristics that make them well adapted. O ver the generations, the characteristics o the population gradually change this is evolution by natural selection. Maj or evolutionary changes are likely to occur over long time periods and many generations, so we should not expect to be able to observe them during our lietime, but there are many examples o smaller but signicant changes that have been observed. The evolution o dark wing colours in moths has been observed in industrial areas with polluted air. Two examples o evolution are described in the next sections o this book: changes to beaks o nches on the Galapagos Islands and the development o antibiotic resistance in bacteria. Avy The impulse to reproduce and pass on characteristics can be very strong. It can cause adult males to carry out infanticide. How could this behaviour pattern have evolved in lions and other species? Female cheetahs mate with two or more males so their litters have multiple paternity. How does this protect the young against infanticide? Daa-baed qe: Evolution in rice plants The bar charts in gure 6 show the results o an investigation o evolution in rice plants. F 1 hybrid plants were bred by crossing together two rice varieties. These hybrids were then grown at ve dierent sites in Japan. Each year the date o fowering was recorded and seed was collected rom the plants, or re-sowing at that site in the ollowing year. F3 F4 F5 F Figure 5 A female cheetahs cubs inherit Sapporo 43 N characteristics from her and from one of the several males with whom she mated Fujisaka 40 N Konasu 36 N single original population planted out at Hiratsuka 35 N Chikugo 33 N Miyazaki 31 N 56 70 84 98 112 126 68 82 96 110 124 138 54 68 82 96 110124138 51 65 79 93 107121 135 days to owering Figure 6 1 Why was the investigation done using hybrids rather than a single pure- bred variety? [2 ] 2 D escribe the changes, shown in the chart, between the F 3 and F 6 generations o rice plants grown at Miyazaki. [2 ] 3 a) S tate the relationship between fowering time and latitude in the F 6 generation. [1 ] b) S uggest a reason or this relationship. 4 a) [1 ] Predict the results i the investigation had been carried on until the F 1 0 generation. [1 ] b) Predict the results o collecting seeds rom F 1 0 plants grown at S apporo and rom F 1 0 plants grown at Miyazaki and sowing them together at Hiratsuka. [3 ] 253 5 E vo l u t i o n an d b i o d i vE r s i t y Galpagos fnches Changes in beaks o fnches on Daphne Major. Pinta (5) Rabida (8) Marchena (4) Genovesa (4) Santiago (10) Daphne Major (2/3) Fernandina (9) Isabela (10) Santa Cruz (9) Santa Fe (5) Santa Maria (8) San Cristbal (7) (a) G. fortis (large beak) Espaola (3) Figure 7 The Galpagos archipelago with the number o species o fnch ound on each island Darwin visited the Galpagos Islands in 1 835 and collected specimens o small birds, which were subsequently identifed as fnches. There are 1 4 species in all. Darwin observed that the sizes and shapes o the beaks o the fnches varied, as did their diet. From the overall similarities between the birds and their distribution over the Galapagos islands (see fgure 7) , Darwin hypothesized that one might really ancy that rom an original paucity o birds in this archipelago, one species had been taken and modifed or dierent ends. There has since been intense research into what have become known as D arwins fnches. In particular, Peter and Rosemary Grant have shown that beak characters and diet are closely related and when one changes, the other does also. A particular ocus o Peter and Rosemary Grants research has been a population o the medium ground fnch, Geospiza fortis, on a small island called D aphne Maj or. O n this island, the small ground fnch, Geospiza fuliginosa, is almost absent. B oth species eed on small seeds, though G. fortis can also eat larger seeds. In the absence o competition rom G. fuliginosa or small seeds, G. fortis is smaller in body size and beak size on D aphne Maj or than on other islands. In 1 977, a drought on D aphne Major caused a shortage o small seeds, so G. fortis ed instead on larger, harder seeds, which the larger-beaked individuals are able to crack open. Most o the population died in that year, with highest mortality 254 (b) G. fortis (small beak) (c) G. magnirostris Figure 8 Variation in beak shape in Galpagos fnches. (a) G. fortis (large beak) . (b) G. fortis (small beak) . (c) G. magnirostris among individuals with shorter beaks. In 1 982 83 there was a severe El Nio event, causing eight months o heavy rain and as a result an increased supply o small, sot seeds and ewer large, hard seeds. G. fortis bred rapidly, in response to the increase in ood availability. With a return to dry weather conditions and greatly reduced supplies o small seeds, breeding stopped until 1 987. In that year, only 3 7 per cent o those alive in 1 983 bred and they were not a random sample o the 1 983 population. In 1 987, G. fortis had longer and narrower beaks than the 1 983 averages, correlating with the reduction in supply o small seeds. Variation in the shape and size o the beaks ( see fgure 8) is mostly due to genes, though the 5 . 2 n At u r A l s E l E c t i o n environment has some eect. The proportion o the variation due to genes is called heritability. Using the heritability o beak length and width and data about the birds that had survived to breed, the changes in mean beak length and width between 1 983 and 1 987 were predicted. The observed results are very close to the predictions. Average beak length was predicted to increase by 1 0 m and actually increased by 6 m. Average beak width was predicted to decrease by 1 3 0 m and actually decreased by 1 2 0 m. O ne o the obj ections to the theory o evolution by natural selection is that signifcant changes caused by natural selection have not been observed actually occurring. It is unreasonable to expect huge changes to have occurred in a species, even i it had been ollowed since D arwins theory was published in 1 85 9, but in the case o G. fortis, signifcant changes have occurred that are clearly linked to natural selection. Daa-baed qe: Galpagos fnches When Peter and Rosemary Grant began to study fnches on the island o D aphne Maj or in 1 973 , there were breeding populations o two species, Geospiza fortis and Geospiza scandens. Geospiza magnirostris established a breeding population on the island in 1 982 , initially with j ust two emales and three males. Figure 9 shows the numbers o G. magnirostris and G. fortis on D aphne Maj or between 1 997 and 2 006. 1500 numbers G. fortis G. magnirostris 1000 500 the changes in the population o G. magnirostris. 2 1998 2000 2002 year 2004 Changes in numbers of G. fortis and G. magnirostris between 1996 and 2006 a) D escribe the changes in the population o G. magnirostris between 1 997 and 2 006. [2 ] b) C ompare the changes in population o G. fortis between 1 997 and 2 006 with spee Yea sma Medm lage 1977 75 10 17 Geospiza fortis 1985 1989 80 77 0.0 5.1 19 16 2004 80 11 8.2 a) O utline the diet o each o the species o fnch on D aphne Maj or. [3 ] b) There was a very severe drought on D aphne Maj or in 2 003 and 2 004. D educe how the diet o the fnches changed during the drought, using the data in the table. 2006 Figure 9 1 D aphne Maj or has an area o 0.3 4 km . 1 km 2 is 1 00 hectares and 1 hectare is 1 00 1 00 m. C alculate the maximum and minimum population densities o G. ortis during 1 9972 006. [4] Table 2 shows the percentages o three types o seed in the diets o the three fnch species on D aphne Maj or. Small seeds are produced by 2 2 plant species, medium seeds by the cactus Opuntia echios, and large seeds, which are very hard, by Tribulus cistoides. 3 0 1996 [3 ] 2 4 [3 ] Figure 1 0 shows an index o beak size o adult G. fortis rom 1 973 to 2 006, with the size in 1 973 assigned the value zero and the sizes in other years shown in comparison to this. Geospiza magnirostris 1985 1989 2004 18 5.9 4.5 0.0 12 26 82 82 69 Geospiza scandens 1977 1985 1989 2004 85 77 23 17 15 22 70 83 0.0 0.0 0.0 0.0 Table 2 255 5 E vo l u t i o n an d b i o d i vE r s i t y c) In the frst severe drought, the mean beak size o G. fortis increased, but in the second drought, it decreased. Using the data in this question, explain how natural selection could cause these changes in beak size in the two droughts. [3 ] 1 beak size index 0.5 0 -0.5 5 The intensity o natural selection on D aphne Maj or was calculated during the two droughts. The calculated values are called selection dierentials. They range rom 1 .08 or beak length during the second drought, to +0. 88 or beak length in the frst drought, with similar selection dierentials or beak width and depth and overall beak size. These are very large selection dierentials, compared to values calculated in other investigations o evolution. Suggest reasons or natural selection on the beak size o G. fortis being unusually intense on the island o D aphne Maj or. [2 ] 6 D iscuss the advantages o investigations o evolution over long periods and the reasons or ew long-term investigations being done. [3 ] -1 -1.5 1975 1980 1985 1990 year 1995 2000 2005 Figure 10 Relative beak size in G. fortis between 1973 and 2006 The graph shows two periods o very rapid change in mean beak size, both o which correspond with droughts on D aphne Maj or. a) S tate two periods o most rapid change in mean beak size o G. fortis. [2 ] b) S uggest two reasons or mean beak size changing most rapidly when there is a drought. [2 ] natural selectio ad atibiotic resistace Use theories to explain natural phenomena: the theory of evolution by natural selection can explain the development of antibiotic resistance in bacteria. Antibiotics were one o the great triumphs o medicine in the 2 0th century. When they were frst introduced, it was expected that they would oer a permanent method o controlling bacterial diseases, but there have been increasing problems o antibiotic resistance in pathogenic bacteria. development o antibiotic resistance is thereore an example o evolution. It can be explained in terms o the theory o natural selection. A scientifc understanding o how antibiotic resistance develops is very useul as it gives an understanding o what should be done to reduce the problem. The ollowing trends have become established: Ater an antibiotic is introduced and used on patients, bacteria showing resistance appear within a ew years. Resistance to the antibiotic spreads to more and more species o pathogenic bacteria. In each species the proportion o inections that are caused by a resistant strain increases. 14 12 % resistant 16 10 8 6 4 2 256 Figure 11 2003 Percentage resistance to ciprofoxacin between 1990 and 2004 2004 2001 2002 1999 2000 1997 1998 1996 1994 1995 1992 1993 1991 1990 0 So, during the time over which antibiotics have been used to treat bacterial diseases there have been cumulative changes in the antibiotic resistance properties o populations o bacteria. The 5 . 2 n At u r A l s E l E c t i o n antibiotic resistnce Evolution of antibiotic resistance in bacteria. Antibiotic resistance is due to genes in bacteria and so it can be inherited. The mechanism that causes antibiotic resistance to become more prevalent or to diminish is summarized in gure 1 2 . The evolution o multiple antibiotic resistance has occurred in j ust a ew decades. This rapid evolution is due to the ollowing causes: population with no antibiotic-resistant bacteria antibiotic resistance gene received from a bacterium in another population population with some antibiotic-resistant bacteria There has been very widespread use o antibiotics, both or treating diseases and in animal eeds used on arms. antibiotic is used therefore there is strong natural selection for resistance B acteria can reproduce very rapidly, with a generation time o less than an hour. population with more antibiotic-resistant bacteria Populations o bacteria are oten huge, increasing the chance o a gene or antibiotic resistance being ormed by mutation. B acteria can pass genes on to other bacteria in several ways, including using plasmids, which allow one species o bacteria to gain antibiotic resistance genes rom another species. antibiotic resistance gene formed by mutation in one bacterium antibiotic is not used therefore there is natural selection (weak) against resistance population with slightly fewer antibiotic-resistant bacteria Figure 12 Evolution o antibiotic resistance Daa-baed qe: Chlortetracycline resistance in soil bacteria 1 a) S tate the relationship between percentage antibiotic resistance and distance rom the animal pen. [1 ] b) E xplain the dierence in antibiotic resistance between populations o bacteria near and ar rom the pen. [4] 3.0 2.5 desistance (%) B acteria were collected rom soil at dierent distances rom a site on a pig arm in Minnesota where manure had been allowed to overfow rom an animal pen and accumulate. The eed given to the pigs on this arm contained subtherapeutic low doses o the antibiotic chlortetracycline, in order to promote aster growth rates. The bacteria were tested to nd out what percentage o them was resistant to this antibiotic. The results are shown in the bar chart. The yellow bars show the percentage o chlortetracycline resistant bacteria that grew on nutrient-rich medium and the orange bars show the percentage on a nutrient- poor medium that encouraged dierent types o bacteria to grow. 2.0 1.5 1.0 0.5 0.0 5m 20 m 100 m distance from animal pen Source: " The efects o subtherapeutic antibiotic use in arm animals on the prolieration and persistence o antibiotic resistance among soil bacteria", Sudeshna Ghosh and Timothy M LaPara, The International Society for Microbial Ecology Journal (2007) 1, 191203 2 Predict whether the percentage antibiotic resistance would have been lower at 200 metres rom the pen than at 1 00 metres. [3] 3 D iscuss the use o subtherapeutic doses o antibiotics in animal eeds. [2 ] 257 5 E vo l u t i o n an d b i o d i vE r s i t y 5.3 classifation o biodiversity udertdig The binomial system o names or species is universal among biologists and has been agreed and developed at a series o congresses. When species are discovered they are given scientifc names using the binomial system. Taxonomists classiy species using a hierarchy o taxa. All organisms are classifed into three domains. The principal taxa or classiying eukaryotes are kingdom, phylum, class, order, amily, genus and species. In a natural classifcation the genus and accompanying higher taxa consist o all the species that have evolved rom one common ancestral species. Taxonomists sometimes reclassiy groups o species when new evidence shows that a previous taxon contains species that have evolved rom dierent ancestral species. Natural classifcations help in identifcation o species and allow the prediction o characteristics shared by species within a group. applictio Classifcation o one plant and one animal species rom domain to species level. External recognition eatures o bryophytes, flicinophytes, conierophytes and angiospermophytes. Recognition eatures o poriera, cnidaria, platyhelminthes, annelida, mollusca and arthropoda, chordata. Recognition o eatures o birds, mammals, amphibians, reptiles and fsh. skill Construction o dichotomous keys or use in identiying specimens. ntre o ciece Cooperation and collaboration between groups o scientists: scientists use the binomial system to identiy a species rather than the many dierent local names. Itertiol coopertio d clifctio Cooperation and collaboration between groups o scientists: scientists use the binomial system to identiy a species rather than the many dierent local names. Recognizable groups of organisms are known to biologists as species. The same species can have many different local names, even within one language. For example, in E ngland the species of plant known to scientists as Arum maculatum has been called lords- and- ladies, cuckoopint, j ack in the pulpit, devils and angels, cows and bulls, willy lily and snakes meat. In French there is also a variety of local names: 258 la chandelle, le pied- de- veau, le manteau de la S ainte- Vierge, la pilette or la vachotte. In S panish there are even more names for this one species of which these are j ust a few: comida de culebra, alcatrax, barba de arn, dragontia menor, hoj as de fuego, vela del diablo and yerba del quemado. The name primaveras is used for Arum maculatum in S panish but for a different plant in other languages. 5 . 3 c l A s s i f i c At i o n o f B i o D i v E r s i t Y Local names may be a valuable part o the culture o an area, but science is an international venture so scientifc names are needed that are understood throughout the world. The binomial system that has developed is a good example o cooperation and collaboration between scientists. The credit or devising our modern system o naming species is given to the Swedish biologist C arl Linnaeus who introduced a system o twopart names in the 1 8th century. This stroke o genius was the basis or the binomial system that is still in use today. In act Linnaeus was mirroring a style o nomenclature that had been used in many languages beore. The style recognizes that there are groups o similar species, so the name or each species in a group consists o a specifc name attached to the group name, as in the Ancient Greek and (used by Threophrastus) , Latin anagallis mas and anagallis femina (used by Pliny) , German weiss Seeblumen and geel Seeblumen (used by Fuchs) , English wild mynte and water mynte (used by Turner) and Malayan jambu bol and jambu chilli (applied by Malays to dierent species o Eugenia) . Figure 1 Arum maculatum development of the binomial system The binomial system of names for species is universal among biologists and has been agreed and developed at a series of congresses. To ensure that all biologists use the same system o names or living organisms, congresses attended by delegates rom around the world are held at regular intervals. There are separate congresses or animals and or plants and ungi. International B otanical C ongresses ( IB C ) were held every year during the late 1 9th century. The IB C held in Genoa in 1 892 proposed that 1 75 3 be taken as the starting point or both genera and species o plants and ungi as this was the year when Linnaeus published Species Plantarum, the book that gave consistent binomials or all species o the plant kingdom then known. The IB C o Vienna in 1 905 accepted by 1 5 0 votes to 1 9 the rule that La nomenclature botanique commence avec Linn, Species Plantarum ( ann. 1 75 3 ) pour les groupes de plantes vasculaires. The 1 9th IB C will be in S henzhen, C hina, in 2 01 7. The frst International Zoological C ongress was held in Paris in 1 889. It was recognized that internationally accepted rules or naming and classiying animal species were needed and these were agreed at this and subsequent congresses. 1 75 8 was chosen as the starting date or valid names o animal species as this was when Linnaeus published Systema Natura in which he gave binomials or all species known then. The current International C ode or Zoological Nomenclature is the 4th edition and there will no doubt be more editions in the uture as scientists refne the methods that they use or naming species. Figure 2 Linnaea borealis. Binomials are often chosen to honour a biologist, or to describe a feature of the organism. Linnaea borealis is named in honour of Carl Linnaeus, the Swedish biologist who introduced the binomial system of nomenclature and named many plants and animals using it 259 5 E vo l u t i o n an d b i o d i vE r s i t y the binomial sysem When species are discovered they are given scientifc names using the binomial system. The system that biologists use is called binomial nomenclature, because the international name o a species consists o two words. An example is Linnaea borealis ( fgure 2 ) . The frst name is the genus name. A genus is a group o species that share certain characteristics. The second name is the species or specifc name. There are various rules about binomial nomenclature: ALLIGATORIDAE mississippiensis Alligator sinensis crocodilus Caiman latirostris yacare Melanosuchus niger palpebrosus Paleosuchus Figure 3 trigonatus Classifcation o the alligator amily The genus name begins with an upper- case ( capital) letter and the species name with a lower-case ( small) letter. In typed or printed text, a binomial is shown in italics. Ater a binomial has been used once in a piece o text, it can be abbreviated to the initial letter o the genus name with the ull species name, or example: L. borealis. The earliest published name or a species, rom 1 75 3 onwards or plants or 1 75 8 or animals, is the correct one. the hierarchy of axa Taxonomists classiy species using a hierarchy o taxa. The word taxon is Greek and means a group o something. The plural is taxa. In biology, species are arranged or classifed into taxa. Every species is classifed into a genus. Genera are grouped into amilies. An example o the genera and species in a amily is shown in fgure 3 . Families are grouped into orders, orders into classes and so on up to the level o kingdom or domain. The taxa orm a hierarchy, as each taxon includes taxa rom the level below. Going up the hierarchy, the taxa include larger and larger numbers o species, which share ewer and ewer eatures. the hree domains All organisms are classifed into three domains. Traditional classifcation systems have recognized two maj or categories o organisms based on cell types: eukaryotes and prokaryotes. This classifcation is now regarded as inappropriate because the prokaryotes have been ound to be very diverse. In particular, when the base sequence o ribosomal RNA was determined, it became apparent that there are two distinct groups o prokaryotes. They were given the names Eubacteria and Archaea. Most classifcation systems thereore now recognize three major categories o organism, Eubacteria, Archaea and Eukaryota. These categories are called domains, so all organisms are classifed into three domains. Table 1 shows some o the eatures that can be used to distinguish between them. Members o the domains are usually reerred to as bacteria, archaeans and eukaryotes. B acteria and eukaryotes are relatively amiliar to most biologists but archaeans are oten less well known. 260 5 . 3 c l A s s i f i c At i o n o f B i o D i v E r s i t Y feaue Baea Histones associated Absent with DNA Presence o introns Rare or absent Structure o cell walls Made o chemical called peptidoglycan Cell membrane Glycerol-ester lipids; dierences unbranched side chains; d-orm o glycerol Dma Ahaea Proteins similar to histones bound to DNA Present in some genes Not made o peptidoglycan Eukaya Present Frequent Not made o peptidoglycan; not always present Glycerol-ether lipids; Glycerol-ester lipids; unbranched side chains; l-orm unbranched side chains; o glycerol d-orm o glycerol Table 1 Archaeans are ound in a broad range o habitats such as the ocean surace, deep ocean sediments and even oil deposits ar below the surace o the Earth. They are also ound in some airly extreme habitats such as water with very high salt concentrations or temperatures close to boiling. The methanogens are obligate anaerobes and give o methane as a waste product o their metabolism. Methanogens live in the intestines o cattle and the guts o termites and are responsible or the production o marsh gas in marshes. Viruses are not classifed in any o the three domains. Although they have genes coding or proteins using the same genetic code as living organisms they have too ew o the characteristics o lie to be regarded as living organisms. Bacteria Archaea Eukaryota Green lamentous Slime bacteria molds Animals Spirochetes Gram Methanobacterium Halophiles Fungi Proteobacteria positives Methanococcus Plants Cyanobacteria Ciliates Flagellates Figure 4 Tree diagram showing relationships between living organisms based on base sequences o ribosomal RNA Ay ideyg a kgdm This is a defnition o the characteristics o organisms in one o the kingdoms. Can you deduce which kingdom it is? Multicellular; cells typically held together by intercellular junctions; extracellular matrix with brous proteins, typically collagens, between two dissimilar epithelia; sexual with production of an egg cell that is fertilized by a smaller, often monociliated, sperm cell; phagotrophic and osmotrophic; without cell wall. Eukaryote classifcation The principal taxa or classiying eukaryotes are kingdom, phylum, class, order, amily, genus and species. E ukaryotes are classifed into kingdoms. Each kingdom is divided up into phyla, which are divided into classes, then orders, amilies and genera. The hierarchy o taxa or classiying eukaryotes is thus kingdom, phylum, class, order, amily, genus and species. Most biologists recognize our kingdoms o eukaryote: plants, animals, ungi and protoctista. The last o these is the most controversial as protoctists are very diverse and should be divided up into more kingdoms. At present there is no consensus on how this should be done. Figure 5 Brown seaweeds have been classifed in the kingdom Protoctista 261 5 E vo l u t i o n an d b i o d i vE r s i t y Examples o classifcatio Classifcation o one plant and one animal species rom domain to species level. Animals and plants are kingdoms o the domain Eukaryota. Table 2 shows the classication o one plant and one animal species rom kingdom down to species. taxon Kingdom Phylum Class Order Family Genus Species Grey wolf Animalia Chordata Mammalia Carnivora Canidae Canis lupus Dae palm Plantae Angiospermophyta Monocotyledoneae Palmales Arecaceae Phoenix dactylifera Table 2 Daa-based quesions: Classiying cartilaginous fsh All the sh shown in gure 6 are in the class C hondrichthyes. They are the most requently ound sh in this class in north- west Europe. 1 S tate the kingdom to which all o the species in gure 6 belong. [1 ] 2 a) Four o the sh in gure 6 are classied in the same genus. D educe which these sh are. [1 ] b) D educe with a reason whether these our sh are in: ( i) the same or dierent species [2 ] ( ii) the same or dierent amilies. [2 ] c) State two characteristics o these our sh that are not possessed by the other our sh. [2 ] 3 Figure 6 Cartilaginous fsh in seas in north-west Europe The other our sh are classied into two orders. D educe, with a reason, how the our sh are split into two orders. [2 ] natural classifcatio In a natural classifcation, the genus and accompanying higher taxa consist o all the species that have evolved rom one common ancestral species. Scientic consensus is to classiy species in a way that most closely ollows the way in which species evolved. Following this convention, all members o a genus or higher taxon should have a common ancestor. This is called a natural classication. Because o the common ancestry we can expect the members o a natural group to share many characteristics. 262 An example o an unnatural or articial classication would be one in which birds, bats and insects are grouped together, because they all fy. Flight evolved separately in these groups and as they do not share a common ancestor they dier in many ways. It would not be appropriate to classiy them together other than to place them 5 . 3 c l A s s i i c At i o n o B i o D i v E r s i t Y all in the animal kingdom and both birds and bats in the phylum C hordata. Plants and ungi were at one time classifed together, presumably because they have cell walls and do not move, but this is an artifcial classifcation as their cell walls evolved separately and molecular research shows that they are no more similar to each other than to animals. It is not always clear which groups o species do share a common ancestor, so natural classifcation can be problematic. C onvergent evolution can make distantly related organisms appear superfcially similar and adaptive radiation can make closely related organisms appear dierent. In the past, natural classifcation was attempted by looking at as many visible characteristics as possible, but new molecular methods have been introduced and these have caused signifcant changes to the classifcation o some groups. More details o this are given later, in sub-topic 5 .4. TOK Wha a fuee he deepme a e eu? Carl Linnaeuss 1753 book Species Plantarum introduced consistent two-part names (binomials) or all species o the vegetable kingdom then known. Thus the binomial Physalis angulata replaced the obsolete phrase-name, Physalis annua ramosissima, ramis angulosis glabris, foliis dentato-serratis. Linnaeus brought the scientifc nomenclature o plants back to the simplicity and brevity o the vernacular nomenclature out o which it had grown. Folk-names or species rarely exceed three words. In groups o species alike enough to have a vernacular group-name, the species are oten distinguished by a single name attached to the group-name, as in the Ancient Greek and (used by Threophrastus), Latin anagallis mas and anagallis emina (used by Pliny), German weiss Seeblumen and geel Seeblumen (used by Fuchs), English wild mynte and water mynte (used by Turner) and Malayan jambu bol and jambu chilli (applied by Malays to dierent species o Eugenia). The International Botanical Congress held in Genoa in 1892 proposed that 1753 be taken as the starting point or both genera and species. This was incorporated in the American Rochester Code o 1883 and in the code used at the Berlin Botaniches Museum and supported by British Museum o Natural History, Harvard University botanists and a group o Swiss and Belgian botanists. The International Botanical Congress o Vienna in 1905 accepted by 150 votes to 19 the rule that La nomenclature botanique commence avec Linn, Species Plantarum (ann. 1753) pour les groupes de plantes vasculaires. 1 Why was Linnaeuss system or naming plants adopted as the international system, rather than any other system? 2 Why do the international rules o nomenclature state that genus and species names must be in Ancient Greek or Latin? 3 Making decisions by voting is rather unusual in science. Why is it done at International Botanical Congresses? What knowledge issues are associated with this method o decision making? reviewing classifcation Taxonomists sometimes reclassiy groups o species when new evidence shows that a previous taxon contains species that have evolved rom dierent ancestral species. S ometimes new evidence shows that members o a group do not share a common ancestor, so the group should be split up into two or more taxa. C onversely species classifed in dierent taxa are sometimes ound to be closely related, so two or more taxa are united, or species are moved rom one genus to another or between higher taxa. The classifcation o humans has caused more controversy than any other species. Using standard taxonomic procedures, humans are assigned to the order Primates and the amily Hominidae. There has been much debate about which, i any, o the great apes to include in this amily. O riginally all the great apes were placed in another amily, 263 5 E vo l u t i o n an d b i o d i vE r s i t y the Pongidae, but research has shown that chimpanzees and gorillas are closer to humans than to orang- utans and so should be in the same amily. This would j ust leave orang- utans in the Pongidae. Most evidence suggests that chimpanzees are closer than gorillas to humans, so i humans and chimpanzees are placed in dierent genera, gorillas should also be in a separate genus. A summary o this scheme or human classication is shown in gure 7. FAMILY GENUS AND SPECIES Figure 7 Pongidae Hominidae Gorilla gorilla (gorilla) Homo sapiens (human) Pan troglodytes (chimpanzee) Pan paniscus (bonobo) Pongo pygmaeus (orang-utan) Classifcation o humans advntges o nturl clssifction Natural classications help in identication o species and allow the prediction o characteristics shared by species within a group. There is great interest at the moment in the biodiversity o the world. Groups o biologists are surveying areas where little research has been done beore, to nd out what species are present. Even in well-known parts o the world new species are sometimes discovered. Natural classication o species is very helpul in research into biodiversity. It has two specic advantages. 1 Identication o species is easier. I a specimen o an organism is ound and it is not obvious what species it is, the specimen can be identied by assigning it rst to its kingdom, then the phylum within the kingdom, class within the phylum and so on down to species level. D ichotomous keys can be used to help with this process. This process would not work so well with an articial classication. For example, i fowering plants were classied according to fower colour and a white- fowered bluebell Hyacinthoides non-scripta was discovered, it would not be identied correctly as the species normally has blue fowers. 2 B ecause all o the members o a group in a natural classication have evolved rom a common ancestral species, they inherit similar characteristics. This allows prediction o the characteristics o species within a group. For example, i a chemical that is useul as a drug is ound in one plant in a genus, this or related chemicals are likely to be ound in other species in the genus. I a new species o bat was discovered, we could make many predictions about it with reasonable certainty that they are correct: the bat will have hair, mammary glands, a placenta, a our- chambered heart and many other mammalian eatures. None o these predictions could be made i bats were classied articially with all other fying organisms. Figure 8 Members o the Hominidae and Pongidae Ativity controlling potato blight Phytophthora infestans, the organism that causes the disease potato blight, has hyphae and was classied as a ungus, but molecular biology has shown that it is not a true ungus and should be classied in a dierent kingdom, possibly the Protoctista. Potato blight has proved to be a difcult disease to control using ungicides. Discuss reasons or this. 264 5 . 3 c l A s s i f i c At i o n o f B i o D i v E r s i t Y dichotomous keys Construction o dichotomous keys or use in identiying specimens D ichotomous keys are oten constructed to use or identiying species within a group. A dichotomy is a division into two; a dichotomous key consists o a numbered series o pairs o descriptions. O ne o these should clearly match the species and the other should clearly be wrong. The eatures that the designer o the key chooses to use in the descriptions should thereore be reliable and easily visible. E ach o the pair o descriptions leads either to another o the numbered pairs o descriptions in the key, or to an identifcation. An example o a key is shown in table 3 . We can use it to identiy the species in fgure 9. In the frst stage o the key, we must decide i hind limbs are visible. They are not, so we are directed to stage 6 o the key. We must now decide i the species has a blowhole. It does not, so it is a dugong or a manatee. A uller key would have another stage to separate dugongs and manatees. 1 Fore and hind limbs visible, can emerge on land ..... 2 Only ore limbs visible, cannot live on land ................ 6 2 Fore and hind limbs have paws ..................................... 3 Fore and hind limbs have fippers ................................. 4 3 Fur is dark ............................................................ sea otters Fur is white ........................................................ polar bears 4 External ear fap visible ........... sea lions and ur seals No external ear fap ........................................................... 5 5 Two long tusks ..................................................... walruses No tusks ............................................................... true seals 6 Mouth breathing, no blowhole ... dugongs and manatees Breathing through blowholes ......................................... 7 7 Two blowholes, no teeth ......................... baleen whales One blowhole, teeth ........ dolphins, porpoises and whales Table 3 Key to groups of marine mammals Ay cug dhmu key Keys are usually designed or use in a particular area. All the groups or species that are ound in that area can be identied using the key. There may be a group o organisms in your area or which a key has never been designed. You could design a key to the trees in the local orest or on your school campus, using lea descriptions or bark descriptions. You could design a key to birds that visit bird-eeding stations in your area. You could design a key to the invertebrates that are associated with one particular plant species. You could design a key to the ootprints o mammals and birds (gure 10) . They are all right ront ootprints and are not shown to scale. bear duck wolf rabbit / hare fox cat dog squirrel deer heron Figure 10 Footprints of mammals and Figure 9 Manatee birds 265 5 E vo l u t i o n an d b i o d i vE r s i t y Plants External recognition eatures o bryophytes, licinophytes, conierophytes and angiospermophytes. All plants are classied together in one kingdom. In the lie cycle o every plant, male and emale gametes are ormed and use together. The zygote ormed develops into an embryo. The way in which this embryo develops depends on the type o plant it is. The dierent types o plants are put into phyla. Most plants are in one o our phyla, but there are other smaller phyla. The Ginkgo biloba tree or Bryophyta Vegetative organs parts o the plant concerned with growth rather than reproduction B ryophyta mosses, liverworts and hornworts Filicinophyta erns C onierophyta coniers Angiospermophyta fowering plants. The external recognition eatures o these phyla are shown in table 4. filiinophyta conierophyta Angiospermophyta Rhizoids but no Roots, stems and leaves are usually present true roots. Some with simple stems and leaves; others have only a thallus No xylem or Vascular tissue tissues with tubular structures used phloem or transport within the plant Xylem and phloem are both present Cambium cells between xylem and phloem that can produce more o these tissues No cambium; no true trees and shrubs Present in coniers and most angiosperms, allowing secondary thickening o stems and roots and development o plants into trees and shrubs Pollen small structures containing male gametes that are dispersed Pollen is not produced Pollen is produced in male cones Ovules contains a emale gamete and develops into a seed ater ertilization No ovaries or ovules Ovules are produced Ovules are enclosed in emale cones inside ovaries in fowers Seeds dispersible unit consisting o an embryo plant and ood reserves, inside a seed coat No seeds Seeds are produced and dispersed Fruits seeds together with a ruit wall developed rom the ovary wall No ruits Table 4 266 example is in one o the smaller phyla. The our main plant phyla are: Pollen is produced by anthers in fowers Fruits produced or dispersal o seeds by mechanical, wind or animal methods 5 . 3 c l A s s i f i c At i o n o f B i o D i v E r s i t Y animl phyl Recognition eatures o poriera, cnidaria, platyhelminthes, annelida, mollusca and arthropoda, chordata. Animals are divided up into over 3 0 phyla, based on their characteristics. Six phyla are eatured in table 5 . Two examples o each are shown in fgure 1 1 . Phyum Muh/au Poriera an sponges, cup sponges, tube sponges, glass sponges No mouth or anus None Internal spicules (sketetal needles) Many pores over the surace through which water is drawn in or lter eeding. Very varied shapes Cnidaria hydras, jellysh, corals, sea anemones Mouth only Radial Sot, but hard corals secrete CaCO 3 Tentacles arranged in rings around the mouth, with stinging cells. Polyps or medusae (jellysh) Platyhelminthes fatworms, fukes, tapeworms Mouth only Bilateral Sot, with no skeleton Flat and thin bodies in the shape o a ribbon. No blood system or system or gas exchange Mollusca bivalves, gastropods, snails, chitons, squid, octopus Mouth and anus Bilateral Most have shell made o CaCO 3 A old in the body wall called the mantle secretes the shell. A hard rasping radula is used or eeding Annelida marine bristleworms, oligochaetes, leeches Mouth and anus Bilateral Internal cavity with fuid under pressure Bodies made up o many ringshaped segments, oten with bristles. Blood vessels oten visible Arthropoda insects, arachnids, crustaceans, myriapods Mouth and anus Bilateral External skeleton made o plates o chitin Segmented bodies and legs or other appendages with joints between the sections Table 5 Characteristics of six animal 1 2 symmey skee ohe exea eg eaue phyla Study the organisms shown in fgure 1 1 and assign each one to its phylum. 3 [7] List the organisms that have: a) j ointed appendages List the organisms that are: b) stinging tentacles a) bilaterally symmetric c) bristles. [3 ] List the organisms that flter eed by pumping water through tubes inside their bodies. [2 ] b) radially symmetric c) not symmetrical in their structure. 4 [3 ] 267 5 E vo l u t i o n an d b i o d i vE r s i t y vertebrates Recognition o eatures o birds, mammals, amphibians, reptiles and fsh. Alcyonium glomeratum Adocia cinerea Nymphon gracilis Pycnogonum littorale Most species o chordate belong to one o fve major classes, each o which contains more than a thousand species. Although the numbers are not certain and new species are still sometimes discovered, there are about 1 0,000 bird species, 9,000 reptiles, 6,000 amphibians and 5 ,700 mammals. All o these classes are outnumbered by the ray-fnned bony fsh, with more than 30,000 species. The recognition eatures o the fve largest classes o chordate are shown in table 6. All o the organisms are vertebrates, because they have a backbone composed o vertebrae. Bony ayfnned fsh Corynactis viridis Polymastia mammiliaris Amphibians reptiles Lepidonotus clara Sot moist skin permeable to water and gases Impermeable skin covered in scales o keratin Skin with Skin has eathers made ollicles with o keratin hair made o keratin Cyanea capillata Gills covered by an operculum, with one gill slit Simple lungs with small olds and moist skin or gas exchange Lungs with extensive olding to increase the surace area Lungs with para-bronchial tubes, ventilated using air sacs No limbs Tetrapods with pentadactyl limbs Fins supported by rays Four legs when adult Loligo forbesii Arenicola marina Eggs and sperm released or external ertilization Remain in water throughout their lie cycle Larval stage that lives in water and adult that usually lives on land Prostheceraeus vittatus Swim bladder Eggs coated containing gas in protective or buoyancy jelly Caprella linearis Four legs (in Two legs and most species) two wings Invertebrate diversity Table 6 Lungs with alveoli, ventilated using ribs and a diaphragm Four legs in most (or two legs and two wings/arms) Sperm passed into the emale or internal ertilization Female lays Most give eggs with hard birth to live young and shells all eed young with milk rom mammary glands Beak but no Teeth o Teeth all o one type, with teeth dierent types with a no living parts living core Female lays eggs with sot shells Do not maintain constant body temperature Gammarus locusta 268 Mammals Scales which are bony plates in the skin Procerodes littoralis Figure 11 Bids Maintain constant body temperature 5 . 