Bi190 Mating Type Interconversion - its.caltech.edubi190/bi190-2011-13.pdf~600 bp Y α ~750 bp Z L...
Transcript of Bi190 Mating Type Interconversion - its.caltech.edubi190/bi190-2011-13.pdf~600 bp Y α ~750 bp Z L...
Bi190 �Mating Type Interconversion
2011 Sternberg, Caltech
Ira Herskowitz
a
α a
a
α
α
a/α zygote
Ascus with 4 spores
haploid cells
-C -N
mating
sporulation
germination +C +N
Saccharomyces cerevisiae (budding yeast) Life Cycle
α- specific genes
a - specific genes
non - specific genes
α mating
a mating
Production of α- factor Agglutination Response to a-factor
Production of a- factor Agglutination Response to α- factor
Response to pheromones
a
α a
a
α
α
a/α zygote
Ascus with 4 spores
haploid cells
-C -N
mating
sporulation
germination +C +N
Saccharomyces cerevisiae (budding yeast) Life Cycle
a a α α
α cells (α-factor producing)
a a α α α α
a a a a α α α α
a/α a/α
a/α a/α
ho HO
Homothallism: single haploid spore gives rise to diploid cells that can undergo meoisis and sporulation
shmoos zygotes
The genetic elements controlling mating type interconversion were defined by natural variants.
In current nomenclature:
S. cerevisiae HMLα MATα HMRa ; HO
S. cerevisiae HMLα MATα HMRa ; ho
S. oviformis HMLa MATα HMRa ; HO
S. norbensis HMLα MATα HMRα ; HO
S. diastaticus HMLα MATα-inc HMRa ; HO
MATa HO MATα ho
a HO Dip(loidizer) a ho a-mater α HO Dip α ho α mater
Tetratype Ascus
HO is necessary for Diploidization
HMLα MATa HMRa ; HO HMLα MATα HMRα ; HO
HMLα MATa HMRα HO Dip HMLα MATa HMRa HO Dip HMLα MATα HMRα HO α HMLα MATα HMRa HO Dip
HMLα MATa HMRα HO Dip HMLα MATa HMRα HO Dip HMLα MATα HMRa HO Dip HMLα MATα HMRa HO Dip
HMRα is necessary for a to α
Dip α
HMLα MATα HMRa
W X Yα ZL W X Yα Z X Ya ZR E EI I
W ~700 bp Ya ~600 bp Yα ~750 bp ZL ~300 bp ZR ~250 bp
CEN III
200 kb 150 kb
HO
HO cuts at ~18 bp site α-inc is the site
Mating Type Interconversion: the molecular level
The event is non-reciprocal --the cassette is lost. Gene Conversion via double-strand break repair
Tests of the cassette model
In 10% of the 10-6 ho switches, There is a deletion, called a-lethal or a Hawthorne deletion:
this is a fusion of MAT to HMRa
HML MAT HMR
Healing:
HO HMLα matα1-5 HMRa Sterile
can switch to an α-mater
Wounding:
HO HMLα-66 MATa HMRa a-mater
switches from a to Ste to a to Ste
Tests of the cassette model
HMLα MATα HMRa
W X Yα ZL W X Yα Z X Ya ZR E EI I
W ~700 bp Ya ~600 bp Yα ~750 bp ZL ~300 bp ZR ~250 bp
CEN III
200 kb 150 kb
HO
HO cuts at ~18 bp site α-inc is the site
Mating Type Interconversion: the molecular level
The event is non-reciprocal --the cassette is lost. Gene Conversion via double-strand break repair
E, essential for silencing I, important for silencing
mutagenize:
ho HMLα matα1-5 HMRa Sterile
pick α-mater
sir1-1 (silent regulator)
analyze: ho HMLα matα1-5 HMRa ; sir1-1 α-mater
ho HMLa matα1-5 HMRa ; sir1-1 Sterile
So, it depends on HML!
