Molecular Cell Biology of the Yeast Saccharomyces cerevisiae

1
Molecular Cell Biology of the Yeast
Saccharomyces cerevisiae

• Lecture I Biology, Genetics, Genomics and
  Proteomics
• Zhang Yi,
• National Institute of Biological Sciences,
  20080511

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Cellular Anatomy
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Life History
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Cell Cycle
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Mitosis V.S Meiosis
a
a
a
a
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Sexual Reproduction Mating
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Mating Type Switch
x
HO knockout renders haploid stable in propagation
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Filamentous Growth
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Cellular Polarity(Morphogenesis)
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Cellular Polarity(Protein Dynamics)
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Cellular Polarity(mRNA Localization Maternal
Effect)
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Phenotype Variegation
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Fermentation
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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

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Chromosomes
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Genetic Nomenclature
Sherman 2002
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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

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Vector Components
Transcription Terminator
Selectible Markers
Multiple Cloning Site (MCS)
Centromere sequence or Plasmid replication origin
Transcription Promoter
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Promoters on Vectors
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Genomic Library
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Classical Mutagenesis
Mutagen (usually toxic drug)
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Transposon Mutagenesis
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PCR-based Gene Engineering
40-50bp homology arm each side
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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

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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

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Strategy in Genetic Mapping
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Conditional Mutant Example
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Complementation
YFG ts (recessive)
Vector with Genomic Fragment Containing YFG
Dead at 37c
Live at 37c
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Non-complementation (Allelic)
YFG ts
Hybrid Mating with Lines having Known Mutations
that have similar phenotype
Known mutant
Dead at 37c
Dead at 37c
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Non-complementation (dominant negative)
YFG ts (DN)
Vector with Genomic Fragment Containing YFG
Dead at 37c
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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
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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)
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Mitotic and Meiotic Mapping
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Genetic Interaction

• Upstream mutantA mutantB phenotype B
• Downstream mutantA mutantB phenotype A
• Enhancer mutantA mutantB enhanced phenotype
  B
• Suppressor mutantA mutantB suppressed
  phenotype B

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Genetic Interaction
Hedgehog
Patched
wg
Wild type
hh
wg
ptc
HSHh
HSWg
ptc,hh
HSPtc
ptc,wg
Drosophila Larvae Pattern (simplified)
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Genetic Interaction
Luck ()
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Suppressor Mechanism
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Suppressor Mechanism
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Suppressor Example
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Suppressor Screen
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Synthetic Lethal Screen
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Plasmid Shuffle
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Hybrid Genetic Interaction Screen (EMAP)
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Genomic Method(Tiling Array)
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Proteomic Methods(Two-hybrid)
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Proteomic Methods(Tandem Affinity Purification)
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Proteomic Methods(Tandem Affinity Purification)
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Combined Proteomic Methods(Y2H and TAP)
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Combined Proteomic/Genomic Method (ChIP-on-Chip)