Bi190Mating Type Interconversion

2007Sternberg, Caltech

Ira Herskowitz

a

αa

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 AgglutinationResponse to a-factor

Production of a- factor AgglutinationResponse to α− factor

Response to pheromones

a

αa

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

aa α α

α cells (α-factor producing)

aa α αα α

aa aaα αα α

a/αa/α

a/αa/α

ho HO

Homothallism: single haploid spore gives rise to diploid cells thatcan undergo meoisis and sporulation

shmoos zygotes

The genetic elements controlling mating type interconversionwere 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 HOMATα ho

a HO Dip(loidizer)a ho a-materα HO Dipα ho α mater

Tetratype Ascus

HO is necessary for Diploidization

HMLα MATa HMRa ; HOHMLα MATα HMRα ; HO

HMLα MATa HMRα HO DipHMLα MATa HMRa HO DipHMLα MATα HMRα HO αHMLα MATα HMRa HO Dip

HMLα MATa HMRα HO DipHMLα MATa HMRα HO DipHMLα MATα HMRa HO DipHMLα MATα HMRa HO Dip

HMRα is necessary for a to α

Dipα

HMLα MATα HMRa

W X Yα ZL W X Yα Z X Ya ZRE EI I

W ~700 bpYa ~600 bpYα ~750 bpZL ~300 bpZR ~250 bp

CEN III

200 kb 150 kb

HO

HO cuts at ~18 bp siteα-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 ZRE EI I

W ~700 bpYa ~600 bpYα ~750 bpZL ~300 bpZR ~250 bp

CEN III

200 kb 150 kb

HO

HO cuts at ~18 bp siteα-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 silencingI, 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

sir1sir2 = mar1sir3 = ste8 = cmtsir4 = ste9

The SIR proteins silence the HML and HMR loci

Jasper Rine:

a sir1-1a sir1-1 Spo+

Thus, HMLα can provide α function

HMLα MATα HMRa

W X Yα ZL W X Yα Z X Ya ZRE EI I

W ~700 bpYa ~600 bpYα ~750 bpZL ~300 bpZR ~250 bp

CEN III

200 kb 150 kb

HO

HO cuts at ~18 bp siteα-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 silencingI, important for silencing

aa α

α

α α

α

Pedigree analysis

mother daughter

mother daughter

a or α but not a/α cells switchOnly mothers switchSwitching in pairsDirectionality: a to α α to a

Strathern, Hicks & Herskowitz (Cell 1979)

Shmoo (G1 arrest at START)

aa α

α

α α

α

Pedigree analysis

mother daughter

mother daughter

a or α but not a/α cells switchOnly mothers switchSwitching in pairsNasmyth (1983)

HO isOff in a/α cellsOff in daughtersOn in late G1

aa α

α

α α

α

Pedigree analysis

aa

α α

aa

α α

SWI+ SIN+

swi5 SIN3+

SWI5+ sin3

mother daughter

mother daughter

HO expression in mother versus daughter cells

ASH1 is a transcriptional repressor only in daughter cell nuclei

ASH1 protein is unstableASH1 mRNA is localized preferentially to daughter cells.

The SHE proteins are necessary for ASH1 mRNA localization

SHE1 (MYO4) encodes a non-muscle myosin

Figure 3. The Effect of the she Mutants on Particle Localization and FormationYeast strains disrupted for each one of the five SHE genes were transformed with the ASH1 reporter RNA and the GFP-MS2fusion 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 formedbright, 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% formedbright, single particles and 16% with signal were localized in the bud. Colocalization (yellow) of a functional myctagged sheprotein (red) with the particle (green). (G) She1myc. (H) She2myc. (I) She3myc. (J–I) She1myc with nonlocalized particle innonbudding 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 thatis activated in a but not α cells.

(see Haber Annual Review of Genetics)

CEN3 MATHML HMRRE

Haber (Ann Rev Genetics 1998)