The document discusses yeast molecular biology and genetics. It provides information on commonly used yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. It discusses yeast genome, life cycle, vectors, cloning, transformation methods, and applications including gene expression and making mutants.

1. References: Science 240, 1439-1443 (1988). Methods in Enzymology 194, 3-77 (1991). Science 274, 546-567 (1996). Yeast molecular biology-yeast vectors, expression of proteins in Yeast

3. S. cerevisiae

6. Genome of diploid Saccharomyce cerevisiae cell Characteristic Chromosomes 2-mm plasmid Mitochondiral Relative amount (%) 85 5 10 Number of copies 2 x 16 60-100 ~50 (8-130) Size (kbp) 14,000 6.318 70-76 Mutants All kinds none Cyt a.a 3 , b Yeast genome

7. Yeast life cycle

8. Separation of spore products by tetrad dissection

9. Sporulation and tetrad dissection

10. Tetrad dissection

11. Analysis of spore products Complement medium Selection medium

12. Yeast Molecular Genetics vectors cloning Making mutants Gene expression

13. Yeast vectors Plasmids Yeast artificial chromosome Origin of replication Selection markers Yeast strains

16. A typical yeast plasmid

18. Yeast cloning vectors

19. URA3 : The gene product of URA3 (orotidine-5’-phosphate decarboxylase) converts 5-FOA (5-fluoroorotic acid) to a toxic product that kills the URA3 cells. LYS2 : The LYS2 gene encodes a-aminoadipate reductase, an enzyme required for lysine biosynthesis. Yeast cells with wild-type LYS2 activity will not grow on media containing  - aminoadipate (  -AA) as a primary nitrogen source. CAN1 : The CAN1 gene encodes an arginine permease. In the absence of arginine, canavanine (arginine analog) is readily incorporated into proteins with lethal consequences; therefore, CAN1 cells are sensitive to canavanine. CYH2 : The CYH2 gene encodes the L29 protein of the yeast ribosome. Cycloheximide blocks translation elongation by interacting with L29. Yeast negative selection systems:

24. Cloning by complementating a temperature sensitive mutant

26. Gene expression in yeast Copy number Promoter Protease problem

31. Gene targeting scheme:

32. Gene targeting scheme: