1. Molecular Cell Biology of the Yeast Saccharomyces cerevisiae Lecture I: Biology, Genetics, Genomics and Proteomics Zhang Yi, National Institute of Biological Sciences, 20080511

2. Cellular Anatomy

3. Life History

4. Cell Cycle

5. Mitosis V.S Meiosis α α a a

6. Sexual Reproduction: Mating

7. Mating Type Switch x HO knockout renders haploid stable in propagation

8. Filamentous Growth

9. Cellular Polarity (Morphogenesis)

10. Cellular Polarity (Protein Dynamics)

11. Cellular Polarity (mRNA Localization & Maternal Effect)

12. Phenotype Variegation

13. Fermentation

14. Growth Conditions Liquid: 30c O/N –Doubling: 90min in YPD and 120min in SC Solid phase: 30c, 2-3 days Medium: YPD/YPG/SC/SD (see ref) Selection: nutrient deficiency, colour, reporter gene expression, temperature sensitivity, chemical resistance, et ceterus

15. Chromosomes

16. Genetic Nomenclature Sherman 2002

17. Routinely Used Genetic Markers HIS3: histidine synthesis, positive selection in his- SC medium, epistatic selection together with 3-AT LEU2: leucine synthesis, positive selection in leu- SC medium. TRP1: tryptophan synthesis, positive selection in trp- SC medium, negative selection on FAA containing medium URA3: uracil synthesis, positive selection in ura- SC medium, negative selection in 5-FOA containing medium ADE2: adenine synthesis, positive selection in ade- SC medium, ade2 colony red coloured in low ade, complete medium CAN1: histidine and arginine transporter, negative selection in canavanine containing medium LacZ: transgenic reporter, colony blue coloured in X-Gal containing medium upon expression

18. Vector Components Transcription Terminator Transcription Promoter Multiple Cloning Site (MCS) Selectible Markers Centromere sequence or Plasmid replication origin

19. Promoters on Vectors

20. Genomic Library

21. Classical Mutagenesis Mutagen (usually toxic drug…)

22. Transposon Mutagenesis

23. PCR-based Gene Engineering 40-50bp homology arm each side

24. Alleles and Phenotypes Wild-type allele: phenotypically normal function (maybe different DNA sequence) Hypermorph: increased function Hypomorph: lowered function Null: no function whatsoever, completely dead Neomorph: novel function gained (…) Conditional: interacting with certain environmental conditions, e.g. can grow at 23c but not at 37c, can only grow in histidine containing medium, etc Dominant: Suppresses allelic phenotype Recessive: Suppressed by allelic phenotype

25. Critical Thinking Concepts Gain-of-Function: a mutation such that confers novel, or enhanced function of a gene –Provides sufficiency Loss-of-Function: a mutation such that lowered or abolished the function of a gene –Provides necessity Epistasis: interaction between genes

26. Strategy in Genetic Mapping

27. Conditional Mutant Example

28. Complementation Vector with Genomic Fragment Containing YFG+ YFG ts (recessive) Dead at 37cLive at 37c

29. Non-complementation (Allelic) Hybrid Mating with Lines having Known Mutations that have similar phenotype YFG ts Dead at 37c Known mutant

30. Non-complementation (dominant negative) Vector with Genomic Fragment Containing YFG+ YFG ts (DN) Dead at 37c

31. Non-complement (2um deficiency) FRTCEN Cir0, mat(alpha) lines with FRT inserted on different chromosomes 2um (FLP+) mutant x FRTCEN 2um (FLP+) mutant x Cir+, mat(a) mutant line

32. Non-complement (2um deficiency) FRTCEN 2um (FLP+) mutant x CEN mutant x FRTCEN FLP CEN mutant x FLP-mediated loss of FRT-inserted chromosome IF the mutant loci is on this FRT-inserted chromosome, it will reveal phenotype (haploid of recessive mutant)

33. Mitotic and Meiotic Mapping

34. Genetic Interaction Upstream: mutantA + mutantB = phenotype B Downstream: mutantA + mutantB = phenotype A Enhancer: mutantA + mutantB = enhanced phenotype B Suppressor: mutantA + mutantB = suppressed phenotype B

35. Genetic Interaction Wild type hh wgptc HedgehogPatchedwg Drosophila Larvae Pattern (simplified) HS:Ptcptc,wg HS:HhHS:Wgptc,hh

36. Genetic Interaction Luck (…)

37. Suppressor Mechanism

39. Suppressor Example

40. Suppressor Screen

41. Synthetic Lethal Screen

42. Plasmid Shuffle

43. Hybrid Genetic Interaction Screen (EMAP)

44. Genomic Method (Tiling Array)

45. Proteomic Methods (Two-hybrid)

46. Proteomic Methods (Tandem Affinity Purification)

48. Combined Proteomic Methods (Y2H and TAP)

49. Combined Proteomic/Genomic Method (ChIP-on-Chip)