4 cl AD i s ti cs 5.4 cad udertdig A clade is a group o organisms that have evolved rom a common ancestor. Evidence or which species are part o a clade can be obtained rom the base sequences o a gene or the corresponding amino acid sequence o a protein. Sequence dierences accumulate gradually so there is a positive correlation between the number o dierences between two species and the time since they diverged rom a common ancestor. Traits can be analogous or homologous. Cladograms are tree diagrams that show the most probable sequence o divergence in clades. Evidence rom cladistics has shown that classifcations o some groups based on structure did not correspond with the evolutionary origins o a group o species. applictio Cladograms including humans and other primates. Reclassifcation o the fgwort amily using evidence rom cladistics. skill Analysis o cladograms to deduce evolutionary relationships. ntre of ciece Falsifcation o theories with one theory being superseded by another: plant amilies have been reclassifed as a result o evidence rom cladistics. Clde A clade is a group o organisms that have evolved rom a common ancestor. S pecies can evolve over time and split to orm new species. This has happened repeatedly with some highly successul species, so that there are now large groups o species all derived rom a common ancestor. These groups o species can be identifed by looking or shared characteristics. A group o organisms evolved rom a common ancestor is called a clade. C lades include all the species alive today, together with the common ancestral species and any species that evolved rom it and then became extinct. They can be very large and include thousands o species, or very small with j ust a ew. For example, birds orm one large clade with about ten thousand living species because they have all evolved rom a common ancestral species. The tree Ginkgo biloba is the only living member o a clade that evolved about 2 70 million years ago. There have been other species in this clade but all are now extinct. 269 5 E vo l u t i o n an d b i o d i vE r s i t y Aciviy the EDGE of Exisence projec The aim o this project is to identiy animal species that have ew or no close relatives and are thereore members o very small clades. The conservation status o these species is then assessed. Lists are prepared o species that are both Evolutionarily Distinct and Globally Endangered, hence the name o the project. Species on these lists can then be targeted or more intense conservation eforts than other species that are either not threatened or have close relatives. In some cases species are the last members o a clade that has existed or tens or hundreds o millions o years and it would be tragic or them to become extinct as a result o human activities. What species on EDGE lists are in your part o the world and what can you do to help conserve them? http://www.edgeoexistence.org/species/ Figure 1 Two species on the EDGE list: Loris tardigradus tardigradus (Horton Plains slender loris) rom Sri Lanka and Bradypus pygmaeus (Pygmy three-toed sloth) rom Isla Escudo de Veraguas, a small island of the coast o Panama Identifying members of a clade Evidence or which species are part o a clade can be obtained rom the base sequences o a gene or the corresponding amino acid sequence o a protein. It is not always obvious which species have evolved from a common ancestor and should therefore be included in a clade. The most obj ective evidence comes from base sequences of genes or amino acid sequences of proteins. S pecies that have a recent common ancestor can be expected to have few differences in base or amino acid sequence. C onversely, species that might look similar in certain respects but diverged from a common ancestor tens of millions of years ago are likely to have many differences. 270 5 . 4 cl AD i s ti cs Moleculr clocks Sequence diferences accumulate gradually so there is a positive correlation between the number o diferences between two species and the time since they diverged rom a common ancestor. D ierences in the base sequence o D NA and thereore in the amino acid sequence o proteins are the result o mutations. They accumulate gradually over long periods o time. There is evidence that mutations occur at a roughly constant rate so they can be used as a molecular clock. The number o dierences in sequence can be used to deduce how long ago species split rom a common ancestor. For example, mitochondrial D NA rom three humans and our related primates has been completely sequenced. From the dierences in base sequence, a hypothetical ancestry has been constructed. It is shown in fgure 2 . Using dierences in base sequence as a molecular clock, these approximate dates or splits between groups have been deduced: European Japanese African Common chimpanzee Pygmy chimpanzee (bonobo) 70, 000 years ago, E uropeanJapanese split Gorilla 1 40, 000 years ago, AricanE uropean/Japanese split Oran -utan 5 , 000, 000 years ago, humanchimpanzee split Figure 2 anlogous nd homologous trits Traits can be analogous or homologous. S imilarities between organisms can either be homologous or analogous. Homologous structures are similar because o similar ancestry; or example the chicken wing, human arm and other pentadactyl orelimbs. Analogous structures are similar because o convergent evolution. The human eye and the octopus eye show similarities in structure and unction but they are analogous because they evolved independently. Problems in distinguishing between homologous and analogous structures have sometimes led to mistakes in classifcation in the past. For this reason the morphology ( orm and structure) o organisms is now rarely used or identiying members o a clade and evidence rom base or amino acid sequences is trusted more. cornea iris lens retina photoreceptors optic nerve Figure 3 The human eye (left) and the octopus eye (right) are analogous because they are quite similar yet evolved independently 271 5 birds non-avian dinosaurs crocodiles lizards snakes turtles E vo l u t i o n an d b i o d i vE r s i t y ancestral species A ancestral species B ancestral species C Figure 4 A cladogram showing the hypothesized relationship between birds and the traditional taxonomic group the reptiles Activity Figure 5 shows an artists impression o two pterosaurs, which were the rst chordates to develop powered fight. They were neither birds nor dinosaurs. Where might pterosaurs have tted into the cladogram shown in gure 4? Cladograms Cladograms are tree diagrams that show the most probable sequence o divergence in clades. A cladogram is a tree diagram based on similarities and dierences between the species in a clade. C ladograms are almost always now based on base or amino acid sequences. C omputer programs have been developed that calculate how species in a clade could have evolved with the smallest number o changes o base or amino acid sequence. This is known as the principle o parsimony and although it does not prove how a clade actually evolved, it can indicate the most probable sequence o divergence in clades. The branching points on cladograms are called nodes. Usually two clades branch o at a node but sometimes there are three or more. The node represents a hypothetical ancestral species that split to orm two or more species. O ption B includes instructions or constructing cladograms rom base sequences using computer sotware. Figure 4 is an example o a cladogram or birds and reptiles. It has been based on morphology, so that extinct groups can be included. B irds, non- avian dinosaurs and ancestral species A orm a clade called dinosauria. B irds, non- avian dinosaurs, crocodiles and ancestral species B are part o a clade called archosaurs. Lizards, snakes and ancestral species C orm a clade called squamates. This cladogram suggests either that birds should be regarded as reptiles or that reptiles should be divided into two or more groups, as some reptiles are more closely related to birds than to other reptiles. Figure 5 Two pterosaurs in fight Primate cladograms Cladograms including humans and other primates. The closest relatives o humans are chimpanzees and bonobos. The entire genome o these three species has been sequenced giving very strong evidence or the construction o a cladogram ( fgure 6) . The numbers on the cladogram are estimates o population sizes and dates when splits occurred. These are based on a molecular clock with a mutation rate o 1 0 9 yr 1 . Figure 7 is a cladogram or primates and the most closely related other groups o mammal. Primates are an order o mammals that have adaptations or climbing trees. Humans, monkeys, baboons, gibbons and lemurs are primates. 272 45,000 4.5 Myr ago 27,000 1 Myr ago 12,000 Bonobo Figure 6 Chimpanzee Human 5 . 4 cl AD i s ti cs Cavies and Coypu anlysis of cldogrms Porcupines Analysis o cladograms to deduce evolutionary relationships. Mice and Rats The pattern o branching in a cladogram is assumed to match the evolutionary origins o each species. The sequence o splits at nodes is thereore a hypothetical sequence in which ancestors o existing clades diverged. I two clades on a cladogram are linked at a node, they are relatively closely related. I two species are only connected via a series o nodes, they are less closely related. Rabbits S ome cladograms include numbers to indicate numbers o dierences in base or amino acid sequence or in genes. B ecause genetic changes are assumed to occur at a relatively constant rate, these numbers can be used to estimate how long ago two clades diverged. This method o estimating times is called a molecular clock. S ome cladograms are drawn to scale according to estimates o how long ago each split occurred. Although cladograms can provide strong evidence or the evolutionary history o a group, they cannot be regarded as proo. C ladograms are constructed on the assumption that the smallest possible number o mutations occurred to account or current base or amino acid sequence dierences. S ometimes this assumption is incorrect and pathways o evolution were more convoluted. It is thereore important to be cautious in analysis o cladograms and where possible compare several versions that have been produced independently using dierent genes. Beavers Chipmunks Primates Treeshrews Figure 7 Avy A adogram for he grea ape The great apes are a amily o primates. The taxonomic name is Hominidae. There are fve species on Earth today, all o which are decreasing in number apart rom humans. Figure 6 is a cladogram or three o the species. Use this inormation to expand the cladogram to include all the great apes: the split between humans and gorillas occurred about 10 million years ago and the split between humans and orangutans about 15 million years ago. Daa-baed queon: Origins of turtles and lizards C ladograms based on morphology suggest that turtles and lizards are not a clade. To test this hypothesis, microRNA genes have been compared or nine species o chordate. The results were used to construct the cladogram in fgure 8. The numbers on the cladogram show which microRNA genes are shared by members o a clade but not members o other clades. For example, there are six microRNA genes ound in humans and short-tailed opossums but not in any o the other chordates on the cladogram. 1 the short- tailed opossum or to the duck-billed platypus. [2 ] 2 C alculate how many microRNA genes are ound in the mammal clade on the cladogram but not in the other clades. [2 ] 3 D iscuss whether the evidence in the cladogram supports the hypothesis that turtles and lizards are not a clade. [3 ] 4 Evaluate the traditional classifcation o tetrapod chordates into amphibians, reptiles, birds and mammals using evidence rom the cladogram. [3 ] D educe, using evidence rom the cladogram, whether humans are more closely related to 273 5 E vo l u t i o n an d b i o d i vE r s i t y African clawed frog 6 Human 340 671 761 885 1251 1397 3 Short-tailed opossum 186 590 873 Duck-billed platypus 19 Zebra nch 1451 1460 1467 1559 1641 1567 1669 1729 1743 1744 1756 1759 1781 1784 1789 1803 2131 2954 1791 1 2964 490 1397 1 Chicken Alligator 1677 4 Painted turtle 5390 5391 5392 5393 Lizard Figure 8 Cladograms and reclassifcation Evidence rom cladistics has shown that classifcations o some groups based on structure did not correspond with the evolutionary origins o a group o species. The construction o cladograms based on base and amino acid sequences only became possible towards the end o the 2 0th century. B eore that the sequence data was not available and computer sotware had not been developed to do the analysis. The construction o cladograms and identifcation o clades is known as cladistics. C ladistics has caused some revolutions in plant and animal classifcation. It is now clear rom cladograms that traditional classifcation based on morphology does not always match the evolutionary origins o groups o species. As a result some groups have been reclassifed. S ome groups have been merged, others have been divided and in some cases species have been transerred rom one group to another. Reclassifcation o groups o organisms is time- consuming and potentially disruptive or biologists, but it is certainly worthwhile. The new classifcations based on cladistics are likely to be much closer to a truly natural classifcation so their predictive value will be higher. They have revealed some unnoticed similarities between groups and also some signifcant dierences between species previously assumed to be similar. 274 5 . 4 cl AD i s ti cs Cladograms and alsifcation Falsifcation o theories with one theory being superseded by another: plant amilies have been reclassifed as a result o evidence rom cladistics. The reclassifcation o plants on the basis o discoveries in cladistics is a good example o an important process in science: the testing o theories and o replacement o theories ound to be alse with new theories. The classifcation o angiospermophytes into amilies based on their morphology was begun by the French botanist Antoine Laurent de Jussieu in Genera plantarum, published in 1 789 and revised repeatedly during the 1 9th century. Classifcation o the fgwort amily Reclassifcation o the fgwort amily using evidence rom cladistics. There are more than 400 amilies o angiosperms. Until recently the eighth largest was the S crophulariaceae, commonly known as the fgwort amily. It was one o the original amilies proposed by de Jussieu in 1 789. He gave it the name S crophulariae and included sixteen genera, based on similarities in their morphology. As more plants were discovered, the amily grew until there were over 2 75 genera, with more than 5 , 000 species. Taxonomists recently investigated the evolutionary origins o the fgwort amily using cladistics. O ne important research proj ect compared the base sequences o three chloroplast genes in a large number o species in genera traditionally assigned to the S crophulariaceae and genera in closely related amilies. It was ound that species in the fgwort amily were not a true clade and that fve clades had incorrectly been combined into one amily. Two small families were merged with the gwort family: the buddleja family, Buddlejaceae and the myoporum family, Myoporaceae Two genera were moved to a newly-created family, the calceolaria family, Calceolariaceae The gwort family Scrophulariaceae Thirteen genera have been transferred to a newly-created family, the lindernia family, Linderniaceae Nearly fty genera have been moved to the plantain family, Plantaginaceae About twelve genera of parasitic plants have been moved to the broomrape family, Orobanchaceae Figure 9 275 5 E vo l u t i o n an d b i o d i vE r s i t y A major reclassifcation has now been carried out. Less than hal o the species have been retained in the amily, which is now only the thirty-sixth largest among the angiosperms. A summary o the Figure 10 Antirrhinum majus has been transerred rom the fgwort amily to the plantain amily 276 changes is shown in fgure 9. This reclassifcation has been welcomed as it was widely appreciated beore that the Scrophulariaceae had been a rag-bag o species rather than a natural group. Figure 11 Scrophularia peregrina has remained in the fgwort amily QuEstion s Questions The bar charts in fgure 1 2 show the growth o three populations o an alga, Ectocarpus siliculosus, at dierent copper concentrations. O ne population came rom an unpolluted environment at Rhosneigr in Wales. The other two came rom the undersides o ships that had been painted with a copper- containing anti- ouling paint. % increase in algal volume 500 4 Which o the ollowing processes are required or copper tolerance to develop in a population? ( i) variation in copper tolerance ( ii) inheritance o copper tolerance ( iii) ailure o algae with lower copper tolerance to survive or reproduce. a) Rhosneigr i) only b) i) and ii) only 0 c) M.V. San Nicholas i) and iii) only d) i) , ii) and iii) . 500 0 M.V. Amama 500 0 0.0 0.01 0.05 0.1 0.5 1.0 5.0 10.0 concentration of copper (mg dm -3 ) 5 In fgure 1 3 , each number represents a species. The closer that two numbers are on the diagram the more similar the two species. The circles represent taxonomic groups. For example, the diagram shows that 2 , 3 , 4 and 5 are in the same genus. Figure 12 1 2 How much higher was the maximum copper concentration tolerated by the algae rom ships than the algae rom an unpolluted environment? a) 0.09 times higher b) 0.1 1 times higher c) 1 .0 times higher d) 1 0 times higher. 1 8 9 10 19 20 21 22 23 What is the reason or results lower than zero on the bar charts? a) Increases in volume were less than 1 00% . d) Results were too small to measure accurately. 3 What was the reason or the dierence in copper tolerance between the algae? a) The algae on the ships absorbed copper. b) The algae can develop copper tolerance and pass it on to their ospring. c) 1112 13 14 15 16 17 18 34 67 24 25 26 27 28 29 30 31 32 33 The volume o algae decreased. b) The algae all died. c) 23 45 The copper in the paint caused mutations. d) The copper in the paint caused natural selection or higher levels o copper tolerance. Figure 13 a) S tate one species that is in a genus with no other species. b) S tate the species that are in a amily with two genera. c) S tate the species that are in an order with two amilies. [1 ] [2 ] [2 ] d) State the species that are in a class with three orders. [2 ] e) D educe whether species 8 is more closely related to species 1 6 or species 6. f) Explain why three concentric circles have been drawn around species 3 4 on the diagram. [2 ] 277 51 E vo l u t i o n an d b i o d i vE r s i t y 6 The map in gure 1 4 shows the distribution in the 1 95 0s o two orms o Biston betularia in B ritain and Ireland. Biston betularia is a species o moth that fies at night. It spends the daytime roosting on the bark o trees. The non-melanic orm has white wings, peppered with black spots. The melanic orm has black wings. B eore the industrial revolution, the melanic orm was very rare. The prevailing wind direction is rom the Atlantic O cean, to the west. a) S tate the maximum and minimum percentages o the melanic orm. Key Non-melanic Melanic [2 ] b) O utline the trends in the distribution o the two orms o Biston betularia, shown in gure 1 4. [2 ] c) Explain how natural selection can cause moths such as Biston betularia to develop camoufaged wing markings. [4] d) S uggest reasons or the distribution o the two orms. 278 [2 ] Figure 14 6 H U m A N p H yS I o l o g y Intrductin Research into human physiology is the foundation of modern medicine. B ody functions are carried out by specialized organ systems. The structure of the wall of the small intestine allows it to move, digest and absorb food. The blood system continuously transports substances to cells and simultaneously collects waste products. The skin and immune system resist the continuous threat of invasion by pathogens. The lungs are actively ventilated to ensure that gas exchange can occur passively. Neurons transmit the message, synapses modulate the message. Hormones are used when signals need to be widely distributed. 6.1 Digestion and absorption Understandin The contraction o circular and longitudinal muscle layers o the small intestine mixes the ood with enzymes and moves it along the gut. The pancreas secretes enzymes into the lumen o the small intestine. Enzymes digest most macromolecules in ood into monomers in the small intestine. Villi increase the surace area o epithelium over which absorption is carried out. Villi absorb monomers ormed by digestion as well as mineral ions and vitamins. Diferent methods o membrane transport are required to absorb diferent nutrients. Aicatins Processes occurring in the small intestine that result in the digestion o starch and transport o the products o digestion to the liver. Use o dialysis tubing to model absorption o digested ood in the intestine. Skis Production o an annotated diagram o the digestive system. Identication o tissue layers in transverse sections o the small intestine viewed with a microscope or in a micrograph. Nature f science Use models as representations o the real world: dialysis tubing can be used to model absorption in the intestine. 279 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy Structure of the digestive system Production of an annotated diagram of the digestive system. The part of the human body used for digestion can be described in simple terms as a tube through which food passes from the mouth to the anus. The role of the digestive system is to break down the diverse mixture of large carbon compounds in food, to yield ions and smaller compounds that can be absorbed. For proteins, lipids and polysaccharides digestion involves several stages that occur in different parts of the gut. D igestion requires surfactants to break up lipid droplets and enzymes to catalyse reactions. Glandular cells in the lining of the stomach and intestines produce some of the enzymes. S urfactants and other enzymes are secreted by accessory glands that have ducts leading to the digestive system. C ontrolled, selective absorption of the nutrients released by digestion takes place in the small intestine and colon, but some small molecules, notably alcohol, diffuse through the stomach lining before reaching the small intestine. Figure 1 is a diagram of the human digestive system. The part of the esophagus that passes through the thorax has been omitted. This diagram can be annotated to indicate the functions of different parts. A summary of functions is given in table 1 below. Structure mouth Mouth Voluntary control of eating and swallowing. Mechanical digestion of food by chewing and mixing with saliva, which contains lubricants and enzymes that start starch digestion Esophagus Movement of food by peristalsis from the mouth to the stomach Stomach Churning and mixing with secreted water and acid which kills foreign bacteria and other pathogens in food, plus initial stages of protein digestion Small intestine Final stages of digestion of lipids, carbohydrates, proteins and nucleic acids, neutralizing stomach acid, plus absorption of nutrients Pancreas Secretion of lipase, amylase and protease Liver Secretion of surfactants in bile to break up lipid droplets Gall bladder Storage and regulated release of bile Large intestine Re-absorption of water, further digestion especially of carbohydrates by symbiotic bacteria, plus formation and storage of feces esophagus gall bladder liver stomach pancreas small intestine large intestine anus Figure 1 280 The human digestive system Function Table 1 6 .1 D i g e S ti o n an D ab S o rpti o n Structure of the wall of the small intestine Identifcation o tissue layers in transverse sections o the small intestine viewed with a microscope or in a micrograph. The wall o the small intestine is made o layers o living tissues, which are usually quite easy to distinguish in sections o the wall. From the outside o the wall going inwards there are our layers: serosa an outer coat muscle layers longitudinal muscle and inside it circular muscle sub-mucosa a tissue layer containing blood and lymph vessels mucosa the lining o the small intestine, with the epithelium that absorbs nutrients on its inner surace. Figure 2 Longitudinal section through the wall o the small intestine. Folds are visible on the inner surace and on these olds are fnger-like projections called villi. All o the our main tissue layers are visible, including both circular and longitudinal parts o the muscle layer. The mucosa is stained darker than the sub-mucosa peristalsis The contraction o circular and longitudinal muscle layers o the small intestine mixes the ood with enzymes and moves it along the gut. The circular and longitudinal muscle in the wall o the gut is smooth muscle rather than striated muscle. It consists o relatively short cells, not elongated fbres. It oten exerts continuous moderate orce, interspersed with short periods o more vigorous contraction, rather than remaining relaxed unless stimulated to contract. Waves o muscle contraction, called peristalsis, pass along the intestine. C ontraction o circular muscles behind the ood constricts the gut to prevent it rom being pushed back towards the mouth. C ontraction o longitudinal muscle where the ood is located moves it on along the gut. The contractions are controlled unconsciously not by the brain but by the enteric nervous system, which is extensive and complex. acvy tssu l dms f h s wll To practice your skill at identiying tissue layers, draw a plan diagram o the tissues in the longitudinal section o the intestine wall in fgure 2. To test your skill urther, draw a plan diagram to predict how the tissues o the small intestine would appear in a transverse section. S wallowed ood moves quickly down the esophagus to the stomach in one continuous peristaltic wave. Peristalsis only occurs in one direction, away rom the mouth. When ood is returned to the mouth rom the stomach during vomiting, abdominal muscles are used rather than the circular and longitudinal muscle in the gut wall. In the intestines the ood is moved only a ew centimetres at a time so the overall progression through the intestine is much slower, allowing time or digestion. The main unction o peristalsis in the intestine is churning o the semi- digested ood to mix it with enzymes and thus speed up the process o digestion. 281 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy pancreatic juice The pancreas secretes enzymes into the lumen of the small intestine. The pancreas contains two types o gland tissue. Small groups o cells secrete the hormones insulin and glucagon into the blood. The remainder o the pancreas synthesizes and secretes digestive enzymes into the gut in response to eating a meal. This is mediated by hormones synthesized and secreted by the stomach and also by the enteric nervous system. The structure o the tissue is shown in fgure 4. Small groups o gland cells cluster round the ends o tubes called ducts, into which the enzymes are secreted. Figure 3 Three-dimensional image showing the wave of muscle contraction (brown) in the esophagus during swallowing. Green indicates when the muscle is exerting less force. Time is shown left to right. At the top the sphincter between the mouth and the esophagus is shown permanently constricted apart from a brief opening when swallowing starts The digestive enzymes are synthesized in pancreatic gland cells on ribosomes on the rough endoplasmic reticulum. They are then processed in the Golgi apparatus and secreted by exocytosis. Ducts within the pancreas merge into larger ducts, fnally orming one pancreatic duct, through which about a litre o pancreatic juice is secreted per day into the lumen o the small intestine. Pancreatic j uice contains enzymes that digest all the three main types o macromolecule ound in ood: amylase to digest starch lipases to digest triglycerides, phospholipids proteases to digest proteins and peptides. Digestion in the small intestine secretory vesicles one acinus Enzymes digest most macromolecules in food into monomers in the small intestine. The enzymes secreted by the pancreas into the lumen o the small intestine carry out these hydrolysis reactions: basement membrane secretory cells wall of duct starch is digested to maltose by amylase triglycerides are digested to atty acids and glycerol or atty acids and monoglycerides by lipase phospholipids are digested to atty acids, glycerol and phosphate by phospholipase proteins and polypeptides are digested to shorter peptides by protease. lumen of duct Figure 4 Arrangement of cells and ducts in a part of the pancreas that secretes digestive enzymes This does not complete the process o digestion into molecules small enough to be absorbed. The wall o the small intestine produces a variety o other enzymes, which digest more substances. S ome enzymes produced by gland cells in the intestine wall may be secreted in intestinal j uice but most remain immobilized in the plasma membrane o epithelium cells lining the intestine. They are active there and continue to be active when the epithelium cells are abraded o the lining and mixed with the semi- digested ood. 282 Nucleases digest D NA and RNA into nucleotides. Maltase digests maltose into glucose. 6 .1 D i g e S ti o n an D ab S o rpti o n Lactase digests lactose into glucose and galactose. Sucrase digests sucrose into glucose and ructose. Exopeptidases are proteases that digest peptides by removing single amino acids either rom the carboxy or amino terminal o the chain until only a dipeptide is let. D ipeptidases digest dipeptides into amino acids. B ecause o the great length o the small intestine, ood takes hours to pass through, allowing time or digestion o most macromolecules to be completed. Some substances remain largely undigested, because humans cannot synthesize the necessary enzymes. C ellulose or example is not digested and passes on to the large intestine as one o the main components o dietary fbre. Villi and the surface area for digestion Figure 5 Cystic fbrosis causes the pancreatic duct to become blocked by mucus. Pills containing synthetic enzymes help digestion in the small intestine. The photograph shows one days supply or a person with cystic fbrosis Villi increase the surface area of epithelium over which absorption is carried out. The process o taking substances into cells and the blood is called absorption. In the human digestive system nutrients are absorbed epithelium principally in the small intestine. The rate o absorption depends on the surace area o the epithelium that carries out the process. The small intestine in adults is approximately seven metres long and layer of microvilli 2 5 3 0 millimetres wide and there are olds on its inner surace, giving on surface of epithelium a large surace area. This area is increased by the presence o villi. Villi are small fnger-like proj ections o the mucosa on the inside o the intestine wall. A villus is between 0. 5 and 1 . 5 mm long and there can be as many as 40 o them per square millimetre o small intestine wall. They increase the surace area by a actor o about 1 0. lacteal (a branch of the lymphatic system) blood capillary goblet cells (secrete mucus) Absorption by villi Villi absorb monomers formed by digestion as well as mineral ions and vitamins. The epithelium that covers the villi must orm a barrier to harmul substances, while at the same time being permeable enough to allow useul nutrients to pass through. Figure 6 Structure o an intestinal villus Villus cells absorb these products o digestion o macromolecules in ood: glucose, ructose, galactose and other monosaccharides any o the twenty amino acids used to make proteins atty acids, monoglycerides and glycerol bases rom digestion o nucleotides. They also absorb substances required by the body and present in oods but not needing digestion: mineral ions such as calcium, potassium and sodium vitamins such as ascorbic acid ( vitamin C ) . Figure 7 Scanning electron micrograph o villi in the small intestine 283 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy S ome harmul substances pass through the epithelium and are subsequently removed rom the blood and detoxied by the liver. S ome harmless but unwanted substances are also absorbed, including many o those that give ood its colour and favour. These pass out in urine. S mall numbers o bacteria pass through the epithelium but are quickly removed rom the blood by phagocytic cells in the liver. methods of absorption Diferent methods o membrane transport are required to absorb diferent nutrients. To be absorbed into the body, nutrients must pass rom the lumen o the small intestine to the capillaries or lacteals in the villi. The nutrients must rst be absorbed into epithelium cells through the exposed part o the plasma membrane that has its surace area enlarged with microvilli. The nutrients must then pass out o this cell through the plasma membrane where it aces inwards towards the lacteal and blood capillaries o the villus. Many dierent mechanisms move nutrients into and out o the villus epithelium cells: simple diusion, acilitated diusion, active transport and exocytosis. These methods can be illustrated using two dierent examples o absorption: triglycerides and glucose. Triglycerides must be digested beore they can be absorbed. The products o digestion are atty acids and monoglycerides, which can be absorbed into villus epithelium cells by simple diusion as they can pass between phospholipids in the plasma membrane. Fatty acids are also absorbed by acilitated diusion as there are atty acid transporters, which are proteins in the membrane o the microvilli. O nce inside the epithelium cells, atty acids are combined with monoglycerides to produce triglycerides, which cannot diuse back out into the lumen. lumen of small intestine interior of villus villus epithelium Na + 3Na + low Na + concentration glucose blood capillary 2K+ glucose fatty acids and monoglycerides lacteal triglyceride Figure 8 284 Methods of absorption in the small intestine lipoprotein 6 .1 D i g e S ti o n an D ab S o rpti o n Triglycerides coalesce with cholesterol to orm droplets with a diameter o about 0. 2 m, which become coated in phospholipids and protein. These lipoprotein particles are released by exocytosis through the plasma membrane on the inner side o the villus epithelium cells. They then either enter the lacteal and are carried away in the lymph, or enter the blood capillaries in the villi. Glucose cannot pass through the plasma membrane by simple diusion because it is polar and thereore hydrophilic. Sodiumpotassium pumps in the inwards- acing part o the plasma membrane pump sodium ions by active transport rom the cytoplasm to the interstitial spaces inside the villus and potassium ions in the opposite direction. This creates a low concentration o sodium ions inside villus epithelium cells. Sodiumglucose co-transporter proteins in the microvilli transer a sodium ion and a glucose molecule together rom the intestinal lumen to the cytoplasm o the epithelium cells. This type o acilitated diusion is passive but it depends on the concentration gradient o sodium ions created by active transport. Glucose channels allow the glucose to move by acilitated diusion rom the cytoplasm to the interstitial spaces inside the villus and on into blood capillaries in the villus. Starch digestion in the small intestine Processes occurring in the small intestine that result in the digestion of starch and transport of the products of digestion to the liver. S tarch digestion illustrates some important processes including catalysis, enzyme specifcity and membrane permeability. S tarch is a macromolecule, composed o many -glucose monomers linked together in plants by condensation reactions. It is a maj or constituent o plant- based oods such as bread, potatoes and pasta. S tarch molecules cannot pass through membranes so must be digested in the small intestine to allow absorption. All o the reactions involved in the digestion o starch are exothermic, but without a catalyst they happen at very slow rates. There are two types o molecule in starch: amylose has unbranched chains o - glucose linked by 1 , 4 bonds; amylopectin has chains o -glucose linked by 1 , 4 bonds, with some 1 , 6 bonds that make the molecule branched. CH 2 OH O OH OH CH 2 OH O OH O O OH OH CH 2 OH O OH OH CH 2 OH O OH CH 2 OH O OH O O OH CH 2 OH O OH O OH O OH Figure 9 Small portion of an amylopectin molecule showing six -glucose molecules, all linked bv 1,4 bonds apart from one 1,6 bond that creates a branch The enzyme that begins the digestion o both orms o starch is amylase. S aliva contains amylase but most starch digestion occurs in the small intestine, catalysed by pancreatic amylase. Any 1 , 4 bond in starch molecules can be broken by this enzyme, as long as there is a chain o at least our glucose monomers. Amylose is thereore 285 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy digested into a mixture o two- and three- glucose ragments called maltose and maltotriose. B ecause o the specicity o its active site, amylase cannot break 1 , 6 bonds in amylopectin. Fragments o the amylopectin molecule containing a 1 , 6 bond that amylase cannot digest are called dextrins. D igestion o starch is completed by three enzymes in the membranes o microvilli on villus epithelium cells. Maltase, glucosidase and dextrinase digest maltose, maltotriose and dextrins into glucose. Glucose is absorbed into villus epithelium cells by co- transport with sodium ions. It then moves by acilitated diusion into the fuid in interstitial spaces inside the villus. The dense network o capillaries close to the epithelium ensures that glucose only has to travel a short distance to enter the blood system. C apillary walls consist o a single layer o thin cells, with pores between adj acent cells, but these capillaries have larger pores than usual, aiding the entry o glucose. B lood carrying glucose and other products o digestion fows though villus capillaries to venules in the sub- mucosa o the wall o the small intestine. The blood in these venules is carried via the hepatic portal vein to the liver, where excess glucose can be absorbed by liver cells and converted to glycogen or storage. Glycogen is similar in structure to amylopectin, but with more 1 , 6 bonds and thereore more extensive branching. modelling physiological processes Use models as representations of the real world: dialysis tubing can be used to model absorption in the intestine. Living systems are complex and when experiments are done on them, many actors can infuence the results. It can be very dicult to control all o the variables and analysis o results becomes dicult. S ometimes it is better to carry out experiments using only parts o systems. For example, much research in physiology has been carried out using clones o cells in tissue culture rather than whole organisms. Another approach is to use a model to represent part o a living system. B ecause it is much simpler, a model can be used to investigate specic aspects o a process. A recent example is the D ynamic Gastric Model, a computer- controlled model o the human stomach that carries out mechanical and chemical digestion o real ood samples. It can be used to investigate the eects o diet, drugs, alcohol and other actors on digestion. A simpler example is the use o dialysis tubing made rom cellulose. Pores in the tubing allow water and small molecules or ions to pass through reely, but not large molecules. These properties 286 Figure 10 The Dynamic Gastric Model with its inventor, Richard Faulks, adjusting the antrum mechanism mimic the wall o the gut, which is also more permeable to small rather than large particles. D ialysis tubing can be used to model absorption by passive diusion and by osmosis. It cannot model active transport and other processes that occur in living cells 6 .1 D i g e S ti o n an D ab S o rpti o n modelling the sall intestine Use of dialysis tubing to model absorption of digested food in the intestine. To make a model o the small intestine, cut a length o dialysis tubing and seal one end by tying a knot in the tubing or tying with a piece o cotton thread. Pour in a suitable mixture o oods and seal the open end by tying with a piece o cotton thread. Two experiments using model intestines made in this way are suggested here: 1 Investigating the need for digestion using a model of the small intestine S et up the apparatus shown in gure 1 1 and leave it or one hour. Results To obtain the results or the experiment, take the bags out o each tube, open them and pour the solutions rom them into separate test tubes rom the liquids in the tubes. You should now have our samples o fuid. D ivide each o these samples into two halves and test one hal or starch and the other hal or sugars. 10 ml of 1% starch solution and 1 ml of 1% amylase solution 10 ml of 1% starch solution and 1 ml of water water maintained at 40C water Figure 11 bags made of dialysis (Visking) tubing water S uggest improvements to the method, or suggest an entirely dierent method o investigating the need or digestion. 2 Investigating membrane permeability using a model of the small intestine C ola drinks contain a mixture o substances with dierent particle sizes. They can be used to represent ood in the small intestine. D ialysis tubing is semi- permeable so can be used to model the wall o the small intestine. Predictions C ola contains glucose, phosphoric acid and caramel, a complex carbohydrate added to produce a brown colour. Predict which o these substances will diuse out o the bag, with reasons or your predictions. Predict whether the bag will gain or lose mass during the experiment. Instructions 1 Make the model intestine with cola inside. 2 Rinse the outside o the bag to wash o any traces o cola and then dry the bag. tube top of bag sealed with cotton thread cola, left to go at before being put into the tube dialysis tubing pure water minimum volume to surround the bag base of bag knotted to prevent leaks Apparatus for showing the need for digestion Record all the results in the way that you think is most appropriate. spotting tile Conclusions and evaluation S tate careully all the conclusions that you can make rom your results. D iscuss the strengths and weaknesses o this method o investigating the need or digestion. pH indicator Figure 12 Apparatus for membrane permeability experiment 287 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy 3 Find the mass o the bag using an electronic balance. 4 When you are ready to start the experiment, place the bag in pure water in a test tube. 5 Test the water around the bag at suitable time intervals. A suggested range is 1 , 2 , 4, 8 and 1 6 minutes. At each time lit the bag up and down a ew times to mix the water in the tube, then do these tests: Look careully at the water to see whether it is still clear or has become brown. Use a dropping pipette to remove a ew drops o the water and test them in a spotting tile with a narrow- range pH indicator. Use a colour chart to work out the pH. D ip a glucose test strip into the water and record the colour that it turns. Instructions vary or these test strips. Follow the instructions and work out the glucose concentration o the water. 