NATIONAL QUALIFICATIONS CURRICULUM SUPPORT
Biology
Unit 1
Tutorials
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Acknowledgements
The publisher gratefully acknowledges permission to use the following sources: image of haemoglobin
from http://commons.wikimedia.org.uk/wiki/File:1GZX_Haemoglobin.png and image of a nucleosome
from http://commons.wikimedia.org/wiki/File:Nucleosome_structure.png both © Richard Wheeler
(Zephyris); image of beta sheets from http://commons.wikimedia.org/wiki/File:PDB_1jy6_EBI.jpg ©
http://www.ebi.ac.uk; image of kinases from http://commons.wikimedia.org/wiki/File:Ch4_kinases.jpg ©
National Institute of General Medical Sciences; image of DNA X-ray from
http://commons.wikimedia.org/wiki/File:ABDNAxrgpj.jpg, ‘Physical Chemistry of Food’, vol. 2, van
Nostrand Reinhold: New York, 1994, I.C. Baianu et al; image of a protein primary structure from
http://commons.wikimedia.org/wiki/File:Protein_primary_structure.svg and image of DNA Exons from
http://commons.wikimedia.org/wiki/File:DNA_exons_introns.gif both © The National Human Genome
Research Institute; image of electrophoresis from http://commons.wikimedia.org/wiki/File:SDSPAGE_Electrophoresis.png © Bensaccount at en.wikipedia; image no 3418 of African sleeping sickness
from http://phil.cdc.gov/phil/details.asp © CDC/Alexander J. da Silva, PhD/Melanie Moser; image no
11820 of Giemsa-stained light photomicrograph revealed the presence of a Trypanosoma brucei parasite,
which was found in a blood smear from http://phil.cdc.gov/phil/details.asp © CDC/Blaine Mathison;
image from Toxicology in Vitro 18 (2004) 1–12, Workshop report, The humane collection of fetal bovine
serum and possibilities for serum-free cell and tissue culture, reprinted from Toxicology in Vitro 18, Vol
1-12, Workshop report, The humane collection of fetal bovine serum and possibilities for serum-free cell
and tissue culture by J. van der Valk,D. Mellor,R. Brands,R. Fischer,F. Gruber,G. Gstraunthaler,L.
Hellebrekers,J. Hyllner,F.H. Jonker,P. Prieto,M. Thalen,V. Baumans, 2004 with permission from Elsevier
http://www.journals.elsevier.com/toxicology-in-vitro/; image from article, Conservation, Variability and
the Modeling of Active Protein Kinases
http://www.plosone.org/article/slideshow.action?uri=info:doi/10.1371/journal.pone.0000982&imageURI
=info:doi/10.1371/journal.pone.0000982.g001 © 2007 Conservation, Variability and the Modeling of
Active Protein Kinases by James D. R. Knight, Bin Qian, David Baker, Rashmi Kothary; image from
article Proteomics of Trypanosoma evansi Infection in Rodents from
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.000979 © 2010 Proteomics of
Trypanosoma evansi Infection in Rodents by Nainita Roy, Rishi Kumar Nageshan, Rani Pallavi, Harshini
Chakravarthy, Syama Chandran, Rajender Kumar, Ashok Kumar Gupta, Raj Kumar Singh, Suresh
Chandra Yadav, Utpal Tatu; image of Signal transduction from
http://commons.wikimedia.org/wiki/File:Signal_transduction_v1.png © Roadnottaken at the English
language Wikipedia
© Crown copyright 2012. You may re-use this information (excluding logos) free of charge in any format
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Contents
Tutorial 1: Proteomics and protein structure 1
4
Tutorial 2: Proteomics and protein structure 2
11
Tutorial 3: Membrane proteins and CF
19
Tutorial 4: Altering signal transduction
23
Tutorial 5: Cell cycle
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TUTORIAL 1
Tutorial 1: Proteomics and protein structure 1
Covalent modification of protein through the ad dition and removal of
phosphate is essential for the control of cellular processes. Kinase enzymes
attach phosphate to other enzymes and thus modulate their activity. The
extract from the journal below details the high structural conservation of
phosphate enzymes.
Read the extract and answer the questions. The answers to these questions
will form the basis of a tutorial discussion. The most important elements of
the paper have been highlighted to aid your understanding.
For the full paper search the open source journal database, Plosone, using the
title Conservation, Variability and the Modeling of Active Protein Kinases .
Citation: Knight JDR, Qian B, Baker D, Kothary R (2007) Conservation,
Variability and the Modeling of Active Protein Kinases. PLoS ONE 2(10):
e982. doi:10.1371/journal.pone.0000982
Abstract
The human proteome is rich with protein kinases, and this richness has made
the kinase of crucial importance in initiating and maintaining cell beh avior.
