BCHM3071/3971

Molecular Biology & Biochemistry - Genes

Course Information

These course outlines are a guide only. They are provided for the information of prospective students. Although every effort is made to ensure the most up to date information is provided, timetables often change each semester due to the availability of rooms and resources. Content (including lecture/practical topics, assessment and textbooks) is also regularly reviewed to ensure relevance and effective learning.

Unit of Study Overview

Most of the recent exciting progress in our understanding of the nature of living systems has come from a combined approach. Modern molecular biology techniques allow us to establish both the structure of genes (and hence the nature of their encoded proteins) and also how gene expression is regulated in response to different physiological stimuli. Biophysical techniques (notably NMR and X-ray crystallography) allow us to define the structure of macromolecules at the molecular level and so reveal important clues as to their functions.

These ideas will be illustrated in this course, which contains accounts of how gene expression in regulated in higher organisms, the consequences when gene expression becomes deranged or when the DNA becomes damaged and the role of RNA in the control processes.

Course Coordinator Contact Details

Mrs Jill Johnston

Room: 410

Telephone: 9351 4248

E-mail: jill.johnston@sydney.edu.au

Professor Iain Campbell

Room: 710

Telephone: 9351 4676

E-mail: iain.campbell@sydney.edu.au

Prerequisites

For BCHM3071
MBLG (1001 or 1901) and 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971 or BCHM2072/2972)] OR [42CP of Intermediate BMedSc units (taken from BMED2801, BMED2802, BMED2803, BMED2804, BMED2805, BMED2806, BMED2807, BMED2808, but including BMED2802 and BMED2804)] OR [18CP of intermediate BMED units (taken from BMED2401, BMED2402, BMED2403, BMED2404,BMED2405, BMED2406, but including BMED2401 and BMED2405) and 6CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971)

For BCHM3971
MBLG (1001 or 1901) and Distinction in 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971 or BCHM2072/2972)] OR [42CP of Intermediate BMedSc units (taken from BMED2801, BMED2802, BMED2803, BMED2804, BMED2805, BMED2806, BMED2807, BMED2808, with Distinction in BMED2802 and BMED2804)] OR [18CP of intermediate BMED units (taken from BMED2401, BMED2402, BMED2403, BMED2404, BMED2405, BMED2406, with Distinction in BMED2401 or BMED2405) and 6CP of Intermediate BCHM/MBLG units (taken from MBLG2071/2971 or BCHM2071/2971) with Distinction in MBLG2071/2971 or BCHM2071/2971

Timetable

1st Lecture: Wednesday 9 am Eastern Avenue LT
2nd Lecture: Friday 9 am Eastern Avenue LT

Practical: Odd weeks, 10:00am - 1:00pm Monday/Tuesday OR 10:00am - 1:00pm Wednesday/Thursday, according to Student Timetable (classes start in Week 3)*
VENUE: All practical classes will be in the Biochemistry 3 lab, Level 4,
Biochemistry and Microbiology building, G08

*Note that it is possible to leave the practical class to attend a lecture in another subject, in which case the practical class will finish at 2:00pm.

Textbooks

For BCHM3071/3971, the recommended textbook is:

Lewin B Genes X (10th edition, Jones & Bartlett, 2010)


Reference texts
Alberts B et al Molecular biology of the cell (5th edition, Garland Science, 2009)

Brown T A Gene Cloning and DNA analysis (6th edition, Blackwell Science, 2010)

Primrose, S B & Twyman, R M & Old R W Principles of Gene Manipulation and Genomics (8th edition, Blackwell Science, 2011)

Lecture Outlines

Gene Architecture and Regulatory Mechanisms
H Nicholas: 11 lectures

1. Genes and Genomes. Revision of characteristics of eukaryotic and prokaryotic genes; defining genes and pseudogenes, examples of gene families; arrangement of genes in different organisms, size and complexity of genes; domain structure and intron/exon boundaries; repeat elements, short and long, retrotransposons, repetitive DNA, chromosomes, centromeres, telomeres.
Experimental evidence: sequencing projects, homology searching.

2. Chromosome structure. CpG islands and distribution of CpG, euchromatin/heterochromatin, histones, structure of the nucleosome, structure of histones, modification of histones.

3. Regulatory Elements in DNA. Defining Promoters, Enhancers, and Locus Control Regions, theoretically and experimentally; mechanisms of promoter action, basal transcriptional apparatus and contact with activators/repressors; models for enhancer action. Experimental techniques: reporter genes, deletion analysis, transfections, DNA-binding assays diseases associated with mutations in control elements.

4. Regulatory Proteins. Cloning regulatory proteins; defining domains for activation, DNA-binding, dimerization; defining DNA-binding specificity; natural evolution and core characteristics of DNA-binding proteins; examples of DNA-binding proteins.
Experimental techniques: comparison of regulatory sequences, affinity purification, mapping domains through deletion analysis, PCR site selection.

