1. komponenta

Lecture type	Total
Lectures	45
Practicum	45

* Load is given in academic hour (1 academic hour = 45 minutes)

LEARNING OUTCOMES:
1. Explain the importance of mutations in genetic analysis and methods how to introduce mutation into the DNA and genome to generate mutants
2. Explain basics of genetic analysis and how to apply it in the research
3. List and explain principles of DNA repair, recombination, transposition, DNA transfer and their role in genome stability
4. List different types of plasmids and explain their use in the research
5. Using lambda phage model, explain different types of gene expression regulation and antiviral mechanisms
6. Explain and contrast the role of regulatory RNA in gene expression in prokaryotes and eukaryotes. Compare miRNA, siRNA, piRNA and crRNA.
7. Explain heat shock response regulation. List the types of chaperones and explain their role in protein folding.


COURSE CONTENT:

LECTURES:

1. Introduction to the course, the concept of E. coli as a model organism, what is a genome map, modern tools for gene and genome analysis, genome organization
2. Mutations and mutagenesis: types of mutations and their properties, types of mutagens, types of reversions and suppressor mutations
3. Mutant isolation, positive and negative selection, generation of deletion mutants in bacteria, in vitro mutagenesis; TALENS, Zn finger nucleases, CRISPR as tools for genome editing in eukaryotes
4. Measuring mutant phenotype, genetic analysis: allelism tests; complementation; epistasis; ordering mutations
5. DNA repair: photoreactivation; methyl directed mismatch repair; nucleotide and base excision repair;
6. DNA repair: recombinational repair; translesion synthesis; SOS response, example of Xeroderma pigmentosum
7. Homologous recombination: molecular models of recombination in prokaryotes; recombination in eukaryotes
8. Plasmids: properties of plasmids; plasmid structure; incompatibility; plasmid replication control mechanisms
9. F plasmid and conjugation: the mechanisms of DNA transfer, Hfr and F prime strains, gene mapping by Hfr crosses
10. Mobile genetic elements: mechanisms and regulation of transposition; transposons and retrotransposons, application of transposons in genetic analysis
11. Bacteriophages: the phage structure, lytic and lysogenic phages, specialized and general transduction
12. The lifecycle of lambda phage, site specific recombination and its application
13. Regulation of heat shock response, types of chaperons in prokaryotes and eukaryotes
14. Restriction and modification: regulation and application; two component mechanism
15. Regulatory RNA in prokaryotes (riboswitch, attenuation, small RNA, antisense RNA, CRISPR) and eukaryotes (miRNA, siRNA, piRNA)

PRACTICAL WORK:
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 ammount of the recC gene transcripts by qPCR. The exercise includes isolation of total RNA, measuring RNA concentration, preparation of dilutions for qPCR and interpretation of the reaction curves.

SEMINAR:
Demonstration of genetic problems and presentation of recent selected papers.

1. J. E. Krebbs, E. S. Goldstein, S. T. Kilpatrick (Lewins Genes XI): Jones and Bartlett learning, 2014
2. L. Snyder, J. E. Peters, T. M. Henkin and W. Champness (Molecular genetics of bacteria): ASM Press, 4th edition, 2013
3. J. W. Dale and S. F. Park (Molecular genetics of bacteria): Wiley; 5th edition, 2010
4. Ivana Ivančić Baće: Molekularna genetika. Upute za laboratorijske vježbe. PMF (skripta)
5. Odabrani znanstveni radovi s najnovijim dostignućima u području molekularne genetike
6. 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
7. 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.

Enrollment :
Passed : Biochemistry 1
Passed : Genetics
Attended : Biochemistry 2

Examination :
Passed : Biochemistry 2

Mandatory course - Regular study - Molecular Biology