Bi190 Mating Type Interconversion

Transcription

1 Ir Herskowitz Bi190 Mting Type Interconversion 2011 Sternberg, Cltech

2 Scchromyces cerevisie (budding yest) Life Cycle mting / zygote sporultion -C -N Ascus with 4 spores germintion +C +N hploid cells

3 specific genes Production of fctor Agglutintion Response to -fctor non specific genes Response to pheromones specific genes mting mting Production of - fctor Agglutintion Response to fctor

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5 Scchromyces cerevisie (budding yest) Life Cycle mting / zygote sporultion -C -N Ascus with 4 spores germintion +C +N hploid cells

6 Homothllism: single hploid spore gives rise to diploid cells tht cn undergo meoisis nd sporultion cells (-fctor producing) shmoos zygotes / / / / ho HO

7 The genetic elements controlling mting type interconversion were defined by nturl vrints. In current nomenclture: S. cerevisie HML MAT HMR ; HO S. cerevisie HML MAT HMR ; ho S. oviformis HML MAT HMR ; HO S. norbensis HML MAT HMR ; HO S. distticus HML MAT-inc HMR ; HO

8 HO is necessry for Diploidiztion MAT MAT HO ho HO Dip(loidizer) ho -mter HO Dip ho mter Tetrtype Ascus

9 HMR is necessry for to HML MAT HMR ; HO HML MAT HMR ; HO Dip HML MAT HMR HO Dip HML MAT HMR HO Dip HML MAT HMR HO HML MAT HMR HO Dip HML MAT HMR HO Dip HML MAT HMR HO Dip HML MAT HMR HO Dip HML MAT HMR HO Dip

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11 Mting Type Interconversion: the moleculr level 200 kb 150 kb HML MAT HMR CEN III HO E I W X Y ZL W X Y Z X Y ZR E I W Y Y Z L Z R ~700 bp ~600 bp ~750 bp ~300 bp ~250 bp HO cuts t ~18 bp site -inc is the site The event is non-reciprocl --the cssette is lost. Gene Conversion vi double-strnd brek repir

12 Tests of the cssette model In 10% of the 10-6 ho switches, There is deletion, clled -lethl or Hwthorne deletion: this is fusion of MAT to HMR HML MAT HMR

13 Tests of the cssette model Heling: HO HML mt1-5 HMR Sterile cn switch to n -mter Wounding: HO HML 66 MAT HMR -mter switches from to Ste to to Ste

14 Mting Type Interconversion: the moleculr level E 200 kb 150 kb HML MAT HMR CEN III HO I W X Y ZL W X Y Z X Y ZR E I W Y Y Z L Z R ~700 bp ~600 bp ~750 bp ~300 bp ~250 bp HO cuts t ~18 bp site -inc is the site E, essentil for silencing I, importnt for silencing The event is non-reciprocl --the cssette is lost. Gene Conversion vi double-strnd brek repir

15 Tests of the cssette model mutgenize: ho HML mt1-5 HMR Sterile pick -mter sir1-1 (silent regultor) nlyze: ho HML mt1-5 HMR ; sir1-1 -mter ho HML mt1-5 HMR ; sir1-1 Sterile So, it depends on HML!

16 Jsper Rine: sir1-1 sir1-1 Spo+ Thus, HML cn provide function sir1 sir2 = mr1 sir3 = ste8 = cmt sir4 = ste9 The SIR proteins silence the HML nd HMR loci

17 Mting Type Interconversion: the moleculr level 200 kb 150 kb HML MAT HMR CEN III HO E I W X Y ZL W X Y Z X Y ZR E I W Y Y Z L Z R ~700 bp ~600 bp ~750 bp ~300 bp ~250 bp HO cuts t ~18 bp site -inc is the site E, essentil for silencing I, importnt for silencing The event is non-reciprocl --the cssette is lost. Gene Conversion vi double-strnd brek repir

18 Pedigree nlysis mother dughter mother dughter Shmoo (G1 rrest t START) or but not / cells switch Only mothers switch Switching in pirs Directionlity: to to Strthern, Hicks & Herskowitz (Cell 1979)

19 Pedigree nlysis mother dughter mother dughter or but not / cells switch Only mothers switch Switching in pirs HO is Off in / cells Off in dughters On in lte G1 Nsmyth (1983)

20 Pedigree nlysis mother dughter mother dughter SWI+ SIN+ swi5 SIN3+ SWI5+ sin3 SWI5 is n ctivtor but is present in both mothers nd dughters SIN3 is repressor tht is ntgonized by SWI5, so we need to find Genes tht regulte SWI5

21 Sil & Herskowitz: Screen for microcolonies in which mother nd dughters switch (1/66,000) mother dughter mother dughter ASH1+ sh1-1 ASH1 is trnscriptionl repressor only in dughter cell nuclei ASH1 protein is unstble nd gene expressed only t M/G1trnsition ASH1 mrna is loclized preferentilly to dughter cells.

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23 HO expression in mother versus dughter cells Nsmyth: SWI5-dependent ctivtors of HO Expression: Screen for genes necessry for HO-lcZ expression but not For GAL1-URS1-HO-lcZ expression 222 swi5 mutnts, 315 she mutnts, 5 complemention groups The SHE proteins re necessry for ASH1 mrna locliztion Trnsport out of mother cells SHE1 (MYO4) encodes non-muscle myosin

24 Figure 3. The Effect of the she Mutnts on Prticle Locliztion nd Formtion Yest strins disrupted for ech one of the five SHE genes were trnsformed with the ASH1 reporter RNA nd the GFP-MS2 fusion protein nd the resultnt prticles observed by epifluorescence fter fixtion. Br, 5 mm. (A) Wild-type cells (K699). Locliztion of the prticle nd its formtion is inhibited in (B) she5 deletion strin (K5205): 36% of cells with signl formed bright, single prticles; pproximtely hlf of the prticles were loclized t the bud neck nd 2% were loclized in the bud. (C) she3 deletion strin (K5235): 6% formed bright, single prticles nd 0% were loclized in the bud. (D) she1 deletion strin (K5209): 16% of cells with signl formed bright, single prticles nd 0% were loclized in the bud. (E) she2 deletion strin (K5547): 0% formed bright, single prticles nd 0% were loclized in the bud. (F) she4 deletion strin (K5560): 32% formed bright, single prticles nd 16% with signl were loclized in the bud. Colocliztion (yellow) of functionl myctgged she protein (red) with the prticle (green). (G) She1myc. (H) She2myc. (I) She3myc. (J I) She1myc with nonloclized prticle in nonbudding cell. (Hlf of the prticles showed colocliztion with She1myc.) Moleculr Cell, Vol. 2, , October, 1998

25 Directionl switching: there is recombintionl enhncer on the left rm of III tht is ctivted in but not cells. (see Hber Annul Review of Genetics) HML RE CEN3 MAT HMR

26 Hber (Ann Rev Genetics 1998)