MBLG2x71 Course Information for mmb web site
Unit of Study Overview
MBLG2071 extends the basic concepts introduced in MBLG1001. It provides a firm
foundation for students wishing to continue in the molecular biosciences as well as for
those students who intend to apply molecular techniques to other biological or
medical questions. MBLG2971 is the advanced option of the course.
The theory component is presented in 23 lectures (2 per week). It explores the
regulation of the flow of genetic information in both eukaryotes and prokaryotes. The
central focus is on the control of replication, transcription and translation and how
these processes can be studied and manipulated in the laboratory. Experiments in
model organisms are provided to illustrate how the field has advanced, together with
discussion of work carried out in human systems and the relevance to human genetic
diseases. The tools of molecular biology are taught within the context of recombinant
DNA.
The practical course (6 five hour sessions; one every fortnight) complements the
theory and builds on the skills learnt in MBLG1001. Specifically students will: use
spectrophotometry for the identification and quantification of nucleic acids, explore
the lac operon system for the investigation of gene expression control, perform PCR
analysis on their own DNA and isolate plasmid DNA. As with MBLG1001, strong
emphasis is placed on the acquisition of generic and technical skills.
The advanced course (MBLG2971) covers the same lecture material as the normal
(MBLG2071) course. The practical course, however, contains a number of different
laboratory experiments designed to cover some of the material in more depth.
Prerequisites
For MBLG2071: MBLG1001 and 12 CP of Junior Chemistry.
For MBLG2971: Distinction in MBLG1001 and 12 CP of Junior Chemistry
Contact Details
Ms Vanessa Gysbers
Room 303, Lab 712,
Biochemistry & Microbiology Building,
Ph. 9351 2511, Email: vanessa@mmb.usyd.edu.au
Lecture Times and Venues
There are 2 lectures a week, on Wednesdays and Fridays in Chemistry Lecture
Theatre 1. Each lecture is presented at 8 am and repeated at 10am.
Practicals: Each student attends 6 practical sessions, one per fortnight. Sessions are
held from 1-6pm, Tue- Fri, in Room 380, Biochemistry & Microbiology Building,
Lecture Topics
1. Introduction: Review of material covered in first year: biopolymers, central dogma,
transcription, translation. While genes and proteins were considered in isolation in first year,
we will now consider their regulation in a genomic context.
2. Transcriptional Regulation I Models of Gene Expression: the Lac Operon. Uses of the
lac operon in Molecular Biology, blue/white colour selection
3. Transcriptional Regulation II Models of Gene Expression: the Trp Operon.
Transcriptional and translational control (attenuation).
4. Eukaryotic transcriptional Regulation RNA polymerases and transcription factors, TBP,
TAFs etc basal transcription, elongation, enhancers
5. Post-transcriptional Processing Splicing (alternative splice sites), poly-adenylation,
transport, stability of mRNA, control of aberrant transcripts
6. Translational & post-translational control Review of prokaryotic translation, extending
to eukaryotic and organelle (mitochondria and chloroplast). Iron-responsive elements, control
of translation, protein stability and degradation, protein trafficking to mitochondria, nucleus,
and membranes.
7. Techniques to measure gene expression Difference between genome and expressed
genes; RNA isolation, Northerns, microarrays, Real time PCR, RNase protection assays,
assessing protein levels.
8. Model Eukaryotes We salute yeast, nematodes, fruit flies and mice in the service of
molecular biology and genetics.
9. Transgenesis Transgenic animals, nuclear transfer and cloning, conditional versus nonconditional transgenics, viral-mediated gene transfer, human gene therapy.
10. Knockout Animals Basic knockouts, Specialized knockouts, genome wide knockouts,
Disease modelling
11. Stem Cells Totipotency, pluripotency,multipotency, embryonic and adult stem cells,
nuclear transfer and cloning.
