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.

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.

Mrs Jill Johnston

Room: 410

Telephone: 9351 4248

FAX: 9351 4726

E-mail: j.johnston@usyd.edu.au

Professor Merlin Crossley

Room: 780

Telephone: 9351 2233

FAX: 9351 4726

E-mail: merlinc@usyd.edu.au

For BCHM3071
(MBLG (1001 or 1901) and 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/MBLG2971or BCHM2071/2971 or BCHM2072/2972)) or (42CP of Intermediate BMedSc units, including BMED2802 and BMED2804)

For BCHM3971
MBLG (1001 or 1901) and Distinction in 12 CP of Intermediate BCHM/MBLG units (taken from MBLG2071/MBLG2971 or BCHM2071/2971 or BCHM2072/2972) or 42CP of Intermediate BMedSc units, with Distinction in BMED2802 and BMED2804.

1st Lecture: Monday 9 am Carslaw Lecture Theatre 275

2nd Lecture: Tuesday 9 am Carslaw Lecture Theatre 275

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.

For BCHM3071/3971, the recommended textbook is:

Lewin B Genes IX (9th edition, Jones & Bartlett, 2008)


Reference texts

Brown T A Gene Cloning and DNA analysis (5th edition, Blackwell Science, 2006)

Primrose, S B & Twyman, R M Principles of Gene Manipulation and Genomics (7th edition, Blackwell Science, 2006)

Gene Architecture and Regulatory Mechanisms
M Crossley: 7 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; CpG islands and distribution of CpG; repeat elements, short and long, retrotransposons, repetitive DNA, chromosomes, centromeres, telomeres.
Experimental evidence: sequencing projects, homology searching.

2. Chromosome structure, euchromatin/heterochromatin, histones, structure of the nucleosome, structure of histones, histone variants, modification of histones, histones in other organisms; nuclear structure, nucleolus, compartmentalization of transcription and translation, transcription factories.
Experimental evidence: structural analysis, yeast genetics, immunofluorescence.

3. Regulatory Elements in DNA. Defining Promoters, Enhancers, and Locus Control Regions, theoretically and experimentally; mechanisms of promoter action, different types of RNA polymerase, basal transcriptional apparatus and contact with activators/repressors; models for enhancer action; position effects, instability of stable expression in cell lines; importance of different elements in different organisms, promoteromics.
Experimental techniques: DNase mapping; reporter genes; deletion analysis, transfections; DNA-binding assays; homology searching; 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: affinity purification; expression screening; one hybrid screening; immunoprecipitation, identification of proteins involved in cancer and genetic diseases; 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: discovery of enzymatic activities, histone deacetylase inhibitors, yeast 2-hybrid system, co-immunoprecipitation, importance of yeast regulatory mechanisms, mating type loci.

6. 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, ecdysone receptor; control circuits and importance of positive versus negative regulation, short range versus long range regulation, insulators in different organisms; importance of viral co-regulators, E1A, Large T Antigen, Rb, and E2F etc.
Experimental techniques: genetic screens for regulatory proteins, protein mapping, functional studies, ligand binding.

7. Transcription factors in development. Biological roles of key regulatory proteins: Pax-6, MyoD, Pit-1, GATA-1, Sry, Rb; identification of target genes; defects arising from mutations in regulatory genes, types of mutations; defects in co-regulatory proteins, CBP, MeCP2.
Experimental techniques: micro-arrays; inducible transcription factors; analysis of mutants.
Validation of Regulatory Processes. Transgenic mice and knockout mice, including conditional knockouts, lox-cre etc, examples where knockouts have been informative or surprising; the problem of genetic redundancy and gene families; stem cells and overall picture of cellular differentiation as a result of gene regulation. Experimental techniques: transgenic technology, knockout approaches.

Techniques in Molecular Biology
D Hancock: 1 lecture

1. Quantitation of gene expression using RT-PCR. Detection of specific DNA sequences using Southern blotting

Gene Regulation in Development
H Nicholas: 4 lectures

1. Molecular development I: transcription factors in lineage specification and differentiation. Introduction to the concept of cell lineage, C. elegans as a model in which to study the molecular events of lineage specification, example of transcription factor cascade in the specification of the worm endoderm, example of transcription factors cascade in the specification of the nervous system and the role of the transcription factor code in controlling terminal differentiation of cell types within this system.

2. Molecular development II: transcription factors in positional specification. Introduction to the concept of positional identity using C. elegans as an example, 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.

3. Epigenetics I: DNA methylation in epigenetics. Review definition of epigenetics from lecture 2, review of methylation as an epigenetic modification of DNA, methylation during development, parental imprinting, examples of imprinting disorders - Prader-Willi and Angelman syndromes, loss of imprinting in cancer.

4. Epigenetics II: X-inactivation. Dosage compensation and random X-inactivation, role of Xist RNA, Tsix-mediated repression of Xist accumulation, polycomb proteins and DNA methylation in the maintenance of X-inactivation, non-random/imprinted X-inactivation in marsupials.

RNA in the control of Information Flow
A Weiss: 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 the Genome
D Gell: 6 lectures

1. DNA damage and mutation. DNA is under constant attack from cellular and environmental sources and damage that is not detected and/or repaired can lead to genomic mutation. The sources of DNA damage and the chemical changes that occur in DNA will be reviewed. A distinction between cytotoxic and mutagenic DNA damage will be made.

2. DNA damage response pathways. The term ‘DNA damage response’ refers to the coordinated cellular response elicited by DNA damage, commonly resulting in resolution of the lesion or programmed cell death. The response involves the steps of 1) damage recognition 2) signal transduction/amplification 3) activation of appropriate effector complexes, including cell cycle regulators and damage repair complexes. Specific examples from this protein machinery will be considered.

3. Molecular mechanisms of excision repair. Chemical modifications of DNA bases are recognised and resolved by a large and diverse family of enzymes constituting the ‘excision repair’ pathways. In general, these involve the removal of damaged bases or nucleotides and re-synthesis of DNA using the information on the undamaged strand. The remarkable molecular mechanisms through which excision repair proteins detect abnormal DNA structures and the biochemical pathways of repair will be discussed. The relative importance of individual pathways in protection from environmental agents and the inhibition of mutagenesis will be reviewed with reference to human disorders.

4. Recombination repair and direct end-joining. . Double-stranded breaks in DNA are particularly harmful form of damage and can be resolved by two pathways: 1) homologous recombination or 2) direct DNA end joining. The molecular machinery comprising these pathways will be outlined along with factors that influence the relative activity of each. Homologous (or generalised) recombination occurs both in meiosis and DNA repair, and will be considered in this context. Direct end joining is also required for maturation of the adaptive immune system.

5. Site-specific recombination and mobile genetic elements. As a complement to discussion of generalised recombination, varied mechanisms and functions of site-specific recombination will be covered. Topics include bacteriophage life cycles, immunoglobulin gene rearrangements, transposons and retro-elements. Many of these have relevance to methods in molecular biology and biotechnology.

6. DNA repair, ageing and cancer. The processes of DNA repair, aging and oncogenesis are discussed in relation to one-another using examples from various human disorders and model genetic systems. Concepts of senescence, molecular clocks tumour progression are introduced.

P1 Purification and Analysis of Plasmid DNA
P2 Gene Analysis using Southern Transfer
P3 Gene Expression using RT-PCR

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