EXPECTED COMPETENCES TO BE ACQUIRED:
1. Explain benefits and shortcommings in work with Escherichia coli.
2. List and compare different types of mutations and explain their properties. Suggest the most likely mutation based on the mutant phenotype.
3. Explain and contrast the advantages and disadvantages of each of the approaches to generate mutants. Design primers for making point and deletion mutant.
4. List and contrast different types of mutagens.
5. Explain types of suppressor mutations, their importance in genetic analyis, name examples.
6. Describe and contrast the advantages and disadvantages of new methods for genome editing in eukaryotes.
7. List and explain DNA repair pathways, the role of DNA repair mechanisms in generation of mutations and viability. Name human diseaeses caused by mutations DNA repair genes.
8. Explain and contrast the mechanisms and homologous recombination pathways in prokaryotes with those in eukaryotes.
9. List and contrast different types of mobile genetic elements and their mechanisms of transfer.
1. Physiology of Escherichia coli and the bacterial genome: features of the genome, bacterial genome annotation, naming genes, useful databases on internet
2. Mutations and mutagenesis: types of mutations; spontaneous and induced mutations; types of mutagens; reversion versus suppression, isolating mutants; in vitro mutagenesis. Useful methods for generating mutants in prokaryotes and eukaryotes.
3. Genetic analysis in bacteria: selecting mutants; allelism test; complementation test; epistasis;
4. DNA repair: specific repair pathways (photo reactivation), general repair pathways (nucleotide excision repair, methyl directed mismatch repair), DNA damage tolerance mechanism (SOS inducible repair, translesion synthesis).
5. Homologous recombination in prokaryotes and eukaryotes: molecular models of recombination; the molecular basis for recombination in E. coli; recombination in eukaryotes
7. Plasmids: plasmid structure, properties of plasmids; plasmid replication control mechanisms; plasmid incompatibility;
8. F-plasmid and conjugation: mechanism of DNA transfer during conjugation; formation of Hfr strains and prime factors; genetic mapping by Hfr crosses
9. Mobile genetic elements: transposons and retrotransposons
10. Bacteriophages: lytic and lysogenic phages; general and specialized transduction; site-specific recombination and families of recombinases
11. Lambda phage life cycle: lytic development; phage integration; phage induction
12. Antiviral mechanisms: restriction and modification; CRISPR.
13. Regulatory small RNAs: miRNA, siRNA, piRNA.
14. Heat shock response and chaperons: protein folding; heat shock regulation, the role in cancer
The practical follows genetic analysis of the recC null mutant which has suppressor mutation in the promoter region.
1. Cloning of the recC gene and its use to complement recC mutation by measuring sensitivity to DNA irradiation. The exercise includes PCR, restriction digest, ligation and transformation by ligation mixture, blue-white selection, and verification of the positive clone by the PCR.
2. Determining the role of suppressor mutation in promoter region of the recC gene in comparison to wt and recC null mutant by measuring the rate of the recC gene expression (transcription) by RT-PCR. The exercise includes isolation of total RNA, measuring RNA concentration, preparation of dilutions for qRT-PCR and interpretation of the reaction curves.
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- Ivana Ivančić Baće: Molekularna genetika. Upute za laboratorijske vježbe. PMF (skripta)
- Friedberg, E.C., Walker, G.C., Siede, E., Wood, R.D., Schultz, R.A. Ellenbergert, T. (DNA repair and mutagenesis): ASM Press, Washington, D.C., 2005
D. L. Nelson, M. M. Cox. Lehninger, Principles of biochemistry. 6th edition., W. H. Freeman & Co. New York 2013.
- Trun, N., Trempy, J., 2004: Fundamental bacterial genetics. Blackwell Publishing, Oxford.Streips, U.N., Yasbin, R.E., 2000: Modern microbial genetics. John Wiley and Sons Inc., New York
- Odabrani znanstveni radovi s najnovijim dostignućima u području molekularne genetike