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Prof. Fahd M. Nasr

Lebanese universityFaculty of sciences I

Department of Natural Sciences

fnasr@ul.edu.lb

B3206Microbial Genetics

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Eukaryotic M. G.The yeast

Saccharomyces cerevisiaeas a genetic model system

Lectures XI and XII

• 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!

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• 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

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MAPK cascades in Yeast• Ste2 or Ste3 GPCR detects the α-factor or a-

factor pheromones

• Heterotrimeric G protein Gpa1/Scg1 Gα, Ste4 Gß, and Ste18 Gγ

• Gßγ dimer activates a MAPK pathway– Ste11 MAPK kinase kinase (MAPKKK)

– Ste7 MAPK kinase (MAPKK)

– Fus3 MAPK by binding the Ste5 scaffold protein

MAPK cascades in Yeast• The Ste4-Ste18 Gßγ dimer also binds Ste20

– A PAK family member MAP4K (MAPKKK kinase)

– Phosphorylate and activate the Ste11 MAPKKK

• Ste4-Ste18 Gßγ dimer– Target both Ste5 and Ste20– Activates the Fus3 MAPK pathway at two levels

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MAPK cascades in Yeast

• Haploid cells a-factor and a-factor mating partner responds to prepare for mating

• Yeast cells of different sex express of different sets of genes– Haploid-specific genes proteins involved in the

response to pheromone as well as the RME1 gene encoding the repressor of meiosis

– a-specific genes a-factor and the gene for the a-factor receptor

– a-specific genes alpha-factor and the gene for the a-factor receptor

Yeast with a sexual life

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

Yeast mating type chromosome diagram

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Structure of MATa and MATalpha alleles

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Genetic determination of yeast cell type

• The mating type allele of the mating type locus MAT on chromosome III

• The mating type locus encodes regulatory proteins– The MATa locus encodes the a1 transcriptional

regulator (a2 has no known function)– The MATa locus encodes the a1 activator and

the a2 repressor• The mating type locus a master regulator

locus controls expression of many genes

Gene expression that determines the mating type

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Mating type

Mating Type Switching in Yeast

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Mating Type Switching in Yeast

Haploids and diploids in nature and laboratory

• In nature, yeast cells always grow as 2n increases their survival

• 2n cells sporulate n spores germinate- Functional copies of all essential genes-This often means that only a single spore (if any)

of a tetrad survives• This single spore MUST find a mating

partner diploid cell• Mating type switch is the solution

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Haploids and dipoids in nature and laboratory

• Following division the mother cell switches mating type and mates with its daughter diploid homozygous for all genes and starts a new clone of cells

• If mating type can be switched and diploid is the preferred form, why then sporulate and have mating types?

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

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

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

• Silent copies of the MAT locus on either side of MAT on chr. III• Gene conversion copies HMLalpha or HMRa to the MAT locus• HO endonuclease mediates switching master regulator of mating

type switching– HO gene transcription is regulated by cell lineage, ploidy and cell cycle– Only mother cells can switch– Daughter cells cannot switch until they have budded off a daughter cell of

their own– Only haploid cells can switch, and only during the G1 phase of the cell

cycle (before replication of DNA)• Expression of HO determines whether a cell switches mating type

– Mother cells express HO during G1– Daughter cells do not express HO– Ectopic expression of HO in daughter cells causes them to switch

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a/a

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3 4

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A B

a b

C D

C Dc d

c d

A Ba bDouble-strand

break model for recombination

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Gene conversion occurs during HR

Haploids can switch mating type• Only MAT locus is active a or a types• Switch two silent mating type loci HMLa and

HMRa on the same chromosome activated by translocation

• Silencing of HMLa and HMRa heterochromatin formation

• Translocation is a gene conversion HO nuclease• HO nuclease cuts within the active mating type

locus in the chromosome• Laboratory yeast strains lack the HO nuclease and

hence have stable haploid phases

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Haploids and dipoids in nature and laboratory

• Yeast genetics grow n cells in the laboratory mating type switch must be prevented laboratory strains are HO mutants

• So how does this switch work?

Mating Type Switch

x HO knockout renders haploid stable in propagation

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Cellular Polarity and Morphogenesis

Cellular Polarity(Protein Dynamics)

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Cellular Polarity

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

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Yeast genetics: the genetic material

• The S. cerevisiae nuclear genome has 16 chromosomes

• A mitochondrial genome and a plasmid, the 2m circle

• Yeast chromosomes contain centromeres and telomeres

• The haploid yeast genome = 12,100 kb

Replicon Size (bp)Chromosome 1 230,218Chromosome 2 813,184Chromosome 3 316,620Chromosome 4 1,531,933Chromosome 5 576,874Chromosome 6 270,161Chromosome 7 1,090,940Chromosome 8 562,643Chromosome 9 439,888

Chromosome 10 745,751Chromosome 11 666,816Chromosome 12 1,078,177Chromosome 13 924,431Chromosome 14 784,333Chromosome 15 1,091,291Chromosome 16 948,066Mitochondrial Chromosome

85,779

Total: 12,157,105

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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

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Replication of eukaryotic genomes

• The yeast Saccharomyces cerevisiae12.1Mb ~300 origins of replication 1 origin/~40kb

• ARS (Autonomously Replication Sequence)

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Strategy to identify replication origins

in yeast cells

Function of ARS and Centromere Sequences

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Identification of Telomeres

The end