molecular cell biology of the yeast saccharomyces cerevisiae n.
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Molecular Cell Biology of the Yeast Saccharomyces cerevisiae. Lecture I: Biology, Genetics, Genomics and Proteomics Zhang Yi, National Institute of Biological Sciences, 20080511. Cellular Anatomy. Life History. Cell Cycle. Mitosis V.S Meiosis. α. α. a. a. Sexual Reproduction: Mating.

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molecular cell biology of the yeast saccharomyces cerevisiae

Molecular Cell Biology of the Yeast Saccharomyces cerevisiae

Lecture I: Biology, Genetics, Genomics and Proteomics

Zhang Yi,

National Institute of Biological Sciences, 20080511

mating type switch
Mating Type Switch

x

HO knockout renders haploid stable in propagation

growth conditions
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
routinely used genetic markers
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
vector components
Vector Components

Selectible Markers

Transcription

Terminator

Multiple Cloning

Site (MCS)

Centromere sequence or

Plasmid replication origin

Transcription

Promoter

classical mutagenesis
Classical Mutagenesis

Mutagen (usually toxic drug…)

pcr based gene engineering
PCR-based Gene Engineering

40-50bp homology arm

each side

alleles and phenotypes
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
critical thinking concepts
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
complementation
Complementation

YFG ts (recessive)

Vector with Genomic Fragment Containing YFG+

Dead at 37c

Live at 37c

non complementation allelic
Non-complementation (Allelic)

YFG ts

Hybrid Mating with Lines having Known Mutations that have similar phenotype

Known mutant

Dead at 37c

Dead at 37c

non complementation dominant negative
Non-complementation (dominant negative)

YFG ts (DN)

Vector with Genomic Fragment Containing YFG+

Dead at 37c

non complement 2um deficiency
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

non complement 2um deficiency1
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)

genetic interaction
Genetic Interaction
  • Upstream: mutantA + mutantB = phenotype B
  • Downstream: mutantA + mutantB = phenotype A
  • Enhancer: mutantA + mutantB = enhanced phenotype B
  • Suppressor: mutantA + mutantB = suppressed phenotype B
genetic interaction1
Genetic Interaction

Hedgehog

Patched

wg

hh

Wild type

ptc

wg

ptc,hh

HS:Hh

HS:Wg

ptc,wg

HS:Ptc

Drosophila Larvae Pattern (simplified)