Elucidating cell signaling networks and manipulating their components to
understand and alter behavior require well designed inhibitors. These
inhibitors are needed in culture to cause and study network perturbations, and
the same compounds can be used as drugs to treat disease. Understanding the
structural biology of protein kinases in detail, including their commonalities,
differences and modes of substrate interaction, is necessary for designing
high quality inhibitors that will be of true use for cell biology and disease
therapy. To this end, we here report on a structural analysis of all available
active-conformation protein kinases, discussing residue conservation, the
novel features of such conservation, unique properties of atypical kinases an d
variability in the context of substrate binding. We also demonstrate how this
information can be used for structure prediction. Our findings will be of use
not only in understanding protein kinase function and evolution, but they
highlight the flaws inherent in kinase drug design as commonly practiced and
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dictate an appropriate strategy for the sophisticated design of specific
inhibitors for use in the laboratory and disease therapy.
Introduction
Protein kinases are the most ubiquitous single family of signaling molecules
in the cell, accounting for approximately 2% of the proteins encoded by the
human genome [1]. The simple mechanism of attaching an ATP -derived
phosphate to a protein involves kinases in every aspect of cell behavior, from
apoptosis to survival, proliferation to differentiation, maturation etc. Protein
kinases provide a unique opportunity for understanding proteins in general by
presenting us with a seeming paradox: wide scale similarity of sequence and
structure combined with a diversity of behavioral consequences to their
activity. The vast majority of protein kinases have readily detectable
sequence similarity, which translates into structure. But even those known
protein kinases that show no significant algorithm-detectable similarity at the
level of sequence are believed to have very typical structures, as is evidenced
by specific examples [2,3]. As they all have a shared function in transferring
the terminal phosphate of ATP to another protein, similarity is
understandable. Evidence to date also suggests a common catalytic
mechanism (the possible exception may be the integrin -linked kinase [4]),
whereby ATP and an active site divalent cation are bound in identical
fashions and phospho- transfer is achieved by a shared set of amino acids.
Studies in yeast [5,6] have shown that kinases can be promiscuous,
phosphorylating hundreds of proteins, but they also have clear specificities.
How is this specificity attained by one family of highly similar proteins? This
paradox suggests the perfection of the kinase as an enzyme: a region ideally
suited for the common function of catalysis, with another region(s) uniquely
modifiable to attain substrate specificity without altering fold, compromising
ligand binding or the subsequent reaction mecha nism. A thorough
understanding of this family of proteins would generate a tremendous
knowledge base for discovering and predicting protein interactions, for
designing highly specific and potent inhibitors, and, as a consequence of
these facts, for understanding the cell and disease.
As protein kinases are the key players in cell signaling, aberrations in their
activity have been directly correlated with numerous disease states (for
example, breast cancer [7] and chronic myeloid leukemia [8]) and made them
potential targets for drug design in many other diseases (for example, Crohn’s
[9] and cerebral vasospasm [10]). This has made the kinase the drug target of
choice [11]. However, there is an inherent flaw in traditional kinase inhibitor
design. Almost all inhibitors target the ATP binding pocket based on a simple
principle: if ATP cannot be bound, phosphorylation cannot occur. Building a
molecule that can occupy this pocket is relatively simple, but since the ATP
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TUTORIAL 1
binding pocket and the regions in its immediate vicinity are the areas of
greatest conservation, building a specific inhibitor is impossible. The inherent
multi-target nature of inhibitors has been demonstrated by Fabian et al. [12],
where the twenty compounds tested had multi -target coverage with only 23%
of the kinome screened. Other ATP binding proteins could very likely display
affinities for these compounds as well, making these inhibitors not just multi kinase but multi-enzyme. In the laboratory, how can the effect of treating
cells with such inhibitors be dissected? And when used for disease, what non intended effects may arise in the targeted cell type or others over the long
term? In the hopes of producing specific inhibitors, what is needed is a new
approach to kinase drug design, one which logically targets the region of
greatest dissimilarity.
True dissimilarity can be known if similarity or conservation is understood in
detail. For this, structure-based comparative approaches are needed to fully
extract the information hidden in the three-dimensional protein-structure
space. Traditional structure-driven alignment studies concentrate on
maximizing fold overlap, and for the highly -similar protein kinase family
which has a largely conserved fold, this can be a useful approach. But it is
not necessarily the correct one, particularly where inhibitor design is
concerned. Due to the information available in a three -di-mensional space,
structures can be aligned in other ways, for example by using geometry
independent of connectivity. Fold can be ignored and focus directed upon
residues free from their covalent associations. The positioning of side -chains
and those functional groups involved in enzymatic catalysis and protein
interactions can be directly overlain for studying similarity and variab ility.
This type of alignment, and not that of fold, is of greater relevance for
understanding protein interactions and therefore in designing small molecules
or peptides to act as inhibitors.