Tests of the cassette model
sir1 sir2 = mar1 sir3 = ste8 = cmt sir4 = ste9
The SIR proteins silence the HML and HMR loci
Jasper Rine:
a sir1-1 a sir1-1 Spo+
Thus, HMLα can provide α function
HMLα MATα HMRa
W X Yα ZL W X Yα Z X Ya ZR E EI I
W ~700 bp Ya ~600 bp Yα ~750 bp ZL ~300 bp ZR ~250 bp
CEN III
200 kb 150 kb
HO
HO cuts at ~18 bp site α-inc is the site
Mating Type Interconversion: the molecular level
The event is non-reciprocal --the cassette is lost. Gene Conversion via double-strand break repair
E, essential for silencing I, important for silencing
a a α
α
α α
α
Pedigree analysis
mother daughter
mother daughter
a or α but not a/α cells switch Only mothers switch Switching in pairs Directionality: a to α α to a
Strathern, Hicks & Herskowitz (Cell 1979)
Shmoo (G1 arrest at START)
a a α
α
α α
α
Pedigree analysis
mother daughter
mother daughter
a or α but not a/α cells switch Only mothers switch Switching in pairs Nasmyth (1983)
HO is Off in a/α cells Off in daughters On in late G1
a a α
α
α α
α
Pedigree analysis
a a
α α
a a
α α
SWI+ SIN+
swi5 SIN3+
SWI5+ sin3
mother daughter
mother daughter
SWI5 is an activator but is present in both mothers and daughters SIN3 is a repressor that is antagonized by SWI5, so we need to find Genes that regulate SWI5
a a α
α
α α
α
Sil & Herskowitz: Screen for microcolonies in which mother and daughters switch (1/66,000)
a a a a
ASH1+
ash1-1
mother daughter
mother daughter
ASH1 is a transcriptional repressor only in daughter cell nuclei ASH1 protein is unstable and gene expressed only at M/G1transition ASH1 mRNA is localized preferentially to daughter cells.
HO expression in mother versus daughter cells
Nasmyth: SWI5-dependent activators of HO Expression: Screen for genes necessary for HO-lacZ expression but not For GAL1-URS1-HO-lacZ expression 222 swi5 mutants, 315 she mutants, 5 complemenation groups
The SHE proteins are necessary for ASH1 mRNA localization Transport out of mother cells
SHE1 (MYO4) encodes a non-muscle myosin
Figure 3. The Effect of the she Mutants on Particle Localization and Formation Yeast strains disrupted for each one of the five SHE genes were transformed with the ASH1 reporter RNA and the GFP-MS2 fusion protein and the resultant particles observed by epifluorescence after fixation. Bar, 5 mm. (A) Wild-type cells (K699). Localization of the particle and its formation is inhibited in (B) she5 deletion strain (K5205): 36% of cells with signal formed bright, single particles; approximately half of the particles were localized at the bud neck and 2% were localized in the bud. (C) she3 deletion strain (K5235): 6% formed bright, single particles and 0% were localized in the bud. (D) she1 deletion strain (K5209): 16% of cells with signal formed bright, single particles and 0% were localized in the bud. (E) she2 deletion strain (K5547): 0% formed bright, single particles and 0% were localized in the bud. (F) she4 deletion strain (K5560): 32% formed bright, single particles and 16% with signal were localized in the bud. Colocalization (yellow) of a functional myctagged she protein (red) with the particle (green). (G) She1myc. (H) She2myc. (I) She3myc. (J–I) She1myc with nonlocalized particle in nonbudding cell. (Half of the particles showed colocalization with She1myc.)
Molecular Cell, Vol. 2, 437–445, October, 1998
Directional switching:
there is a recombinational enhancer on the left arm of III that is activated in a but not α cells.
(see Haber Annual Review of Genetics)
CEN3 MAT HML HMR RE
Haber (Ann Rev Genetics 1998)