6 Ater testing the water or the last time, remove the bag, dry it and fnd its mass again with the electronic balance. Conclusions a) Explain the conclusions that you can draw about the permeability o the dialysis tubing rom the tests o the water and rom the change in mass o the bag. [5 ] b) C ompare and contrast the dialysis tubing and the plasma membranes that carry out absorption in villus epithelium cells in the wall o the intestine. [5 ] c) Use the results o your experiment to predict the direction o movement o water by osmosis across villus epithelium cells. [5 ] TOK What are some o the variables that afect perspectives as to what is normal? In some adult humans, levels o lactase are too low to digest lactose in milk adequately. Instead, lactose passes through the small intestine into the large intestine, where bacteria eed on it, producing carbon dioxide, hydrogen and methane. These gases cause some unpleasant symptoms, discouraging consumption o milk. The condition is known as lactose intolerance. It has sometimes in the past been regarded as an abnormal condition, or even as a disease, but it could be argued that lactose intolerance is the normal human condition. The rst argument or this view is a biological one. Female mammals produce milk to eed their young ofspring. When a young mammal is weaned, solid oods replace milk and lactase secretion declines. Humans who 288 continue to consume milk into adulthood are thereore unusual. Inability to consume milk because o lactose intolerance should not thereore be regarded as abnormal. The second argument is a simple mathematical one: a high proportion o humans are lactose intolerant. The third argument is evolutionary. Our ancestors were almost certainly all lactose intolerant, so this is the natural or normal state. Lactose tolerance appears to have evolved separately in at least three centres: Northern Europe, parts o Arabia, the Sahara and eastern Sudan, and parts o East Arica inhabited by the Tutsi and Maasai peoples. Elsewhere, tolerance is probably due to migration rom these centres. 6 . 2 t h e b l o o D S yS t e m 6.2 t d ss Understanding Arteries convey blood at high pressure rom the ventricles to the tissues o the body. Arteries have muscle and elastic bres in their walls. The muscle and elastic bres assist in maintaining blood pressure between pump cycles. Blood fows through tissues in capillaries with permeable walls that allow exchange o materials between cells in the tissue and the blood in the capillary. Veins collect blood at low pressure rom the tissues o the body and return it to the atria o the heart. Valves in veins and the heart ensure circulation o blood by preventing backfow. There is a separate circulation or the lungs. The heartbeat is initiated by a group o specialized muscle cells in the right atrium called the sinoatrial node. The sinoatrial node acts as a pacemaker. The sinoatrial node sends out an electrical signal that stimulates contraction as it is propagated through the walls o the atria and then the walls o the ventricles. The heart rate can be increased or decreased by impulses brought to the heart through two nerves rom the medulla o the brain. Epinephrine increases the heart rate to prepare or vigorous physical activity. Applications William Harveys discovery o the circulation o the blood with the heart acting as the pump. Causes and consequences o occlusion o the coronary arteries. Pressure changes in the let atrium, let ventricle and aorta during the cardiac cycle. Skills Identication o blood vessels as arteries, capillaries or veins rom the structure o their walls. Recognition o the chambers and valves o the heart and the blood vessels connected to it in dissected hearts or in diagrams o heart structure. Nature of science Theories are regarded as uncertain: William Harvey overturned theories developed by the ancient Greek philosopher Galen on movement o blood in the body. 289 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy William Harvey and the circulatin f bld William Harveys discovery of the circulation of the blood with the heart acting as the pump. William Harvey is usually credited with the discovery o the circulation o the blood as he combined earlier discoveries with his own research ndings to produce a convincing overall theory or blood fow in the body. He overcame widespread opposition by publishing his results and also by touring Europe to demonstrate experiments that alsied previous theories and provided evidence or his theory. As a result his theory became generally accepted. published his theory about the circulation o blood in 1 62 8. It was not until 1 660, ater his death, that blood was seen fowing rom arteries to veins though capillaries as he had predicted. Harvey demonstrated that blood fow through the larger vessels is unidirectional, with valves to prevent backfow. He also showed that the rate o fow through maj or vessels was ar too high or blood to be consumed in the body ater being pumped out by the heart, as earlier theories proposed. It must thereore return to the heart and be recycled. Harvey showed that the heart pumps blood out in the arteries and it returns in veins. He predicted the presence o numerous ne vessels too small to be seen with contemporary equipment that linked arteries to veins in the tissues o the body. B lood capillaries are too narrow to be seen with the naked eye or with a hand lens. Microscopes had not been invented by the time that Harvey Figure 1 Harveys experiment to demonstrate that blood fow in the veins o the arm is unidirectional overturning ancient theries in science Theories are regarded as uncertain: William Harvey overturned theories developed by the ancient Greek philosopher Galen on movement of blood in the body. D uring the Renaissance, interest was reawakened in the classical writings o Greece and Rome. This stimulated literature and the arts, but in some ways it hampered progress in science. It became almost impossible to question the doctrines o such writers as Aristotle, Hippocrates, Ptolemy and Galen. According to Galen, blood is ormed in the liver and is pumped to and ro between the liver and the right ventricle o the heart. A little blood passes into the let ventricle, where it meets air rom the lungs and becomes vital spirits. The 290 vital spirits are distributed to the body by the arteries. S ome o the vital spirits fow to the brain, to be converted into animal spirits, which are then distributed by the nerves to the body. William Harvey was unwilling to accept these doctrines without evidence. He made careul observations and did experiments, rom which he deduced that blood circulates through the pulmonary and systemic circulations. He predicted the existence o capillaries, linking arteries and veins, even though the lenses o the time were not powerul enough or him to see them. 6 . 2 t h e b l o o D S yS t e m The ollowing extract is rom Harveys book On the Generation of Animals, published in 1 65 1 when he was 73 . And hence it is that without the due admonition of the senses, without frequent observation and reiterated experiment, our mind goes astray after phantoms and appearances. Diligent observation is therefore requisite in every science, and the senses are frequently to be appealed to. We are, I say, to strive after personal experience, not to rely of the experience of others: without which no one can properly become a student of any branch of natural science. I would not have you therefore, gentle reader, to take anything on trust from me concerning the Generation of Animals: I appeal to your own eyes as my witness and judge. The method of pursuing truth commonly pursued at this time therefore is to be held erroneous and almost foolish, in which so many enquire what things others have said, and omit to ask whether the things themselves be actually so or not. Arteries Arteries convey blood at high pressure rom the ventricles to the tissues o the body. Arteries are vessels that convey blood rom the heart to the tissues o the body. The main pumping chambers o the heart are the ventricles. They have thick strong muscle in their walls that pumps blood into the arteries, reaching a high pressure at the peak o each pumping cycle. The artery walls work with the heart to acilitate and control blood fow. E lastic and muscle tissue in the walls are used to do this. E lastic tissue contains elastin bres, which store the energy that stretches them at the peak o each pumping cycle. Their recoil helps propel the blood on down the artery. C ontraction o smooth muscle in the artery wall determines the diameter o the lumen and to some extent the rigidity o the arteries, thus controlling the overall fow through them. Both the elastic and muscular tissues contribute to the toughness o the walls, which have to be strong to withstand the constantly changing and intermittently high blood pressure without bulging outwards (aneurysm) or bursting. The bloods progress along major arteries is thus pulsatile, not continuous. The pulse refects each heartbeat and can easily be elt in arteries that pass near the body surace, including those in the wrist and the neck. E ach organ o the body is supplied with blood by one or more arteries. For example, each kidney is supplied by a renal artery and the liver by the hepatic artery. The powerul, continuously active muscles o the heart itsel are supplied with blood by coronary arteries. Artery walls Arteries have muscle and elastic fbres in their walls. The wall o the artery is composed o several layers: tunica externa a tough outer layer o connective tissue tunica media a thick layer containing smooth muscle and elastic bres made o the protein elastin tunica intima a smooth endothelium orming the lining o the artery. acivi Discussin qusins n Wii hrvs ds 1 William Harvey reused to accept doctrines without evidence. Are there academic contexts where it is reasonable to accept doctrines on the basis o authority rather than evidence gathered rom primary sources? 2 Harvey welcomed questions and criticisms o his theories when teaching anatomy classes. Suggest why he might have done this. 3 Can you think o examples o the phantoms and appearances that Harvey reers to? 4 Why does Harvey recommend reiteration o experiments? 5 Harvey practised as a doctor, but ater the publication in 1628 o his work on the circulation o the blood, ar ewer patients consulted him. Why might this have been? 291 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy tunica externa tunica media tunica intima (endothelium) lumen Figure 3 Structure of an artery activity mesuring blood pressures Because arteries are distensible, blood pressure in those that pass near the body surace can be measured relatively easily. A common method is to infate an arm cu until it squeezes the tissues (skin, supercial at as well as the vessels themselves) enough to stop blood fow. The pressure is then released slowly until fow resumes and the operator or instrument can hear the pulse again. The pressures at which blood fow stops and resumes are the systolic and diastolic pressures. They are measured with a pressure monitor. According to the American Heart Association the desired blood pressures or adults o 18 years or older measured in this way are: systolic 90-119 mmHg diastolic 60-79 mmHg Figure 4 Blood pressure monitor 292 Figure 2 The cardiovascular system. The main artery that supplies oxygenated blood to the tissues of the body is the aorta, shown as the red vessel that emerges from the heart and forms an arch with branches carrying blood to the arms and head. The aorta continues through the thorax and abdomen, with branches serving the liver, kidneys, intestines and other organs Arterial blood pressure The muscle and elastic bres assist in maintaining blood pressure between pump cycles. The blood entering an artery rom the heart is at high pressure. The peak pressure reached in an artery is called the systolic pressure. It pushes the wall o the artery outwards, widening the lumen and stretching elastic bres in the wall, thus storing potential energy. At the end o each heartbeat the pressure in the arteries alls suciently or the stretched elastic bres to squeeze the blood in the lumen. This mechanism saves energy and prevents the minimum pressure inside the artery, called the diastolic pressure, rom becoming too low. B ecause it is relatively high, blood fow in the arteries is relatively steady and continuous although driven by a pulsating heart. The circular muscles in the wall o the artery orm a ring so when they contract, in a process called vasoconstriction, the circumerence is reduced and the lumen is narrowed. Vasoconstriction increases blood pressure in the arteries. B ranches o arteries called arterioles have a particularly high density o muscle cells that respond to various hormone and neural signals to control blood fow to downstream tissues. Vasoconstriction o arterioles restricts blood fow to the part o the body that they supply and the opposite process, called vasodilation, increases it. 6 . 2 t h e b l o o D S yS t e m Capillaries Blood fows through tissues in capillaries with permeable walls that allow exchange o materials between cells in the tissue and the blood in the capillary. C apillaries are the narrowest blood vessels with diameter o about 1 0 m. They branch and rej oin repeatedly to orm a capillary network with a huge total length. C apillaries transport blood through almost all tissues in the body. Two exceptions are the tissues o the lens and the cornea in the eye which must be transparent so cannot contain any blood vessels. The density o capillary networks varies in other tissues but all active cells in the body are close to a capillary. The capillary wall consists o one layer o very thin endothelium cells, coated by a lter- like protein gel, with pores between the cells. The wall is thus very permeable and allows part o the plasma to leak out and orm tissue fuid. Plasma is the fuid in which the blood cells are suspended. Tissue fuid contains oxygen, glucose and all other substances in blood plasma apart rom large protein molecules, which cannot pass through the capillary wall. The fuid fows between the cells in a tissue, allowing the cells to absorb useul substances and excrete waste products. The tissue fuid then re- enters the capillary network. acivi bruiss Bruises are caused by damage to capillary walls and leakage o plasma and blood cells into spaces between cells in a tissue. The capillaries are quickly repaired, hemoglobin is broken down to green and yellow bile pigments which are transported away and phagocytes remove the remains o the blood cells by endocytosis. When you next have a bruise, make observations over the days ater the injury to ollow the healing process and the rate at which hemoglobin is removed. The permeabilities o capillary walls dier between tissues, enabling particular proteins and other large particles to reach certain tissues but not others. Permeabilities can also change over time and capillaries repair and remodel themselves continually in response to the needs o tissues that they peruse. Veins Veins collect blood at low pressure rom the tissues o the body and return it to the atria o the heart. Veins transport blood rom capillary networks back to the atria o the heart. B y now the blood is at much lower pressure than it was in the arteries. Veins do not thereore need to have as thick a wall as arteries and the wall contains ar ewer muscle and elastic bres. They can thereore dilate to become much wider and thus hold more blood than arteries. Around 80% o a sedentary persons blood is in the veins though this proportion alls during vigorous exercise. B lood fow in veins is assisted by gravity and by pressures exerted on them by other tissues especially skeletal muscles. C ontraction makes a muscle shorter and wider so it squeezes on adjacent veins like a pump. Walking, sitting or even just dgeting greatly improves venous blood fow. E ach part o the body is served by one or more veins. For example blood is carried rom the arms in the subclavian veins and rom the head in the j ugular veins. The hepatic portal vein is unusual because it does not carry blood back to the heart. It carries blood rom the stomach and intestines to the liver. It is regarded as a portal vein rather than an artery because the blood it carries is at low pressure so it is relatively thin. 293 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy activity Stnding on your hed Pocket valves and vein walls become less efcient with age, causing poor venous return to the heart. Have you ever perormed gymnastic moves such as headstands or handstands, or experienced very high g-orces on a ride at an amusement park? Young people can mostly do any o these activities easily but older people may not be able to. What is the explanation? Valves in veins Valves in veins and the heart ensure circulation o blood by preventing backow. B lood pressure in veins is sometimes so low that there is a danger o backfow towards the capillaries and insucient return o blood to the heart. To maintain circulation, veins contain pocket valves, consisting o three cup-shaped faps o tissue. I blood starts to fow backwards, it gets caught in the faps o the pocket valve, which ll with blood, blocking the lumen o the vein. When blood fows towards the heart, it pushes the faps to the sides o the vein. The pocket valve thereore opens and blood can fow reely. These valves allow blood to fow in one direction only and make ecient use o the intermittent and oten transient pressures provided by muscular and postural changes. They ensure that blood circulates in the body rather than fowing to and ro. Identifying blood vessels Identication o blood vessels as arteries, capillaries or veins rom the structure o their walls. B lood vessels can be identied as arteries, capillaries or veins by looking at their structure. Table 1 below gives dierences that may be useul. artery Figure 5 Which veins in this gymnast will need valves to help with venous return? and vein in transverse section. The tunica externa and tunica intima are stained more darkly than the tunica media. Clotted blood is visible in both vessels Vein Diameter Larger than 10 m Around 10 m Variable but much larger than 10 m Relative thickness o wall and diameter o lumen Relatively thick wall and narrow lumen Extremely thin wall Relatively thin wall with variable but oten wide lumen Number o layers in wall Three layers, tunica externa, media and intima. These layers may be sub-divided to orm more layers Only one layer the tunica intima which is an endothelium consisting o a single layer o very thin cells Three layers tunica externa, media and intima Muscle and elastic bres in the wall Abundant None Small amounts Valves None None Present in many veins Figure 6 Artery 294 Cpillry Table 1 6 . 2 t h e b l o o D S yS t e m The double circulation lungs There is a separate circulation for the lungs. pulmonary circulation There are valves in the veins and heart that ensure a one- way fow, so blood circulates through arteries, capillaries and veins. Fish have a single circulation. B lood is pumped at high pressure to their gills to be oxygenated. Ater fowing through the gills the blood still has enough pressure to fow directly, but relatively slowly, to other organs o the body and then back to the heart. In contrast, the lungs used by mammals or gas exchange are supplied with blood by a separate circulation. B lood capillaries in lungs cannot withstand high pressures so blood is pumped to them at relatively low pressure. Ater passing through the capillaries o the lungs the pressure o the blood is low, so it must return to the heart to be pumped again beore it goes to other organs. Humans thereore have two separate circulations: heart systemic circulation the pulmonary circulation, to and rom the lungs the systemic circulation, to and rom all other organs, including the heart muscles. other organs Figure 7 Figure 7 shows the double circulation in a simplied orm. The pulmonary circulation receives deoxygenated blood that has returned rom the systemic circulation, and the systemic circulation receives blood that has been oxygenated by the pulmonary circulation. It is thereore essential that blood fowing to and rom these two circulations is not mixed. The heart is thereore a double pump, delivering blood under dierent pressures separately to the two circulations. semilunar valve Heart structure Recognition of the chambers and valves of the heart and the blood vessels connected to it in dissected hearts or in diagrams of heart structure. aorta pulmonary artery vena cavae pulmonary veins The heart has two sides, let and right, that pump blood to the systemic and pulmonary circulations. Each side o the heart has two chambers, a ventricle that pumps blood out into the arteries and an atrium that collects blood rom the veins and passes it to the ventricle. Each side o the heart has two valves, an atrioventricular valve between the atrium and the ventricle and a semilunar valve between the ventricle and the artery. The double circulation O xygenated blood fows into the let side o the heart through the pulmonary veins rom the lungs and out through the aorta. semilunar valve atrioventricular valve right atrium left ventricle right ventricle septum Figure 8 Structure of the heart 295 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy D eoxygenated blood fows into the let side o the heart through the vena cava and out in the pulmonary arteries. The heart is a complicated three- dimensional structure. The best way to learn about its structure is by doing a dissection. A resh specimen o a mammalian heart, with blood vessels still attached, a dissecting dish or board and dissecting instruments are needed. 1 Arteries and veins Tidy up the blood vessels attached to the heart by removing membranes and other tissue rom around them. Identiy the thick- walled arteries and the thin-walled veins. 4 Left ventricle Identiy the let ventricle. It has a smooth wall, with a tree- like pattern o blood vessels. Using a sharp scalpel, make an incision as shown by the dashed line X in gure 9. This should open up the let ventricle. Look at the thick muscular wall that you have cut through. 5 Atrioventricular valve Extend the incision urther towards the atrium i necessary until you can see the two thin faps o the atrioventricular valve. Tendons attached to the sides o the let ventricle prevent the valve inverting into the atrium. 6 Left atrium and pulmonary vein 2 Pulmonary artery and aorta Push a glass rod or other blunt- ended instrument into the heart through the arteries and eel through the wall o the heart to where the end o the rod has reached. Identiy the pulmonary artery, through which you will reach the thinner- walled right ventricle, and the aorta, through which you will reach the thicker- walled let ventricle. Identiy the let atrium. It will look surprisingly small as there is no blood inside it. The outer surace o its wall has a wrinkled appearance. Extend the incision that you have already made, either with the scalpel or with scissors, to cut through the wall o the let atrium as ar as the pulmonary vein. Look at the thin wall o the atrium and the opening o the pulmonary vein or veins ( there may be two) . 3 Dorsal and ventral sides 7 Aorta Lay the heart so that the aorta is behind the pulmonary artery, as in gure 9. The ventral side is now uppermost and the dorsal side underneath. The dorsal side o an animal is its back. Find the aorta again and measure the diameter o its lumen, in millimetres. Using scissors, cut through the wall o the aorta, starting at its end and working towards the let ventricle. Look at the smooth inner surace o the aorta and try stretching the wall to see how tough it is. 8 Semilunar valve aorta pulmonary artery right artrium left atrium Where the aorta exits the let ventricle, there will be three cup- shaped faps in the wall. These orm the semilunar valve. Try pushing a blunt instrument into the faps to see how blood fowing backwards pushes the faps together, closing the valve. X 9 Coronary artery coronary artery Y Figure 9 296 Ventral view of the exterior of the heart Look careully at the inner surace o the aorta, near the semilunar valve. A small hole should be visible, which is the opening to the coronary arteries. Measure the diameter o the lumen o this artery. The coronary arteries supply the wall o the heart with oxygen and nutrients. 6 . 2 t h e b l o o D S yS t e m 10 Septum right ventricle Figure 10 septum left ventricle Make a transverse section through the heart near the base o the ventricles, along the dotted line marked Y in gure 9. Measure the thickness in millimetres o the walls o the let and right ventricles and o the septum between them ( gure 1 0) . The septum contains conducting bres, which help to stimulate the ventricles to contract. Transverse section through the ventricles Atherosclerosis Causes and consequences of occlusion of the coronary arteries. One o the commonest current health problems is atherosclerosis, the development o atty tissue called atheroma in the artery wall adjacent to the endothelium. Low density lipoproteins (LD L) containing ats and cholesterol accumulate and phagocytes are then attracted by signals rom endothelium cells and smooth muscle. The phagocytes engul the ats and cholesterol by endocytosis and grow very large. Smooth muscle cells migrate to orm a tough cap over the atheroma. The artery wall bulges into the lumen narrowing it and thus impeding blood fow. S mall traces o atheroma are normally visible in childrens arteries by the age o ten, but do not aect health. In some older people atherosclerosis becomes much more advanced but oten goes unnoticed until a maj or artery becomes so blocked that the tissues it supplies become compromised. C oronary occlusion is a narrowing o the arteries that supply blood containing oxygen and nutrients to the heart muscle. Lack o oxygen (anoxia) causes pain, known as angina, and impairs the muscles ability to contract, so the heart beats aster as it tries to maintain blood circulation with some o its muscle out o action. The brous cap covering atheromas sometimes ruptures, which stimulates the ormation o blood clots that can block arteries supplying blood to the heart and cause acute heart problems. This is described in sub-topic 6.3. The causes o atherosclerosis are not yet ully understood. Various actors have been shown to be associated with an increased risk o atheroma but are not the sole causes o the condition: high blood concentrations o LD L ( low density lipoprotein) chronic high blood glucose concentrations, due to overeating, obesity or diabetes acivi Srucur nd funcin f r Discuss the answers to these questions. 1 Why are the walls of the atria thinner than the walls of the ventricles? 2 What prevents the atrioventricular valve from being pushed into the atrium when the ventricle contracts? 3 Why is the left ventricle wall thicker than the right ventricle wall? 4 Does the left side of the heart pump oxygenated or deoxygenated blood? 5 Why does the wall of the heart need its own supply of blood, brought by the coronary arteries? 6 Does the right side of the heart pump a greater volume of blood per minute, a smaller volume, or the same volume as the left? 297 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy activity Crnitine nd coronry occusion A chemical called carnitine that is ound in certain oods is converted into TMAO by bacteria in the gut. Find out what oods contain the highest concentrations o carnitine and discuss whether this nding should infuence dietary advice. chronic high blood pressure due to smoking, stress or any other cause consumption o trans ats, which damage the endothelium o the artery. There are also some more recent theories that include microbes: inection o the artery wall with Chlamydia pneumoniae production o trimethylamine N- oxide ( TMAO ) by microbes in the intestine. Figure 11 A normal artery (left) has a much wider lumen than an artery that is occluded by atheroma (right) The sinoatrial node The heartbeat is initiated by a group o specialized muscle cells in the right atrium called the sinoatrial node. The heart is unique in the body as its muscles can contract without stimulation rom motor neurons. The contraction is called myogenic, meaning that it is generated in the muscle itsel. The membrane o a heart muscle cell depolarizes when the cell contracts and this activates adj acent cells, so they also contract. A group o cells thereore contracts almost simultaneously at the rate o the astest. Figure 12 298 The sinoatrial node The region o the heart with the astest rate o spontaneous beating is a small group o special muscle cells in the wall o the right atrium, called the sinoatrial node. These cells have ew o the proteins that cause contraction in other muscle cells, but they have extensive membranes. The sinoatrial node thereore initiates each heartbeat, because the membranes o its cells are the frst to depolarize in each cardiac cycle. 6 . 2 t h e b l o o D S yS t e m Initiating the heartbeat The sinoatrial node acts as a pacemaker. B ecause the sinoatrial node initiates each heartbeat, it sets the pace or the beating o the heart and is oten called the pacemaker. I it becomes deective, its output may be regulated or even replaced entirely by an artifcial pacemaker. This is an electronic device, placed under the skin with electrodes implanted in the wall o the heart that initiate each heartbeat in place o the sinoatrial node. Atrial and ventricular contraction The sinoatrial node sends out an electrical signal that stimulates contraction as it is propagated through the walls o the atria and then the walls o the ventricles. The sinoatrial node initiates a heartbeat by contracting and simultaneously sends out an electrical signal that spreads throughout the walls o the atria. This can happen because there are interconnections between adjacent fbres across which the electrical signal can be propagated. Also the fbres are branched so each fbre passes the signal on to several others. It takes less than a tenth o a second or all cells in the atria to receive the signal. This propagation o the electrical signal causes the whole o both let and right atria to contract. Ater a time delay o about 0. 1 seconds, the electrical signal is conveyed to the ventricles. The time delay allows time or the atria to pump the blood that they are holding into the ventricles. The signal is then propagated throughout the walls o the ventricles, stimulating them to contract and pump blood out into the arteries. D etails o the electrical stimulation o the heartbeat are included in O ption D . Figure 13 Heart monitor displaying the heart rate, the electrical activity of the heart and the percentage saturation with oxygen of the blood TOK Wa ars r in ica dcisin aking: inn r cnsquncs? There are some circumstances in which prolonging the lie o an individual who is sufering brings in to question the role o the physician. Sometimes, an active pacemaker may be involved in prolonging the lie o a patient and the physician receives a request to deactivate the device. This will accelerate the pace o the patients death. Euthanasia involves taking active steps to end the lie o a patient and it is illegal in many jurisdictions. However, there is a widely accepted practice o withdrawing lie-sustaining interventions such as dialysis, mechanical ventilation, or tube eeding rom terminally ill patients. This is oten a decision o the amily o the patient. The withdrawal o lie support is seen as distinct rom euthanasia because the patient dies o their condition rather than the active steps to end the patients lie in the case o euthanasia. However, the distinction can be subtle. The consequence is the same: the death o the patient. The intent can be the same: to end the patients sufering. Yet in many jurisdictions, one action is illegal and the other is not. 299 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy The cardiac cycle Pressure changes in the left atrium, left ventricle and aorta during the cardiac cycle. 0.1 5 0.4 seconds The pressure in the ventricles rises above the pressure in the arteries so the semilunar valves open and blood is pumped rom the ventricles into the arteries, transiently maximizing the arterial blood pressure. The pressure changes in the atrium and ventricle o the heart and the aorta during a cardiac cycle are shown in gure 1 5 . To understand them it is necessary to appreciate what occurs at each stage o the cycle. Figure 1 4 below summarizes the events, with timings assuming a heart rate o 7 5 beats per minute. Typical volumes o blood are shown and also an indication o the direction o blood fow to or rom a chamber o the heart. 0.0 0.1 seconds The atria contract causing a rapid but relatively small pressure increase, which pumps blood rom the atria to the ventricles, through the open atrioventricular valves. 0.4 0.45 seconds The contraction o the ventricular muscles wanes and pressure inside the ventricles rapidly drops below the pressure in the arteries, causing the semilunar valves to close. The semilunar valves are closed and blood pressure in the arteries gradually drops to its minimum as blood continues to fow along them but no more is pumped in. The atrioventricular valves remain closed. 0.45 0.8 seconds Pressure in the ventricles drops below the pressure in the atria so the atrioventricular valves open. 0.1 0.1 5 seconds The ventricles contract, with a rapid pressure build up that causes the atrioventricular valves to close. Pressure slowly rises in the atria as blood drains into them rom the veins and they ll. The semilunar valves remain closed. B lood rom the veins drains into the atria and rom there into the ventricles, causing a slow increase in pressure. vein 25 ml atrium relaxing atrium contracts 25 ml atrioventricular valve valve open atrium atrioventricular valve closed ventricle contracting 70 ml ventricle relaxing ventricle semilunar valve artery atrium relaxing 45 ml atrioventricular valve open ventricle relaxing valve closed valve open semilunar valve closed diastolic systolic diastolic tissues of the body 0 0.1 0.15 0.4 0.45 time (seconds) 0.8 Figure 14 One cardiac cycle is represented on the diagram, starting on the let with contraction o the atrium. Vertical arrows show fows o blood to and rom the atrium and ventricle 300 6 . 2 t h e b l o o D S yS t e m D-sd qusins: Heart action and blood pressures 1 D educe when blood is being pumped rom the atrium to the ventricle. Give both the start and the end times. [2 ] 2 Deduce when the ventricle starts to contract. [1 ] 3 The atrioventricular valve is the valve between the atrium and the ventricle. S tate when the atrioventricular valve closes. [1 ] 4 The semilunar valve is the valve between the ventricle and the artery. S tate when the semilunar valve opens. [1 ] 5 D educe when the semilunar valve closes. [1 ] 6 D educe when blood is being pumped rom the ventricle to the artery. Give both the start and the end times. pressure / mm Hg Figure 1 5 shows the pressures in the atrium, ventricle and artery on one side o the heart, during one second in the lie o the heart. ventricle 120 artery 100 80 60 40 20 7 atrium [2 ] D educe when the volume o blood in the ventricle is: 0 20 0 a) at a maximum [1 ] b) at a minimum. [1 ] 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 time / s Figure 15 Pressure changes during the cardiac cycle Changing the heart rate acivi The heart rate can be increased or decreased by impulses brought to the heart through two nerves rom the medulla o the brain. lisning r sunds The sinoatrial node that sets the rhythm or the beating o the heart responds to signals rom outside the heart. These include signals rom branches o two nerves originating in a region in the medulla o the brain called the cardiovascular centre. S ignals rom one o the nerves cause the pacemaker to increase the requency o heartbeats. In healthy young people the rate can increase to three times the resting rate. S ignals rom the other nerve decrease the rate. These two nerve branches act rather like the throttle and brake o a car. Sounds produced by blood fow can be heard with a simple tube or stethoscope placed on the chest near the heart. The consequences o this whole cardiac cycle or the fow o blood out o the heart can be elt as the pulse in a peripheral artery. (a) The cardiovascular centre receives inputs rom receptors that monitor blood pressure and its pH and oxygen concentration. The pH o the blood refects its carbon dioxide concentration. Low blood pressure, low oxygen concentration and low pH all suggest that the heart rate needs to speed up, to increase the fow rate o blood to the tissues, deliver more oxygen and remove more carbon dioxide. High blood pressure, high oxygen concentration and high pH are all indicators that the heart rate may need to slow down. (b) Figure 16 Taking the pulse: (a) radial pulse (b) carotid pulse 301 61 HUM C EAN LLPBHI YS O LO I OGLO Y GY Epinephrine Epinephrine increases the heart rate to prepare or vigorous physical activity. The sinoatrial node also responds to epinephrine in the blood, by increasing the heart rate. This hormone is also sometimes called adrenalin and is produced by the adrenal glands. The secretion o epinephrine is controlled by the brain and rises when vigorous physical activity may be necessary because o a threat or opportunity. S o epinephrine has the nickname ght or fight hormone . Figure 17 Adventure sports such as rock climbing cause epinephrine secretion In the past when humans were hunter- gatherers rather than armers, epinephrine would have been secreted when humans were hunting or prey or when threatened by a predator. In the modern world athletes oten use pre- race routines to stimulate adrenalin secretion so that their heart rate is already increased when vigorous physical activity begins. 6.3 Defence against infectious disease Understanding The skin and mucous membranes orm a 302 primary deence against pathogens that cause inectious disease. Cuts in the skin are sealed by blood clotting. Clotting actors are released rom platelets. The cascade results in the rapid conversion o brinogen to brin by thrombin. Ingestion o pathogens by phagocytic white blood cells gives non-specic immunity to diseases. Production o antibodies by lymphocytes in response to particular pathogens gives specic immunity. Antibiotics block processes that occur in prokaryotic cells but not in eukaryotic cells. Viral diseases cannot be treated using antibiotics because they lack a metabolism. Some strains o bacteria have evolved with genes which coner resistance to antibiotics and some strains o bacteria have multiple resistance. Applications Causes and consequences o blood clot ormation in coronary arteries. Efects o HIV on the immune system and methods o transmission. Florey and Chains experiments to test penicillin on bacterial inections in mice. Nature of science Risks associated with scientic research: Florey and Chains tests on the saety o penicillin would not be compliant with current protocols on testing. 6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e Skin as a barrier to infection The skin and mucous membranes orm a primary deence against pathogens that cause inectious disease. There are many different microbes in the environment that can grow inside the human body and cause a disease. S ome microorganisms are opportunistic and although they can invade the body they also commonly live outside it. O thers are specialized and can only survive inside a human body. Microbes that cause disease are called pathogens. The primary defence of the body against pathogens is the skin. Its outermost layer is tough and provides a physical barrier against the entry of pathogens and protection against physical and chemical damage. S ebaceous glands are associated with hair follicles and they secrete a chemical called sebum, which maintains skin moisture and slightly lowers skin pH. The lower pH inhibits the growth of bacteria and fungi. Mucous membranes are a thinner and softer type of skin that is found in areas such as the nasal passages and other airways, the head of the penis and foreskin and the vagina. The mucus that these areas of skin secrete is a sticky solution of glycoproteins. Mucus acts as a physical barrier; pathogens and harmful particles are trapped in it and either swallowed or expelled. It also has antiseptic properties because of the presence of the anti-bacterial enzyme lysozyme. Cuts and clots Figure 1 Scanning electron micrograph of bacteria on the surface of teeth. Mucous membranes in the mouth prevent these and other microbes from invading body tissues acvy im hm sk A digital microscope can be used to produce images o the diferent types o skin covering the human body. Figure 2 shows our images produced in this way. Cuts in the skin are sealed by blood clotting. When the skin is cut, blood vessels in it are severed and start to bleed. The bleeding usually stops after a short time because of a process called clotting. The blood emerging from a cut changes from being a liquid to a semi- solid gel. This seals up the wound and prevents further loss of blood and blood pressure. C lotting is also important because cuts breach the barrier to infection provided by the skin. C lots prevent entry of pathogens until new tissue has grown to heal the cut. platelets and blood clotting Clotting actors are released rom platelets. B lood clotting involves a cascade of reactions, each of which produces a catalyst for the next reaction. As a result blood clots very rapidly. It is important that clotting is under strict control, because if it occurs inside blood vessels the resulting clots can cause blockages. The process of clotting only occurs if platelets release clotting factors. Platelets are cellular fragments that circulate in the blood. They are smaller than either red or white blood cells. When a cut or other inj ury involving damage to blood vessels occurs, platelets aggregate at the site forming a temporary plug. They then release the clotting factors that trigger off the clotting process. Figure 2 303 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy Fibrin production The cascade results in the rapid conversion o fbrinogen to fbrin by thrombin. platelets lymphocyte red blood cell phagocyte The cascade o reactions that occurs ater the release o clotting actors rom platelets quickly results in the production o an enzyme called thrombin. Thrombin in turn converts the soluble protein fbrinogen into the insoluble fbrin. The fbrin orms a mesh in cuts that traps more platelets and also blood cells. The resulting clot is initially a gel, but i exposed to the air it dries to orm a hard scab. Figure 4 shows red blood cells trapped in this fbrous mesh. Figure 3 Cells and cell ragments rom blood. Lymphocytes and phagocytes are types o white blood cell Coronary thrombosis Causes and consequences o blood clot ormation in coronary arteries. In patients with coronary heart disease, blood clots sometimes orm in the coronary arteries. These arteries branch o rom the aorta close to the semilunar valve. They carry blood to the wall o the heart, supplying the oxygen and glucose needed by cardiac muscle fbres or cell respiration. The medical name or a blood clot is a thrombus. Coronary thrombosis is the ormation o blood clots in the coronary arteries. Figure 4 Scanning electron micrograph o clotted blood with fbrin and trapped blood cells I the coronary arteries become blocked by a blood clot, part o the heart is deprived o oxygen and nutrients. C ardiac muscle cells are then unable to produce sufcient ATP by aerobic respiration and their contractions become irregular and uncoordinated. The wall o the heart makes quivering movements called fbrillation that do not pump blood eectively. This condition can prove atal unless it resolves naturally or through medical intervention. Atherosclerosis causes occlusion in the coronary arteries. Where atheroma develops the endothelium o the arteries tends to become damaged and roughened; especially, the artery wall is hardened by deposition o calcium salts. Patches o atheroma sometimes rupture causing a lesion. C oronary occlusion, damage to the capillary epithelium, hardening o arteries and rupture o atheroma all increase the risk o coronary thrombosis. There are some well-known actors that are correlated with an increased risk o coronary thrombosis and heart attacks: Figure 5 Early intervention during a heart attack can save the patients lie so it is important to know what to do by being trained 304 smoking high blood cholesterol concentration high blood pressure diabetes obesity lack o exercise. O course correlation does not prove causation, but doctors nonetheless advise patients to avoid these risk actors i possible. 