Understanding the similar/conserved and dissimilar/non -conserved aspects of
protein kinases allows for effective drug design. In addition, conservational
studies will aid especially in structure prediction. There are at least 518
known human protein kinases [1] and deriving crystal structures for them all
would involve a great deal of time and effort. As all known protein kinases
have similar structures, homology-driven approaches to structure prediction
that incorporate knowledge of conservation should prove fertile. Having a
reliable predicted structural kinome would be of great practical use.
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Figure 1 The structure of protein kinase A
(PKA). PKA is shown in its active
conformation with ATP in green sticks and
Mn2+ as black spheres. β-strands, helices and
loops are labeled as in Knighton et al. [45].
The active site is situated between the small
and large lobes, located above and below
ATP respectively. CL: catalytic loop; MPL:
magnesium- positioning loop.
Figure 2 Multiple kinase alignment.
The fifteen active-conformation kinase
structures listed in Table 1 were aligned
using our modified Procrustes approach.
Shown in green sticks is the ATP or
ATP analog molecule of each structure.
Each kinase is colored uniquely.
Table 1 Active-conformation kinase structures
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Figure 3 A kinase consensus structure. Each sphere represents a conserved residue.
Red indicates full conservation of a particular amino acid in all fifteen kinase
structures; orange, conservation in thirteen or fourteen structures; and yellow,
conservation in eleven or twelve structures. Blue spheres indicate full conservation
of an amino-acid category. The ATP molecule of protein kinase A is shown in green
sticks. (A) The consensus structure consisting of the forty -four points listed in Table
2. (B) The consensus structure overlaid on the multiple alignment.
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TUTORIAL 1
Questions
1.
What does a kinase enzyme do?
2.
What protein structures are visible in Figure 1?
3.
What is the prosthetic group of protein kinase A in Figure 1?
4.
For simplicity, bonding is not shown in all of the ribbon diagrams of
kinase. What type of bonding would be present in the secondary and
tertiary structures visible?
5.
Compare Figure 1 and Figure 2. What do these pictures indicate?
6.
Using Table 1 identify how many kinases have a human origin.
7.
Mutation to the DNA of one kinase in Table 1 would increase your
chances of developing cancer. Name this kinase.
8.
What does Figure 3 indicate about the active site of kinase enzymes?
9.
How could this information be used in the design of a drug?
10.
If drugs are developed in this way, what might the benefits be to
patients?
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Answers
1.
What does a kinase enzyme do?
Phosphorylates other enzymes through the addition of phosphate.
2.
What protein structures are visible in Figure 1?
Alpha-helix, beta-sheets, primary, strucuture polypeptide, prosthetic
group.
3.
What is the prosthetic group of protein kinase A in Figure 1?
Mn 2+
4.
For simplicity, bonding is not shown in all of the ribbon diagrams of
kinase. What type of bonding would be present in the visible secondary
and tertiary structures?
Hydrogen bonding, alpha-helix and beta-sheets, hydrogen bonding in
tertiary, hydrophobic interactions, ionic, Van der Waals, sulphur
bridges.
5.
Compare Figure 1 and Figure 2. What do these pictures indicate?
High commonality in structure between kinase enzymes.
6.
Using Table 1 identify how many kinases have a human origin.
7
7.
Mutation to the DNA of one kinase in Table 1 would increase your
chances of developing cancer. Name this kinase.
Pim-1
8.
What does Figure 3 indicate about the active site of kinase enzymes?
High conservation of residues in active site s as all of these enzymes are
catalysts for phosphorylation. The limited variation is likely to be
related to each enzyme’s substrate specificity.
9.
How could this information be used in the design of a drug?
By designing drugs that target the specific residues that differ between
different kinase enzymes, drugs will have a more specific action.
10.
If drugs are developed in this way, what might the benefits be to
patients?
Tailor-made drugs could target the specific residues of specific faulty
enzymes. Specific drugs are likely to be more effective and have fewer
side effects.
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TUTORIAL 2
Tutorial 2: Proteomics and protein structure 2
Proteomics is a rapidly expanding field of biology. One area of particular
significance is its use in the identification of proteins produced by pathogens
during their lifecycle that could be used as identification markers and
potential targets for specific drugs and vaccines to combat infection.
The lifecycles of parasites are often complex involving many specific stages
depending on which host and stage of their li fecycle they are in. As a result,
the number of proteins expressed is extensive.
Figure 1 Trypanosoma lifecycle. Centers for Disease Control and Prevention (CDC)
Public Health Image Library.
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TUTORIAL 2
Figure 2 Trypanosoma brucei parastite in human blood. Centers for Disease Control
and Prevention (CDC) Public Health Image Library
The parasite Trypanomsa evansi has a similar lifecycle to T. brucei. The
excerpts from the journal below discuss the identification of specific proteins
unique to specific stages of the parasite’s lifecycle identified through SDS gel
electrophoresis and mass spectrometry analysis .
