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Mating Test

Mating Test. a/  or sterile?. Mating Pheromone Halo Assay. WT. paf1 . mal13 . yvh1 . pkh2 . srb2 . rpb4 . Pheromone Halo Test. Mating type Switching. HO Strain. Two Models for Mating Type Switching. MAT a or MAT . mat . STE. ste . Silencers. Mating Type Switching.

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Mating Test

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  1. Mating Test a/ or sterile?

  2. Mating Pheromone Halo Assay WT paf1 mal13 yvh1 pkh2 srb2 rpb4

  3. Pheromone Halo Test

  4. Mating type Switching HO Strain

  5. Two Models for Mating Type Switching MATa or MAT mat STE ste

  6. Silencers Mating Type Switching

  7. Silencing of the Mating Type Loci

  8. Mate Switching is asymmetric Switch

  9. Mating-Type Switching is cell-type dependent

  10. Regulation of HO WT ----> HO swi -----> ho swi/sin ----> HO

  11. Repression in daughter cells

  12. Cell cycle regulation of HO WT ----> HO swi -----> ho swi/sin ----> HO SWI5 exp. HO expression

  13. Synthetic Lethality If over express CLN2 then suppress lethality

  14. Cell Type MAT Gene Expression (genotype) Mating SPO transcript SGaSGHSGSSG 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 + + + -

  15. Regulation of mating type

  16. 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 Transcpt Activator sterile AGA1 cell agglutination sterile (bilaterally) FUS1 cell fusion sterile (bilaterally) KAR3 nuclear fusion heterokaryotic SST2 recovery from arrest -factor sensitive KSS1 or FUS3 X --->Y

  17. Processing of  pheromone - Mating factor Mutants Lethal kex2 kex1 ste13 -specific, Not sterile in a cells

  18. Genotype Phenotype gpa1 = const. ste4- = sterile gpa1ste4- = sterile Genotype Phenotype STE4GF = constit. ste12 = sterile STE4GFste12= sterile Genotype Phenotype STE11GF = constit ste4 = sterile STE11GFste4 = constit

  19. Fus3 Far1 Fus1/Fus2 Aga1/2 Ste2/3 Mfa1/2 Kar1

  20. 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 transcriptionfactor TFIID

  21. Promoter Fusion Assays

  22. Checkpoint

  23. Meiosis is blocked by the pachytene checkpoint

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