6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e phagocytes Ingestion o pathogens by phagocytic white blood cells gives non-specifc immunity to diseases. I microorganisms get past the physical barriers o skin and mucous membranes and enter the body, white blood cells provide the next line o deence. There are many dierent types o white blood cell. Some are phagocytes that squeeze out through pores in the walls o capillaries and move to sites o inection. There they engul pathogens by endocytosis and digest them with enzymes rom lysosomes. When wounds become inected, large numbers o phagocytes are attracted, resulting in the ormation o a white liquid called pus. Antibody roduction Production o antibodies by lymphocytes in response to particular pathogens gives specifc immunity. I microorganisms get past the physical barriers o the skin and invade the body, proteins and other molecules on the surace o pathogens are recognized as oreign by the body and they stimulate a specifc immune response. Any chemical that stimulates an immune response is reerred to as an antigen. The specifc immune response is the production o antibodies in response to a particular pathogen. The antibodies bind to an antigen on that pathogen. Antibodies are produced by types o white blood cell called lymphocytes. Each lymphocyte produces j ust one type o antibody, but our bodies can produce a vast array o dierent antibodies. This is because we have small numbers o lymphocytes or producing each o the many types o antibody. There are thereore too ew lymphocytes initially to produce enough antibodies to control a pathogen that has not previously inected the body. However, antigens on the pathogen stimulate cell division o the small group o lymphocytes that produce the appropriate type o antibody. A large clone o lymphocytes called plasma cells are produced within a ew days and they secrete large enough quantities o the antibody to control the pathogen and clear the inection. Antibodies are large proteins that have two unctional regions: a hypervariable region that binds to a specifc antigen and another region that helps the body to fght the pathogen in one o a number o ways, including these: making a pathogen more recognizable to phagocytes so they are more readily enguled preventing viruses rom docking to host cells so that they cannot enter the cells. Antibodies only persist in the body or a ew weeks or months and the plasma cells that produce them are also gradually lost ater the inection has been overcome and the antigens associated with it are no longer present. However, some o the lymphocytes produced during an inection are not active plasma cells but instead become memory cells Figure 6 Avian infuenza viruses. In this electron micrograph o a virus in transverse section, alse colour has been used to distinguish the protein coat that is recognized as antigens by the immune system (purple) rom the DNA o the virus (green) 305 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy that are very long-lived. These memory cells remain inactive unless the same pathogen inects the body again, in which case they become active and divide to produce plasma cells very rapidly. Immunity to an inectious disease involves either having antibodies against the pathogen, or memory cells that allow rapid production o the antibody. Human immunodefciency virus Efects o HIV on the immune system and methods o transmission. The production o antibodies by the immune system is a complex process and includes dierent types o lymphocyte, including helper T- cells. The human immunodefciency virus ( HIV) invades and destroys helper T-cells. The consequence is a progressive loss o the capacity to produce antibodies. In the early stages o inection, the immune system makes antibodies against HIV. I these can be detected in a persons body, they are said to be HIV- positive. HIV is a retrovirus that has genes made o RNA and uses reverse transcriptase to make D NA copies o its genes once it has entered a host cell. The rate at which helper T-cells are destroyed by HIV varies considerably and can be slowed down by using anti-retroviral drugs. In most HIV- positive patients antibody production eventually becomes so ineective that a group o opportunistic inections strike, which would be easily ought o by a healthy immune system. S everal o these are normally so rare that they are marker diseases or the latter stages o HIV inection, or example Kaposis sarcoma. A collection o several diseases or conditions existing together is called a syndrome. When the syndrome o conditions due to HIV is present, the person is said to have acquired immune defciency syndrome ( AID S ) . AID S spreads by HIV inection. The virus only survives outside the body or a short time and inection normally only occurs i there is blood to blood contact between inected and uninected people. There are various ways in which this can occur: sexual intercourse, during which abrasions to the mucous membranes o the penis and vagina can cause minor bleeding transusion o inected blood, or blood products such as Factor VIII sharing o hypodermic needles by intravenous drug users. Antibiotics Antibiotics block processes that occur in prokaryotic cells but not in eukaryotic cells. An antibiotic is a chemical that inhibits the growth o microorganisms. Most antibiotics are antibacterial. They block processes that occur in prokaryotes but not in eukaryotes and can thereore be used to kill bacteria inside the body without causing harm to human cells. The processes targeted by antibiotics are bacterial D NA replication, transcription, translation, ribosome unction and cell wall ormation. Figure 7 Fleming's petri dish which frst showed the inhibition o bacterial growth by penicillin rom a mycelium o Penicillium 306 Many antibacterial antibiotics were discovered in saprotrophic ungi. These ungi compete with saprotrophic bacteria or the dead organic matter on which they both eed. B y secreting antibacterial antibiotics, saprotrophic ungi inhibit the growth o their bacterial competitors. An example is penicillin. It is produced by some strains o the Penicillium ungus, but only when nutrients are scarce and competition with bacteria would be harmul. 6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e Testing penicillin Florey and Chains experiments to test penicillin on bacterial inections in mice. Howard Florey and Ernst C hain ormed a research team in O xord in the late 1 93 0s that investigated the use o chemical substances to control bacterial inections. The most promising o these was penicillin, discovered by Alexander Fleming in 1 92 8. Florey and C hains team developed a method o growing the ungus Penicillium in liquid culture in conditions that stimulated it to secrete penicillin. They also developed methods or producing reasonably pure samples o penicillin rom the cultures. acvy Wrld aiDS Dy The red AIDS awareness ribbon is an international symbol o awareness and support or those living with HIV. It is worn on World AIDS Day each year December 1st. Are you aware how many people in your area are afected and what can be done to support them? The penicillin killed bacteria on agar plates, but they needed to test whether it would control bacterial inections in humans. They frst tested it on mice. E ight mice were deliberately inected with Streptococcus bacteria that cause death rom pneumonia. Four o the inected mice were given inj ections with penicillin. Within 2 4 hours all the untreated mice were dead but the our given penicillin were healthy. Florey and C hain decided that they should next do tests on human patients, which required much larger quantities. When enough penicillin had been produced, a 43 - year-old policeman was chosen or the frst human test. He had an acute and liethreatening bacterial inection caused by a scratch on the ace rom a thorn on a rose bush. He was given penicillin or our days and his condition improved considerably, but supplies o penicillin ran out and he suered a relapse and died rom the inection. Larger quantities o penicillin were produced and fve more patients with acute inections were tested. All were cured o their inections, but sadly one o them died. He was a small child who had an inection behind the eye. This had weakened the wall o the artery carrying blood to the brain and although cured o the inection, the child died suddenly o brain hemorrhage when the artery burst. Pharmaceutical companies in the United S tates then began to produce penicillin in much larger quantities, allowing more extensive testing, which confrmed that it was a highly eective treatment or many previously incurable bacterial inections. Figure 8 Penicillin the green ball represents a variable part of the molecule 307 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy penicillin and drug testing Risks associated with scientifc research: Florey and Chains tests on the saety o penicillin would not be compliant with current protocols on testing. When any new drug is introduced there are risks that it will prove to be ineffective in some or all patients or that it will cause harmful side effects. These risks are minimized by strict protocols that pharmaceutical companies must follow. Initial tests are performed on animals and then on small numbers of healthy humans. O nly if a drug passes these tests is it tested on patients with the disease that the drug is intended to treat. The last tests involve very large numbers of patients to test whether the drug is effective in all patients and to check that there are no severe or common side effects. There are some famous cases of drugs causing problems during testing or after release. Thalidomide was introduced in the 1 95 0s as a treatment for various mild conditions but when it was found to relieve morning sickness in pregnant women it was prescribed for that purpose. The side effects of the drug on the fetus had not been tested and more than 1 0, 000 children were born with birth deformities before the problem was recognized. In 2 006 six healthy volunteers were given TGN1 41 2 , a new protein developed for treatment of autoimmune diseases and leukemia. All six rapidly became very ill and suffered multiple organ failure. Although the volunteers recovered, they may have suffered long- term damage to their immune systems. It is very unlikely that Florey and C hain would have been allowed to carry out tests on a new drug today with the methods that they used for penicillin. They tested the drug on human patients after only a very brief period of animal testing. Penicillin was a new type of drug and there could easily have been severe side effects. Also the samples that they were using were not pure and there could have been side effects from the impurities. On the other hand, the patients that they used were all on the point of death and several were cured of their infections as a result of the experimental treatment. B ecause of expeditious testing with greater risk-taking than would now be allowed, penicillin was introduced far more quickly than would be possible today. D uring the D -day landings in June 1 944 penicillin was used to treat wounded soldiers and the number of deaths from bacterial infection was greatly reduced. Figure 9 Wounded US troops on Omaha beach 6 June 1944 Viruses and antibiotics Viral diseases cannot be treated using antibiotics because they lack a metabolism. Viruses are non- living and can only reproduce when they are inside living cells. They use the chemical processes of a living host cell, instead of having a metabolism of their own. They do not have their own means of transcription or protein synthesis and they rely on the 308 6 . 3 D e Fe n Ce ag ai n S t i n Fe Cti o u S D i S e aS e host cells enzymes or ATP synthesis and other metabolic pathways. These processes cannot be targeted by drugs as the host cell would also be damaged. All o the commonly used antibiotics such as penicillin, streptomycin, chloramphenicol and tetracycline control bacterial inections and are not eective against viruses. Not only is it inappropriate or doctors to prescribe them or a viral inection, but it contributes to the overuse o antibiotics and increases in antibiotic resistance in bacteria. There are a ew viral enzymes which can be used as targets or drugs to control viruses without harming the host cell. O nly a ew drugs have been discovered or developed to control viruses in this way. These are known as antivirals rather than antibiotics. acvy Dssh bw bcrl d vrl fcs How can a doctor distinguish between bacterial and viral infections, without prescribing an antibiotic and seeing if it cures the infection? Resistance to antibiotics Some strains of bacteria have evolved with genes which confer resistance to antibiotics and some strains of bacteria have multiple resistance. In 2 01 3 the governments chie medical ofcer or England, S ally D avies, said this: The danger posed by growing resistance to antibiotics should be ranked along with terrorism on a list of threats to the nation. If we dont take action, then we may all be back in an almost 1 9th-century environment where infections kill us as a result of routine operations. We wont be able to do a lot of our cancer treatments or organ transplants. Figure 10 Many viruses cause a common cold. Children lack immunity to most of them so frequently catch a cold. Antibiotics do not cure them The development o resistance to antibiotics by natural selection is described in sub- topic 5 .2 . S trains o bacteria with resistance are usually discovered soon ater the introduction o an antibiotic. This is not o huge concern unless a strain develops multiple resistance, or example methicillin-resistant Staphylococcus aureus ( MRS A) which has inected the blood or surgical wounds o hospital patients and resists all commonly used antibiotics. Another example o this problem is multidrug-resistant tuberculosis ( MD R-TB ) . The WHO has reported more than 3 00, 000 cases worldwide per year with the disease reaching epidemic proportions in some areas. Antibiotic resistance is an avoidable problem. These measures are required: doctors prescribing antibiotics only or serious bacterial inections patients completing courses o antibiotics to eliminate inections completely hospital sta maintaining high standards o hygiene to prevent crossinection armers not using antibiotics in animal eeds to stimulate growth pharmaceutical companies developing new types o antibiotic no new types have been introduced since the 1 980s. 309 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy Data-based questions: Antibiotic resistance 1 a) D escribe the pattern o erythromycin resistance over the period rom 1 992 to 2 002 . [3] [2 ] Evaluate the claim that reduction in the use o erythromycin has led to a reduction in the incidence o antibiotic resistance in S. pyogenes. [3 ] 20 15 10 2002 2001 2000 1999 1997 1998 1995 0 1996 5 1993 3 C alculate the percentage dierence in antibiotic resistance between 2002 and 1 992. 1994 The data in fgure 1 1 shows the incidence in Finland, over a 1 0-year period, o Streptococcus pyogenes strains that are resistant to the antibiotic erythromycin. S. pyogenes is responsible or the condition known as strep throat. 2 1992 In the early 1 990s, Finnish public health authorities began discouraging the use o the antibiotic erythromycin or URIs in response to rising bacterial resistance to the antibiotic, and the national erythromycin consumption per capita dropped by 43 per cent. b) Suggest a reason or the pattern shown. [2 ] % antibiotic resistance B acterial resistance to antibiotics is a direct consequence o the overuse o these drugs. In the US A, currently more than hal o the doctor visits or upper respiratory tract inections ( URIs) are prescribed antibiotics, despite knowledge that most URIs are caused by viruses. year Figure 11 The incidence of Streptococcus pyogenes strains that are resistant to the antibiotic erythromycin over a 10-year period in Finland 6.4 gas exchane Understanding Ventilation maintains concentration gradients 310 o oxygen and carbon dioxide between air in alveoli and blood fowing in adjacent capillaries. Type I pneumocytes are extremely thin alveolar cells that are adapted to carry out gas exchange. Type II pneumocytes secrete a solution containing suractant that creates a moist surace inside the alveoli to prevent the sides o the alveolus adhering to each other by reducing surace tension. Air is carried to the lungs in the trachea and bronchi and then to the alveoli in bronchioles. Muscle contractions cause the pressure changes inside the thorax that orce air in and out o the lungs to ventilate them. Dierent muscles are required or inspiration and expiration because muscles only do work when they contract. Applications External and internal intercostal muscles, and diaphragm and abdominal muscles as examples o antagonistic muscle action. Causes and consequences o lung cancer. Causes and consequences o emphysema. Skills Monitoring o ventilation in humans at rest and ater mild and vigorous exercise. (Practical 6) Nature of science Obtain evidence or theories: epidemiological studies have contributed to our understanding o the causes o lung cancer. 6 . 4 g aS e xCh an g e Ventilation Ventilation maintains concentration gradients o oxygen and carbon dioxide between air in alveoli and blood fowing in adjacent capillaries. All organisms absorb one gas rom the environment and release a dierent one. This process is called gas exchange. Leaves absorb carbon dioxide to use in photosynthesis and release the oxygen produced by this process. Humans absorb oxygen or use in cell respiration and release the carbon dioxide produced by this process. Terrestrial organisms exchange gases with the air. In humans gas exchange occurs in small air sacs called alveoli inside the lungs ( gure 1 ) . type I pneumocytes in alveolus wall phagocyte 10 network of blood capillaries 0 m type II pneumocytes in alveolus wall Figure 1 Gas exchange happens by diusion between air in the alveoli and blood fowing in the adj acent capillaries. The gases only diuse because there is a concentration gradient: the air in the alveolus has a higher concentration o oxygen and a lower concentration o carbon dioxide than the blood in the capillary. To maintain these concentration gradients resh air must be pumped into the alveoli and stale air must be removed. This process is called ventilation. 311 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy Data-based questions: Concentration gradients Figure 2 shows the typical composition o atmospheric air, air in the alveoli and gases dissolved in air returning to the lungs in the pulmonary arteries. oxygen carbon dioxide nitrogen 1 E xplain why the oxygen concentration in the alveoli is not as high as in resh air that is inhaled. [2 ] 2 a) C alculate the dierence in oxygen concentration between air in the alveolus and blood arriving at the alveolus. [1 ] b) D educe the process caused by this concentration dierence. 700 598 partial pressure / mm Hg 600 570 570 565 c) 500 400 300 200 3 atmospheric air that is inhaled 40 air in alveoli 40 45 [2 ] d) D espite the high concentration o nitrogen in air in alveoli, little or none diuses rom the air to the blood. S uggest reasons or this. [2 ] 120 105 100 0 (i) C alculate the dierence in carbon dioxide concentration between air inhaled and air exhaled. [1 ] (ii) Explain this dierence. 159 [1 ] 27 blood travelling air exhaled to alveoli Figure 2 Partial pressures of gases in the pulmonary system Ventilation experiments Monitoring of ventilation in humans at rest and after mild and vigorous exercise. (Practical 6) In an investigation o the eect o exercise on ventilation, the type or intensity o exercise is the independent variable and the ventilation parameter that is measured is the dependent variable. A simple approach or the independent variable is to choose levels o activity ranging rom inactive to very active, such as lying down, sitting and standing, walking, j ogging and sprinting. A more quantitative approach is to do the same activity at dierent measured rates, or example running at dierent speeds on a treadmill. This allows the ventilation parameters to be correlated with work rate in j oules per minute during exercise. Ventilation o the lungs is carried out by drawing some resh air into the lungs and then expelling some o the stale air rom the lungs. The volume o air drawn in and expelled is the tidal volume. The number o times that air is drawn in or expelled per minute is the ventilation rate. 312 Either or both o these can be the dependent variable in an investigation o the eect o exercise on ventilation rate. They should be measured ater carrying on an activity or long enough to reach a constant rate. The example methods given below include a simple and a more advanced technique that could be used or the investigation. 1 Ventilation rate The most straightorward way to measure ventilation rate is by simple observation. C ount the number o times air is exhaled or inhaled in a minute. B reathing should be maintained at a natural rate, which is as slow as possible without getting out o breath. Ventilation rate can also be measured by data logging. An infatable chest belt is placed around the thorax and air is pumped in with a bladder. A dierential pressure sensor is then used to measure 6 . 4 g aS e xCh an g e pressure variations inside the belt due to chest expansions. The rate o ventilations can be deduced and the relative size o ventilations may also be recorded. 2 Tidal volume S imple apparatus is shown in gure 3 . O ne normal breath is exhaled through the delivery tube into a vessel and the volume is measured. It is not sae to use this apparatus or repeatedly inhaling and exhaling air as the C O 2 concentration will rise too high. To ensure that the experimental design is rigorous, all variables apart rom the independent and dependent variables should be kept constant. Ventilation parameters should be measured several times at all levels o exercise with each person in the trial. As many dierent people as possible should be tested. bell jar with graduations delivery tube S pecially designed spirometers are available or use with data logging. They measure fow rate into and out o the lungs and rom these measurements lung volumes can be deduced. pneumatic trough Figure 3 Type I pneumocytes bronchiole Type I pneumocytes are extremely thin alveolar cells that are adapted to carry out gas exchange. The lungs contain huge numbers o alveoli with a very large total surace area or diusion. The wall o each alveolus consists o a single layer o cells, called the epithelium. Most o the cells in this epithelium are Type I pneumocytes. They are fattened cells, with the thickness o only about 0.1 5 m o cytoplasm. The wall o the adj acent capillaries also consists o a single layer o very thin cells. The air in the alveolus and the blood in the alveolar capillaries are thereore less than 0. 5 m apart. The distance over which oxygen and carbon dioxide has to diuse is thereore very small, which is an adaptation to increase the rate o gas exchange. 0.25 mm alveolus Type II pneumocytes epithelium of alveolus wall nucleus of epithelium cell Type II pneumocytes secrete a solution containing surfactant that creates a moist surface inside the alveoli to prevent the sides of the alveolus adhering to each other by reducing surface tension. Type II pneumocytes are rounded cells that occupy about 5 % o the alveolar surace area. They secrete a fuid which coats the inner surace o the alveoli. This lm o moisture allows oxygen in the alveolus to dissolve and then diuse to the blood in the alveolar capillaries. It also provides an area rom which carbon dioxide can evaporate into the air and be exhaled. basement membrane endothelium of capillary alveolus blood plasma erythrocyte 1 m Figure 4 Structure of alveoli 313 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy air in alveolus water surface monolayer of surfactant Figure 5 Pulmonary suractant molecules on the surace o the flm o moisture lining the alveoli trachea intercostal muscle The fuid secreted by the Type II pneumocytes contains a pulmonary suractant. Its molecules have a structure similar to that o phospholipids in cell membranes. They orm a monolayer on the surace o the moisture lining the alveoli, with the hydrophilic heads acing the water and the hydrophobic tails acing the air. This reduces the surace tension and prevents the water rom causing the sides o the alveoli to adhere when air is exhaled rom the lungs. This helps to prevent collapse o the lung. Premature babies are oten born with insucient pulmonary suractant and can suer rom inant respiratory distress syndrome. Treatment involves giving the baby oxygen and also one or more doses o suractant, extracted rom animal lungs. right bronchus Airways for ventilation right lung diaphragm Air is carried to the lungs in the trachea and bronchi and then to the alveoli in bronchioles. bronchioles ribs Air enters the ventilation system through the nose or mouth and then passes down the trachea. This has rings o cartilage in its wall to keep it open even when air pressure inside is low or pressure in surrounding tissues is high. The trachea divides to orm two bronchi, also with walls strengthened with cartilage. O ne bronchus leads to each lung. Figure 6 The ventilation system (a) inspiration ribs Inside the lungs the bronchi divide repeatedly to orm a tree-like structure o narrower airways, called bronchioles. The bronchioles have smooth muscle bres in their walls, allowing the width o these airways to vary. At the end o the narrowest bronchioles are groups o alveoli, where gas exchange occurs. vertebral column ribs diaphragm pressure changes during ventilation (b) expiration Muscle contractions cause the pressure changes inside the thorax that force air in and out of the lungs to ventilate them. air movement ribcage movement diaphragm movement Figure 7 Ventilation o the lungs 314 Ventilation o the lungs involves some basic physics. I particles o gas spread out to occupy a larger volume, the pressure o the gas becomes lower. C onversely, i a gas is compressed to occupy a smaller volume, the pressure rises. I gas is ree to move, it will always fow rom regions o higher pressure to regions o lower pressure. 6 . 4 g aS e xCh an g e D uring ventilation, muscle contractions cause the pressure inside the thorax to drop below atmospheric pressure. As a consequence, air is drawn into the lungs rom the atmosphere ( inspiration) until the lung pressure has risen to atmospheric pressure. Muscle contractions then cause pressure inside the thorax to rise above atmospheric, so air is orced out rom the lungs to the atmosphere ( expiration) . Antagonistic muscles Dierent muscles are required or inspiration and expiration because muscles only do work when they contract. Muscles can be in two states: contracting and relaxing. Muscles do work when they contract by exerting a pulling orce ( tension) that causes a particular movement. They become shorter when they do this. Muscles lengthen while they are relaxing, but this happens passively they do not lengthen themselves. Most muscles are pulled into an elongated state by the contraction o another muscle. They do not exert a pushing orce (compression) while relaxing so do no work at this time. Muscles thereore can only cause movement in one direction. I movement in opposite directions is needed at dierent times, at least two muscles will be required. When one muscle contracts and causes a movement, the second muscle relaxes and is elongated by the frst. The opposite movement is caused by the second muscle contracting while the frst relaxes. When muscles work together in this way they are known as an antagonistic pair o muscles. Figure 8 Diferent muscles are used or bending the leg at the knee and or the opposite movement o straightening it Inspiration and expiration involve opposite movements, so dierent muscles are required, working as antagonistic pairs. Antagonistic muscle action in ventilation External and internal intercostal muscles, and diaphragm and abdominal muscles as examples o antagonistic muscle action. Ventilation involves two pairs o opposite movements that change the volume and thereore the pressure inside the thorax: Diaphragm isprto Moves downwards and fattens eprto Moves upwards and becomes more domed Ribcage Moves upwards and outwards Moves downwards and inwards Antagonistic pairs o muscles are needed to cause these movements. Volume and pressure changes isprto The volume inside the thorax increases and consequently the pressure decreases eprto The volume inside the thorax decreases and consequently the pressure increases 315 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy Movement of the diaphragm Movement of the ribcage Diaphragm The diaphragm contracts and so it moves downwards and pushes the abdomen wall out The diaphragm relaxes so it can be pushed upwards into a more domed shape Abdomen wall muscles Muscles in the abdomen wall relax allowing pressure from the diaphragm to push it out Muscles in the abdomen wall contract pushing the abdominal organs and diaphragm upwards External intercostal muscles The external intercostal muscles contract, pulling the ribcage upwards and outwards The external intercostal muscles relax and are pulled back into their elongated state. Internal intercostal muscles The internal intercostal muscles relax and are pulled back into their elongated state The internal intercostal muscles contract, pulling the ribcage inwards and downwards Epidemiology Obtain evidence for theories: epidemiological studies have contributed to our understanding of the causes of lung cancer. Epidemiology is the study o the incidence and causes o disease. Most epidemiological studies are observational rather than experimental because it is rarely possible to investigate the causes o disease in human populations by carrying out experiments. As in other felds o scientifc research, theories about the causes o a disease are proposed. To obtain evidence or or against a theory, survey data is collected that allows the association between the disease and its theoretical cause to be tested. For example, to test the theory that smoking causes lung cancer, the smoking habits o people who have developed lung cancer and people who have not are needed. Examples o very large epidemiological surveys that provided strong evidence or a link between smoking and lung cancer are included in sub-topic 1 .6. A correlation between a risk actor and a disease does not prove that the actor causes the disease. There are usually conounding actors which also have an eect on the incidence. They can cause spurious associations between a disease and a actor that does not cause it. For example, an association has repeatedly been ound by epidemiologists between leanness and an increased risk o lung cancer. C areul analysis showed that among smokers leanness is not signifcantly associated with an increased risk. Smoking reduces appetite and so is associated with leanness and o course smoking is a cause o lung cancer. This explains the spurious association between leanness and lung cancer. To try to compensate or conounding actors it is usually necessary to collect data on many actors apart rom the one being investigated. This allows statistical procedures to be carried out to take account o conounding actors and try to isolate the eect o single actors. Age and sex are almost always recorded and sometimes epidemiological surveys include only males or emales or only people in a specifc age range. Causes of lung cancer Causes and consequences of lung cancer. Lung cancer is the most common cancer in the world, both in terms o the number o cases and the number o deaths due to the disease. The 316 general causes o cancer are described in subtopic 1 .6. The specifc causes o lung cancer are considered here. 6 . 4 g aS e xCh an g e and smoke rom burning coal, wood or other organic matter. Figure 9 A large tumour (red) is visible in the right lung. The tumour is a bronchial carcinoma Smoking causes about 87% o cases. Tobacco smoke contains many mutagenic chemicals. As every cigarette carries a risk, the incidence o lung cancer increases with the number smoked per day and the number o years o smoking. Passive smoking causes about 3% o cases. This happens when non-smokers inhale tobacco smoke exhaled by smokers. The number o cases will decline in countries where smoking is banned indoors and in public places. Air pollution probably causes about 5 % o lung cancers. The sources o air pollution that are most signifcant are diesel exhaust umes, nitrogen oxides rom all vehicle exhaust umes Radon gas causes signifcant numbers o cases in some parts o the world. It is a radioactive gas that leaks out o certain rocks such as granite. It accumulates in badly ventilated buildings and people then inhale it. Asbestos, silica and some other solids can cause lung cancer i dust or other particles o them are inhaled. This usually happens on construction sites or in quarries, mines or actories. The consequences o lung cancer are oten very severe. Some o them can be used to help diagnose the disease: difculties with breathing, persistent coughing, coughing up blood, chest pain, loss o appetite, weight loss and general atigue. In many patients the tumour is already large when it is discovered and may also have metastasized, with secondary tumours in the brain or elsewhere. Mortality rates are high. O nly 1 5 % o patients with lung cancer survive or more than 5 years. I a tumour is discovered early enough, all or part o the aected lung may be removed surgically. This is usually combined with one or more courses o chemotherapy. O ther patients are treated with radiotherapy. The minority o patients who are cured o lung cancer, but have lost some o their lung tissue, are likely to continue to have pain, breathing difculties, atigue and also anxiety about the possible return o the disease. Emphysema Causes and consequences of emphysema. In healthy lung tissue each bronchiole leads to a group o small thin-walled alveoli. In a patient with emphysema these are replaced by a smaller number o larger air sacs with much thicker walls. The total surace area or gas exchange is considerably reduced and the distance over which diusion o gases occurs is increased, and so gas exchange is thereore much less eective. The lungs also become less elastic, so ventilation is more difcult. The molecular mechanisms involved are not ully understood, though there is some evidence or these theories: Phagocytes inside alveoli normally prevent lung inections by engulfng bacteria and produce elastase, a protein-digesting enzyme, to kill them inside the vesicles ormed by endocytosis. An enzyme inhibitor called alpha 1 -antitrypsin (A1 AT) usually prevents elastase and other proteases rom digesting lung tissue. In smokers, the number o phagocytes in the lungs increases and they produce more elastase. Genetic actors aect the quantity and eectiveness o A1 AT produced in the lungs. 317 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy In about 3 0% o smokers digestion o proteins in the alveolus wall by the increased quantity o proteases is not prevented and alveolus walls are weakened and eventually destroyed. Emphysema is a chronic disease because the damage to alveoli is usually irreversible. It causes low oxygen saturation in the blood and higher than normal carbon dioxide concentrations. As a result the patient lacks energy and may eventually fnd even tasks such as climbing stairs too onerous. In mild cases there is shortness o breath during vigorous exercise but eventually even mild activity causes it. Ventilation is laboured and tends to be more rapid than normal. Data-based questions: Emphysema and gas exchange Figure 1 0 shows healthy lung tissue and tissue rom a lung with emphysema, at the same magnifcation. S moking usually causes emphysema. B reathing polluted air makes the disease worse. 1 2 3 Figure 10 Healthy lung tissue (top) and lung tissue showing emphysema (bottom) 318 a) Place a ruler across each micrograph and count how many times the edge o the ruler crosses a gas exchange surace. Repeat this several times or each micrograph, in such a way that the results are comparable. S tate your results using suitable units. [3 ] b) Explain the conclusions that you draw rom the results. [3 ] Explain why people who have emphysema eel tired all the time. [3 ] S uggest why people with emphysema oten have an enlarged and strained right side o the heart. [1 ] 6 . 5 n e u r o n S an D S yn apS e S 6.5 ns d sss Understanding Applications Neurons transmit electrical impulses. Secretion and reabsorption o acetylcholine by The myelination o nerve bres allows or neurons at synapses. Blocking o synaptic transmission at cholinergic synapses in insects by binding o neonicotinoid pesticides to acetylcholine receptors. saltatory conduction. Neurons pump sodium and potassium ions across their membranes to generate a resting potential. An action potential consists o depolarization and repolarization o the neuron. Nerve impulses are action potentials propagated along the axons o neurons. Propagation o nerve impulses is the result o local currents that cause each successive part o the axon to reach the threshold potential. Synapses are junctions between neurons and between neurons and receptor or efector cells. When pre-synaptic neurons are depolarized they release a neurotransmitter into the synapse. A nerve impulse is only initiated i the threshold potential is reached. Skills Analysis o oscilloscope traces showing resting potentials and action potentials. Nature of science Cooperation and collaboration between groups o scientists: biologists are contributing to research into memory and learning. Neurons Neurons transmit electrical impulses. Two systems o the body are used or internal communication: the endocrine system and the nervous system. The endocrine system consists o glands that release hormones. The nervous system consists o nerve cells called neurons. There are about 8 5 billion neurons in the human nervous system. Neurons help with internal communication by transmitting nerve impulses. A nerve impulse is an electrical signal. Neurons have a cell body with cytoplasm and a nucleus but they also have narrow outgrowths called nerve fbres along which nerve impulses travel. D endrites are short branched nerve fbres, or examples those used to transmit impulses between neurons in one part o the brain or spinal cord. Axons are very elongated nerve fbres, or example those that transmit impulses rom the tips o the toes or the fngers to the spinal cord. 319 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy cell body axon skeletal muscle (eector) dendrites Figure 1 Neuron with dendrites that transmit impulses to the cell body and an axon that transmits impulses a considerable distance to muscle fbres myelinated nerve fbres The myelination o nerve fbres allows or saltatory conduction. The basic structure o a nerve fbre along which a nerve impulse is transmitted is very simple: the fbre is cylindrical in shape, with a plasma membrane enclosing a narrow region o cytoplasm. The diameter in most cases is about 1 m, though some nerve fbres are wider than this. A nerve fbre with this simple structure conducts nerve impulses at a speed o about 1 metre per second. Figure 2 Nerve fbres (axons) transmitting electrical impulses to and rom the central nervous system are grouped into bundles myelin nucleus of node of sheath Schwann cell Ranvier axon S ome nerve fbres are coated along most o their length by a material called myelin. It consists o many layers o phospholipid bilayer. S pecial cells called S chwann cells deposit the myelin by growing round and round the nerve fbre. E ach time they grow around the nerve fbre a double layer o phospholipid bilayer is deposited. There may be 2 0 or more layers when the S chwann cell stops growing. There is a gap between the myelin deposited by adj acent S chwann cells. The gap is called a node o Ranvier. In myelinated nerve fbres the nerve impulse can j ump rom one node o Ranvier to the next. This is called saltatory conduction. It is much quicker than continuous transmission along a nerve fbre so myelinated nerve fbres transmit nerve impulses much more rapidly than unmyelinated nerve fbres. The speed can be as much as 1 00 metres per second. Figure 3 Detail o a myelinated nerve fbre showing the gaps between adjacent Schwann cells (nodes o Ranvier) Figure 4 Transverse section o axon showing the myelin sheath ormed by the Schwann cell's membrane wrapped round the axon many times (red) 320 6 . 