Read the excerpts below and answer the questions. Your responses will form
part of a discussion about the techniques used and the significance of this
research.
For the full paper search the open source journal database, Plosone, using the
title below.
Proteomics of Trypanosoma evansi Infection in Rodents
Citation: Roy N, Nageshan RK, Pallavi R, Chakravarthy H, Chandran S, et al.
(2010) Proteomics of Trypanosoma evansi Infection in Rodents. PLoS ONE
5(3): e9796. doi:10.1371/journal.pone.0009796
Abstract
Background
Trypanosoma evansi infections, commonly called ‘surra’, cause significant
economic losses to livestock industry. While this infection is mainly
restricted to large animals such as camels, donkeys and equines, recent
reports indicate their ability to infect humans. There are no World Animal
Health Organization (WAHO) prescribed diagnostic tests or vaccines
available against this disease and the available drugs show significant
toxicity. There is an urgent need to develop improved methods of di agnosis
and control measures for this disease. Unlike its related human parasites T.
brucei and T. cruzi whose genomes have been fully sequenced T. evansi
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genome sequence remains unavailable and very little efforts are being made
to develop improved methods of prevention, diagnosis and treatment. With a
view to identify potential diagnostic markers and drug targets we have
studied the clinical proteome of T. evansi infection using mass spectrometry
(MS).
Methodology/Principal Findings
Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of
Flight (QTOF) mass spectrometry we have identified over 160 proteins
expressed by T. evansi in mice infected with camel isolate. Homology driven
searches for protein identification from MS/MS data led to most of the
matches arising from related Trypanosoma species. Proteins identified
belonged to various functional categories including metabolic enzymes; DNA
metabolism; transcription; translation as well as cell -cell communication and
signal transduction. TCA cycle enzymes were strikingly missing, possibly
suggesting their low abundances. The clinical proteome revealed the presence
of known and potential drug targets such as oligopeptidases, kinases, cysteine
proteases and more.
Conclusions/Significance
Previous proteomic studies on Trypanosomal infections, including human
parasites T. brucei and T. cruzi, have been carried out from lab grown
cultures. For T. evansi infection this is indeed the first ever proteomic study
reported thus far. In addition to providing a glimpse into the biology of this
neglected disease, our study is the first step towards identification of
diagnostic biomarkers, novel drug targets as well as potential vaccine
candidates to fight against T. evansi infections.
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TUTORIAL 2
Stages involved in processing the parasites
to determine the proteins present
Figure 3 Purified parasites observed at
406 magnification.
Figure 4 Parasites were
lysed and proteins were
fractionated on 10% SDS
PAGE.
Figure 5 Functional classifications of identified proteins. Pie chart showing
different functional classes of proteins which includes metabolic enzymes,
cytoskeletal proteins, proteins involved in synthesis, signal transduction proteins,
nucleic acid associated proteins, protein involved in virulence, chaperones and co chaperones, proteins involved in deciding protein fate, protein s involved in
trafficking, hypothetical proteins, proteases and peptidases, transport proteins,
kinases and phosphatases and proteins with unknown functions.
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TUTORIAL 2
Figure 6 Representation of proteins according to their cellular localisation in
T. evansi. All the proteins identified have been categorised based on their homology
to related Trypanosomal species and their known localizations in those species.
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TUTORIAL 2
Questions
1.
What is a protozoan parasite?
2.
What is the vector of T. brucei?
3.
What was the in vivo host for T. evansi in the investigation?
4.
T. brucei is described as being an obligate parasite. What does this
mean?
5.
What structure does T. brucei have to allow it to move (see Figure 2)?
6.
Suggest a technique that could be used to separate out and purify T.
evansi in Figure 3 from a sample of blood and explain how this
technique works.
7.
The gel in Figure 4 was produced using electrophoresis. Describe and
explain how this technique works and how the protein fragments from
T. evansi were isolated.
8.
Using Figure 4 identify between which regi ons the most intensive bands
of protein appeared in the gel.
9.
The gel in Figure 4 was cut into 26 contiguous gel slices an d each slice
was processed using gel trypsin digestion. What would this process do?
10.
The proteins identified were compared against k nown proteins in
Trypanosoma. This led to the identification of 166 proteins. What was
the number of transport proteins identified?
11.
Using Figures 5 and 6 suggest a possible target protein(s) for future
vaccine development. Explain why you have chosen thi s protein.
12.
Using Figures 5 and 6 suggest which protein(s) you would use as the
basis of an identification technique for T. evansi in a blood sample of an
infected animal. Explain why you have chosen this protein.
13.
What other stages of the lifecycle have still to be analysed?