5. Mechanism of action regulatory proteins. DNA-binding domains and co-regulator contact domains; enzymatic co-regulators HATs, HDACs, Methylases (DNA and protein), ubiquitination, importance of lysine, SWI/SNF and chromatin remodelling.
Experimental evidence: yeast 2-hybrid system, co-immunoprecipitation, discovery of enzymatic activities, histone deacetylase inhibitors.

6. The histone code. Writing and reading the code, domains involved in recognising protein modifications, the maintenance of gene expression states, the link to DNA methylation. Regulating the regulators: post-translational modifications, control of cellular localization, control by partner availability, small molecules and inducible transcription factors, androgen receptor, artificial regulatory proteins.

7. Transcription factors in development. Control circuits in differentiation, biological roles of key regulatory proteins: Pax-6, MyoD, GATA-1, Sry, AR. Experimental techniques: transgenic technology, knockout approaches, RNA interference

8. Transcription factors in positional specification. Drosophila as a model organism, role of Hox genes in positional specification, conservation of Hox genes from worm to man, genomic arrangement and relationship to function, role of trithorax and polycomb proteins in the maintenance of developmentally regulated genes through cell division, “cellular memory” as an epigenetic phenomenon..

9. Epigenetics in human health and disease. Epigenetic regulation of gene expression in the nervous system, epigenetics and cancer, epigenomics.

Experimental techniques: chromatin immunoprecipitation, bisulfite sequencing.


10. Parental imprinting. Imprint acquisition and erasure during development, imprinted genes and embryonic/neonatal growth, role of methylation in imprinting, examples of imprinting disorders – Prader-Willi and Angelman syndromes.

11. X-inactivation. Dosage compensation and random X-inactivation, role of Xist RNA, TsSix-mediated repression of Xist accumulation, polycomb proteins and DNA methylation in the maintenance of X-inactivation, non-random/imprinted X-inactivation in marsupials.

Techniques in Molecular Biology
D Hancock: 1 lecture

1. Quantitation of gene expression using RT-PCR.

RNA in the control of Information Flow
P Waterhouse: 6 lectures

1. Splicing engines I. Overview of alternative splicing. The spliceosome, snRNPs, lariats and splicing endonucleases.

2. Splicing engines II. snRNP complexes. Key spliceosome participants. Interplay of splicing and sequences.

3. RNA stability. Roles of structure and sequence. Comparison of RNA destruction paths. Surveillance and multiple activities in RNA degradation.

4. Catalytic RNA. Ribozyme reactions. The RNA world. Group I and II introns. Self-cleavage sites of viroids and virusoids. Hammerheads. RNA editing and guide RNAs.

5. microRNA (miRNA) and small interfering RNA (siRNA). Origins and context. Double-stranded RNA, Dicer, siRNA and RNA-induced silencing complexes. Multiple human miRNAs.

6. RNAi therapies. Comparison of dsRNA and antisense in worms. Limitations of gene silencing. Gene targets. Modes of synthesis and delivery. siRNA therapeutic options.

Maintenance of Genomic Integrity
A Weiss: 6 lectures

1. Homologous recombination. How do our chromosomes contribute to genetic diversity? How does homologous recombination affect what we inherit from our parents and what happens when it goes wrong?

2 Molecular machines in recombination. DNA recombination events do not simply involve nucleic acids – they rely on the interplay of molecular machines. Typically these protein machines coordinate their activities to manage remarkably precise processes.

3 Short-range repair of DNA. Telomeres truncate with age while our DNA constantly needs repair. UV light, chemicals and radiation can cause local damage and contribute to an aged appearance and cancer.

4 Model systems for DNA repair. How is specific DNA repaired at the molecular level? Part of the process adapts familiar features from recombination. Other parts of the process rely heavily on the careful alignment and restoration of DNA by comparing strands and using proteins that bind these DNA segments.

5 Mobile elements. Transposable elements are an important source of variation in nearly all genomes. Transposons can supply their own sequences to effect this process but can also rely on endogenous molecular machinery. They can proceed through the movement of DNA or move through DNA copies of RNA intermediates. On this basis, the host genome is in a regular state of flux.

6 What happens when our DNA changes? What happens when our DNA changes? Accidental recombination can be life-altering and life-threatening. Xeroderma pigmentosum is an inherited disorder that dramatically reduces a person’s ability to repair damaged DNA. Genetic testing of BRCA1/2 is profoundly influencing the diagnosis of specific hereditary breast-ovarian cancer syndromes. Yet experimental knowledge of how to precisely recombine DNA, particularly using Cre and Lox, can teach us a lot about how genes function.

Practical Course

P1 Genetic Manipulation and DNA Analysis (2 weeks)
P2 Southern Transfer and DNA Hybridization (2 weeks) BCHM3071
P3 Control of Gene Expression in C. elegans using siRNA (2 weeks) BCHM3971
P4 RT-PCR (2 weeks)

Assessment

Lecture course: 50% (end-of-semester examination)
Practical course: 50% (25% in-semester practical work, 25% end-of-semester examination)