5 n e u r o n S an D S yn apS e S Resting potentials Neurons pump sodium and potassium ions across their membranes to generate a resting potential. uid outside neuron Na + Na+ channel closed Na + Sodiumpotassium pumps transer sodium (Na + ) and potassium (K + ) ions across the membrane. Na+ ions are pumped out and K+ ions are pumped in. The numbers o ions pumped is unequal when three Na+ ions are pumped out, only two K+ ions are pumped in, creating concentration gradients or both ions. Also the membrane is about 5 0 times more permeable to K + ions than Na + ions, so K + ions leak back across the membrane aster than Na + ions. As a result, the Na + concentration gradient across the membrane is steeper than the K + gradient, creating a charge imbalance. K+ Na + Na + A neuron that is not transmitting a signal has a potential dierence or voltage across its membrane that is called the resting potential. This potential is due to an imbalance o positive and negative charges across the membrane. Na + Na + K+ Na + Na + Na + Na+ /K+ pump K+ - K+ channel closed K+ - K+ K+ K+ - - K+ K+ - - Na+ K+ K+ K+ protein K+ K+ K+ K+ K+ K+ K+ cytoplasm Figure 5 The resting potential is generated by the sodiumpotassium pump In addition to this, there are proteins inside the nerve fbre that are negatively charged ( organic anions) , which increases the charge imbalance. These actors together give the neuron a resting membrane potential o about - 70 mV. Action potentials An action potential consists of depolarization and repolarization of the neuron. An action potential is a rapid change in membrane potential, consisting o two phases: depolarization a change rom negative to positive repolarization a change back rom positive to negative. D epolarization is due to the opening o sodium channels in the membrane, allowing Na + ions to diuse into the neuron down the concentration gradient. The entry o Na + ions reverses the charge imbalance across the membrane, so the inside is positive relative to the outside. This raises the membrane potential to a positive value o about + 3 0 mV. 321 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy uid outside neuron uid outside neuron Na+ Na + Na+ channel open Na + K+ Na+ K+ Na+ Na + channel closed Na+ Na+ K+ K+ Na+ K+ K K+ Na + - - K+ + K - K+ Na + Na + - K+ Na + K+ protein K K+ K+ Na+ + K+ channel closed Na+ Na + K+ - - K+ K+ K+ Na+ /K+ pump Na + /K+ pump - K+ K+ K+ + + Na+ Na + Na+ Na K+ K+ K+ K+ Na + - K+ K+ Na+ Na + K+ Na + K K+ - Na+ K+ + K+ - K+ protein Na + Na+ - K+ K+ Na + Na+ + -K K+ - K+ K+ cytoplasm cytoplasm Figure 6 Neuron depolarizing impulse movement + + + + + + + + + A B + cell membrane cytoplasm + + + + + + + + Figure 7 Neuron repolarizing Repolarization happens rapidly after depolarization and is due to the closing of the sodium channels and opening of potassium channels in the membrane. This allows potassium ions to diffuse out of the neuron, down their concentration gradient, which makes the inside of the cell negative again relative to the outside. The potassium channels remain open until the membrane has fallen to a potential close to - 7 0 mV. The diffusion of potassium repolarizes the neuron, but it does not restore the resting potential as the concentration gradients of sodium and potassium ions have not yet been re- established. This takes a few milliseconds and the neuron can then transmit another nerve impulse. Na Na++ proagation of action otentials + + + + + + + C + + ++ NaNa K+ + + + + + + D + + + Na + Na + K+ + + + + + + E + + + Na + Na + Figure 8 Action along axons 322 potentials are propagated K+ channel open Nerve impulses are action potentials propagated along the axons of neurons. A nerve impulse is an action potential that starts at one end of a neuron and is then propagated along the axon to the other end of the neuron. The propagation of the action potential happens because the ion movements that depolarize one part of the neuron trigger depolarization in the neighbouring part of the neuron. Nerve impulses always move in one direction along neurons in humans and other vertebrates. This is because an impulse can only be initiated at one terminal of a neuron and can only be passed on to other neurons or Na + 6 . 5 n e u r o n S an D S yn apS e S dierent cell types at the other terminal. Also, there is a reractive period ater a depolarization that prevents propagation o an action potential backwards along an axon. loca currents Propagation o nerve impulses is the result o local currents that cause each successive part o the axon to reach the threshold potential. The propagation o an action potential along an axon is due to movements o sodium ions. D epolarization o part o the axon is due to diusion o sodium ions into the axon through sodium channels. This reduces the concentration o sodium ions outside the axon and increases it inside. The depolarized part o the axon thereore has dierent sodium ion concentrations to the neighbouring part o the axon that has not yet depolarized. As a result, sodium ions diuse between these regions both inside and outside the axon. Inside the axon there is a higher sodium ion concentration in the depolarized part o the axon so sodium ions diuse along inside the axon to the neighbouring part that is still polarized. O utside the axon the concentration gradient is in the opposite direction so sodium ions diuse rom the polarized part back to the part that has j ust depolarized. These movements are shown in fgure 1 0. They are called local currents. Local currents reduce the concentration gradient in the part o the neuron that has not yet depolarized. This makes the membrane potential rise rom the resting potential o - 70mV to about - 5 0 mV. Sodium channels in the axon membrane are voltage-gated and open when a membrane potential o - 5 0mV is reached. This is thereore known as the threshold potential. Opening o the sodium channels causes depolarization. activit ns i s m d mfsh Anemonesh have a nervous system similar to ours, with a central nervous system and neurons that transmit nerve impulses in one direction only. Sea anemones have no central nervous system. Their neurons orm a simple network and will transmit impulses in either direction along their nerve bres. They both protect each other rom predators more efectively than they can themselves. Explain how they do this. Figure 9 Anemonefsh among the tentacles o a sea anemone Thus local currents cause a wave o depolarization and then repolarization to be propagated along the axon at a rate o between one and a hundred ( or more) metres per second. impulse movement + N a d i u s i o n outside inside N a + d i u s i o n part that has just depolarized (action potential) Figure 10 membrane part that has not yet depolarized (resting potential) Local currents 323 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy action potential peak Analysing oscilloscope traces 0 re po la riz at io n de po la riz at io n potential dierence across membrane (mV) +35 50 70 undershoot Analysis o oscilloscope traces showing resting potentials and action potentials. threshold potential resting potential 0 1 2 3 45 6 7 time/ms stimulus Figure 11 Changes in membrane polarity during an action potential Membrane potentials in neurons can be measured by placing electrodes on each side o the membrane. The potentials can be displayed using an oscilloscope. The display is similar to a graph with time on the x- axis and the membrane potential on the y- axis. I there is a resting potential, a horizontal line appears on the oscilloscope screen at a level o - 7 0 mV, assuming that this is the resting potential o the neuron. I an action potential occurs, a narrow spike is seen, with the rising and alling phases showing the depolarization and repolarization. The oscilloscope trace may also show the potential rising beore the depolarization until the threshold potential is reached. The repolarization does not usually return the membrane potential to - 70 mV immediately and there is a phase in which the potential changes gradually until the resting potential is reached. Data-based questions: Analysing an oscilloscope trace The oscilloscope trace in gure 1 2 was taken rom a digital oscilloscope. It shows an action potential in a mouse hippocampal pyramidal neuron that happened ater the neuron was stimulated with a pulse o current. 1 2 membrane voltage (mV) 3 50 50 time (ms) D educe with a reason the threshold potential needed to open voltage- gated sodium channels in this neuron. [2 ] Estimate the time taken or the depolarization, and the repolarization. [2 ] Predict the time taken rom the end o the depolarization or the resting potential to be regained. [2 ] 5 D iscuss how many action potentials could be stimulated per second in this neuron. [2 ] S uggest a reason or the membrane potential rising briefy at the end o the repolarization. [1 ] 50 0 [1 ] 4 0 resting potential S tate the resting potential o the mouse hippocampal pyramidal neuron. 100 6 Figure 12 Synapses Synapses are junctions between neurons and between neurons and receptor or efector cells. Synapses are junctions between cells in the nervous system. In sense organs there are synapses between sensory receptor cells and neurons. In both the brain and spinal cord there are immense numbers o synapses between neurons. In muscles and glands there are synapses between neurons and 324 6 . 5 n e u r o n S an D S yn apS e S muscle bres or secretory cells. Muscles and glands are sometimes called eectors, because they eect (carry out) a response to a stimulus. C hemicals called neurotransmitters are used to send signals across synapses. This system is used at all synapses where the pre- synaptic and post- synaptic cells are separated by a fuid-lled gap, so electrical impulses cannot pass across. This gap is called the synaptic clet and is only about 2 0 nm wide. Synaptic transmission When pre-synaptic neurons are depolarized they release a neurotransmitter into the synapse. S ynaptic transmission occurs very rapidly as a result o these events: A nerve impulse is propagated along the pre-synaptic neuron until it reaches the end o the neuron and the pre-synaptic membrane. D epolarization o the pre-synaptic membrane causes calcium ions ( C a 2+ ) to diuse through channels in the membrane into the neuron. Infux o calcium causes vesicles containing neurotransmitter to move to the pre-synaptic membrane and use with it. Neurotransmitter is released into the synaptic clet by exocytosis. The neurotransmitter diuses across the synaptic clet and binds to receptors on the post- synaptic membrane. pre-synaptic cell nerve impulse pre-synaptic membrane neurotransmitter (e.g. acetylcholine) Sodium ions diuse down their concentration gradient into the post- synaptic neuron, causing the postsynaptic membrane to reach the threshold potential. The neurotransmitter is rapidly broken down and removed rom the synaptic clet. synaptic cleft 20nm approximately neurotransmitter activates receptors The binding o the neurotransmitter to the receptors causes adj acent sodium ion channels to open. An action potential is triggered in the post- synaptic membrane and is propagated on along the neuron. synaptic knob synaptic vesicles Electron micrograph o a synapse. False colour has been used to indicate the pre-synaptic neuron (purple) with vesicles o neurotransmitter (blue) and the post-synaptic neuron (pink) . The narrowness o the synaptic clet is visible Ca 2+ diuses into knob Figure 13 ion channel opened post-synaptic membrane post-synaptic cell Figure 14 A nerve impulse is propagated across a synapse by the release, difusion and post-synaptic binding o neurotransmitter Dt-bsd qstis: Parkinsons disease D opamine is one o the many neurotransmitters that are used at synapses in the brain. In Parkinsons disease, there is a loss o dopaminesecreting neurons, which causes slowness in initiating movement, muscular rigidity and in many cases shaking. Figure 1 5 shows the metabolic pathways involved in the ormation and breakdown o dopamine. 1 Explain how symptoms o Parkinsons disease are relieved by giving the ollowing drugs: a) L- D O PA [1 ] 325 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy 2 b) selegeline, which is an inhibitor o monoamine oxidase- B ( MAO - B ) [1 ] c) tolcapone, which is an inhibitor o catechol- O - methyl transerase ( C O MT) [1 ] tyrosine COOH tyrosine hydroxylase HO CH 2 CH NH 2 (FOOD) COMT HO COOH CH 2 CH NH 2 HO CH 3 O d) ropinirole, which is an agonist o dopamine [1 ] e) safnamide, which inhibits reuptake o dopamine by pre-synaptic neurons. [1 ] HO L-DOPA CH 2 CH 2 NH 2 HO HO D iscuss how a cure or Parkinsons disease might in the uture be developed by: a) stem cell therapy [3 ] b) gene therapy. [2 ] HO CH 3 O CH 2 COOH COMT HO COOH CH 2 CH NH 2 HO dopa dopamine decarboxylase HO MAO-B O CH 2 C H aldehyde dehydrogenase CH 2 COOH HO Figure 15 The formation and breakdown of L-DOPA and dopamine. The enzymes catalysing each step are shown in red Acetylcholine Secretion and reabsorption of acetylcholine by neurons at synapses. Acetylcholine is used as the neurotransmitter in many synapses, including synapses between neurons and muscle fbres. It is produced in the pre- synaptic neuron by combining choline, absorbed rom the diet, with an acetyl group produced during aerobic respiration. The acetylcholine is loaded into vesicles and then released into the synaptic clet during synaptic transmission. choline acetyl group Figure 16 Acetylcholine The receptors or acetylcholine in the post- synaptic membrane have a binding site to which acetylcholine will bind. The acetylcholine only remains bound to the receptor or a short time, during which only one action potential is initiated in the post- synaptic neuron. This is because the enzyme acetylcholinesterase is present in the synaptic clet and rapidly breaks acetylcholine down into choline and acetate. The choline is reabsorbed into the pre- synaptic neuron, where it is converted back into active neurotransmitter by recombining it with an acetyl group. Neonicotinoids Blocking of synaptic transmission at cholinergic synapses in insects by binding of neonicotinoid pesticides to acetylcholine receptors. Neonicotinoids are synthetic compounds similar to nicotine. They bind to the acetylcholine receptor in cholinergic synapses in the central nervous system o insects. Acetylcholinesterase does not 326 6 . 5 n e u r o n S an D S yn apS e S break down neonicotinoids, so the binding is irreversible. The receptors are blocked, so acetylcholine is unable to bind and synaptic transmission is prevented. The consequence in insects is paralysis and death. Neonicotinoids are thereore very eective insecticides. O ne o the advantages o neonicotinoids as pesticides is that they are not highly toxic to humans and other mammals. This is because a much greater proportion o synapses in the central nervous system are cholinergic in insects than in mammals and also because neonicotinoids bind much less strongly to acetylcholine receptors in mammals than insects. Neonicotinoid pesticides are now used on huge areas o crops. In particular one neonicotinoid, imidacloprid, is the most widely used insecticide in the world. However, concerns have been raised about the eects o these insecticides on honeybees and other benefcial insects. There has been considerable controversy over this and the evidence o harm is disputed by the manuacturers and some government agencies. Threshold potentials A nerve impulse is only initiated i the threshold potential is reached. activit rsch dts ictiids There are currently intense research eforts to try to discover whether neonicotinoids are to blame or collapses in honeybee colonies. What are the most recent research ndings and do they suggest that these insecticides should be banned? Figure 17 Research has shown that the neonicotinoid pesticide imidacloprid reduces growth of bumblebee colonies Nerve impulses ollow an all-or-nothing principle. An action potential is only initiated i the threshold potential is reached, because only at this potential do voltage-gated sodium channels start to open, causing depolarization. The opening o some sodium channels and the inward diusion o sodium ions increases the membrane potential causing more sodium channels to open there is a positive eedback eect. I the threshold potential is reached there will thereore always be a ull depolarization. At a synapse, the amount o neurotransmitter secreted ollowing depolarization o the pre-synaptic membrane may not be enough to cause the threshold potential to be reached in the post-synaptic membrane. The post-synaptic membrane does not then depolarize. The sodium ions that have entered the post-synaptic neuron are pumped out by sodium potassium pumps and the post-synaptic membrane returns to the resting potential. A typical post- synaptic neuron in the brain or spinal cord has synapses not j ust with one but with many pre- synaptic neurons. It may be necessary or several o these to release neurotransmitter at the same time or the threshold potential to be reached and a nerve impulse to be initiated in the post- synaptic neuron. This type o mechanism can be used to process inormation rom dierent sources in the body to help in decision- making. 327 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy Research into memory and learning Cooperation and collaboration between groups of scientists: biologists are contributing to research into memory and learning. Higher unctions o the brain including memory and learning are only partly understood at present and are being researched very actively. They have traditionally been investigated by psychologists but increasingly the techniques o molecular biology and biochemistry are being used to unravel the mechanisms at work. O ther branches o science are also making important contributions, including biophysics, medicine, pharmacology and computer science. Figure 18 Many synapses are visible in this scanning electron micrograph between the cell body o one post-synaptic neuron and a large number o diferent pre-synaptic neurons (blue) The C entre or Neural C ircuits and B ehaviour at O xord University is an excellent example o collaboration between scientists with dierent areas o expertise. The our group leaders o the research team and the area o science that they originally studied are: Proessor Gero Miesenbck medicine and physiology D r Martin B ooth engineering and optical microscopy D r Korneel Hens chemistry and biochemistry Proessor S cott Waddell genetics, molecular biology and neurobiology. The centre specializes in research techniques known as optogenetics. Neurons are genetically engineered to emit light during synaptic transmission or an action potential, making activity in specifc neurons in brain tissue visible. They are also engineered so specifc neurons in brain tissue respond to a light signal with an action potential. This allows patterns o activity in the neurons o living brain tissue to be studied. Figure 19 Memory and learning are unctions o the cerebrumthe olded upper part o the brain 328 There are many research groups in universities throughout the world that are investigating memory, learning and other brain unctions. Although there is sometimes competition between scientists to be the frst group to make a discovery, there is also a strongly collaborative element to scientifc research. This extends across scientifc disciplines and national boundaries. Success in understanding how the brain works will undoubtedly be the achievement o many groups o scientists in many countries throughout the world. 6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n 6.6 hs, sss d dc Understanding Insulin and glucagon are secreted by and cells in the pancreas to control blood glucose concentration. Thyroxin is secreted by the thyroid gland to regulate the metabolic rate and help control body temperature. Leptin is secreted by cells in adipose tissue and acts on the hypothalamus o the brain to inhibit appetite. Melatonin is secreted by the pineal gland to control circadian rhythms. A gene on the Y chromosome causes embryonic gonads to develop as testes and secrete testosterone. Testosterone causes prenatal development o male genitalia and both sperm production and development o male secondary sexual characteristics during puberty. Estrogen and progesterone cause prenatal development o emale reproductive organs and emale secondary sexual characteristics during puberty. The menstrual cycle is controlled by negative and positive eedback mechanisms involving ovarian and pituitary hormones. Applications Causes and treatment o type I and type II diabetes. Testing o leptin on patients with clinical obesity and reasons or the ailure to control the disease. Causes o jet lag and use o melatonin to alleviate it. The use in IVF o drugs to suspend the normal secretion o hormones, ollowed by the use o artifcial doses o hormones to induce superovulation and establish a pregnancy. William Harveys investigation o sexual reproduction in deer. Skills Annotate diagrams o the male and emale reproductive system to show names o structures and their unctions. Nature of science Developments in scientifc research ollow improvements in apparatus: William Harvey was hampered in his observational research into reproduction by lack o equipment. The microscope was invented 17 years ater his death. Control of blood glucose concentration Insulin and glucagon are secreted by and cells in the pancreas to control blood glucose concentration. C ells in the pancreas respond to changes in blood glucose levels. If the glucose concentration deviates substantially from the set point of about 5 mmol L - 1 , homeostatic mechanisms mediated by the pancreatic hormones insulin and glucagon are initiated. 329 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy The pancreas is eectively two glands in one organ. Most o the pancreas is exocrine glandular tissue that secretes digestive enzymes into ducts leading to the small intestine. There are small regions o endocrine tissue called islets o Langerhans dotted through the pancreas that secrete hormones directly into the blood stream. The two cell types in the islets o Langerhans secrete dierent hormones. Figure 1 Fluorescent light micrograph of the pancreas showing two islets of Langerhans surrounded by exocrine gland tissue. Alpha cells in the islets are stained yellow and beta cells are stained red Alpha cells ( cells) synthesize and secrete glucagon i the blood glucose level alls below the set point. This hormone stimulates breakdown o glycogen into glucose in liver cells and its release into the blood, increasing the concentration. B eta cells ( cells) synthesize insulin and secrete it when the blood glucose concentration rises above the set point. This hormone stimulates uptake o glucose by various tissues, particularly skeletal muscle and liver, in which it also stimulates the conversion o glucose to glycogen. Insulin thereore reduces blood glucose concentration. Like most hormones, insulin is broken down by the cells it acts upon, so its secretion must be ongoing. S ecretion begins within minutes o eating and may continue or several hours ater a meal. Diabetes Causes and treatment of type I and type II diabetes. Diabetes is the condition where a person has consistently elevated blood glucose levels even during prolonged asting, leading to the presence o glucose in the urine. C ontinuously elevated glucose damages tissues, particularly their proteins. It also impairs water reabsorption rom urine while it is orming in the kidney, resulting in an increase in the volume o urine and body dehydration. I a person needs to urinate more requently, is constantly thirsty, eels tired and craves sugary drinks, they should test or glucose in the urine to check whether they have developed diabetes. There are two main types o this disease: 330 Type I diabetes, or early- onset diabetes, is characterized by an inability to produce sufcient quantities o insulin. It is an autoimmune disease arising rom the destruction o beta cells in the islets o Langerhans by the bodys own immune system. In children and young people the more severe and obvious symptoms o the disease usually start rather suddenly. The causes o this and other autoimmune diseases are still being researched. Type II diabetes, sometimes called late- onset diabetes, is characterized by an inability to process or respond to insulin because o a defciency o insulin receptors or glucose transporters on target cells. O nset is slow and the disease may go unnoticed or many years. Until the last ew decades, this orm o diabetes was very rare in people under 5 0 and common only in the over 65 s. The causes o this orm o diabetes are not well understood but the main risk actors are sugary, atty diets, prolonged obesity due to habitual overeating and lack o exercise, together with genetic actors that aect energy metabolism. The treatment o the two types o diabetes is dierent: Type I diabetes is treated by testing the blood glucose concentration regularly and inj ecting insulin when it is too high or likely to become too high. Inj ections are oten done beore a meal to prevent a peak o blood glucose as the ood is digested and absorbed. Timing is very important because insulin molecules do not last long in the blood. B etter treatments are being developed using implanted devices that can release exogenous insulin into the blood as and when it is necessary. A permanent cure may be achievable by coaxing stem cells to become ully unctional replacement beta cells. 6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n Type II diabetes is treated by adj usting the diet to reduce the peaks and troughs o blood glucose. S mall amounts o ood should be eaten requently rather than inrequent large meals. Foods with high sugar content should be avoided. S tarchy ood should only be eaten i it has a low glycemic index, indicating that it is digested slowly. High- fbre oods should be included to slow the digestion o other oods. S trenuous exercise and weight loss are benefcial as they improve insulin uptake and action. acvy The glucose tolerance test is a method used to diagnose diabetes. In this test, the patient drinks a concentrated glucose solution. The blood glucose concentration is monitored to determine the length o time required or excess glucose to be cleared rom the blood. Fds f y ii dbcs concentration / mg 100 cm 3 D-bsd qss: The glucose tolerance test 400 350 300 250 200 150 100 50 0 Discuss which o the oods in fgure 2 are suitable or a person with type II diabetes. They should be oods with a low glycemic index. diabetic unaected 0 0.5 1 2 3 4 time after glucose ingestion / h 5 Figure 3 A person with diabetes and an unafected person give very diferent responses to the glucose tolerance test With reerence to fgure 3 , compare the person with normal glucose metabolism to the person with diabetes with respect to: a) The concentration o glucose at time zero, i.e. beore the consumption o the glucose drink. b) The length o time required to return to the level at time zero. c) The maximum glucose level reached. Figure 2 d) The time beore glucose levels start to all. tyx Thyroxin is secreted by the thyroid gland to regulate the metabolic rate and help control body temperature. The hormone thyroxin is secreted by the thyroid gland in the neck. Its chemical structure is unusual as the thyroxin molecule contains our atoms o iodine. Prolonged defciency o iodine in the diet thereore prevents the synthesis o thyroxin. This hormone is also unusual as almost all cells in the body are targets. Thyroxin regulates the bodys metabolic rate, so all cells need to respond but the most metabolically active, such as liver, muscle and brain are the main targets. Higher metabolic rate supports more protein synthesis and growth and it increases the generation o body heat. In a person with normal physiology, cooling triggers increased thyroxin secretion by the thyroid gland, which stimulates heat production so body temperature rises. 331 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy Thyroxin thus regulates the metabolic rate and also helps to control body temperature. The importance o thyroxin is revealed by the eects o thyroxin defciency ( hypothyroidism) : Figure 4 Structure of thyroxin with atoms of lack o energy and eeling tired all the time orgetulness and depression weight gain despite loss o appetite as less glucose and at are being broken down to release energy by cell respiration eeling cold all the time because less heat is being generated constipation because contractions o muscle in the wall o the gut slow down. impaired brain development in children. iodine shown purple leptin Leptin is secreted by cells in adipose tissue and acts on the hypothalamus of the brain to inhibit appetite. Leptin is a protein hormone secreted by adipose cells ( at storage cells) . The concentration o leptin in the blood is controlled by ood intake and the amount o adipose tissue in the body. The target o this hormone is groups o cells in the hypothalamus o the brain that contribute to the control o appetite. Leptin binds to receptors in the membrane o these cells. I adipose tissue increases, blood leptin concentrations rise, causing long- term appetite inhibition and reduced ood intake. Figure 5 Mouse with obesity due to lack of leptin and a mouse with normal body mass The importance o this system was demonstrated by research with a strain o mice discovered in the 1 95 0s that eed ravenously, become inactive and gain body weight, mainly through increased adipose tissue. They grow to a body weight o about 1 00 grams, compared with wild type mice o 2 02 5 grams. B reeding experiments showed that the obese mice had two copies o a recessive allele, ob. In the early 1 990s it was shown that the wild- type allele o this gene supported the synthesis o a new hormone that was named leptin. Adipose cells in mice that have two recessive ob alleles cannot produce leptin. When ob/ob mice were inj ected with leptin their appetite declined, energy expenditure increased and body mass dropped by 3 0% in a month. leptin and obesity Testing of leptin on patients with clinical obesity and reasons for the failure to control the disease. The discovery that obesity in mice could be caused by a lack o leptin and cured by leptin injections soon led to attempts to treat obesity in humans in this way. Amgen, a biotechnology company based in C aliornia, paid $2 0 million or the commercial rights to leptin and a large clinical trial was carried 332 out. Seventy-three obese volunteers injected themselves either with one o several leptin doses or with a placebo. A double blind procedure was used, so neither the researchers nor the volunteers knew who was injecting leptin until the results were analysed. 6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n The leptin inj ections induced skin irritation and swelling and only 47 patients completed the trial. The eight patients receiving the highest dose lost 7.1 kg o body mass on average compared with a loss o 1 .3 kg in the 1 2 volunteers who were inj ecting the placebo. However, in the group receiving the highest dose the results varied very widely rom a loss o 1 5 kg to a gain o 5 kg. Also any body mass lost during the trial was usually regained rapidly aterwards. S uch disappointing outcomes are requent in drug research the physiology o humans is dierent in many ways rom mice and other rodents. In contrast to ob/ob mice, most obese humans have exceptionally high blood leptin concentrations. The target cells in the hypothalamus may have become resistant to leptin so ail to respond to it, even at high concentrations. Appetite is thereore not inhibited and ood intake is excessive. More adipose tissue develops, causing a rise in blood leptin concentration but the leptin resistance prevents inhibition o appetite. Inj ection o extra leptin inevitably ails to control obesity i the cause is leptin resistance, j ust as insulin inj ections alone are ineective with early- stage type II diabetes. A very small proportion o cases o obesity in humans are due to mutations in the genes or leptin synthesis or its various receptors on target cells. Trials in people with such obesity have shown signifcant weight loss while the leptin injections are continuing. However leptin is a short-lived protein and has to be injected several times a day and consequently most o those oered this treatment have reused it. Also leptin has been shown to aect the development and unctioning o the reproductive system, so injections are not suitable in children and young adults. All in all leptin has not ulflled its early promise as a means o solving the human obesity problem. melatonin Melatonin is secreted by the pineal gland to control circadian rhythms. Humans are adapted to live in a 2 4- hour cycle and have rhythms in behaviour that ft this cycle. These are known as circadian rhythms. They can continue even i a person is placed experimentally in continuous light or darkness because an internal system is used to control the rhythm. C ircadian rhythms in humans depend on two groups o cells in the hypothalamus called the suprachiasmatic nuclei ( S C N) . These cells set a daily rhythm even i grown in culture with no external cues about the time o day. In the brain they control the secretion o the hormone melatonin by the pineal gland. Melatonin secretion increases in the evening and drops to a low level at dawn and as the hormone is rapidly removed rom the blood by the liver, blood concentrations rise and all rapidly in response to these changes in secretion. The most obvious eect o melatonin is the sleep- wake cycle. High melatonin levels cause eelings o drowsiness and promote sleep through the night. Falling melatonin levels encourage waking at the end o the night. Experiments have shown that melatonin contributes to the nighttime drop in core body temperature, as blocking the rise in melatonin levels reduces it and giving melatonin artifcially during the day causes a drop in core temperature. Melatonin receptors have been discovered in the kidney, suggesting that decreased urine production at night may be another eect o this hormone. When humans are placed experimentally in an environment without light cues indicating the time o day, the S C N and pineal gland usually Figure 6 Until a baby is about three months old it does not develop a regular day-night rhythm o melatonin secretion so sleep patterns do not ft those o the babys parents 333 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy maintain a rhythm o slightly longer than 2 4 hours. This indicates that timing o the rhythm is normally adj usted by a ew minutes or so each day. A special type o ganglion cell in the retina o the eye detects light o wavelength 460480 nm and passes impulses to cells in the S C N. This indicates to the SC N the timing o dusk and dawn and allows it to adj ust melatonin secretion so that it corresponds to the day- night cycle. Jet lag and melatonin Causes of jet lag and use of melatonin to alleviate it. Jet lag is a common experience or someone who has crossed three or more time zones during air travel. The symptoms are diculty in remaining awake during daylight hours and diculty sleeping through the night, atigue, irritability, headaches and indigestion. The causes are easy to understand: the S C N and pineal gland are continuing to set a circadian rhythm to suit the timing o day and night at the point o departure rather than the destination. Jet lag only lasts or a ew days, during which impulses sent by ganglion cells in the retina to the SC N when they detect light help the body to adj ust to the new regime. Melatonin is sometimes used to try to prevent or reduce j et lag. It is taken orally at the time when sleep should ideally be commencing. Most trials o melatonin have shown that it is eective at promoting sleep and helping to reduce j et lag, especially i fying eastwards and crossing ve or more time zones. Sex determination in males A gene on the Y chromosome causes embryonic gonads to develop as testes and secrete testosterone. Human reproduction involves the usion o a sperm rom a male with an egg rom a emale. Initially the development o the embryo is the same in all embryos and embryonic gonads develop that could either become ovaries or testes. The developmental pathway o the embryonic gonads and thereby the whole baby depends on the presence or absence o one gene. I the gene SRY is present, the embryonic gonads develop into testes. This gene is located on the Y chromosome, so is only present in 5 0% o embryos. S RY codes or a D NA- binding protein called TD F ( testis determining actor) . TD F stimulates the expression o other genes that cause testis development. 5 0% o embryos have two X chromosomes and no Y so they do not have a copy o the S RY gene. TD F is thereore not produced and the embryonic gonads develop as ovaries. Testosterone Testosterone causes prenatal development of male genitalia and both sperm production and development of male secondary sexual characteristics during puberty. Figure 7 334 X and Y chromosomes The testes develop rom the embryonic gonads in about the eighth week o pregnancy, at the time when the embryo is becoming a etus and is about 3 0mm long. The testes develop testosterone-secreting cells at an early stage and these produce testosterone until about the teenth week 6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n o pregnancy. D uring the weeks o secretion, testosterone causes male genitalia to develop, which are shown in fgure 8. At puberty the secretion o testosterone increases. This stimulates sperm production in the testes, which is the primary sexual characteristic o males. Testosterone also causes the development o secondary sexual characteristics during puberty such as enlargement o the penis, growth o pubic hair and deepening o the voice due to growth o the larynx. Sex deterination in feales Estrogen and progesterone cause prenatal development of female reproductive organs and female secondary sexual characteristics during puberty. I the gene S RY is not present in an embryo because there is no Y chromosome, the embryonic gonads develop as ovaries. Testosterone is thereore not secreted, but the two emale hormones, estrogen and progesterone, are always present in pregnancy. At frst they are secreted by the mothers ovaries and later by the placenta. In the absence o etal testosterone and the presence o maternal estrogen and progesterone, emale reproductive organs develop which are shown in fgure 9. During puberty the secretion o estrogen and progesterone increases, causing the development o emale secondary sexual characteristics. These include enlargement o the breasts and growth o pubic and underarm hair. male and feale reproductive systes Annotate diagrams of the male and female reproductive system to show names of structures and their functions. The tables on the next page indicate unctions that should be included when diagrams o male and emale reproductive systems are annotated. seminal vesicle bladder bladder sperm duct sperm duct prostate gland seminal vesicle erectile tissue penis prostate gland penis epididymis testis epididymis urethra urethra scrotum testis scrotum Figure 8 foreskin Male reproductive system in front and side view 335 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy oviduct ovary opening to oviduct oviduct ovary uterus uterus cervix bladder vagina urethra large intestine vulva vagina cervix labia (vulva) Figure 9 Female reproductive system in front and side view male reproductive syste Feale reproductive syste Testis Produce sperm and testosterone Ovary Produce eggs, estrogen and progesterone Scrotum Hold testes at lower than core body temperature Oviduct Epididymis Store sperm until ejaculation Collect eggs at ovulation, provide a site or ertilization then move the embryo to the uterus Sperm duct Transer sperm during ejaculation Uterus Provide or the needs o the embryo and then etus during pregnancy Seminal vesicle and prostate gland Secrete fuid containing alkali, proteins and ructose that is added to sperm to make semen Cervix Protect the etus during pregnancy and then dilate to provide a birth canal Urethra Transer semen during ejaculation and urine during urination Vagina Stimulate penis to cause ejaculation and provide a birth canal Penis Penetrate the vagina or ejaculation o semen near the cervix Vulva Protect internal parts o the emale reproductive system menstrual cycle The menstrual cycle is controlled by negative and positive eedback mechanisms involving ovarian and pituitary hormones. The menstrual cycle occurs in most women rom puberty until the menopause, apart rom during pregnancies. E ach time the cycle occurs it gives the chance o a pregnancy. The frst hal o the menstrual cycle is called the ollicular phase because a group o ollicles is developing in the ovary. In each ollicle an egg is stimulated to grow. At the same time the lining o the uterus ( endometrium) is repaired and starts to thicken. The most developed ollicle breaks open, releasing its egg into the oviduct. The other ollicles degenerate. The second hal o the cycle is called the luteal phase because the wall o the ollicle that released an egg becomes a body called the corpus luteum. C ontinued development o the endometrium prepares 336 6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n it or the implantation o an embryo. I ertilization does not occur the corpus luteum in the ovary breaks down. The thickening o the endometrium in the uterus also breaks down and is shed during menstruation. TOK Figure 1 0 shows hormone levels in a woman over a 3 6- day period, including one complete menstrual cycle. The pattern o changes is typical or a woman who is not pregnant. The hormone levels are measured in mass per millilitre. The actual masses are very small, so progesterone, FS H and LH are measured in nanograms ( ng) and estrogen is measured in picograms ( pg) . Figure 1 0 also shows the state o the ovary and o the endometrium. Human eggs can be obtained by using FSH to stimulate the ovaries, then collecting eggs rom the ovaries using a micropipette. Women have sometimes undergone this procedure to produce eggs or donation to another woman who is unable to produce eggs hersel. t w x d vs w jdgg ly f c? The our hormones in fgure 1 0 all help to control the menstrual cycle by both negative and positive eedback. FS H and LH are protein hormones produced by the pituitary gland that bind to FS H and LH receptors in the membranes o ollicle cells. E strogen and progesterone are ovarian hormones, produced by the wall o the ollicle and corpus LH FSH 800 600 400 200 menstruation menstruation hormone level /ng ml 1 1000 follicle starting to develop 400 corpus luteum follicle nearly mature 8 progesterone estrogen 300 6 200 4 100 2 0 26 28 2 4 5 8 days of menstrual cycle thickness of endometrium ovulation 28 Figure 10 10 12 14 16 18 20 22 24 26 28 7 14 21 2 4 progesterone level/ng ml 1 estrogen level/pg ml 1 0 Recently stem-cell researchers have used eggs in therapeutic cloning experiments. The nucleus o an egg is removed and replaced with a nucleus rom an adult. I the resulting cell developed as an embryo, stem cells could be removed rom it and cloned. It might then be possible to produce tissues or organs or transplanting to the adult who donated the nucleus. There would be no danger o tissue rejection because the stem cells would be genetically identical to the recipient. There is a shortage o eggs both or donation to other women and or research. In 2006, scientists in England got permission to ofer women cut-price IVF treatment, i they were willing to donate some eggs or research. In Sweden only travel and other direct expenses can be paid to egg donors, and in Japan egg donation is banned altogether. 1 Is there a distinction to be drawn between donating eggs or therapeutic cloning experiments and donating eggs to a woman who is unable to produce eggs hersel, or example because her ovaries have been removed? Can the same act be judged diferently depending on motives? 