Now follow the link to Proteome Technologies and Cancer
(http://proteomics.cancer.gov/whatisproteomics/videotutorial) to watch the
video tutorial on the future of proteomics in cancer research, identification
and treatment. What does this mean for the future the disease treatment?
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TUTORIAL 2
Answers
1.
What is a protozoan parasite?
Unicellular eukaryotic heterotrophic organism.
2.
What is the vector of T. brucei?
Tsetse fly
3.
What was the in vivo host for T. evansi in the investigation?
Mice
4.
T. brucei is described as being an obligate parasite. What does this
mean?
It cannot survive outside the host.
5.
What structure does T. brucei have to allow it to move (see Figure 2)?
Flagellum
6.
Suggest a technique that could be used to separate out and purify T.
evansi in Figure 3 from a sample of blood and explain how this
technique works.
Chromatography or centrifugation.
7.
The gel in Figure 4 was produced using electrophoresis. Describe and
explain how this technique works and how the protein fragments from
T. evansi were isolated.
Separate by charge and size of protein, placed in an electric current, gel
is porous, largest fragments migrate slowest and move the shortest
distance in the gel, and vice versa.
8.
Using Figure 4 identify between which regions the most intensive bands
of protein appeared in the gel.
66–45 kDa
9.
The gel in Figure 4 was cut into 26 contiguous gel slices and each slice
was processed using gel trypsin digestion. What would this process do?
The slicing isolates the proteins into 26 different categories. Trypsin
digestion breaks the proteins in each group into peptides for further
analysis and identification.
10.
The proteins identified were compared against known proteins in
Trypanosoma. This led to the identification of 166 proteins. What was
the number of transport proteins identified?
6–7
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TUTORIAL 2
11.
Using Figures 5 and 6 suggest a possible target protein for future
vaccine development. Explain why you have chosen this protein.
Any reasonable answer justified. For example, it would be appropriate
to focus on proteins that are found in the parasite but not in the host.
Alternatively a focus on proteins associated with virulence or the
flagellum might be particularly appropriate.
12.
Using Figures 5 and 6 suggest which protein(s) you would use as the
basis of an identification technique for T. evansi in a blood sample of an
infected animal. Explain why you have chosen this protein.
Any reasonable answer justified. For example, again it would be
appropriate to focus on proteins found in the parasite but not in the
host. Surface proteins associated with the membrane or flagellum might
be particularly appopriate.
13.
What other stages of the lifecycle and therefore proteins have still to be
analysed?
Vector-based stages of life cycle.
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TUTORIAL 3
Tutorial 3: Membrane proteins and CF
Introduction
Cystic fibrosis (CF) is an autosomal recessive condition that aff ects over
9000 people in the UK, with 1 in 25 of the population thought to be carriers.
In the European Union 1 in 2000–3000 newborns is found to be affected by
CF. Individuals produce thick sticky mucus that blocks airways, the digestive
tract and the reproductive system. This results in loss of lung function and
increased risk of respiratory infection. Digestive problems arise through
blockage of the pancreatic ducts resulting in poor release of digestive
enzymes. Sterility can also be an issue through bl ockages in the reproductive
tract. Prognosis can be poor although improving. Mean life expectancy is now
in the mid-30s whereas 40 years ago many children died in infancy or their
early teens. There is as yet no cure.
The aim of this tutorial is to develop skills in the use of scientific literature
and group discussion through developing an understanding of the following :
1.
2.
3.
4.
The biology and pathology of cystic fibrosis.
The role of cystic fibrosis trans-membrane conductance regulator
(CFTR) in normal cells.
Mutations of the CFTR gene and CF.
CF gene therapy.
Targeted web/downloadable resources
General (1–2)
http://ghr.nlm.nih.gov/condition/cystic-fibrosis
http://www.cff.org/AboutCF/Faqs/
http://www.nhs.uk/conditions/Cystic-fibrosishttp://www.cftrust.org.uk)/
CFTR (2–4)
http://ghr.nlm.nih.gov/gene/CFTR
http://www.rcjournal.com/contents/05.09/05.09.0595.pdf
http://www.colorado.edu/MCDB/MCDB4600/1ReviewPhenotype.pdf
CF gene therapy (4)
http://www.nature.com/gt/journal/v9/n20/pdf/3301791a.pdf
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TUTORIAL 3
Links to syllabus
Unit 1: Cells and Proteins
c) Membrane proteins
(i) Movement of molecules across membranes: ion
channels/CFTR/mutation/cystic fibrosis
Outline
Learners should review the general links prior to the class session and review
the syllabus on Movement of molecules across membrane.
It is suggested that the teacher guides the tutorial discussion through the
following threads. These threads are for teacher guidance.