12. Signalling pathways that control eukaryotic gene expression Overview of major
receptor classes-cell surface and cytoplasmic, receptor coupled-signal transduction pathways,
down-modulation and negative regulation of receptor signalling
13. Gene regulation during eukaryote development Mechanisms for establishing
differential gene expression, control of mRNA localization, cell-to-cell contact and secreted
cell signaling
14. Review. Review of the themes of the last 6 lectures, covering exam style questions.
15. Introduction to the structure of the Genome Review DNA structure with A, B and Z of
DNA. DNA packging. Chromosome length and diversity, differences between eukaryotic and
prokaryotic chromosomes, packaging proteins e.g. histones and the chromosome packaging.
Heterochromatin and euchromatin and their relationship to transcription.
16. Genomic sequence complexity Size and complexity, C-value paradox, melting and
annealing DNA, CoT curves and different classes of DNA. Introduction to the complexity of
the genome: protein-coding portions of the human genome and non-coding RNA.
Transcription of rRNA and tRNA by RNA pol I and III. Other non-coding RNAs, snoRNAs,
miRNAs, natural antisense RNAs and their processing and function.
17. Eukaryotic replication Starting with the E. coli replication fork covered in MBLG1001,
initiation and termination in bacteria. Eukaryotic replication with multiple, linear chromosomes,
the eukaryotic cell cycle, the link between DNA replication and the cell cycle; both prokaryotic
and eukaryotic. Mitotic cell division revisited (prophase, metaphase etc)
18. Cell cycle regulation The elucidation of the cell cycle in eukaryotes, check points in the
cell cycle, introduction to immortal cell lines, the constraints normal cells face with cell division,
stem cells, differentiation and proliferation, Meiosis review
19. Homologous recombination and meiosis From the concept of cross-over covered by
Dr Lyons in MBLG1001....What is happening at a molecular level? Models of homolgous
recombination, recombination in eukaryotes, genetic consequences, site-specific
recombination, VDJ recombination.
20. Mutation The need for change, replication errors (nature of mutations, tautomers of
bases, escape of proofreading), mutation rates, spontaneous DNA damage (hydrolysis and
deamination), environmental DNA damage (alkylation, oxidation and radiation), mutagenic
DNA damage (base analogues and intercalating agents). Types of errors (insertion, deletion,
substitution)
21. DNA Repair Importance of repairing the genome. repair of DNA damage (direct reversal
(photoreactivation, methyltransferase), correcting cytosine deamination
22. Working with DNA experimentally PCR and DNA sequencing: how DNA polymerases
are used in molecular biology; Klenow and Taq.
23. Review
24. Exam Preparation Tutorial and past paper questions
Practical Course Outline
Introduces students to the use of UV
spectrophotometry to identify and quantify bases and
nucleic acids and biochemicals.
1
UV Spectrophotometry
2
Gene Expression I
Investigates regulation of the lac operon in E. coli,
using IPTG, lactose, glucose and protein synthesis
inhibitors.
3
Gene Expression II
Students design their own experiment to examine a
particular aspect of the lac operon system.
4
DNA Fingerprinting I
5
6
Specific loci in students’ DNA amplified by the
polymerase chain reaction.
DNA Fingerprinting II & Plasmid Gel electrophoresis analysis of PCR products and
Isolation
miniprep isolation of plasmid DNA.
Plasmid Analysis
Gel electrophoresis analysis of plasmid isolates.
Text Books
‘Molecular Biology of the Gene’ by Watson et al, 5th Edition.
Also suitable are any of the recent Biochemistry Textbooks that are all entitled
‘Biochemistry’ but identified by their different authors: e.g. Garrett & Grisham, Voet
& Voet or Stryer. Any of these textbooks would be a good alternative for those
wishing to continue with Biochemistry in second semester.
Assessment
The lecture component of the course is worth ~50% of the final mark for this course.
The material is assessed in the exam.
The practical component contributes ~50% to the final mark and is assessed both
with in-semester tasks and in the final end-of-semester exam. ‘Theory of practical’
questions comprise ~30%- of the written exam.
Tasks: One 2.5 hour exam including ‘Theory’ and ‘Theory of Practical’ questions, 3
in-semester hand-in laboratory assignments, continuous assessment during practical
classes and laboratory book hand-in for final assessment.