28 The menstrual cycle 337 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy luteum. They are absorbed by many cells in the emale body, where they infuence gene expression and thereore development. FS H rises to a peak towards the end o the menstrual cycle and stimulates the development o ollicles, each containing an oocyte and ollicular fuid. FS H also stimulates secretion o estrogen by the ollicle wall. E strogen rises to a peak towards the end o the ollicular phase. It stimulates the repair and thickening o the endometrium ater menstruation and an increase in FS H receptors that make the ollicles more receptive to FS H, boosting estrogen production ( positive eedback) . When it reaches high levels estrogen inhibits the secretion o FS H ( negative eedback) and stimulates LH secretion. LH rises to a sudden and sharp peak towards the end o the ollicular phase. It stimulates the completion o meiosis in the oocyte and partial digestion o the ollicle wall allowing it to burst open at ovulation. LH also promotes the development o the wall o the ollicle ater ovulation into the corpus luteum which secretes estrogen ( positive eedback) and progesterone. Progesterone levels rise at the start o the luteal phase, reach a peak and then drop back to a low level by the end o this phase. Progesterone promotes the thickening and maintenance o the endometrium. It also inhibits FSH and LH secretion by the pituitary gland ( negative eedback) . Data-based questions: The female athlete triad 1 a) O utline the relationship between number o menstrual cycles per year and bone density. [3 ] b) Compare the results or the neck o the emur with the results or the trochanter. [3] 338 2 3 Explain the reasons or some o the runners having: a) higher bone density than the mean [2 ] b) lower bone density than the mean. [4] a) S uggest reasons or emale athletes having ew or no menstrual cycles. [2 ] b) Suggest one reason or eating disorders and low body weight in emale athletes. [1 ] t-score (SD) The emale athlete triad is a syndrome consisting o three interrelated disorders that can aect emale athletes: osteoporosis, disordered eating and menstrual disorders. O steoporosis is reduced bone mineral density. It can be caused by a diet low in calcium, vitamin D or energy, or by low estrogen levels. Figure 1 1 shows the bone mineral density in two parts o the emur or emale runners who had dierent numbers o menstrual cycles per year. The t- score is the number o standard deviations above or below mean peak bone mass or young women. 1 neck of femur 0.5 trochanter of femur 0 0.5 1 menstrual cycles per year 03 410 1113 Figure 11 Bone mass in women grouped by number of menstrual cycles 6 . 6 h o r m o n e S , h o m e o S ta S i S an D r e pr o D u Cti o n In vitro fertilization The use in IVF o drugs to suspend the normal secretion o hormones, ollowed by the use o artifcial doses o hormones to induce superovulation and establish a pregnancy. The natural method o ertilization in humans is in vivo, meaning that it occurs inside the living tissues o the body. Fertilization can also happen outside the body in careully controlled laboratory conditions. This is called in vitro ertilization, almost always abbreviated to IVF. This procedure has been used extensively to overcome ertility problems in either the male or emale parent. There are several dierent protocols or IVF, but the rst stage is usually down- regulation. The woman takes a drug each day, usually as a nasal spray, to stop her pituitary gland secreting FSH or LH. S ecretion o estrogen and progesterone thereore also stops. This suspends the normal menstrual cycle and allows doctors to control the timing and amount o egg production in the womans ovaries. Intramuscular inj ections o FS H and LH are then given daily or about ten days, to stimulate ollicles to develop. The FS H inj ections give a much higher concentration o this hormone than during a normal menstrual cycle and as a consequence ar more ollicles develop than usual. Twelve is not unusual and there can be as many twenty ollicles. This stage o IVF is thereore called superovulation. When the ollicles are 1 8 mm in diameter they are stimulated to mature by an inj ection o HC G, another hormone that is normally secreted by the embryo. A micropipette mounted on an ultrasound scanner is passed through the uterus wall to wash eggs out o the ollicles. Each egg is mixed with 5 0, 000 to 1 00, 000 sperm cells in sterile conditions in a shallow dish, which is then incubated at 3 7 C until the next day. I ertilization is successul then one or more embryos are placed in the uterus when they are about 48 hours old. Because the woman has not gone through a normal menstrual cycle extra progesterone is usually given as a tablet placed in the vagina, to ensure that the uterus lining is maintained. I the embryos implant and continue to grow then the pregnancy that ollows is no dierent rom a pregnancy that began by natural conception. William Harvey and sexual reproduction William Harveys investigation o sexual reproduction in deer. William Harvey is chiefy remembered or his discovery o the circulation o the blood, but he also had a lielong obsession with how lie is transmitted rom generation to generation and pioneered research into sexual reproduction. He was taught the seed and soil theory o Aristotle, according to which the male produces a seed, which orms an egg when it mixes with menstrual blood. The egg develops into a etus inside the mother. William Harvey tested Aristotles theory using a natural experiment. Deer are seasonal breeders and only become sexually active during the autumn. Harvey examined the uterus o emale deer during the mating season by slaughtering and dissecting them. He expected to nd eggs developing in the uterus immediately ater mating, but only ound signs o anything developing in emales two or more months ater the start o the mating season. Figure 12 IVF allows the earliest stages in a human life to be seen. This micrograph shows a zygote formed by fertilization. The nuclei of the egg and sperm are visible in the centre of the zygote. There is a protective layer of gel around the zygote called the fertilization membrane 339 61 Hum C Ean LLpBHI ys O LO i oGlo Y gy He regarded his experiments with deer as proo that Aristotles theory o reproduction was alse and concluded the etus doth neither proceed rom the seed o male or emale in coition, nor yet rom any commixture o that seed. Although Aristotles seed and soil theory was alse, Harveys conclusion that the etus did not result rom events during coitus ( sexual intercourse) was also alse. Harvey was well aware that he had not discovered the basis o sexual reproduction: neither the philosophers nor the physicians o yesterday or today have satisactorily explained, or solved the problem o Aristotle. Figure 13 William Harveys book on the reproduction of animals Exercitationes de Generatione Animalium published in 1651 Improvements in apparatus and research breakthroughs Developments in scientifc research ollow improvements in apparatus: William Harvey was hampered in his observational research into reproduction by lack o equipment. The microscope was invented seventeen years ater his death. Harvey was understandably reluctant to publish his research into sexual reproduction, but he did eventually do so in 1 65 1 when he was 73 years old in his work Exercitationes de Generatione Animalium. He knew that he had not solved the mystery o sexual reproduction: When I plainly see nothing at all doth remain in the uterus ater coition, ... no more than remains in the braine ater sensation, ... I have invented this Fable. Let the learned and ingenious ock o men consider o it; let the supercilious reject it: and or the scofng ticklish generation, let them laugh their swinge. Because I say, there is no sensible thing in the uterus ater coition; and yet there is a necessity, that something should be there, which may render the animal ruitul. 340 William Harvey ailed to solve the mystery because eective microscopes were not available when he was working, so usion o gametes and subsequent embryo development remained undiscovered. He was unlucky with his choice o experimental animal because embryos in the deer that he used remain microscopically small or an unusually long period. Microscopes were invented seventeen years ater Harveys death, allowing the discovery o sperm, eggs and early stage embryos. Scientifc research has oten been hampered or a time by defciencies in apparatus, with discoveries only being made ollowing improvements. This will continue into the uture and we can look orward to urther transormations in our understanding o the natural world as new techniques and technology are invented. QueStion S Questions 1 Using the data in table 1 : a) outline the relationship between the age of the mother and the success rate of IVF [3 ] b) outline the relationship between the number of embryos transferred and the chance of having a baby as a result of IVF c) accidents during the daytime as a result of disrupted sleep and tiredness. Figure 1 5 shows the percentage oxygen saturation of arterial blood during a night of sleep in a patient with severe obstructive sleep apnea. 100 70 2 100 70 3 100 70 4 100 70 5 100 70 6 100 70 7 100 70 8 100 70 [3 ] discuss how many embryos fertility centres should be allowed to transfer. [4] hours prcg f rgcs r iVF cycl ag f ccrdg h mbr f mbrys rsfrrd mhr 1 2 3 single single twins single twins triplets < 30 10.4 20.1 9.0 17.5 3.6 0.4 3034 13.4 21.8 7.9 18.2 7.8 0.6 3539 19.1 19.1 5.0 17.4 5.6 0.6 > 39 4.1 12.5 3.5 12.7 1.7 0.1 Table 1 2 1 Figure 1 4 shows variations in liver glycogen over the course of one day. O2% 0 a) E xplain the variation in liver glycogen. [3 ] a) liver glycogen level an evening snack 8:00 12:00 16:00 20:00 24:00 time of da breakfast 4:00 8:00 S ometimes the ventilation of the lungs stops. This is called apnea. O ne possible cause is the blockage of the airways by the soft palate during sleep. This is called obstructive sleep apnea. It has some potentially harmful consequences, including an increased risk of 50 60 Explain the causes of falls in saturation. [2] ( ii) E xplain the causes of rises in saturation. [2 ] ( iii) C alculate how long each cycle of falling and rising saturation takes. [2 ] b) Estimate the minimum oxygen saturation that the patient experienced during the night, and when it occurred. [2 ] Figure 14 3 30 40 minutes Hour 8 shows a typical pattern due to obstructive sleep apnea. (i) dinner 20 Figure 15 b) E valuate the contribution of glycogen to blood sugar homeostasis. [2 ] lunch 10 c) 4 D educe the sleep patterns of the patient during the night when the trace was taken. [2 ] The action potential of a squid axon was recorded, with the axon in normal sea water. The axon was then placed in water with a Na + concentration of one- third of that of sea water. 341 61 h u m an p h yS i o lo g y The action potential was recorded again. Figure 1 6 shows these recordings. a) Using only the data in gure 1 7, outline the eect o reduced Na + concentration on: membrabe potential (mV) ( i) +40 ( ii) the duration o the action potential. sea water +20 -20 33% c) D iscuss the eect o reduced Na concentration on the time taken to return to the resting potential. [2 ] -40 -60 -80 2 Geneticists discovered a mutant variety o ruit fy that shakes vigorously when anaesthetized with ether. Studies have shown that the shaker mutant has K + channels that do not unction properly. Figure 1 7 shows action potentials in normal ruit fies and in shaker mutants. 40 wild-type drosophila normal action potential 0 -40 4 8 40 12 16 shaker mutant abnormal action potential 0 -40 4 Figure 17 [3 ] + Figure 16 membrabe potential/mV [2 ] b) Explain the eects o reduced Na concentration on the action potential. 0 time (ms) 342 [2 ] + 1 5 the magnitude o depolarization 8 12 time (ms) 16 d) C ompare the action potentials o shaker and normal ruit fies. [3 ] e) E xplain the dierences between the action potentials. 7 N U CLE I C ACI D S ( AH L) Introduction The discovery of the structure of D NA revolutionized biology. Information stored in a coded form in D NA is copied onto mRNA. The structure of D NA is ideally suited to its function. Information transferred from D NA to mRNA is translated into an amino acid sequence. 7.1 DNA structure and replication Understanding DNA structure suggested a mechanism or DNA replication. Nucleosomes help to supercoil the DNA. DNA replication is continuous on the leading strand and discontinuous on the lagging strand. DNA replication is carried out by a complex system o enzymes. DNA polymerases can only add nucleotides to the 3 end o a primer. Some regions o DNA do not code or proteins but have other important unctions. Nature of science Making careul observations: Rosalind Franklins X-ray diraction provided crucial evidence that DNA is a double helix. Applications Rosalind Franklins and Maurice Wilkins investigation o DNA structure by X-ray diraction. Tandem repeats are used in DNA profling. Use o nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation o samples or base sequencing. Skills Analysis o results o the Hershey and Chase experiment providing evidence that DNA is the genetic material. Utilization o molecular visualization sotware to analyse the association between protein and DNA within a nucleosome. 343 7 N U C L E I C AC I D S ( AH L ) The HersheyChase experiment Analysis of the results of the HersheyChase experiment providing evidence that DNA is the genetic material. From the late 1 800s, scientists were convinced that chromosomes played a role in heredity and that the hereditary material had a chemical nature. Aware that chromosomes were composed o both protein and nucleic acid, both molecules were contenders to be the genetic material. Until the 1 940s, the view that protein was the hereditary material was avoured, as it was a class o macromolecules that had great variety due to twenty naturally occurring sub-units as opposed to our nucleotide sub- units. Further, many specifc unctions had been identifed or proteins. Variety and specifcity o unction were two properties that were expected to be essential requirements or the hereditary material. Alred Hershey and Martha C hase wanted to ascertain whether the genetic material o viruses was protein or D NA. In the 1 95 0s, it was known that viruses are inectious particles which transorm cells into virus-producing actories by becoming bound to host cells and inj ecting their genetic material. The non-genetic portion o the virus remains outside the cell. An inected cell then manuactures large numbers o new viruses and bursts, releasing them to the environment ( see fgure 1 ) . Viruses are oten specifc to a certain cell type. The virus they chose to work with was the T2 bacteriophage because o its very simple structure. It has a coat composed entirely o protein while D NA is ound inside the coat. DNA protein Figure 1 Coloured transmission electron micrograph (TEM) o T2 viruses (blue) bound to an Escherichia coli bacterium. Each virus consists o a large DNA-containing head and a tail composed o a central sheath with several fbres. The fbres attach to the host cell surace, and the virus DNA is injected into the cell through the sheath. It instructs the host to build copies o the virus (blue, in cell) Figure 2 Diagram illustrating the structure o the T2 virus Data-based questions: The HersheyChase experiment Alred Hershey and Martha C hase were two scientists who worked to resolve the debate over the chemical nature o the genetic material. In their experiment, they took advantage o the act that D NA contains phosphorus but not sulphur while proteins contain sulphur but not phosphorus. They cultured viruses that contained proteins with radioactive ( 3 5 S ) sulphur and they separately cultured viruses that contained D NA with radioactive ( 3 2 P) 344 phosphorus. They inected bacteria separately with the two types o viruses. They used a blender to separate the non- genetic component o the virus rom the cell and then centriuged the culture solution to concentrate the cells in a pellet. The cells were expected to have the radioactive genetic component o the virus in them. They measured the radioactivity in the pellet and the supernatant. Figure 3 represents the process and results o the experiment. 7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N radioactive protein ( 35 S) protein coat with 35 S bacteria virus radioactivity ( 35 S) in supernatant bacterium radioactive DNA ( 32 P) virus DNA with 32 P bacteria bacterium radioactivity ( 32 P) in pellet Questions b) Explain why the genetic material should be ound in the pellet and not the supernatant. c) D etermine the percentage o the remains in the supernatant. 32 P that d) D etermine the percentage o 35 S that remains in the supernatant. e) D iscuss the evidence that D NA is the chemical which transorms the bacteria into inected cells. % of isotope in supernatant a) Explain what a supernatant is. percentage of isotope in supernatant after 8 minutes agitation 100% 80% 60% 40% 20% 0% 35 S 32 P Figure 3 X-ay dfan an a vdn ma Making careul observations: Rosalind Franklins X-ray difraction provided crucial evidence that DNA was a helix. Two names are usually remembered in connection with the discovery o D NA, C rick and Watson. Flashes o insight led to their success, but they could not have achieved it without skilled experimental work and careul observations by other scientists. O ne o these was Erwin C harga. His research into the percentage base composition o D NA is described in the data- based question in sub-topic 2 .6 ( page 1 07) . Another key fgure in the discovery o D NA was Rosalind Franklin. In 1 95 0, she became a research associate in the biophysics unit at Kings C ollege, London. The unit was already investigating the structure o D NA by X- ray diraction. Franklin had already become skilled in techniques o crystallography and X- ray diraction while researching other carbon compounds at an institute in Paris. 345 7 N U C L E I C AC I D S ( AH L ) At Kings C ollege she improved the resolution o a camera, so she could make more detailed measurements o the X-ray diraction patterns than had previously been possible. She also produced high quality samples o D NA with the molecules aligned in narrow bres. B y careul control o humidity two types o pure sample could be produced and as Franklin was unsure which represented the normal structure o D NA, she investigated both. Soon ater starting work at Kings C ollege, Franklin had obtained the sharpest X- ray diraction images o D NA in existence. They have been described as amongst the most beautiul X- ray photographs o any substance ever taken. Their implications are described in the next section. She was unwilling to publish her ndings until there was strong evidence. S he thereore embarked on a rigorous analysis o the diraction patterns that allowed her to calculate the dimensions o the D NA helix. Without Franklins knowledge or permission, James Watson was shown the best diraction pattern and the calculations based on it. B eore Franklin could publish her results C rick and Watson had used them to build their model o D NA structure. It is widely accepted that Rosalind Franklin deserved a Nobel Prize or her research, but this never happened. C rick and Watson were awarded prizes in 1 962 , but she died o cancer in 1 95 8, aged thirty-seven. Nobel Prizes cannot be awarded posthumously, but Rosalind Franklin is remembered more than many prize winners. What we can remember rom her lie is that discoveries may sometimes be made through serendipity or fashes o insight, but the real oundations o science are rigorous experimental techniques and diligent observation. Rosalind Franklins investigation of DNA structure Rosalind Franklin and Maurice Wilkins investigation o DNA structure by X-ray difraction. I a beam o X- rays is directed at a material, most o it passes through but some is scattered by the particles in the material. This scattering is called diraction. The wavelength o X- rays makes them particularly sensitive to diraction by the particles in biological molecules including D NA. In a crystal the particles are arranged in a regular repeating pattern, so the diraction occurs in a regular way. D NA cannot be crystallized but the molecules were arranged in an orderly enough array in Franklins samples or a diraction pattern to be obtained, rather than random scattering. An X-ray detector is placed close to the sample to collect the scattered rays. The sample can be rotated in three dierent dimensions to investigate the pattern o scattering. D iraction patterns can be recorded using X-ray lm. Franklin developed a high resolution camera containing X- ray lm to obtain very clear images o diraction patterns rom D NA. Figure 4 shows the most amous o these diraction patterns. 346 Figure 4 Rosalind Franklins X-ray difraction photograph o DNA From the diraction pattern in gure 4 Franklin was able to make a series o deductions about the structure o D NA: The cross in the centre o the pattern indicated that the molecule was helical in shape. The angle o the cross shape showed the pitch ( steepness o angle) o the helix. 7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N The distance between the horizontal bars showed turns o the helix to be 3 .4 nm apart. The distance between the middle o the diraction pattern and the top showed that there was a repeating structure within the molecule, with a distance o 0. 3 4 nm between the repeats. This turned out to be the vertical distance between adj acent base pairs in the helix. These deductions that were made rom the X-ray diraction pattern o DNA were critically important in the discovery o the structure o DNA. The Watson and Crick model suggested semiconservative replication DNA structure suggested a mechanism or DNA replication. S everal lines o experimental evidence came together to lead to the knowledge o the structure o D NA: molecular modelling pioneered by the Nobel prize winner Linus Pauling, X- ray diraction patterns discerned rom the careul photographs o Rosalind Franklin and the base composition studies o Erwin C harga. B ut insight and imagination played a role as well. O ne o Watson and C ricks frst models had the sugar- phosphate strands wrapped around one another with the nitrogen bases acing outwards. Rosalind Franklin countered this model with the knowledge that the nitrogen bases were relatively hydrophobic in comparison to the sugarphosphate backbone and would likely point in to the centre o the helix. Franklins X-ray diraction studies showed that the DNA helix was tightly packed so when Watson and C rick built their models, their choices required the bases to ft together such that the strands were not too ar apart. As they trialled various models, Watson and C rick ound the tight packing they were looking or would occur i a pyrimidine was paired with a purine and i the bases were upside down in relation to one another. In addition to being structurally similar, adenine has a surplus negative charge and thymine has a surplus positive charge so that pairing was electrically compatible. Pairing cytosine with guanine allows or the ormation o three hydrogen bonds which enhances stability. O nce the model was proposed, the complementary base pairing immediately suggested a mechanism by which D NA replication could occur one o the key requirements that any structural model would have to address. The WatsonC rick model led to the hypothesis o semiconservative replication. The role of nucleosomes in DNA packing Nucleosomes help to supercoil DNA. One dierence between eukaryotic DNA and bacterial DNA is that eukaryotic DNA is associated with proteins called histones. Most groups o prokaryotes have D NA that is not associated with histones, or proteins like histones. For this reason, prokaryotic DNA is reerred to as being naked. toK Wha n d n hav whn h and dn dn fy mah xmna vdn? Chargaf wrote about his observations: the results serve to disprove the tetranucleotide hypothesis. It is, however, noteworthy - whether this is more than accidental, cannot yet be said - that in all deoxypentose nucleic acids examined thus ar the molar ratios o total purines to total pyrimidines and also o adenine to thymine and o guanine to cytosine were not ar rom 1 H. H. Bauer, author o the book Scientifc Literacy and the Myth o the Scientifc Method, argues that Chargaf needed to: stick his neck out beyond the actual results and say that they mean exact equality and hence some sort o pairing in the molecular structure . Watson and Crick, on the other hand were speculating and theorizing about the molecular nature and biological unctions o DNA and they postulated a structure in which the equalities are exactly one and the deviation orm this in the data could be regarded as experimental error. Ideas and theory turned out to be a better guide than raw data. Histones are used by the cell to package the D NA into structures called nucleosomes. A nucleosome consists o a central core o eight histone 347 7 N U C L E I C AC I D S ( AH L ) H1 histone proteins with D NA coiled around the proteins. The eight proteins, or octamer, consist o two copies o our dierent types o histones. A short section o linker D NA connects one nucleosome to the next. An additional histone protein molecule, called H1 , serves to bind the D NA to the core particle ( fgure 5 ) . DNA nucleosome 30nm bre Figure 5 The association o histones with the D NA contributes to a pattern known as supercoiling. An analogy is i you twist an elastic band repeatedly eventually it orms an additional pattern o coils. S upercoiling allows a great length o D NA to be packed into a much smaller space within the nucleus. The nucleosome is an adaptation that acilitates the packing o the large genomes that eukaryotes possess. The H1 histone binds in such a way to orm a structure called the 3 0 nm fbre that acilitates urther packing. Visualizing nucleosomes Activity Determining packing ratio Packing ratio is defned as the length o DNA divided by the length into which it is packaged. Use the inormation below to estimate the packing ratio o: Utilization o molecular visualization sotware to analyse the association between protein and DNA within a nucleosome. Visit the protein data bank at http://www.rcsb.org/pdb/home/home.do or download the image o a nucleosome rom the companion website or this textbook. 1 Rotate the molecule to see the two copies o each histone protein. In fgure 6, they are identifed by the tails that extend rom the core. Each protein has such a tail that extends out rom the core. 2 Note also the approximately 1 5 0 bp o D NA wrapped nearly twice around the octamer core. 3 Note the N- terminal tail that proj ects rom the histone core or each protein. C hemical modifcation o this tail is involved in regulating gene expression. 4 Visualize the positively charged amino acids on the nucleosome core. S uggest how they play a role in the association o the protein core with the negatively charged D NA. (a) a nucleosome; and (b) chromosome 22 (one o the smallest human chromosomes) . 348 The distance between base pairs is 0.34 nm. There is approximately 200 bp o DNA coiled around a nucleosome. A nucleosome is approximately 10 nm long. There is an estimated 5.0 10 7 total base pairs (bp) present in the shortest human autosome (chromosome 22). Chromosome 22 in its most condensed orm is approximately 2 m long. Figure 6 7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N Daa-bad qn: Apoptosis and the length of DNA between nucleosomes Under natural conditions, programmed cell death sometimes occurs. This is known as apoptosis and it plays an important role in such processes as metamorphosis and embryological development. O ne mechanism involved in this auto-destruction is the digestion o D NA by enzymes called D NAases. The D NA associated with the nucleosome is normally not as accessible to the D NAase as the linking sections. D NA gets digested into ragments o lengths equal to multiples o the distance between nucleosomes. Origin 2000 bp 1500 bp 1000 bp 750 bp 500 bp The let hand column o fgure 7 shows the results o separation by gel electrophoresis o the D NA released by the action o D NAase on rat liver cells. The right column represents ragments used as a reerence called a ladder. 250 bp Figure 7 O nce the D NA had been cut, nucleosomes were digested by protease. 1 ( iii) the length o D NA between two linker D NA regions with three nucleosomes between them. Identiy on the diagram the ragment that represents: ( i) the length o D NA between the two sections o linker D NA on either side o one nucleosome; ( ii) the length o D NA between two linker D NA regions with two nucleosomes between them; 2 D educe the length o D NA associated with a nucleosome. 3 S uggest how the pattern in the lethand column would change i very high concentrations o D NAase were applied to the cells. The leading strand and the lagging strand DNA replication is continuous on the leading strand and discontinuous on the lagging strand. B ecause the two strands o the D NA double helix are arranged in an anti-parallel ashion, synthesis on the two strands occurs in very dierent ways. One strand, the leading strand, is made continuously ollowing the ork as it opens. The other strand, known as the lagging strand, is made in ragments moving away rom the replication ork. New ragments are created on the lagging strand as the replication ork exposes more o the template strand. These ragments are called Okazaki ragments. Proteins involved in replication DNA replication is carried out by a complex system of enzymes. Replication involves the ormation and movement o the replication ork and synthesis o the leading and lagging strands. Proteins are involved as enzymes at each stage but also serve a number o other unctions. 349 7 N U C L E I C AC I D S ( AH L ) The enzyme helicase unwinds the D NA at the replication ork and the enzyme topoisomerase releases the strain that develops ahead o the helicase. Single-stranded binding proteins keep the strands apart long enough to allow the template strand to be copied. S tarting replication requires an RNA primer. Note that on the lagging strand there are a number o primers but there is j ust one on the leading strand. The enzyme D NA primase creates one RNA primer on the leading strand and many RNA primers on the lagging strand. The RNA primer is necessary to initiate the activity o D NA polymerase. D NA polymerase is responsible or covalently linking the deoxyribonucleotide monophosphate to the 3 end o the growing strand. D ierent organisms have dierent kinds o D NA polymerases, each with dierent unctions such as proo- reading, polymerization and removal o RNA primers once they are no longer needed. D NA ligase connects the gaps between ragments. DNA topoisomerase leading strand DNA polymerase 5 3 parental DNA primase RNA primer DNA helicase DNA ligase DNA polymerase 3 lagging 5 strand Figure 8 The direction of replication DNA polymerases can only add nucleotides to the 3 end of a primer Within D NA molecules, D NA replication begins at sites called origins o replication. In prokaryotes there is one site and in eukaryotes there are many. Replication occurs in both directions away rom the origin. The result appears as a replication bubble in electron micrographs. The fve carbons o the deoxyribose sugar have a number ( see fgure 9) . phosphate nitrogen base O 5 CH 2 4 H 1 H H 2 3 OH 350 OH T H Figure 9 DNA growing strand deoxyribose sugar C A template strand DNA H Figure 10 C A G T G G C 5 end OH base 3 end sugar phosphate 7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N The phosphate group o new D NA nucleotides is added to the the 3 carbon o the deoxyribose o the nucleotide at the end o the chain. Replication thereore occurs in the 5 to 3 direction. Non-coding regions o DNA have important unctions Some regions o DNA do not code or proteins but have other important unctions. The cellular machinery operates according to a genetic code. D NA is used as a guide or the production o polypeptides using the genetic code. However, only some D NA sequences code or the production o polypeptides. These are called coding sequences. There are a number o non- coding sequences ound in genomes. S ome o them have unctions, such as those sequences that are used as a guide to produce tRNA and rRNA. S ome non- coding regions play a role in the regulation o gene expression such as enhancers and silencers. In sub-topic 7.2 we will explore non-coding sequences called introns. toK t wha xn d n hav a nq nby whn a dmay? Molecular biologist Elizabeth Blackburn is one o the most renowned original researchers in the feld o telomeres. She shared the Nobel Prize in Physiology or medicine or her co-discovery o telomerase. She made headlines in 2004 when she was dismissed rom the Presidents Council on Bioethics ater objecting to the councils call or a ban on stem cell research and or criticizing the suppression o relevant scientifc evidence in its fnal report. Most o the eukaryotic genome is non- coding. Within the genome, especially in eukaryotes, repetitive sequences can be common. There are two types o repetitive sequences: moderately repetitive sequences and highly repetitive sequences ( satellite D NA) . Together they can orm between 5 and 60 per cent o the genome. In humans, nearly 60% o the D NA consists o repetitive sequences. O ne such area o repetitive sequences occurs on the ends o eukaryotic chromosomes called telomeres. The telomere serves a protective unction. D uring interphase, the enzymes that replicate D NA cannot continue replication all the way to the end o the chromosome. I cells went through the cell cycle without telomeres, they would lose the genes at the end o the chromosomes. S acrifcing the repetitive sequences ound in telomeres serves a protective unction. Figure 11 False colour scanning electron micrograph with telomeres coloured pink. The grey region in the centre is the centromere which also consists of non-coding repetitive sequences DNA profling Tandem repeats are used in DNA profling. A variable number tandem repeat ( VNTR) is a short nucleotide sequence that shows variations between individuals in terms o the number o times the sequence is repeated. E ach variety can be inherited as an allele. Analysis o VNTR allele combinations in individuals is the basis behind D NA profling or use in such applications as genealogical investigations. A locus is the physical location o a heritable element on the chromosome. In the hypothetical example shown in fgure 1 2, locus A has a VNTR o the sequence AT and locus B has a VNTR o the sequence TC G. In the two individuals shown, there are two dierent alleles (varieties) o locus A, two repeats (allele A2) and our repeats (allele A4) . In the same individuals, there are three alleles or locus B , three repeats (allele B 3) , our repeats (allele B 4) and fve repeats (allele B 5 ) . The asterisk mark indicates where the restriction enzyme would cut. The D NA profle that would result is shown in the lower part o fgure 1 2 . Note that the two individuals have some bands in common and some unique bands. 351 7 N U C L E I C AC I D S ( AH L ) Genealogists deduce paternal lineage by analysing short tandem repeats rom the Y-chromosome, and deduce maternal lineage by analysing mitochondrial D NA variations in single nucleotides at specifc locations called hypervariable regions. individual # 1 individual # 2 locus A allele A2 (2 repeats) AT AT locus A allele A4 (4 repeats) allele A2 (2 repeats) AT AT allele A2 (2 repeats) AT AT AT AT AT AT locus B allele B3 (3 repeats) TCG TCG TCG locus B allele B3 (3 repeats) TCG TCG TCG allele B4 (4 repeats) TCG TCG TCG TCG allele B5 (5 repeats) TCG TCG TCG TCG TCG DNA prole origin B5 B4 B3 B3 A4 A2 individual #1 A2 individual #2 Figure 12 Activity Analysis o a DNA profle involving alleles o short tandem repeats o DNA A logarithm is an alternative way to express an exponent. For example, log 1,000 = log 10 3 =3 log 100 = log 10 2 =2 In biology, very large changes in a variable are easier to represent graphically i logarithms are used. Figure 13 Gel electrophoresis. The outside columns represent ladders of known length. The two inside columns represent samples of unknown length 352 In the example (fgure 13), DNA ragments were separated using gel electrophoresis. The ragments vary in size rom 100 bp (base pairs) up to 5,000 bp. The two outside columns o the gel represent ladders, i.e. mixtures o DNA ragments o known size. These were used to obtain the data in table 1 and create the plot shown in fgure 14. The other inner columns shown in fgure 13 are unknowns. Knwn add fagmn z (b) 5,000 2,000 850 400 100 base pairs 7. 1 D N A s t r u c t u r e A N D r e p l i c At i o N Dan mvd (mm) 58 96 150 200 250 10 3 10 2 Table 1 1 10 4 Using fgure 1 4 determine the size o D NA ragments in the two centre digests: Fagmn Dan Fagmn Dan z (b) mvd (mm) z (b) mvd (mm) (mn 2) (mn 2) (mn 3) (mn 3) 60 70 70 160 130 200 10 1 50 100 150 200 250 distance / mm Figure 14 Distance moved as a function of fragment size in gel electrophoresis. Notice that the y-axis scale on this graph goes up in powers of 10. This is a logarithmic scale Daa-bad qn: Analysis o DNA profles using D1S80 O ne commonly studied D NA locus is a VNTR named D 1 S 80. D 1 S 80 is located on human chromosome 1 . This locus is composed o repeating units o 1 6- nucleotide- long segments o D NA. The number o repeats varies rom one individual to the next with 2 9 known alleles ranging rom 1 5 repeats to 41 . In the image o a D NA profle ( fgure 1 5 ) the outside and inside lanes represent ladders representing multiples o one hundred and twenty- three bp. a) Identiy the lengths o the ragments represented by each o the bands in the ladder. b) Using a ruler measure the distance between the origin and the band. Use the length and distance data, to create a standard curve using a logarithmic graph. c) Measure the distance travelled by each band rom the origin. Figure 15 d) Using the standard curve, estimate the lengths o the bands in each individual. e) Estimate the number o repeats represented by each band. f) It is unclear whether the individual in lane 7 has two dierent copies o the same allele or dierent alleles. S uggest what could be done to urther resolve the genotype o the fnal individual. 353 7 N U C L E I C AC I D S ( AH L ) DNA sequencing Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing. The determination o the sequence o bases in a genome is carried out most commonly using a method that employs fuoresence. Many copies o the unknown D NA that is to be sequenced are placed into test tubes with all o the raw materials including deoxyribonucleotides and the enzymes necessary to carry out replication. In addition very small quantities o dideoxyribonucleotides that have been labelled with dierent fuorescent markers are added. The dideoxyribonucleotides will be incorporated into some o the new D NA, but when they are incorporated, they will stop the replication at precisely the point where they were added. The ragments are separated by length using electrophoresis. The sequence o bases can be automatically analysed by comparing the colour o the fuorescence with the length o the ragment. DNA to be sequenced A T A G A C T A G C C primer extension reactions: ddA reaction: ddC reaction: TACTATGCC AG A TACTATGCCAG A ATG A ATGATAC primer ddG reaction: ddT reaction: TACTATGCC AG A TACTATGCCAG A ATG ATACG ATGAT C T A to computer T mixture of nucleotides containing rare dideoxyribonucleotides (ddn) replication stops when a ddn is incorporated column electrophoresis electropherogram T A A G A G A C C G T A T C A T T G ddn that is on the end of the fragment detector laser Figure 16 354 C ????????? 123456789 7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N 7.2 tann and n xn Understanding Gene expression is regulated by proteins that bind to specifc base sequences in DNA. The environment o a cell and o an organism has an impact on gene expression. Nucleosomes help to regulate transcription in eukaryotes. Transcription occurs in a 5' to 3' direction. Eukaryotic cells modiy mRNA ater transcription. Splicing o mRNA increases the number o dierent proteins an organism can produce. Applications The promoter as an example o non-coding DNA with a unction. Skills Analysis o changes in DNA methylation patterns. Nature of science Looking or patterns, trends and discrepancies: there is mounting evidence that the environment can trigger heritable changes in epigenetic actors. The function of the promoter The promoter as an example o non-coding DNA with a unction. Only some DNA sequences code or the production o polypeptides. These are called coding sequences. There are a number o non-coding sequences ound in genomes. Some o them have unctions, such as those sequences that produce tRNA and rRNA. Some non-coding regions play a role in the regulation o gene expression such as enhancers and silencers. The promoter is a sequence that is located near a gene. It is the binding site o RNA polymerase, the enzyme that catalyses the ormation o the covalent bond between nucleotides during the synthesis o RNA. The promoter is not transcribed but plays a role in transcription. Regulation of gene expression by proteins Gene expression is regulated by proteins that bind to specifc base sequences in DNA. S ome proteins are always necessary or the survival o the organism and are thereore expressed in an unregulated ashion. O ther proteins need to be produced at certain times and in certain amounts; i.e., their expression must be regulated. Gene expression is regulated in prokaryotes as a consequence o variations in environmental actors. For example, the genes responsible or the absorption and metabolism o lactose by E.coli are expressed in the presence o lactose and are not expressed in the absence o lactose. In this case, the breakdown o lactose results in regulation o gene expression by negative eedback. In the presence o lactose a repressor protein is deactivated ( fgure 1 ) . O nce the lactose has been broken 355 7 N U C L E I C AC I D S ( AH L ) lactose not in the environment; repressor blocks transcription down, the repressor protein is no longer deactivated and proceeds to block the expression o lactose metabolism genes. As in prokaryotes, eukaryotic genes are regulated in response to variations in environmental conditions. E ach cell o a multicellular eukaryotic organism expresses only a raction o its genes. p ro m o te r lactose present in the environment; repressor deactivated; genes involved in lactose use are transcribed promoter There are a number o proteins whose binding to D NA regulates transcription. These include enhancers, silencers and promoter-proximal elements. Unlike the promoter sequence, the sequences linked to regulatory transcription actors are unique to the gene. RNA polymerase -galactosidase - The regulation o eukaryotic gene expression is also a critical part o cellular dierentiation as well as the process o development. This is seen in the passage o an insect through its lie cycle stages or in human embryological development. + transacetylase permease - - Figure 1 lactose Regulatory sequences on the D NA which increase the rate o transcription when proteins bind to them are called enhancers. Those sequences on the D NA which decrease the rate o transcription when proteins bind to them are called silencers. While enhancers and silencers can be distant rom the promoter, another series o sequences called promoter-proximal elements are nearer to the promoter and binding o proteins to them is also necessary to initiate transcription. The impact of the environment on gene expression The environment of a cell and of an organism has an impact on gene expression. In the history o Western thought, much debate has gone in to the naturenurture debate. This is a debate centred on the extent to which a particular human behaviour or phenotype should be attributed to the environment or to heredity. Much eort has gone into twin studies especially or twins raised apart. Data-based questions: Identical twin studies Twin studies have been used to identiy the relative infuence o genetic actors and environmental actors in the onset o disease ( gure 2 ) . Identical twins have 1 00% o the same D NA while raternal twins have approximately 5 0% o the same D NA. Questions 1 2 3 356 D etermine the percentage o identical twins where both have diabetes. [2 ] Explain why a higher percentage o identical twins sharing a trait suggests that a genetic component contributes to the onset o the trait. [3 ] With reerence to any our conditions, discuss the relative role o the environment and genetics in the onset o the condition. [3 ] percent of twin pairs who share the trait 0% 100% greater height genetic inuence reading disability autism Alzheimers schizophrenia alcoholism bipolar disorder hypertension diabetes multiple sclerosis breast cancer Crohns disease stroke rheumatoid arthritis Figure 2 identical twins greater fraternal twins environmental inuence 7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N The infuence o the environment on gene expression or some traits is unequivocal. Environmental actors can aect gene expression such as the production o skin pigmentation during exposure to sunlight in humans. Avy exlan h a lu an f sam a In embryonic development, the embryo contains an uneven distribution o chemicals called morphogens. C oncentrations o the morphogens aect gene expression contributing to dierent patterns o gene expression and thus dierent ates o the embryonic cells depending on their position in the embryo. In coat colour in cats, the C gene codes or the production o the enzyme tyrosinase, the rst step in the production o pigment. A mutant allele o the gene, cs allows normal pigment production only at temperatures below body temperature. This mutant allele has been selected or in the selective breeding o S iamese cats. At higher temperatures, the protein product is inactive or less active, resulting in less pigment. Nucleosomes regulate transcription Nucleosomes help to regulate transcription in eukaryotes. E ukaryotic D NA is associated with proteins called histones. C hemical modication o the tails o histones is an important actor in determining whether a gene will be expressed or not. A number o dierent types o modication can occur to the tails o histones including the addition o an acetyl group, the addition o a methyl group or the addition o a phosphate group. O C H3 C - Acetyl group C H 3 - Methyl group For example, residues o the amino acid lysine on histone tails can have acetyl groups either removed or added. Normally the lysine residues on histone tails bear a positive charge that can bind to the negatively charged D NA to orm a condensed structure that inhibits transcription. Histone acetylation neutralizes these positive charges allowing a less condensed structure with higher levels o transcription. C hemical modication o histone tails can either activate or deactivate genes by decreasing or increasing the accessibility o the gene to transcription actors. G C M M C T G A M C T G G A C M T G A C M NH 2 CH 3 C Analysing methylation patterns Analysis of changes in DNA methylation patterns The addition o methyl groups directly to D NA is thought to play a role in gene expression. Whereas methylation o histones can promote or inhibit transcription, direct methylation o D NA tends to decrease gene expression. The amount o D NA methylation varies during a lietime and is aected by environmental actors. N C C C O N Figure 3 DNA methylation is the addition of a methyl group (green M) to the DNA base cytosine 357 7 N U C L E I C AC I D S ( AH L ) Data-basd qustions: Changes in methylation pattern with age in identical twins One study compared the methylation patterns o 3-year-old identical twins with 50-year-old identical twins. Methylation patterns were dyed red on one chromosome or one twin and dyed green or the other twin on the same chromosome. Chromosome pairs in each set o twins were digitally superimposed. The result would be a yellow colour i the patterns were the same. Dierences in patterns on the two chromosomes results in mixed patterns o green and red patches. This was done or our o the twenty-three chromosome pairs in the genome. 