What is cystic fibrosis? (general links + prior syllabus)
 Pathology
o Sites affected in human body
o Mode of pathology, eg increased mucus production
o Affected organs
o Prognosis
o Molecular basis of pathology
 Mutation in CFTR gene (multiple mutations identified) results in
failure of cAMP-controlled ATP-gated chloride (Cl – ) channels
 Normally Cl – flows out through gated channels when triggered. Na 2+
influx also decreases due to change in membrane potential via other
channels
 Either through failure of expression or structural mutation the
channel becomes non-functional
 Cl – becomes trapped inside the cell increasing the negative potential.
Na + influx increases due to this
 This increased NaCl in epithelial cell cytoplasm causes water inflow
from mucus secreted to outside epithelium
 Water loss from mucus causes it to become thicker and stickier
 Thickened mucus in airways causes blockages and cannot be moved
by cilia. Bacteria become trapped in epithelium, resulting in
increased infection rates.
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TUTORIAL 3
 Cell biology and target sites of possible mutations of CFTR gene
o Class 1–6 mutations
 http://www.rcjournal.com/contents/05.09/05.09.0595.pdf (p 596)
 http://www.colorado.edu/MCDB/MCDB4600/1ReviewPhenotype.pdf
(pp 473–476)
 Discuss different mutations and their ability to affect normal cell
function.
 ΔF508 is the most common mutation in CF, resulting in a class 2
mutation (60–70% of cases)
Use of materials by learners
The notes here are for the guidance of teachers dealing with learners in a
tutorial setting.
It is envisage that the general links be issued prior to the lesson to all learners
with the initial aims 1–4. Learners should be asked to familiarise themselves
with CF.
Subgroups should be issued with the following and tasked to report back to
the class. If learners are suitably motivated t his could be set as a chance to
collaborate outside the classroom; if not time will have to be set aside during
teaching lessons for group discussion and reporting back.
Molecular basis of pathology group
 http://ghr.nlm.nih.gov/gene/CFTR
 http://www.rcjournal.com/contents/05.09/05.09.0595.pdf
 http://www.colorado.edu/MCDB/MCDB4600/1ReviewPhenotype.pdf
This subgroup should be asked to identify the normal and abnormal CFTR
gene product and relate its normal and undamaged function to what they
know of CF and gated ion channels.
Cell biology and target sites of possible mutations of CFTR gene group
(including class of mutation (1–6)
 (http://www.rcjournal.com/contents/05.09/05.09.0595.pdf (p 596)
 http://www.colorado.edu/MCDB/MCDB4600/1ReviewPhenotype.pdf (pp
473–476)
This subgroup should be asked to identify the possible classes of CFTR gene
mutation and relate their consequences to receptor expression and/or
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TUTORIAL 3
function. ΔF508 is the most common mutation in CF, resulting in a class 2
mutation (60–70% of cases) and should be discussed.
Gene therapy (advanced tutorial extension)
Much scope here. This is possibly a tutorial in its own right.
 The Nature paper
http://www.nature.com/gt/journal/v9/n20/pdf/3301791a.pdf provides a
detailed review of the state of gene therapy as of 2002. Although very
technical in parts learners should be guided to look for the following
themes.
o What is gene therapy?
o Barriers to gene insertion
o Insertion mechanisms (vectors) for non-damaged CFTR
 Viruses
 Why used
 Problems
 Endocytosis
 What is it?
 Why used?
o Issues with gene therapy
 Safety/ethics
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UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
TUTORIAL 4
Tutorial 4: Altering signal transduction
Introduction
Membrane proteins are vitally important in signal transduction, but what
happens if the signal is changed or blocked? Learners will look at some of the
consequences in relation to genetic disease, toxins and drugs.
Aims
To develop skills in the use of scientific literature and group discussion.
To illustrate the importance of membrane protein interactions in genetic
disease.
To illustrate the importance of membrane proteins in drug/toxin interactions.
Resources/task group area
Group 1: Enzyme-linked receptors
Genetic disease: achondroplasia and the FGF 3 receptor gene
http://ghr.nlm.nih.gov/condition=achondroplasia
http://www.gghjournal.com/volume22/4/featureArticle.cfm
Groups 2: Synapses, ion channels and their function/malfunction
Local anaesthetics
http://www.biologymad.com/nervoussystem/synapses.htm
http://bja.oxfordjournals.org/content/89/1/52.full
Group 3: Synapses, ion channels and their function/malfunction
Neurotoxins
http://www.biologymad.com/nervoussystem/synapses.htm
http://faculty.washington.edu/chudler/toxin1.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210964/
UNIT 1 (AH, BIOLOGY)
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TUTORIAL 4
Outline
Learners should review the resource links prior to the class session and
review the syllabus on Movement of molecules across membranes.
Subgroups should be issued with the appropriate resource links and tasked to
report back to the class on their tasks. If learners are suitably motivated this
could be set as a chance to collaborate outside the classroom; if not time will
have to be set aside during teaching lessons for research/group discussion and
reporting back.
