Prof. Fahd M. Nasr

Lebanese universityFaculty of sciences

fnasr@ul.edu.lb

https://yeastwonderfulworld.wordpress.com/

Mighty Yeasts

Yeast sexual lifeMating type switch by gene

conversion

• Under nitrogen starvation diploid cells meiosis and sporulation an ascus with four haploid spores

• Although unicellular distinguish different cell types with different genetic programmes– Haploid MATa versus MATa– Haploid versus Diploid (MATa/a)– Spores– Mothers and daughters

Yeast has a sexual life!

• Pheromone response signal transduction pathway sexual communication

• Controls the response of yeast cells to a- or a-factor• Specific pheromone receptor binds a- or a-factor• Pheromone binding

– Stimulates reorientation of the cell towards the source of the pheromone (the mating partners)

– Stimulates a signalling cascade MAP kinase pathway– This signalling pathway causes cell cycle arrest

• The pathway controls expression of genes important for mating

Yeast with a sexual life

MAPK cascades in Yeast• Gpa1 Gα subunit plays three roles

– No pheromone signalingGpa1 binds to the Ste4-Ste18 Gßγ dimer prevent activation of the MAPK pathway

– Upon activation Gpa1 binds the Fus3 MAPK to positively regulate cell growth and cellular fusion

– Positive role in signaling via an interaction with Scp160

– RGS protein Sst2 accelerates Gpa1 GTPase activity and desensitize the cells to the pheromone present in the growth environment

MAPK cascades in Yeast

Signal Transduction Pathways• Overlap and integration of signal transduction pathways

Yeast Pheromone Signaling Pathway

Humans : MAPK signaling pathway : growth control -> tumor developmenthttp://www.bio.davidson.edu/courses/Immunology/Flash/MAPK.html

Cell attachment, cell fusion and nuclear fusion in an electron micrograph

Yeast mating type chromosome diagram

Structure of MATa and MATalpha alleles

Mating type

Model for the STE2 gene regulation

Mating Type Switching in Yeast

Mating Type Switching in Yeast

Haploids and dipoids in nature and laboratory

• Several reasons– Spores are hardy and survive very harsh

conditions– Sporulation is a way to "clean" the genome

from accumulated mutations– Meiosis is a way to generate new combinations

of alleles – New allele combinations with a partner from a

different tetrad

Mating Type Conversion: heterothallic strains of yeast switch sex each generation!

a/a

So what regulates HO expression?

• Transcription of HO– in the late G1 phase of the cell cycle– is determined by binding of cell- cycle box

factor CCBF to the cell-cycle box – CCBF = Swi4p and Swi6p– Cell-cycle box sequences 12bp sequence

motif that is repeated 10 times in URS2– CCBF is active only during G1

Two regulators HO gene

Transcriptional Regulation

SWI/SNF

SWI5

RNA Pol IITATA BPGENERAL TFs

Ash1p: a negative regulator of HO expression in daughter cells

• Found in 3 different mutant screening strategies– Bobola et al. (1996) also looked for mutations that

restore HO expression in a she1 mutant background– SHE1 (Swi5p-dependent HO expression) is essential for

HO expression in mother cells– but encodes an unconventional myosin– therefore may be involved in localization of negative

regulator (repressor) of HO expression– Then loss of negative regulator would restore HO

expression in she1 mutants

Recombination enhancer structure

Mating type switching is initiated by a ds-break in the recipient site (MAT). The break point is a 24 bp sequence recognized by HO endonuclease.

3 4

1 23'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

A B

a b

C D

C Dc d

c d

A Ba bDouble-strand

break model for recombination

3 4

3'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

3'5'

3'5'

5'3'

5'3'

1 23'5'

3'5'

5'3'

5'3'

1 + 2

3 + 4

1 + 4

2 + 3

Resolution of theheteroduplex with twoHolliday junctions

A B

a b

C D

c DC D

C d

A Ba b

A B

a b

C d

C Dc D

C D

A Ba b

A b

a B

C D

c DC D

C d

A ba B

a B

A b

C D

c DC D

C d

a BA b

(H1)

(H2)

(H3)

(H4)

(H3 and H4) CO(H1 and H2) no CO

a Bc D

C Da B

A b

C D

C d

A b

A B

a b

C D

C D

c d

c d

A B

a b

Cen

trom

ere

a BC D

C Da B

A B

a b

C D

C D

c d

c d

A B

a b

A b

C D

C D

A b

Sister chromatids

Cen

trom

ere

Cen

trom

ere

Cen

trom

ere

Sister chromatids

Non-sister chromatids

Gene conversion leads to unusual ratiosof 3C:1c or 3D:1d during meiosis

Gene conversion occurs during HR

HO endonuclease is

transcriptionally regulated

via three different systems

Cellular Polarity and Morphogenesis

Cellular Polarity(Protein Dynamics)

Cellular Polarity

Cellular Polarity

Cellular PolaritymRNA Localization & Maternal

Effect

Why yeast as genetics model

• Basic cellular mechanisms conserved• Unicellular• Grow on readily controlled, defined media• Ideal life cycle• Very compact genome• Quick to map a phenotype producing gene• Single gene deletion mutants• One third of the genes have counterparts in

human

Yeast chromosomes

The S. cerevisiae genome of 12.1 Mb has 6000 genes, almost all uninterrupted

Yeast Genetic InformationInheritance Mendelian Non-Mendelian

Nucleic acid Double-stranded DNA Double-stranded RNA

Location Nucleus Cytoplasm

Genetic determinant

Chrs 2-mm plasmid

Mito. DNA RNA viruses

L-A M L-BC T W

Relative amount

85% 5% 10% 80% 10% 9% 0.5% 0.5%

Nb of copies 2 sets of 16 60-100 ~50 (8-130) 103 170 150 10 10

Size (kb) 12,100 6.318 70-76 4.576

1.8 4.6 2.7 2.25

Deficiencies in mutants

All kinds None Cytochromes a.a3 and b

Killer toxin None

Wild type YFG1 cir+ r+ KIL-k1

Mutant of variant

yfg1-1 cir0 r- KIL-o

Function of ARS and Centromere Sequences

Identification of Telomeres

The end