A1258568791HIsMn

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Mating Test
a/? or sterile?
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Mating Pheromone Halo Assay
WT
paf1?
mal13?
yvh1?
pkh2?
srb2?
rpb4?
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Pheromone Halo Test
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Mating type Switching
HO Strain
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Two Models for Mating Type Switching
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Mating Type Switching
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Silencing of the Mating Type Loci
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Switching is asymmetric
Switch
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Mating-Type Switching is cell-type dependent
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Regulation of HO
WT ----gt HO swi -----gt ho swi/sin ----gt HO
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Repression in daughter cells
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Cell cycle regulation of HO
WT ----gt HO swi -----gt ho swi/sin ----gt HO
SWI5 exp.
HO expression
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Synthetic Lethality
If over express CLN2 then suppress lethality
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Cell Type MAT Gene Expression (genotype) Mat
ing SPO transcript ?SG aSG HSG SSG MATa mates
with ? - a1 - - MAT? mates with a
- ?1 and ?2 - - MATa/MAT? nonmater
a1 and ?2 - - - mat?1
(?2) nonmater - ?2 - - - mat?2
(?1) nonmater - ?1 - ?mat mates
with ??alf - none -
- MATa/?mat mates with ? - a1 -
- MAT?/?mat mates with a - ?1 and ?2 -
- MATa/mat?1 nonmater a1 and ?2 - -
- MATa/mat?2 bimater (weak) - a1 and ?1
-
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Regulation of mating type
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Gain of Function Gene Function Null
Phenotype Phenotype MFA1,2 a factor
pheromone a-specific sterile MF?1,2 ?-factor
pheromone ?-specific sterile BAR1 ?-factor
degradation ?-factor sensitive STE6 pheromone
secretion sterile STE2 ?-factor
receptor a-specific sterile STE3 a-factor
receptor ?-specific sterile GPA1 G?
subunit constitutive (lethal) sterile
STE4 G? subunit sterile constitutive
(lethal) STE18 G? subunit sterile STE5 Nucleation
factor sterile FUS3 protein kinase sterile (if
kss1-) KSS1 protein kinase sterile (if
fus3-) STE7 protein kinase sterile STE11 protein
kinase sterile constitutive (lethal) STE12 Transcp
t Activator sterile AGA1 cell agglutination steri
le (bilaterally) FUS1 cell fusion sterile
(bilaterally) KAR3 nuclear fusion heterokaryotic S
ST2 recovery from arrest ?-factor sensitive
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Processing of ? pheromone - Mating factor
Mutants
Lethal
kex2
kex1
ste13
?-specific, Not sterile in a cells
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Genotype Phenotype gpa1 const. ste4-
sterile gpa1 ste4- sterile
Genotype Phenotype STE4GF constit. ste12
sterile STE4GF ste12 sterile
Genotype Phenotype STE11GF constit ste4
sterile STE11GFste4 constit
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Fus3 Far1
Fus1/Fus2
Aga1/2 Ste2/3 Mfa1/2
Kar1
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SPT Screen
SPT1 - HIR2, Histone transcriptional
regulator SPT2 - SIN1, HMG chromatin protein that
regulates HO SPT3, 7, 8 - Components of the
nucleosomal histone acetyltransferase SAGA
complex, SPT4, 5, 6 - involved in chromatin
structure, influence expression of many
genes SPT10 - CRE1- amplifies the magnitude of
transcriptional regulation at various loci. SPT11
- HTA1 - Histone H2A SPT12 - HTB1 - Histone
H2B SPT13 - GAL11 - Component of RNA polymerase
holoenzyme, part mediator complex SPT14 - GPI3
N-acetylglucosaminyltransferase, required for GPI
anchor synthesis SPT15 - TBP1 TATA-binding
component of RNA polymerase transcription factor
TFIID
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Promoter Fusion Assays
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Checkpoint
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Meiosis is blocked by the pachytene checkpoint