Task areas
Group 1: Enzyme-linked receptors
 Achondroplasia is an autosomal dominant condition caused by a mutation
of the FGF 3 receptor.
- What role does FGF and the FGF 3 receptor have in normal cells?
- Why does inheritance of one FGF 3 -mutated allele cause genetic disease
and why are homozygous mutations nearly always lethal?
Group 2: Synapses, ion channels and their function/malfunction
 The use of ion channels in nerve action potentials should be consolidated
here by extending knowledge of the roles of ion channels at synapses using
the general link.
- The manipulation of ion channels in the relief of pain should be
researched.
- The effect of various drugs acting on neural receptors/channels should
be researched.
Group 3: Synapses, ion channels and their function/malfunction
 The use of ion channels in nerve action potentials should be consolidated
here by extending knowledge of the roles of ion channels at synapses using
the general link.
- The catastrophic effects of neurotoxins should be investigated and their
mode of action at the cell surface examined.
24
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
TUTORIAL 5
Tutorial 5: Cell cycle
Teacher’s notes
Investigating the nature of the eukaryotic cell cycle: Notes for teachers
Section 1
(a)
The full address for the website is:
www.biology.arizona.edu/cell_bio/activities/cell_cycle/cell_cycle.html
If learners do not have access to the internet then they can use the Onion Cell
Data Table (enclosed) to complete part (a). Alternatively, images of the cells
could be printed off from the website and given to the learners in sets of 36
with the answers printed on the back.
Number of
cells
Percentage
of cells
(1dp)
Interphase
20
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Total
36
55.6
27.7
8.3
5.6
2.8
100
(b)
If the sample of onion cells was compared with other samples it is
likely that the numbers of cells would vary between samples.
Converting the numbers into percentages controls for this variation and
allows valid comparisons to be made between the samples.
(c)
Mitotic index
(d)
= (no. of cells undergoing mitosis/total no. of cells in
sample) × 100
= (16/36) × 100
= 44.4%
(ii)
Or subtract the percentage of cells in interphase from 100.
(i)
Error bars have been drawn. Note that these are not standard
deviation or standard error of the mean error bars.
UNIT 1 (AH, BIOLOGY)
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25
TUTORIAL 5
(ii)
For interphase, prophase and metaphase the duration of the phase
as a percentage of the total length of the cell cycle (24 hours) is
approximately the same as the percentage of cells identified in
each stage in the table that the learners have completed. The
percentages for anaphase and telophase are not in such close
agreement. The evidence may not be valid as the source of the
onion root tip cells that the tabulated percentages are based on is
unknown, ie were the cells grown in vitro or in vivo?
(iii) A comparison of the mitotic indices of different samples of cells
tells us how rapidly cell division is proceeding. Samples with a
higher mitotic index contain cells that are dividing at a faster rate
than cells in samples with a lower mitotic index.
(iv) The distance separating the error bars for the times spent in
interphase and prophase is large.
(v)
A statistical test would need to be done.
(In this case a t test such as the one described in the Learning
Activity for Unit 3 (c) Evaluating Data Analysis would be
inappropriate. This is because it is likely that a within-group
comparison has been made to produce the data, ie the cells
observed in each stage are the same. This is the reason why the
error bars drawn do not represent the standard error of the mean
(SEM): it could be possible for SEM error bars to overlap greatly,
but for there to be a significant difference in the time each
individual cell spends in the two stages).
Section 2
In Section 1 learners saw how they can distinguish between cells in
interphase and the stages of mitosis by using microscopy. Before learners
read the background information to Section 2 it is worth challenging them to
think about and discuss how it would be possible to distinguish between cells
in the G 1 , S and G 2 phases of the cell cycle.
The questions in this section should be viewed as points for discussio n rather
than items of assessment.
3
(a)
(i)
H-thymidine contains a radioactive isotope of hydrogen. Cells
containing 3 H-thymidine can be visualised using autoradiography.
Bromo-deoxyuridine (BrdU), which is an artificial thymidine
analogue, can be labelled with fluorescent anti-BrdU antibodies.
26
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
TUTORIAL 5
(b)
(ii)
The percentage of cells that is labelled is proportional to the
duration of S phase as a percentage of the duration of the whole
cell cycle.
(i)
The more DNA a cell contains, the more DNA there is av ailable
for molecules of dye to bind to and the higher the fluorescence
value of the cell will be.
(ii)
Cells with a relative DNA content of 2 contain twice as much
DNA as those with a relative DNA content of 1 so they must have
replicated their DNA.
Cells with a relative DNA content of 1 have not replicated their
DNA.
Cells with an intermediate relative DNA content are in the process
of replicating their DNA.