1 2 3 Explain the reason or yellow coloration i the methylation pattern is the same in the two twins. [3 ] Identiy the chromosome with the least changes as twins age. [1 ] Identiy the chromosomes with the most changes as twins age. [1 ] 4 Explain how these dierences could arise. [3 ] 5 Predict with a reason whether identical twins will become more or less similar to each other in their characteristics as they grow older. [2 ] Figure 4 epigntics Looking for patterns, trends and discrepancies: there is mounting evidence that the environment can trigger heritable changes in epigenetic factors. The chemical modifcations o chromatin that impact gene expression, including acetylation, methylation and phosphorylation o amino acid tails o histones (fgure 5 ) as well as methylation o D NA(fgure 6) , all have an impact on gene expression and thus impact the visible characteristics o an individual (fgure 7) . These chemical modifcations are called epigenetic tags. There is mounting evidence that the chemical modifcations that occur to the hereditary material in one generation might, in certain circumstances, be passed on to the next generation both at the cellular as well as whole organism level. The sum o all the epigenetic tags constitutes the epigenome. D ierent cells have their own methylation pattern so that a unique set o proteins will be produced in order or that cell to perorm its unction. D uring cell division, the methylation pattern will be passed over to the daughter cell. In other words, the environment is aecting inheritance. 358 Sperm and eggs develop rom cells with epigenetic tags. When two reproductive cells meet, the epigenome is erased through a process called reprogramming. Ac M P acetylation methylation phosphorylation Figure 5 Histone modifcations NH 2 NH 2 C N C O C H N C N C CH H Figure 6 DNA methylation O Me C N H CH 7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N About 1 % o the epigenome is not erased and survives yielding a result called imprinting. For example, when a mammalian mother has gestational diabetes, the high levels o glucose in the etal circulation trigger epigenetic changes in the daughters D NA such that she is predisposed to develop gestational diabetes hersel. transcription possible gene switched on active (open) chromatin unmethylated cytosines (white circles) acetylated histones gene switched o silent (condensed) chromatin methylated cytosines (red circles) deacetylated histones transcription prevented Figure 7 The diagram compares the chemical modifcations that prevent transcription with the chemical modifcations that allow transcription The direction o transcription Transcription occurs in a 5' to 3' direction. The synthesis o mRNA occurs in three stages: initiation, elongation and termination. Transcription begins near a site in the D NA called the promoter. O nce binding o the RNA polymerase occurs, the D NA is unwound by the RNA polymerase orming an open complex. The RNA polymerase slides along the D NA, synthesizing a single strand o RNA. base RNA growing strand OH C U A template strand OH DNA OH G OH OH OH A C T G G 3 end C 5 end sugar phosphate Figure 8 Post-transcriptional modifcation Eukaryotic cells modify mRNA after transcription. The regulation o gene expression can occur at several points. Transcription, translation and post-translational regulation occur in both eukaryotes and prokaryotes. However, most regulation o prokaryotic gene expression occurs at transcription. In addition, post-transcriptional modifcation o RNA is a method o gene expression that does not occur in prokaryotes. Figure 9 Coloured transmission electron micrograph o DNA transcription coupled with translation in the bacterium Escherichia coli. During transcription, complementary messenger ribonucleic acid (mRNA) strands ( green) are synthesized using DNA (pink) as a template and immediately translated by ribosomes (blue) 359 7 N U C L E I C AC I D S ( AH L ) a) O H N H H H OH HO O N H2N CH 2 O O H H P O O O P O O O P O O base CH 2 O H H O OH H N N O O CH 3 H P O O CH 2 5 7-methylguanosine cap exon b) intron exon 5 3 pre-mRNA spliceosome sn RNPs exon toK Hw d he crieria fr judgmen used change he cnclusins drawn frm he same daa? Estimates o the number o genes ound in the human genome fuctuated wildly in the time between 2000 and 2007. Reported as high as 120,000 in 2000, the current consensus view is that there are approximately 20,500. The reason or the uncertainty was due to the dierent criteria used or searching used by dierent gene-nding programs. Dening the criteria was problematic because: small genes are diicult to detect; 360 because o mRNA splicing, one gene can code or several protein products; some genes are nonprotein coding and two genes can overlap. exon 5 3 excised intron 5 3 mature mRNA c) 5 A A poly A tail consisting of 100200 adenine nucleotides is added after transcription. A A A A 3 poly A tail Figure 10 O ne o the most signifcant dierences between eukaryotes and prokaryotes is the absence o a nuclear membrane surrounding the genetic material in prokaryotes. The absence o a compartment in prokaryotes means that transcription and translation can be coupled. The separation o the location o transcription and translation into separate compartments in eukaryotes allows or signifcant post-transcriptional modifcation to occur beore the mature transcript exits the nucleus. An example would be the removal o intervening sequences, or introns, rom the RNA transcript. Prokaryotic DNA does not contain introns. In eukaryotes, the immediate product o mRNA transcription is reerred to as pre- mRNA, as it must go through several stages o posttranscriptional modifcation to become mature mRNA. O ne o these stages is called RNA splicing, shown in fgure 1 1 b. Interspersed throughout the mRNA are sequences that will not 3 7. 2 t r A N s c r i p t i o N A N D g e N e e X p r e s s i o N contribute to the ormation o the polypeptide. They are reerred to as intervening sequences, or introns. These introns must be removed. The remaining coding portions o the mRNA are called exons. These will be spliced together to orm the mature mRNA. Post-transcriptional modication also includes the addition o a 5 cap that usually occurs beore transcription has been completed (see gure 1 1 a) . A poly-A tail is added ater the transcript has been made (see gure 1 1 c) . mRNA splicing Splicing o mRNA increases the number o diferent proteins an organism can produce. Alternative splicing is a process during gene expression whereby a single gene codes or multiple proteins. This occurs in genes with multiple exons. A particular exon may or may not be included in the nal messenger RNA. As a result, the proteins translated rom alternatively spliced mRNAs will dier in their amino acid sequence and possibly in their biological unctions. In mammals, the protein tropomyosin is encoded by a gene that has eleven exons. Tropomyosin pre- mRNA is spliced dierently in dierent tissues resulting in ve dierent orms o the protein. For example, in skeletal muscle, exon 2 is missing rom the mRNA and in smooth muscle, exons 3 and 1 0 are not present. In ruit fies, the Dscam protein is involved in guiding growing nerve cells to their targets. Research has shown that there are potentially 3 8, 000 dierent mRNAs possible based on the number o dierent introns in the gene that could be spliced alternatively. 361 7 N U C L E I C AC I D S ( AH L ) 7.3 translaion Understanding Initiation o translation involves assembly o the components that carry out the process. Synthesis o the polypeptide involves a repeated cycle o events. Disassembly o the components ollows termination o translation. Free ribosomes synthesize proteins or use primarily within the cell. Bound ribosomes synthesize proteins primarily or secretion or or use in lysosomes. Translation can occur immediately ater transcription in prokaryotes due to the absence o a nuclear membrane. The sequence and number o amino acids in the polypeptide is the primary structure. The secondary structure is the ormation o alpha helices and beta pleated sheets stabilized by hydrogen bonding. The tertiary structure is the urther olding o the polypeptide stabilized by interactions between R groups. The quaternary structure exists in proteins with more than one polypeptide chain. Applications tRNA-activating enzymes illustrate enzyme- substrate specifcity and the role o phosphorylation. Skills The use o molecular visualization sotware to analyse the structure o eukaryotic ribosomes and a tRNA molecule. Identifcation o polysomes in an electron micrograph. Nature of science Developments in scientifc research ollow improvements in computing: the use o computers has enabled scientists to make advances in bioinormatics applications such as locating genes within genomes and identiying conserved sequences. The structure of the ribosome The use o molecular visualization sotware to analyse the structure o eukaryotic ribosomes and a tRNA molecule. Ribosome structure includes: 362 Proteins and ribosomal RNA molecules (rRNA) . Two sub-units, one large and one small. Three binding sites or tRNA on the surace o the ribosome. Two tRNA molecules can bind at the same time to the ribosome. There is a binding site or mRNA on the surace o the ribosome. E ach ribosome has three tRNA binding sites the E or exit site, the P or peptidyl site and the A or aminoacyl site ( see fgure 1 ) . The protein data bank ( PD B ) is a public database containing data regarding the threedimensional structure or a large number o biological molecules. In 2 000, structural biologists Venkatraman Ramakrishnan, Thomas A. S teitz and Ada E . Yonath made the frst data about 7. 3 t r A N s l At i o N position of growing polypeptide tRNA structure A C large sub-unit 5 binding sites for tRNA 3 C double stranded sections linked by base pairing site for attaching an amino acid loop of seven nucleotides small sub-unit 5 extra loop 3 position of mRNA loop of eight nucleotides Figure 1 ribosome subunits available through the PD B . In 2 009, they received a Nobel Prize or their work on the structure o ribosomes. Visit the protein databank to obtain images o the Thermus thermophilus ribosome ( images 1 j go and 1 giy) , or download these images rom the companion website to the textbook. Using Jmol, rotate the image to visualize the small sub-unit and the large sub- unit. In the image in fgure 2 , an mRNA molecule is coloured yellow. The pink, purple and blue areas in the image represent the three tRNA binding sites with tRNA molecules bound. anticodon loop anticodon Figure 3 a triplet o bases called the anticodon which is part o a loop o seven unpaired bases two other loops the base sequence C C A at the 3 ' end which orms a site or attaching an amino acid. Visit the PD B to obtain an image o a tRNA molecule or download the image rom the companion site to this book to explore the structure in a programme such as Jmol. Figure 4 shows such an image. The parts marked green represent the amino acid binding site and the anticodon. The part in purple shows a region o the molecule where a triplet o bases are hydrogen bonded. This is shown in the second image. Figure 2 The generalized structure o a tRNA molecule is shown in fgure 3 . All tRNA molecules have: sections that become double-stranded by base pairing, creating loops Figure 4 Whole view of a tRNA molecule with a close-up of a triplet of bases connected by hydrogen bonds 363 7 N U C L E I C AC I D S ( AH L ) tRNA-activating enzymes tRNA-activating enzymes illustrate enzyme-substrate specifcity and the role o phosphorylation. Each tRNA molecule is recognized by a tRNAactivating enzyme that attaches a specifc amino acid to the tRNA, using ATP or energy. The base sequence o tRNA molecules varies and this causes some variability in structure. Activation o a tRNA molecule involves the attachment o an amino acid to the 3' terminal o the tRNA by an enzyme called a tRNA-activating enzyme. There are twenty dierent tRNA-activating enzymes that are each specifc to one o the 2 0 amino acids and the correct tRNA molecule. The active site o the activating enzyme is specifc to both the correct amino acid and the correct tRNA. Energy rom ATP is needed or the attachment o amino acids. Once ATP and an amino acid are attached to the active site o the enzyme, the amino acid is activated by the ormation o a bond between the enzyme and adenosine monophosphate (AMP) . Then the activated amino acid is covalently attached to the tRNA. Energy rom this bond is later used to link the amino acid to the growing polypeptide chain during translation. tRNA ATP charged tRNA P P P amino acid P aminoacyl-tRNA synthetase P Pi pyrophosphate A specic amino acid and ATP bind to the enzyme The amino acid is a activated by the hydrolysis of ATP and covalent bonding of AMP P AMP The correct tRNA binds to the active site. The amino acid binds to the attachment site on the tRNA and AMP is released The activated tRNA is released Figure 5 Initiation of translation Met 3 U 5 A A C U G 5 3 To begin the process o translation, an mRNA molecule binds to the small ribosomal subunit at an mRNA binding site. An initiator tRNA molecule carrying methionine then binds at the start codon AUG . initiator tRNA 3 5 start codon mRNA binding site Figure 6 364 Initiation o translation involves assembly o the components that carry out the process. The large ribosomal subunit then binds to the small one. small ribosomal subunit The initiator tRNA is in the P site. The next codon signals another tRNA to bind. It occupies the A site. A peptide bond is ormed between the amino acids in the P and A site. 7. 3 t r A N s l At i o N P site Met E peptide bond forming large ribosomal subunit E A E 3 3 5 P A site site 5 P A Figure 8 Figure 7 Elongation of the polypeptide Synthesis of the polypeptide involves a repeated cycle of events. Following initiation, elongation occurs through a series of repeated steps. The ribosome translocates three bases along the mRNA, moving the tRNA in the P site to the E site, freeing it and allowing a tRNA with the appropriate anticodon to bind to the next codon and occupy the vacant A site. E E 3 P A 5 P A site site Figure 9 Termination of translation Disassembly of the components follows termination of translation. The process continues until a stop codon is reached when the free polypeptide is released. Note the direction of movement along the mRNA is from the 5 end to the 3 end. free polypeptide 3 5 3 5 stop codon (UAG, UAA, or UGA) Figure 10 365 7 N U C L E I C AC I D S ( AH L ) Free ribosomes toK Hw d wrds acquire heir meaning? Is a ribosome an organelle? Karl August Mbius is credited as the frst to establish the analogy between cellular substructures with defned unctions and the organs o the body. Early usage varied rom reerring only to the reproductive structures o protists, later ocusing on propulsion structures and later even including extracellular structures such as cell walls. The original defnition o an organelle as a subcellular unctional unit in general has emerged as the dominant defnition, and this would include ribosomes. A criterion in this case or defning an organelle is whether it can be isolated by a process known as cellular ractionation. Others limit the term to membrane-bound cell compartments and some cell biologists choose to limit the term even urther to those structures that originated rom endosymbiotic bacteria. Free ribosomes synthesize proteins or use primarily within the cell. In eukaryotes, proteins unction in a particular cellular compartment. Proteins are synthesized either in the cytoplasm or at the endoplasmic reticulum depending on the fnal destination o the protein. Translation occurs more commonly in the cytosol. Proteins destined or use in the cytoplasm, mitochondria and chloroplasts are synthesized by ribosomes ree in the cytoplasm. Bound ribosomes Bound ribosomes synthesize proteins primarily or secretion or or use in lysosomes. In eukaryotic cells, thousands o proteins are made. In many cases, proteins perorm a unction within a specifc compartment o the cell or they are secreted. Proteins must thereore be sorted so that they end up in their correct location. Proteins that are destined or use in the ER, the Golgi apparatus, lysosomes, the plasma membrane or outside the cell are synthesized by ribosomes bound to the ER. Whether the ribosome is ree in the cytosol or bound to the E R depends on the presence o a signal sequence on the polypeptide vesicle containing polypeptide ribosome mRNA signal sequence signal recognition protein (SRP) polypeptide SRP receptor lumen of ER Figure 11 366 ER membrane 7. 3 t r A N s l At i o N being translated. It is the frst part o the polypeptide translated. As the signal sequence is created it becomes bound to a signal recognition protein that stops the translation until it can bind to a receptor on the surace o the E R. O nce this happens, translation begins again with the polypeptide moving into the lumen o the E R as it is created. The coupling o transcription and translation in prokaryotes Translation can occur immediately ater transcription in prokaryotes due to the absence o a nuclear membrane. In eukaryotes, cellular unctions are compartmentalized whereas in prokaryotes they are not. O nce transcription is complete in eukaryotes, the transcript is modifed in several ways beore exiting the nucleus. Thus there is a delay between transcription and translation due to compartmentalization. In prokaryotes, as soon as the mRNA is transcribed, translation begins. Identifcation o polysomes Identifcation o polysomes in an electron micrograph. Polysomes are structures visible in an electron microscope. They appear as beads on a string. They represent multiple ribosomes attached to a single mRNA molecule. B ecause translation and transcription occur in the same compartment in prokaryotes, as soon as the mRNA is transcribed, translation begins. Thus, multiple polysomes are visible associated with one gene. In eukaryotes, polysomes occur in both the cytoplasm and next to the E R. Figure 12 Strings of polysomes attached to a DNA molecule in a prokaryote. The arrow designates where investigators believe RNA polymerase is sitting at, or near, the initiation site for a gene 367 7 N U C L E I C AC I D S ( AH L ) polypeptide ribosome mRNA Figure 13 The image shows multiple ribosomes translating a single mRNA molecule within the cytoplasm at the same time. The beginning of the mRNA is to the right (at the arrow) . The polypeptides being synthesized get longer and longer, the closer the end of the mRNA the ribosomes get Bioinformatics Developments in scientifc research ollow improvements in computing: the use o computers has enabled scientists to make advances in bioinormatics applications such as locating genes within genomes and identiying conserved sequences. B ioinormatics involves the use o computers to store and analyse the huge amounts o data being generated by the sequencing o genomes and the identication o gene and protein sequences. Such inormation is oten amassed in databases, or example, GenB ank ( a US -based database) , the D D B J ( D NA databank o Japan) or the nucleotide sequence database maintained by the E MB L ( the European Molecular B iology Laboratory) , which then become accessible to the global community including scientists and the general public. The unctions o conserved sequences are oten investigated in model organisms such as E. coli, yeast ( S. cerevisiae) , ruit fies ( D. melanogaster) , a soil roundworm C. elegans, thale cress A. thalania and mice M. musculus. These particular organisms are oten used because, along with humans, their entire genomes have been sequenced. Functions are oten discovered by knockout studies where the conserved gene is disrupted or altered and the impact on the organisms phenotype is observed. A scientist studying a particular genetic disorder in humans might identiy sequence similarities that exist in people with the disorder. They might then search or homologous sequences in other organisms. These sequences might have a common ancestral origin but have accumulated dierences over time due to random mutation. To carry out the search or a homologous nucleotide or amino acid sequence, the scientist would conduct a B LAS T search. The acronym stands or basic local alignment search tool. Sometimes the homologous sequences are identical or nearly identical across species. These are called conserved sequences. The act that they are conserved across species suggests they play a unctional role. 368 Figure 14 Examples of model organisms In addition to the B LAS T program, there are other sotware programs available. C lustalW can be used to align homologous sequences to search or changes. PhyloWin can be used to construct evolutionary trees based on sequence similarities. 7. 3 t r A N s l At i o N Primary structure The sequence and number of amino acids in the polypeptide is the primary structure. A chain of amino acids is called a polypeptide. Given that the 20 commonly occurring amino acids can be combined in any sequence, it should not be surprising that there is a huge diversity of proteins. The sequence of amino acids in a polypeptide is termed its primary structure. Daa-baed quen The hemoglobin molecule transports oxygen in the blood. It consists of 4 polypeptide chains. In human adults the molecule has two kinds of chains, alpha chains and beta chains, and there are two each. The alpha chain has 1 41 amino acid residues and the beta chain has 1 46 amino acid residues. The primary sequence of both polypeptides is shown below. The single residue in the beta chain marked in blue is the site of a mutation in sickle cell anemia. In the mutation, the glutamic acid is replaced by valine. alpha chain: 1 val * leu ser pro ala asp lys thr asn val lys ala ala trp gly lys val gly ala his ala gly glu tyr gly ala glu ala leu glu arg met phe leu ser phe pro thr thr lys thr tyr phe pro his phe * asp leu ser his gly ser ala * * * * * gln val lys gly his gly lys lys val ala asp ala leu thr asn ala val ala his val asp asp met pro asn ala leu ser ala leu ser asp leu his ala his lys leu arg val asp pro val asp phe lys leu leu ser his cys leu leu val thr leu ala ala his leu pro ala glu phe thr pro ala val his ala ser leu asp lys phe leu ala ser val ser thr val leu thr ser lys tyr arg 1 41 beta chain: 1 val his leu thr pro glu glu lys ser ala val thr ala leu trp gly lys val asn * * val asp glu val gly gly glu ala leu gly arg leu leu val val tyr pro trp thr gln arg phe phe glu ser phe gly asp leu ser thr pro asp ala val met gly asn pro lys val lys ala his gly lys lys val leu gly ala phe ser asp gly leu ala his leu asp asn leu lys gly thr phe ala thr leu ser glu leu his cys asp lys leu his val asp pro glu asn phe arg leu leu gly asn val leu val cys val leu ala his his phe gly lys glu phe thr pro pro val gln ala ala tyr gln lys val val ala gly val ala asp ala leu ala his lys tyr his 1 46 C ompare the primary structure of the two polypeptides. The asterix ( *) symbols indicates locations where sections of the amino acid sequence are missing to facilitate comparison. [4] Secondary structure The secondary structure is the formation of alpha helices and beta pleated sheets stabilized by hydrogen bonding. B ecause the chain of amino acids in a polypeptide has polar covalent bonds within its backbone, it tends to fold in such a way that hydrogen bonds form between the carboxyl ( C = O ) group of one residue and the amino group ( NH) group of an amino acid in another part of the chain. This results in the formation of patterns within the polypeptide called secondary structures. The - helix and the - pleated sheet are examples of secondary structures. 369 7 N U C L E I C AC I D S ( AH L ) (a) alpha helix H N C H N C HO N C O (b) beta pleated sheet O N O H C N O C C C H C C O C C H O N C N H C C N H hydrogen bond O C C N Figure 15 The structure of insulin showing three areas where the -helix can be seen. It also shows the quaternary structure of insulin, i.e. the relative positions of the two polypeptides N C C H C N O H O O H C CN C N C C N C C C C N N C C H O H H O O C H O CN O H C H O O C N C H H C N O O C N C H C H CN O O C C O Figure 16 Two examples of protein secondary structure Tertiary structure The tertiary structure is the further folding of the polypeptide stabilized by interactions between R groups. Tertiary structure reers to the overall three- dimensional shape o the protein ( fgure 1 8) . This shape is a consequence o the interaction o R- groups with one another and with the surrounding water medium. There are several dierent types o interaction. Positively charged R-groups will interact with negatively charged R-groups. Hydrophobic amino acids will orientate themselves toward the centre o the polypeptide to avoid contact with water, while hydrophilic amino acids will orientate themselves outward. Polar R- groups will orm hydrogen bonds with other polar R-groups. The R-group o the amino acid cysteine can orm a covalent bond with the R- group o another cysteine orming what is called a disulphide bridge. H3C H3C CH 2 hydrogen OH bond O OH C CH 2 hydrophobic interaction CH CH 3 CH 3 CH CH 2 S polypeptide backbone S CH 2 disulphide bridge O Figure 17 Collagenthe quaternary structure consists of three polypeptides wound together to fom a tough, rope-like protein 370 CH 2 CH 2 CH 2 CH 2 O NH 3 ionic bond C CH 2 Figure 18 R-group interactions contribute to tertiary structure 7. 3 t r A N s l At i o N Quartenary structure beta chain beta chain The quaternary structure exists in proteins with more than one polypeptide chain. Proteins can be ormed rom a single polypeptide chain or rom more than one polypeptide chain. Lysozyme is composed o a single chain, so lysozyme is both a polypeptide and a protein. Insulin is ormed rom two polypeptides, and hemoglobin is made up o our chains. Quaternary structure reers to the way polypeptides ft together when there is more than one chain. It also reers to the addition o non- polypeptide components. The quaternary structure o the hemoglobin molecule consists o our polypeptide chains and our heme groups. alpha chain heme alpha chain Figure 19 The biological activity o a protein is related to its primary, secondary, tertiary and quaternary structure. C ertain treatments such as exposure to high temperatures, or changes in pH can cause alterations in the structure o a protein and thereore disrupt its biological activity. When a protein has permanently lost its structure it is said to be denatured. The quaternary structure of hemoglobin in adults consists of four chains: two -chains and two -chains. Each subunit contains a molecule called a heme group Daa-baed quen or the changes in hemoglobin type during development and ater birth. [3 ] Hemoglobin is a protein composed o two pairs o globin subunits. During the process o development rom conception through to 6 months ater birth, human hemoglobin changes in composition. Adult hemoglobin consists o two alpha- and two betaglobin subunits. Four other polypeptides are ound during development: zeta, delta, epsilon and gamma. b) C ompare changes in the amount o the gamma- globin gene with beta- globin. [3 ] c) D etermine the composition o the hemoglobin at 1 0 weeks o gestation and at 6 months o age. [2 ] d) S tate the source o oxygen or the etus. [1 ] e) The dierent types o hemoglobin have dierent afnities or oxygen. Suggest reasons alpha-globin gamma-globin beta-globin delta-globin epsilon-globin zeta-globin % hemoglobin Figure 2 0 illustrates the changes in hemoglobin composition during gestation and ater birth in a human. a) S tate which two subunits are present in highest amounts early in gestation. [1 ] Key 50 40 30 20 10 0 10 20 30 Weeks of gestation 40 Birth 2 4 6 Month of age Figure 20 371 37 N U C L E I C AC I D S Questions 1 D ierent samples o bacteria were supplied with radioactive nucleoside triphosphates or a series o times ( 1 0, 3 0 or 60 seconds) . This was the pulse period. This was ollowed by adding a large excess o non-radioactive nucleoside triphosphates or a longer period o time. This is called the chase period. The appearance o radioactive nucleotides ( incorporated during the pulse) in parts o the product D NA give an indication o the process o converting intermediates to fnal products. 2 B A C D NA was isolated rom the bacterial cells, denatured ( separated into two strands by heat) and centriuged to separate molecules by size. The closer to the top o the centriuge tube, the smaller the molecule. D a) C ompare the sample that was pulsed or 1 0 seconds with the sample that was pulsed or 3 0 seconds. [2 ] E Figure 22 b) Explain why the sample that was pulsed or 3 0 seconds provides evidence or the presence o both a leading strand and many lagging strands. a) What part o the nucleotide is labelled A? [1 ] c) Explain why the sample that was pulsed or 60 seconds provides evidence or the activity o D NA ligase. Radioactivity cpm / 0.1 m 1 With reerence to Figure 2 2 , answer the ollowing questions. 6,000 b) What kind o bond orms between the structures labelled B ? [1 ] c) What kind o bond is indicated by label C ? [1 ] d) What sub-unit is indicated by label D ? [1 ] e) What sub-unit is indicated by label E? [1 ] 60 sec 3 5,000 4,000 Reer to fgure 2 3 when answering the ollowing questions. V CH 2 OH 30 sec I O H 3,000 IV 2,000 H H H OH II 1,000 10 sec OH H III 0 Figure 21 372 0 1 2 3 Distance from top Figure 23 a) S tate what molecule is represented. [1 ] b) S tate whether the molecule would be ound in D NA or RNA. [1 ] c) S tate the part o the molecule to which phosphates bind. [1 ] d) Identiy the part o the molecule that reers to the 3 end. [1 ] 8 M ETAB O LI SM , CE LL RE SPI RATI O N AN D PH O TO SYN TH E SI S ( AH L) CE LL B I O LO GY Introduction Life is sustained by a complex web of chemical reactions inside cells. These metabolic reactions are regulated in response to the needs of the cell and the organism. Energy is converted to a usable form in cell respiration. In photosynthesis light energy is converted into chemical energy and a huge diversity of carbon compounds is produced. 8.1 Metabolism Understanding Metabolic pathways consist o chains and cycles o enzyme-catalysed reactions. Enzymes lower the activation energy o the chemical reactions that they catalyse. Enzyme inhibitors can be competitive or non-competitive. Metabolic pathways can be controlled by end-product inhibition. Applications End-product inhibition o the pathway that converts threonine to isoleucine. Use o databases to identiy potential new anti-malarial drugs. Skills Distinguishing diferent types o inhibition rom graphs at specied substrate concentration. Calculating and plotting rates o reaction rom raw experimental results. Nature of science Developments in scientic research ollow improvements in computing: developments in bioinormatics, such as the interrogation o databases, have acilitated research into metabolic pathways. 373 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) initial TREAD substrate BREAD BREED BLEED intermediates BLEND BLIND BLINK end product Figure 1 Word game analogy for metabolic pathways Metabolic pathways Metabolic pathways consist of chains and cycles of enzyme-catalysed reactions. The word metabolism was introduced in the 1 9th century by the German cytologist and physiologist Theodor S chwann, to reer to the chemical changes that take place in living cells. It is now known that a huge range o chemical reactions occur in cells, catalysed by over 5 , 000 dierent types o enzyme. Although metabolism is very complex, there are some common patterns. 1 Most chemical changes happen not in one large j ump, but in a sequence o small steps, together orming what is called a metabolic pathway. The word game in fgure 1 is an analogy. 2 Most metabolic pathways involve a chain o reactions. Figure 2 shows a reaction chain that is used by cells to convert phenylalanine into umarate and acetoacetate, which can be used as energy sources in respiration. Phenylalanine causes severe health problems i there is an excess o it in the blood. 3 S ome metabolic pathways orm a cycle rather than a chain. In this type o pathway, the end product o one reaction is the reactant that starts the rest o the pathway. phenylalanine I tyrosine II hydroxyphenylpyruvate III homogentisate input: 3 CO 2 IV RuBP 4-maleylacetoacetate V 3 ADP 3-PGA Calvin cycle 3 ATP NADH + H + NAD + FADH 2 4-fumarylacetoacetate 5 G3P 6 G3P Figure 2 Example of a metabolic pathway output: 1 G3P Krebs cycle 6 ATP FAD 6 ADP + P 6 NADPH C 4 compound C 6 compound NADH + H + + NAD NAD + + NADH + H CO 2 C5 compound 6 NADP+ VI fumarate + acetoacetate acetyl group C 2 glucose and other compounds CO 2 Figure 3 Enzymes and activation energy Enzymes lower the activation energy of the chemical reactions that they catalyse. C hemical reactions are not single- step processes. S ubstrates have to pass through a transition state beore they are converted into products. E nergy is required to reach the transition state, and although energy is released in going rom the transition state to the product, some energy must be put in to reach the transition state. This is called the activation energy. The activation energy is used to break or weaken bonds in the substrates. Figure 4 shows these energy 374 8 . 1 M e Tab O li s M changes for an exergonic ( energy releasing) reaction that is and is not catalysed by an enzyme. (b) transition state energy activation energy transition state energy (a) substrate activation energy substrate product product progress of reaction progress of reaction Figure 4 Graphs showing activation energy (a) without an enzyme and (b) with an enzyme When an enzyme catalyses a reaction, the substrate binds to the active site and is altered to reach the transition state. It is then converted into the products, which separate from the active site. This binding lowers the overall energy level of the transition state. The activation energy of the reaction is therefore reduced. The net amount of energy released by the reaction is unchanged by the involvement of the enzyme. However as the activation energy is reduced, the rate of the reaction is greatly increased, typically by a factor of a million or more. Types of enzyme inhibitors Enzyme inhibitors can be competitive or non-competitive. Some chemical substances bind to enzymes and reduce the activity of the enzyme. They are therefore known as inhibitors. The two main types are competitive and non- competitive inhibitors. C ompetitive inhibitors interfere with the active site so that the substrate cannot bind. Non- competitive inhibitors bind at a location other than the active site. This results in a change of shape in the enzyme so that the enzyme cannot bind to the substrate. Table 1 shows examples of each type. substrate competitive inhibitor active site is blocked by competitor Figure 6 non-competitive inhibitor binding of inhibitor changes shape of active site no inhibition Figure 5 A molecular model o the restriction enzyme EcoRV (purple and pink) bound to a DNA molecule (deoxyribonucleic acid, yellow and orange) . Restriction enzymes, also known as restriction endonucleases, recognize specifc nucleotide sequences and cut the DNA at these sites. They are ound in bacteria and archaea and are thought to have evolved as a deence against viral inection TOK To wht xtnt houd thc contrn th dvopmnt of knowdg n cnc? Sarin was a chemical developed as an insectide beore being applied is a chemical weapon. It is a competitive inhibitor o the neurotransmitter acetylcholinesterase. Chemical weapons would not exist without the activities o scientists. In act, the name Sarin is an acronym o the surnames o the scientists who frst synthesized it. Fritz Haber received the 1918 Nobel Prize or Chemistry or his work in developing the chemistry behind the industrial production o ammonia ertilizer. Some scientists boycotted the award ceremony because Haber had been instrumental in encouraging and developing the use o chlorine gas in the First World War. Haber is quoted as saying: "During peace time a scientist belongs to the World, but during war time he belongs to his country." 375 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) enzym dihydropteroate synthetase sutrat inhtor para-aminobenzoate bndng suladiazine OC N SO 2 O N N H H 2N H 2N phosphoructokinase The inhibitor binds reversibly to the enzymes active site. While it remains bound, substrates cannot bind. This is competitive inhibition. ructose-6-phosphate xylitol-5-phosphate P OH OH CH 2 CH 2 C H C H C H O H2C C The inhibitor binds reversibly to a site away rom the active site. While it remains bound, the active site is distorted and substrate cannot bind. This is non-competitive inhibition. OH P CH 2 C H OH HO H OH OH C C OH H Table 1 Examples of each type of inhibitor Efects o enzyme inhibitors Distinguishing diferent types o inhibition rom graphs at specied substrate concentration. The orange line represents the effect of substrate concentration on enzyme activity in the absence of an inhibitor. The red line shows the effect of substrate concentration on the rate of reaction when a competitive inhibitor is present. When the concentration of substrate begins to exceed the amount of inhibitor, the maximum rate of the uninhibited enzyme can be achieved; however, it takes a much higher concentration of substrate to achieve this maximum rate. The blue line shows the effect of substrate concentration on the rate of reaction when a non- competitive inhibitor is present. In the presence of a non- competitive inhibitor, the enzyme does not reach the same maximum rate because the binding of the non- competitive 376 inhibitor prevents some of the enzymes from being able to react regardless of substrate concentration. Those enzymes that do not bind inhibitors follow the same pattern as the normal enzyme. It takes approximately the same concentration of enzyme to reach the maximum rate, but the maximum rate is lower than the uninhibited enzyme. maximum rate of reaction rate of reaction Figure 7 represents the effect of substrate concentration on the rate of an enzyme controlled reaction. normal enzyme competitive inhibitor non-competitive inhibitor substrate concentration Figure 7 8 . 