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Molecular Cell Biology of the Yeast Saccharomyces cerevisiae

Molecular Cell Biology of the Yeast Saccharomyces cerevisiae

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Molecular Cell Biology of the Yeast Saccharomyces cerevisiae

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

  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 YFG ts (recessive) Vector with Genomic Fragment Containing YFG+ Dead at 37c Live at 37c

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

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

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

  32. Non-complement(2um deficiency) x mutant CEN FRT CEN FLP x 2um (FLP+) mutant CEN FRT CEN x mutant CEN 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 Hedgehog Patched wg hh Wild type ptc wg ptc,hh HS:Hh HS:Wg ptc,wg HS:Ptc Drosophila Larvae Pattern (simplified)

  36. Genetic Interaction Luck (…)

  37. Suppressor Mechanism

  38. 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)

  47. Proteomic Methods(Tandem Affinity Purification)

  48. Combined Proteomic Methods(Y2H and TAP)

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