(iii) The more cells that are found to be in a particular phase (as given
by the relative content of DNA in the cells) the greater the length
of the phase relative to the others.
The distribution of cells shown on the graph indicates that G 1 is the longest
phase of the cell cycle. Conclusions cannot be made about the length of S
phase compared to the G 2 + M phases because the relative numbers of cells in
each phase cannot be discerned from a visual inspection of the graph.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
27
TUTORIAL 5
Onion cell data table
Interphase
20
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Onion cell data table
Interphase
20
Number
of cells
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Onion cell data table
Interphase
20
Number
of cells
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Onion cell data table
Interphase
20
Number
of cells
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Onion cell data table
Interphase
20
Number
of cells
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Onion cell data table
Interphase
20
Number
of cells
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Onion cell data table
Interphase
20
Number
of cells
Prophase
10
Metaphase
3
Anaphase
2
Telophase
1
Number
of cells
28
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
TUTORIAL 5
Learner’s notes
Investigating the nature of the eukaryotic cell cycle
In this activity you will learn about how the progression of events in the cell
cycle has been established.
Section 1: Observing the stages of mitosis in onion root tip cells
Our understanding of the events of the cell cycle initially came from using
microscopes to observe the appearance of cells taken from areas where
growth occurs, eg meristematic tissue.
The appearance of the nucleus of a eukaryotic cell allows us to tell whether
the cell is in interphase or undergoing mitosis, and which stage of mitosis it
is at. These stages are shown in the diagrams and images below.
Stages of the cell cycle
Interphase
Prophase
Mitosis
Metaphase
Anaphase
Telophase
Go to the URL: http://tinyurl.com/56669
(a)
Read through the background information then click on the ‘Next’
button to progress.
You will be presented with a set of onion root tip cells to classify. Use
the diagrams and images above to help you make your decisions. When
you have finished, record your data in the table below and calculate the
totals and the percentages:
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
29
TUTORIAL 5
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total
Number of
cells
Percentage
of cells
(1dp)
If you do not have access to the internet use the Onion Cell Data Sheet to
complete the table.
(b)
Explain why it is good practice to express the numbers of cells as
percentages.
(c)
The mitotic index of a cell sample is calculated as the number of cells
undergoing mitosis in a sample as a percentage of the total number of
cells present in the sample.
(d)
(i)
Calculate the mitotic index of the sample of onion cells you have
examined. Give your answer to one decimal place.
(ii)
Is there more than one way to use the data in your table to
calculate the mitotic index?
The graph below shows the length of time spent by cultured onion root
tip cells in interphase and the four stages of mitosis:
Error bars = mean ±
standard error
difference
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UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
TUTORIAL 5
(i)
Explain how the appearance of the graph suggests that multiple
measurements of the stage durations were made.
(ii)
What evidence from the graph and your tabulated data supports
the hypothesis that the percentage of cells identified in each stage
of mitosis is a valid indicator of the relative duration of each
stage? Is this evidence valid?
(iii) In view of your answer to (ii) what type of conclusions can be
made by comparing the sizes of the mitotic indices of different
samples of cells?
One conclusion made from the data was that the cells spent a
significantly longer time in interphase than in prophase.
(iv) Describe the evidence from the graph that supports this
conclusion.
(v)
What else would have to be done with the data to confirm that the
observed difference between the duration of two stages is unlikely
to be due to chance?
Section 2: Investigating the DNA content of a proliferating cell
It is not possible to recognise cells that are in the DNA synthesis (S) phase of
the cell cycle by visual inspection using a microscope. Cultured cells in S
phase can, however, be detected by briefly exposing them to molecules such
as 3 H-thymidine or bromo-deoxyuridine (BrdU) that the cells will incorporate
into newly synthesised DNA. Only cells in S phase take up these molecules
and become labelled.
Cells in culture can also be exposed to chemical dyes that fluoresce when
bound to DNA. The cells are passed through an instrument c alled a flow
cytometer (literally: ‘cell measurer’) that measures the quantity of light
emitted by each cell (the fluorescence value) and counts the number of cells
with each fluorescence value.
UNIT 1 (AH, BIOLOGY)
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31
TUTORIAL 5
The graph below shows the results obtained from analysing a population of
proliferating cells.
(a)
(b)
(i)
3
(ii)
How could the relative duration of S phase in a culture of cells
labelled with 3 H-thymidine or BrdU be estimated?
(i)
What is the likely relationship between the fluorescence value of
a cell and the relative quantity of DNA that it contains?
(ii)
Identify which phases of the cell cycle t he cells in areas A, B and
C on the graph are in. Justify your answers.
H-thymidine and BrdU are not fluorescent. What properties could
they have that would allow cells that have incorporated them into
their DNA to be detected?
(iii) How can the distribution of cells shown on the graph be used to
make conclusions about the relative duration of the phases they
are in?
32
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012