1 M e Tab O li s M End-product inhibition Metabolic pathways can be controlled by end-product inhibition. Many enzymes are regulated by chemical substances that bind to special sites on the enzyme away rom the active site. These are called allosteric interactions and the binding site is called an allosteric site. In many cases, the enzyme that is regulated catalyses one o the rst reactions in a metabolic pathway and the substance that binds to the allosteric site is the end product o the pathway. The end product acts as an inhibitor. The pathway works rapidly in cells with a shortage o end product but can be switched o completely in cells where there is an excess. An example of end-product inhibition initial substrate (threonine) threonine in active site active site no longer binds to threonine enzyme 1 (threonine deaminase) intermediate A enzyme 2 isoleucine in allosteric site End-product inhibition o the pathway that converts threonine to isoleucine. Through a series o ve reactions, the amino acid threonine is converted to isoleucine. As the concentration o isoleucine builds up, it binds to the allosteric site o the rst enzyme in the chain, threonine deaminase, thus acting as a non- competitive inhibitor ( gure 8) . feedback inhibition To see why this is such an economical way to control metabolic pathways, we need to understand how the concentration o the product o a reaction can infuence the rate o reaction. Reactions oten do not go to completion instead an equilibrium position is reached with a characteristic ratio o substrates and products. So, i the concentration o products increases, a reaction will eventually slow down and stop. This eect reverberates back through a metabolic pathway when the end product accumulates, with all the intermediates accumulating. Endproduct inhibition prevents this build-up o intermediate products. intermediate B enzyme 3 intermediate C enzyme 4 intermediate D enzyme 5 end product (isoleucine) Figure 8 Investigating metabolism through bioinformatics Developments in scientifc research ollow improvements in computing: developments in bioinormatics, such as the interrogation o databases, have acilitated research into metabolic pathways. Computers have increased the capacity o scientists to organize, store, retrieve and analyse biological data. Bioinormatics is an approach whereby multiple research groups can add inormation to a database enabling other groups to query the database. O ne promising bioinormatics technique that has acilitated research into metabolic pathways is reerred to as chemogenomics. S ometimes when a chemical binds to a target site, it can signicantly alter metabolic activity. S cientists looking to develop new drugs test massive libraries o chemicals individually on a range o related organisms. For each organism a range o target sites are identied and a range o chemicals which are known to work on those sites are tested. O ne researcher called chemogenomics the chemical universe tested against the target universe . 377 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) Chemogenomics applied to malaria drugs Use of databases to identify potential new anti-malarial drugs. Malaria is a disease caused by the pathogen Plasmodium falciparum. The increasing resistance o P. falciparum to anti-malarial drugs such as chloroquine, the dependence o all new drug combinations on a narrow range o medicines and increasing global eorts to eradicate malaria all drive the need to develop new anti-malarial drugs. Plasmodium falciparum strain 3 D 7 is a variety o the malarial parasite or which the genome has been sequenced. In one study, approximately 3 1 0, 000 chemicals were screened against a chloroquine- sensitive 3 D 7 strain and the chloroquine-resistant K1 strain to see i these chemicals inhibited metabolism. O ther related and unrelated organisms, including human cell lines, were also screened. O ne promising outcome was the identifcation o 1 9 new chemicals that inhibit the enzymes normally targeted by anti-malarial drugs and 1 5 chemicals that bind to a total o 61 dierent malarial proteins. This provides other scientists with possible lines o investigation in the search or new anti-malarials. Calculating rates of reaction Calculating and plotting rates of reaction from raw experimental results. A large number o dierent protocols are available or investigating enzyme activity. D etermining the rate o an enzyme-controlled reaction involves measuring either the rate o disappearance o a substrate or the rate o appearance o a product. Sometimes this will require conversion o units to yield a rate unit which should include s - 1 . Data-basd qustions: The efectiveness o enzymes The degree to which enzymes increase the rate o reactions varies greatly. B y calculating the ratio between the rate o reactions with and without an enzyme catalyst, the afnity between an enzyme and its substrate can be estimated. Table 2 shows the rates o our reactions with and without an enzyme. The ratio between these rates has been calculated or one o the reactions. 1 S tate which enzyme catalyses the reaction with the slowest rate in the absence o an enzyme. [1 ] enzym State which enzyme catalyses its reaction at the most rapid rate. [1 ] 3 C alculate the ratios between the rate o reaction with and without an enzyme or ketosteroid isomerase, nuclease and O MP decarboxylase. [3 ] 4 D iscuss which o the enzymes is the more eective catalyst. [3 ] 5 Explain how the enzymes increase the rate o the reactions that they catalyse. Rat without nzym/s 1 Rat with nzym/s 1 Ratio btwn rat with and without nzym Carbonic anhydrase 1.3 10 1 1.0 10 6 7.7 10 6 Ketosteroid isomerase 1.7 10 7 6.4 10 4 Nuclease 1.7 10 13 9.5 10 6 OMP decarboxylase 2.8 10 16 3.9 10 8 Table 2 378 2 [2 ] 8 . 1 M e Tab O li s M oxygen/% Dt-d quton: Calculating rates of reaction 22.0 21.5 21.0 20.5 20.0 19.5 19.0 18.5 18.0 actvty For each o the ollowing enzyme experiments, describe how the rate o reaction can be determined: 0 10 51C 20 4C 30 40 time/s 21C 50 60 70 34C Figure 9 Percentage of oxygen concentration over time at various temperatures after adding catalase to a 1.5% hydrogen peroxide solution Ten drops o a commercial catalase solution were added to our reaction vessels containing a 1 .5 % hydrogen peroxide solution. Each o the solutions had been kept at a dierent temperature. The % oxygen in the reaction vessel was determined using a data logger in a set-up similar to fgure 1 0. Figure 10 1 E xplain the variation in the % oxygen at time zero. 2 Determine the rate o reaction at each temperature using the graph. 3 C onstruct a scatter plot o reaction rate versus temperature. ) Paper discs soaked in the enzyme catalase are added to diferent concentrations o hydrogen peroxide. The reaction produces oxygen bubbles. ) Lipase catalyses the breakdown o triglycerides to atty acids and water. The pH o the reaction solution will lower as the reaction proceeds. c) Papain is a protease that can be extracted rom pineapple ruits. Gelatin cubes will be digested by papain. d) Catechol oxidase converts catechol to a yellow pigment in cut ruit. It can be extracted rom bananas. The yellow pigment reacts with oxygen in the air to turn brown. 379 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) 8.2 Cell respiration Understanding Applications Cell respiration involves the oxidation and reduction o compounds. Phosphorylation o molecules makes them less stable. In glycolysis, glucose is converted to pyruvate. Glycolysis gives a small net gain o ATP without the use o oxygen. In aerobic cell respiration pyruvate is decarboxylated and oxidized. In the link reaction pyruvate is converted into acetyl coenzyme A. In the Krebs cycle, the oxidation o acetyl groups is coupled to the reduction o hydrogen carriers, liberating carbon dioxide. Energy released by oxidation reactions is carried to the cristae o the inner mitochondrial membrane by reduced NAD and FAD. Transer o electrons between carriers in the electron transport chain is coupled to proton pumping. In chemiosmosis protons difuse through ATP synthase to generate ATP. Oxygen is needed to bind with the ree protons to orm water to maintain the hydrogen gradient. The structure o the mitochondrion is adapted to the unction it perorms. Electron tomography used to produce images o active mitochondria. Skills Analysis o diagrams o the pathways o aerobic respiration to deduce where decarboxylation and oxidation reactions occur. Annotation o a diagram to indicate the adaptations o a mitochondrion to its unction. Nature of science Paradigm shits: the chemiosmotic theory led to a paradigm shit in the eld o bioenergetics. Oxidation and reduction Cell respiration involves the oxidation and reduction o compounds. O xidation and reduction are chemical processes that always occur together. This happens because they involve transfer of electrons from one substance to another. O xidation is the loss of electrons from a substance and reduction is the gain of electrons. A useful example to help visualize this in the laboratory is in the B enedicts test, a test for certain types of sugar. The test involves the 380 8 . 2 C e l l R e s p i R aT i O n use o copper sulphate solution, containing copper ions with a charge o two positive ( C u 2 + ) . C u 2+ oten imparts a blue or green colour to solutions. These copper ions are reduced and become atoms o copper by being given electrons. C opper atoms are insoluble and orm a red or orange precipitate. The electrons come rom sugar molecules, which are thereore oxidized. Electron carriers are substances that can accept and give up electrons as required. They oten link oxidations and reductions in cells. The main electron carrier in respiration is NAD ( nicotinamide adenine dinucleotide) . In photosynthesis a phosphorylated version o NAD is used, NAD P ( nicotinamide adenine dinucleotide phosphate) . The structure o the NAD molecule is shown in fgure 1 . adenine base ribose sugar phosphates ribose sugar The equation below shows the basic reaction. NAD + 2 electrons reduced NAD The chemical details are a little more complicated. NAD initially has one positive charge and exists as NAD + . It accepts two electrons in the ollowing way: two hydrogen atoms are removed rom the substance that is being reduced. O ne o the hydrogen atoms is split into a proton and an electron. The NAD + accepts the electron, and the proton ( H + ) is released. The NAD accepts both the electron and proton o the other hydrogen atom. The reaction can be shown in two ways: nicotinamide base Figure 1 Structure of NAD NAD + + 2 H + + 2 electrons ( 2e ) NAD H + H + NAD + + 2 H NAD H + H + This reaction demonstrates that reduction can be achieved by accepting atoms o hydrogen, because they have an electron. O xidation can thereore be achieved by losing hydrogen atoms. O xidation and reduction can also occur through loss or gain o atoms o oxygen. There are ewer examples o this in biochemical processes, perhaps because in the early evolution o lie oxygen was absent rom the atmosphere. A ew types o bacteria can oxidize hydrocarbons using oxygen: 1 O C H C H O H C 7 H 1 5 C H 3 + _ 7 15 2 2 2 n- octane n- octanol Nitriying bacteria oxidize nitrite ions to nitrate. 1 O NO NO -2 + _ 3 2 2 Adding oxygen atoms to a molecule or ion is oxidation, because the oxygen atoms have a high afnity or electrons and so tend to draw them away rom other parts o the molecule or ion. In a similar way, losing oxygen atoms is reduction. Phosphorylation Phosphorylation of molecules makes them less stable. Phosphorylation is the addition o a phosphate molecule ( PO 3) to 4 an organic molecule. B iochemists indicate that certain amino acid sequences tend to act as binding sites or the phosphate molecule on proteins. For many reactions, the purpose o phosphorylation is to make 381 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) the phosphorylated molecule more unstable; i.e., more likely to react. Phosphorylation can be said to activate the molecule. The hydrolysis o ATP releases energy to the environment and is thereore termed an exergonic reaction. Many chemical reactions in the body are endergonic ( energy absorbing) and thereore do not proceed spontaneously unless coupled with an exergonic reaction that releases more energy. For example, depicted below is the frst reaction in the series o reactions known as glycolysis. Glucose-6- phosphate Glucose ATP AD P The conversion o glucose to glucose-6- phosphate is endergonic and the hydrolysis o ATP is exergonic. B ecause the reactions are coupled, the combined reaction proceeds spontaneously. Many metabolic reactions are coupled to the hydrolysis o ATP. Glycolysis and ATP Glycolysis gives a small net gain of ATP without the use of oxygen. The most signifcant consequence o glycolysis is the production o a small yield o ATP without the use o any oxygen, by converting sugar into pyruvate. This cannot be done as a single- step process and instead is an example o a metabolic pathway, composed o many small steps. The frst o these may seem rather perverse: ATP is used up in phosphorylating sugar. Glucose ATP AD P Glucose- 6- phosphate Fructose6phosphate Fructose- 1 , 6- bisphosphate ATP AD P However, these phosphorylation reactions reduce the activation energy required or the reactions that ollow and so make them much more likely to occur. Pyruvate is a product of glycolysis In glycolysis, glucose is converted to pyruvate. In the next step, the ructose bisphosphate is split to orm two molecules o triose phosphate. E ach o these triose phosphates is then oxidized to glycerate- 3 - phosphate in a reaction that yields enough energy to make ATP. This oxidation is carried out by removing hydrogen. Note that it is hydrogen atoms that are removed. I only hydrogen ions were removed ( H + ) , no electrons would be removed and it would not be an oxidation. The hydrogen is accepted by NAD + , which becomes NAD H + H + . In the fnal stages o glycolysis, the phosphate group is transerred to AD P to produce more ATP and also pyruvate. These stages are summarized in the equation below, which occurs twice per glucose. 382 8 . 2 C e l l R e s p i R aT i O n NAD + NAD H + H + triose phosphate glycerate- 3 - phosphate The fate of pyruvate Glucose In aerobic cell respiration pyruvate is decarboxylated and oxidized. reduced NAD pyruvate ATP Pyruvate Two molecules o pyruvate are produced in glycolysis per molecule o glucose. I oxygen is available, this pyruvate is absorbed into the mitochondrion, where it is ully oxidized. 2 C H 3 C O C O O H + 5 O 2 6C O 2 + 4H 2 O Glycolysis reduced NAD Link reaction Acetyl CoA reduced FAD Electron transport Oxidative reduced phosphorylation NAD Chemiosmosis Krebs cycle ATP As with glycolysis, this is not a single-step process. C arbon and oxygen are removed in the orm o carbon dioxide, in reactions called decarboxylations. The oxidation o pyruvate is achieved by the removal o pairs ATP o hydrogen atoms. The hydrogen carrier NAD + , and a Figure 2 A summary of aerobic respiration related compound called FAD , accept these hydrogen atoms and pass them on to the electron transport chain where oxidative phosphorylation will occur. These reactions are summarized in fgure 2 . O The link reaction In the link reaction pyruvate is converted into acetyl coenzyme A. In the Krebs cycle, the oxidation of acetyl groups is coupled to the reduction of hydrogen carriers. This cycle has several names but is oten called the Krebs cycle, in honour o the biochemist who was awarded the Nobel Prize or its discovery. The link reaction involves one decarboxylation and one oxidation. There are two more decarboxylations and our more oxidations in the Krebs cycle. I glucose as oxidized by burning in air, energy would be released as heat. Most o the energy released in the oxidations o the link reaction and the Krebs cycle is used to reduce hydrogen carriers ( NAD + and FAD ) . O S CoA C O C O CH 3 CO 2 NAD + reduced NAD CH 3 Figure 3 The link reaction The frst step, represented by fgure 3 , occurs ater the pyruvate, which has been produced in the cytoplasm, is shuttled into the mitochondrial matrix. O nce there, the pyruvate is decarboxylated and oxidized to orm an acetyl group. Two high energy electrons are removed rom pyruvate. These react with NAD + to produce reduced NAD . This is called the link reaction, because it links glycolysis with the cycle o reactions that ollow. The Krebs cycle CoA-SH C CO 2 pyruvic acid NAD + reduced NAD acetyl-CoA citric acid (6C) CoA OAA (4C) reduced NAD NAD + reduced NAD CO 2 NAD + CO 2 NAD + reduced NAD FADH 2 FAD ATP ADP+ i P Figure 4 Summary of the Krebs cycle 383 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) TOK What kinds o explanations do scientists ofer, and how do these explanations compare with those ofered in other areas o knowledge? Hans Krebs was awarded the Nobel Prize in 1953. The two nal paragraphs o the lecture that he gave on this occasion are reproduced here. The reactions o the cycle have been ound to occur in representatives o all orms o lie, rom unicellular bacteria and protozoa to the highest mammals. The study o intermediary metabolism shows that the basic metabolic processes, in particular those providing energy and those leading to the synthesis o cell constituents, are also shared by all orms o lie. The existence o common eatures in dierent orms o lie indicates some relationship between the dierent organisms, and according to the concept o evolution these relations stem rom the circumstance that the higher organisms, in the course o millions o years, have gradually evolved rom simpler ones. The concept o evolution postulates that living organisms have common roots, and in turn the existence o common eatures is powerul support or the concept o evolution. The presence o the same mechanism o energy production in all orms o lie suggests two other inerences: frstly that the mechanism o energy production has arisen very early in the evolutionary process; and secondly that lie, in its present orms, has arisen only once. 1 Outline the argument or similarities o metabolism as evidence or evolution. 2 Are there any alternative explanations or the similarities? 384 The energy thereore remains in chemical orm and can be passed on to the nal part o aerobic cell respiration: oxidative phosphorylation. For every turn o the cycle, the production o reduced NAD occurs three times, decarboxylation occurs twice and the reduction o FAD occurs once. O ne molecule o ATP is also generated. Oxidative phosphorylation Energy released by oxidation reactions is carried to the cristae o the mitochondria by reduced NAD and FAD. In aerobic respiration, there are several points where energy released by oxidation reactions is coupled to the reduction o mainly NAD but also FAD . Reduced NAD is produced during glycolysis, the link reaction and the Krebs cycle. FAD H 2 is produced during the Krebs cycle. The nal part o aerobic respiration is called oxidative phosphorylation, because AD P is phosphorylated to produce ATP, using energy released by oxidation. The substances oxidized include the FAD H 2 generated in the Krebs cycle and the reduced NAD generated in glycolysis, the link reaction and the Krebs cycle. Thus these molecules are used to carry the energy released in these stages to the mitochondrial cristae. The electron transport chain Transer o electrons between carriers in the electron transport chain is coupled to proton pumping. The nal part o aerobic respiration is called oxidative phosphorylation, because AD P is phosphorylated to produce ATP, using energy released by oxidation. The main substance oxidized is reduced NAD . The energy is not released in a single large step, but in a series o small steps, carried out by a chain o electron carriers. Reduced NAD and FAD H 2 donate their electrons to electron carriers. As the electrons are passed rom carrier to carrier, energy is utilized to transer protons across the inner membrane rom the matrix into the intermembrane space. The protons then fow through ATP synthase down their concentration gradient providing the energy needed to make ATP. Chemiosmosis In chemiosmosis protons difuse through ATP synthase to generate ATP. The mechanism used to couple the release o energy by oxidation to ATP production remained a mystery or many years, but is now known to be chemiosmosis. This happens in the inner mitochondrion membrane. It is called chemiosmosis because a chemical substance ( H + ) moves across a membrane, down the concentration gradient. This releases the energy needed or the enzyme ATP synthase to make ATP. The main steps in the process are as ollows ( also see gure 5 ) . 8 . 2 C e l l R e s p i R aT i O n NAD H + H + supplies pairs o hydrogen atoms to the rst carrier in the chain, with the NAD + returning to the matrix. The hydrogen atoms are split, to release two electrons, which pass rom carrier to carrier in the chain. Energy is released as the electrons pass rom carrier to carrier, and three o these use this energy to transer protons (H + ) across the inner mitochondrial membrane, rom the matrix to the intermembrane space. As electrons continue to fow along the chain and more and more protons are pumped across the inner mitochondrial membrane, a concentration gradient o protons builds up. This proton gradient is a store o potential energy. To allow electrons to continue to fow, they must be transerred to a terminal electron acceptor at the end o the chain. In aerobic respiration this is oxygen, which briefy becomes O 2 , but then combines with two H + ions rom the matrix to become water. Protons pass back rom the intermembrane space to the matrix through ATP synthase. As they are moving down the concentration gradient, energy is released and this is used by ATP synthase to phosphorylate AD P. inter inner mitochondrial membrane space membrane matrix NADH + H + H+ NAD + 2e - FADH 2 H+ FAD H 2O H+ H+ 2H + O2O2 H+ ATP ADP +Pi low H + concentration The role of oxygen Oxygen is needed to bind with the free protons to form water to maintain the hydrogen gradient. H+ high H + concentration Figure 5 Summary of oxidative phosphorylation O xygen is the nal electron acceptor in the mitochondrial electron transport chain. The reduction o the oxygen molecule involves both accepting electrons and orming a covalent bond with hydrogen. B y using up hydrogen, the proton gradient across the inner mitochondrial membrane is maintained so that chemiosmosis can continue. Dt-bd quto: Oxygen consumption by mitochondria Figure 6 shows the results o an experiment in which mitochondria were extracted rom liver cells and were kept in a fuid medium, in which oxygen levels were monitored. Pyruvate was added at point I on the graph, and AD P was added at points II, III and IV. 1 Explain why oxygen consumption by the mitochondria could not begin unless pyruvate had been added. [3 ] 2 D educe what prevented oxygen consumption between points I and II. [2 ] Predict, with reasons, what would have happened i AD P had not been added at point III. [2 ] D iscuss the possible reasons or oxygen consumption not being resumed ater AD P was added at point IV. [3 ] oxygen saturation / % 3 I II 100 III 4 50 IV 0 time Figure 6 Results of oxygen consumption experiment 385 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) The chemiosmotic theory Paradigm shits: the chemiosmotic theory produced a paradigm shit in the feld o bioenergetics. In 1 961 Peter Mitchell proposed the chemiosmotic hypothesis to explain the coupling o electron transport in the inner mitochondrial membrane to ATP synthesis. His hypothesis was a radical departure rom previous hypotheses and only ater many years was it generally accepted. He was awarded the Nobel Prize or Chemistry in 1 978 and part o the Banquet Speech that he gave is reproduced here: Emile Zola described a work o art as a corner o nature seen through a temperament. The philosopher Karl Popper, the economist F.A. Hayek and the art historian K.H. Gombrich have shown that the creative process in science and art consists o two main activities: an imaginative jumping orward to a new abstraction or simplifed representation, ollowed by a critical looking back to see how nature appears in the light o the new vision. The imaginative leap orward is a hazardous, unreasonable activity. Reason can be used only when looking critically back. Moreover, in the experimental sciences, the scientifc raternity must test a new theory The fnal outcome cannot be known, either to the originator o a new theory, or to his colleagues and critics, who are bent on alsiying it. Thus, the scientifc innovator may eel all the more lonely and uncertain. On the other hand, aced with a new theory, the members o the scientifc establishment are oten more vulnerable than the lonely innovator. For, i the innovator should happen to be right, the ensuing upheaval o the established order may be very painul and uncongenial to those who have long committed themselves to develop and serve it. Such, I believe, has been the case in the feld o knowledge with which my work has been involved. Naturally I have been deeply moved, and not a little astonished, by the accidents o ortune that have brought me to this point. Structure and function in the mitochondrion Examine fgure 7 showing an electron micrograph o a mitochondrion and a drawing representing that mitochondrion. The structure o the mitochondrion is adapted to the unction it perorms. The mitochondrion is a semi- autonomous organelle in that it can grow and reproduce itsel but it still depends on the rest o the cell or resources and is otherwise part o the cellular system. 70S ribosomes and a naked loop o D NA are ound within the mitochondrial matrix. There is oten a clear relationship between the structures o the parts o living organisms and the unctions they perorm. This can be explained in terms o natural selection and evolution. The mitochondrion can be used as an example. I mitochondrial structure varied, those organisms with the mitochondria that produced ATP most efciently would have an advantage. They would have an increased chance o survival and would tend to produce more ospring. These ospring would inherit the type o mitochondria that produce ATP more efciently. I this trend continued, the structure o mitochondria would gradually evolve to become more and more efcient. This is called adaptation a change in structure so that something carries out its unction more efciently. 386 to destruction, i possible. Meanwhile, the creator o a theory may have a very lonely time, especially i his colleagues fnd his views o nature unamiliar and difcult to appreciate. The mitochondrion is the site o aerobic respiration. The outer mitochondrial membrane separates the contents o the mitochondrion rom the rest o the cell creating a compartment specialized or the biochemical reactions o aerobic respiration. The inner mitochondrial membrane is the site o oxidative phosphorylation. It contains electron transport chains and ATP synthase, which carry out oxidative phosphorylation. Cristae are tubular projections o the inner membrane which increase the surace area available or oxidative phosphorylation. The intermembrane space is the location where protons build up as a consequence o the electron 8 . 2 C e l l R e s p i R aT i O n transport chain. The proton build-up is used to produce ATP via the ATP synthase. The volume o the space is small, so a concentration gradient across the inner membrane can be built up rapidly. The matrix is the site o the Krebs cycle and the link reaction. The matrix fuid contains the enzymes necessary to support these reaction systems. Annotating a diagram of a mitochondrion Annotation o a diagram to indicate the adaptations o a mitochondrion to its unction. Outer mitochondrial membrane separates the contents of the mitochondrion Matrix from the rest of the cell, creating a cellular contains enzymes for the Krebs cycle and the link reaction compartment with ideal conditions for Intermembrane space aerobic respiration Proteins are pumped Inner mitochondrial into this space by the membrane contains electron transport chain. electron transport The space is small so the chanins and ATP synthase concentration builds up quickly Cristae are projections of the inner membrane Ribosome DNA which increase the surface area available for for expression of oxidative phosphorylation mitochondrial genes Figure 7 actvty 0.1m a) b) d) c) Figure 8 Electron micrographs of mitochondria: (a) from a bean plant (b) from mouse liver (c) from axolotl sperm (d) from bat pancreas Study the electron micrographs in gure 8 and then answer the multiple-choice questions. 1 The fuid-lled centre o the mitochondrion is called the matrix. What separates the matrix rom the cytoplasm around the mitochondrion? 80S ribosomes. Which o these hypotheses is consistent with this observation? (i) Protein is synthesized in the mitochondrion. (ii) Ribosomes in mitochondria have evolved rom ribosomes in bacteria. ) One wall. c) Two membranes. (iii) Ribosomes are produced by aerobic cell respiration. b) One membrane. d) One wall and one membrane. ) (i) only c) (i) and (ii) b) (ii) only d) (i) , (ii) and (iii) 2 The mitochondrion matrix contains 70S ribosomes, whereas the cytoplasm o eukaryotic cells contains 387 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) Mitochondrial membranes are dynamic Electron tomography used to produce images o active mitochondria. Ideas in science sometimes change gradually. B ut sometimes they remain stable or years or even decades and then undergo a sudden change. This can be due to the insight or enthusiasm o a particular scientist, or team. The development o new techniques can sometimes be the stimulus. The technique o electron tomography has recently allowed three-dimensional images o the interior o mitochondria to be made. O ne o the leaders in this feld is D r. C armen Mannella, ormer D irector, D ivision o Molecular Medicine, Wadsworth C enter, Albany NY: Resource or Visualization o B iological C omplexity. He recently gave this brie comment on developments in our understanding o mitochondrial structure and unction. The new take-home message about the mitochondrial inner membrane is that the cristae are not simple inoldings but are invaginations, dening micro-compartments in the organelle. The cristae originate at narrow openings (crista junctions) that likely restrict diusion o proteins and metabolites between the compartments. The membranes are not only very fexible but also dynamic, undergoing usion and ssion in response to changes in metabolism and physiological stimuli. The working hypothesis is that the observed changes in membrane shape (topology) are not random and passive but rather a specic mechanism by which mitochondrial unction is regulated by changes in internal diusion pathways, e.g., allowing more ecient utilization o ADP. It appears that there are specic proteins and lipids that actively regulate the topology o the inner membrane. This is a bit speculative at the time but it gives a sense o where things are headed in the eld. Figure 9 Three images of the inner mitochondrial membrane of mitochondria from liver cells show the dynamic nature of this membrane TOK There are some scientic elds that depend entirely upon technology or their existence, or example, spectroscopy, radio or X-ray astronomy. What are the knowledge implications o this? Could there be problems o knowledge that are unknown now, because the technology needed to reveal them does not exist yet? 388 activity Answer the ollowing questions with respect to the three images in gure 9 . ) The diameter o the mitochondrion was 700 nm. Calculate the magnication o the image. [3] b) Electron tomography has shown that cristae are dynamic structures and that the volume o the intracristal compartment increases when the mitochondrion is active in electron transport. Suggest how electron transport could cause an increase in the volume o fuid inside the cristae. [2] c) Junctions between the cristae and boundary region o the inner mitochondrial membrane can have the shape o slots or tubes and can be narrow or wide. Suggest how narrow tubular connections could help in ATP synthesis by one o the cristae in a mitochondrion. [2] 8 . 3 ph O TO s yn Th e s i s 8.3 potot Understanding Light-dependent reactions take place in the intermembrane space o the thylakoids. Reduced NADP and ATP are produced in the light-dependent reactions. Light-independent reactions take place in the stroma. Absorption o light by photosystems generates excited electrons. Photolysis o water generates electrons or use in the light-dependent reactions. Transer o excited electrons occurs between carriers in thylakoid membranes. Excited electrons rom Photosystem II are used to generate a proton gradient. ATP synthase in thylakoids generates ATP using the proton gradient. Excited electrons rom Photosystem I are used to reduce NADP. In the light-independent reactions a carboxylase catalyses the carboxylation o ribulose bisphosphate. Glycerate 3-phosphate is reduced to triose phosphate using reduced NADP and ATP. Triose phosphate is used to regenerate RuBP and produce carbohydrates. Ribulose bisphosphate is reormed using ATP. The structure o the chloroplast is adapted to its unction in photosynthesis. Applications Calvins experiment to elucidate the carboxylation o RuBP. Skills Annotation o a diagram to indicate the adaptations o a chloroplast to its unction. Nature of science Developments in scientifc research ollow improvements in apparatus: sources o 1 4 C and autoradiography enabled Calvin to elucidate the pathways o carbon fxation. Location of light-dependent reactions Light-dependent reactions take place in the intermembrane space o the thylakoids. Research into photosynthesis has shown that it consists of two very different parts, one of which uses light directly ( light- dependent reactions) and the other does not use light directly ( light-independent 389 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) reactions) . The light- independent reactions can only carry on in darkness or a ew seconds because they depend on substances produced by the light-dependent reactions which rapidly run out. The chloroplast has an outer membrane and an inner membrane. The inner membrane encloses a third system o interconnected membranes called the thylakoid membranes. Within the thylakoid is a compartment called the thylakoid space. The light-dependent reactions take place in the thylakoid space and across the thylakoid membranes. Data-based questions: Freeze-fracture images of chloroplasts I chloroplasts are rozen rapidly in liquid nitrogen and then split, they racture across planes o weakness. These planes o weakness are usually the centres o membranes, between the two layers o phospholipid, where there are no hydrogen bonds attracting water molecules to each other. Structures within the membrane such as the photosystems are then visible in electron micrographs ( see fgure 1 ) . 1 2 3 D escribe the evidence, visible in the electron micrograph, or chloroplasts having many layers o membrane. [2 ] Explain how photosystems become visible as lumps in reeze- racture electron micrographs o chloroplasts. [2 ] S ome membranes contain large particles arranged in rectangular arrays. These are Photosystem II. They have a diameter o 1 8 nm. C alculate the magnifcation o the electron micrograph. [3 ] 4 Other membranes visible in the electron micrograph contain a variety o other structures. Use the inormation on the ollowing pages to deduce what these are. [3] Figure 1 Freeze-fracture electron micrograph of spinach chloroplast The products of the light-dependent reactions Reduced NADP and ATP are produced in the light-dependent reactions. Light energy is converted into chemical energy in the orm o ATP and reduced NAD P in the light reacations. The ATP and reduced NAD P serve as energy sources or the light- independent reactions. The location of the light-independent reactions Light-independent reactions take place in the stroma. The inner membrane o the chloroplast encloses a compartment called the stroma. This is a thick protein-rich medium containing enzymes or use in the light-independent reactions, also known as the C alvin 390 8 . 3 ph O TO s yn Th e s i s cycle. In the light-independent reactions the C alvin cycle is an anabolic pathway that requires endergonic reactions to be coupled to the hydrolysis o ATP and the oxidation o reduced NAD P. Figure 2 summarizes the processes o both the light- dependent and lightindependent reactions. outer membrane of chloroplast inner membrane of chloroplast CO 2 thylakoid membrane light energy thylakoid space P1 + ADP ATP Calvin cycle NADP NADPH + H + sugars 2e H 2O light-independent reactions - photolysis - photoactivation - electron transport - chemiosmosis - ATP synthesis - reduction of NADP 2H + + 1 2 O2 light-independent reactions - carbon xation - carboxylation of RuBP - production of triose phosphate - ATP and NADPH as energy sources - ATP used to regenerate RuBP - ATP used to produce carbohydrates Figure 2 Photoactivation Absorption of light by photosystems generates excited electrons. C hlorophyll and the accessory pigments are grouped together in large light- harvesting arrays called photosystems. These photosystems are located in the thylakoids, an arrangement o membranes inside the chloroplast. There are two types o light- harvesting arrays, called Photosystems I and II. In addition to light- harvesting arrays, the photosystems have reaction centres ( fgure 3 ) . B oth types o photosystem contain many chlorophyll molecules, which absorb light energy and pass it to two special chlorophyll molecules in the reaction centre o the photosystem. Like other chlorophylls, when these special chlorophyll molecules absorb the energy rom a photon o light an electron within the molecule becomes excited. The chlorophyll is then p hotoactivated. The chlorophylls at the reaction centre have the special property o being able to donate excited electrons to an electron acceptor. 391 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) Photosystem II light light harvesting array Rather conusingly, Photosystem II, rather than Photosystem I, is where the light- dependent reactions o photosynthesis begin. The electron acceptor or this photosystem is called plastoquinone. It collects two excited electrons rom Photosystem II and then moves away to another position in the membrane. Plastoquinone is hydrophobic, so although it is not in a xed position, it remains within the membrane. reaction centre primary acceptor e- plastoquinone Absorption o two photons o light causes the production transfer of electrons pigment molecules chlorophyll molecules that transfer electrons Figure 3 Diagram showing the relationship between the light-harvesting array, the reaction centre and plastoquinone o one reduced plastoquinone, with one o the chlorophylls at the reaction centre having lost two electrons to a plastoquinone molecule. Photosystem II can repeat this process, to produce a second reduced plastoquinone, so the chlorophyll at the reaction centre has lost our electrons and two plastoquinone molecules have been reduced. Photolysis Photolysis of water generates electrons for use in the light-dependent reactions. O nce the plastoquinone becomes reduced, the chlorophyll in the reaction centre is then a powerul oxidizing agent and causes the water molecules nearest to it to split and give up electrons, to replace those that it has lost: 2 H 2 O O 2 + 4H + + 4e The splitting o water, called photolysis, is how oxygen is generated in photosynthesis. O xygen is a waste product and diuses away. The useul product o Photosystem II is the reduced plastoquinone, which not only carries a pair o electrons, but also much o the energy absorbed rom light. This energy drives all the subsequent reactions o photosynthesis. The electron transport chain Transfer of excited electrons occurs between carriers in thylakoid membranes. The production o ATP, using energy derived rom light is called photophosphorylation. It is carried out by the thylakoids. These are regular stacks o membranes, with very small fuid-lled spaces inside ( see gure 4) . The thylakoid membranes contain the ollowing structures: Figure 4 Electron micrograph of thylakoids 75,000 392 Photosystem II ATP synthase a chain o electron carriers Photosystem I. Reduced plastoquinone is needed, carrying the pair o excited electrons rom the reaction centre o Photosystem II. Plastoquinone carries the electrons to the start o the chain o electron carriers. 8 . 3 ph O TO s yn Th e s i s The proton gradient Excited electrons from Photosystem II are used to generate a proton gradient. O nce plastoquinone transers its electrons, the electrons are then passed rom carrier to carrier in this chain. As the electrons pass, energy is released, which is used to pump protons across the thylakoid membrane, into the space inside the thylakoids. A concentration gradient o protons develops across the thylakoid membrane, which is a store o potential energy. Photolysis, which takes place in the fuid inside the thylakoids, also contributes to the proton gradient. stroma (low H + concentration) Photosystem II light 2 H+ cytochrome complex light NADP+ reductase Photosystem I Fd NADP+ + H + NADPH Pq H2O thylakoid space (high H + concentration) 1 2 O2 +2 H + Pc 2 H+ to Calvin cycle stroma (low H + concentration) thylakoid membrane ATP synthase ADP + P1 ATP H+ Figure 5 Chemiosmosis ATP synthase in thylakoids generates ATP using the proton gradient. The protons can travel back across the membrane, down the concentration gradient, by passing through the enzyme ATP synthase. The energy released by the passage o protons down their concentration gradient is used to make ATP rom AD P and inorganic phosphate. This method o producing ATP is strikingly similar to the process that occurs inside the mitochondrion and is given the same name: chemiosmosis. When the electrons reach the end o the chain o carriers they are passed to plastocyanin, a water-soluble electron acceptor in the fuid inside the thylakoids. Reduced plastocyanin is needed in the next stage o photosynthesis. 393 8 M E TAB O LI S M , C E LL R E S P I R AT I O N AN D P H O TO S YN T H E S I S ( AH L ) Data-based questions: Evidence for chemiosmosis One o the rst experiments to give evidence or ATP production by chemiosmosis was perormed in the summer o 1 966 by Andr Jagendor. Thylakoids were incubated or several hours in darkness, in acids with a pH ranging rom 3.8 to 5.2. The lower the pH o an acid, the higher its concentration o protons. During the incubation, protons diused into the space inside the thylakoids, until the concentrations inside and outside were equal. The thylakoids were then transerred, still in darkness, into a solution o ADP and phosphate that was more alkaline. There was a brie burst o ATP production by the thylakoids. The graph shows the yield o ATP at three acid incubation pHs and a range o pHs o the ADP solution. 1 ATP production / mol 3.8 a) D escribe the relationship between pH o ADP solution and ATP yield, when acid incubation was at pH 3 .8. [2 ] b) Explain why the pH o the AD P solution aects the ATP yield. [2 ] 2 4.8 Explain the eect o changing the pH o acid incubation on the yield o ATP. [2 ] Explain why there was only a short burst o ATP production. [2 ] Explain the reason or perorming the experiment in darkness. [2 ] 5.2 3 6.5 7.0 7.5 8.0 pH of ADP solution 8.5 Figure 6 Results of Jagendorf experiment 4 Reduction of NADP Excited electrons from Photosystem I are used to reduce NADP. The remaining parts o the light-dependent reactions involve Photosystem I. The useul product o these reactions is reduced NAD P, which is needed in the light- independent reactions o photosynthesis. Reduced NAD P has a similar role to reduced NAD in cell respiration: it carries a pair o electrons that can be used to carry out reduction reactions. uid in thylakoid H 2O 2H + thylakoid membrane Photosystem II 2e - 1 2 O2 plastocyanin uid outside thylakoid plastoquinone electron transport chain Photosystem II ferredoxin NADP Figure 7 Summary of the lightdependent reactions of photosynthesis 394 C hlorophyll molecules within Photosystem I absorb light energy and pass it to the special two chlorophyll molecules in the reaction centre. This raises an electron in one o the chlorophylls to a high energy level. As with Photosystem II, this is called photoactivation. The excited electron passes along a chain o carriers in Photosystem I, at the end o which it is
