The mating type locus Chr. III

The MAT locus information The MAT locus can encode three regulatory peptides: - a1 is encoded by the MATa allele -  1 and  2 are encoded by the MAT  allele Three regulatory activities:  1,  2, and a1-  2.

Sterile mutants can monitor the MAT status Mutations have been identified at several loci that produce a non-mating phenotype, called sterile (STE). The sterile mutations fall into three classes: 1. sterility only in a cells - STE2, the a pheromone receptor 2. sterility only in  cells - STE3, the  pheromone receptor 3. sterility in both a and  cells - STE12, the general pheromone-responsive transcription factor

Saccharomyces as a model system How do cells generate a mitotically stable, complex, specific cell type?  same DNA, but different gene expression states. How do cells respond to environmental change or information from other cells?  decision-making algorithms. How do cells maintain an undifferentiated state “stem cell”?  non-equivalence of daughter cells at mitosis.

Sterile mutants can monitor the MAT status The STE genes can be used to track the effects of mutations at other loci, such as MAT. STE response be measured as fertility/sterility (mating). Or, reporter gene constructs made with the transcriptional response elements from STE genes can drive the E. coli  -galactosidase gene. The reporter gene is visualized on screens by the ability to metabolize XGAL to a blue color, giving blue (gene active) or white (gene inactive) colonies. STE12 response  -galactosidase

MAT regulation in  cells When the  allele is present at MAT, two genes are expressed: MAT  1 and MAT  2, Mutations in  1 affect only  -specific genes, such as STE3. MAT  1 mutants prevent normal expression of STE3. They do not affect other haploid specific genes or a-specific genes.   1 is a positive regulator of  -specific genes Mutations in  2 allow the expression of a-specific genes, even in a MAT  cell.   2 is a negative regulator of a-specific genes Consequently, in a MAT  cell the  genes are expressed while the a genes are not.

Genetic elements of yeast

The yeast genome –First eukaryotic genome sequenced, April 1996 –Consortium effort, US / EU –16 well characterized chromosomes –PFGE separation of chromosomes –Chr. I (230 kb) chr. IV (1532 kb) –13 Mb (3.5 x coli) –6183 ORFs > 99 aa –72% coding ! (<2% human) –Average ORF 1450 bp –Few introns (<4% of ORFs) –1/3 of ORFs characterized –1/3 of ORFs have homologies, motifs –1/3 of ORFs have unknown function –120 rRNA copies of 9137 bp on chr. XII –262 tRNAs

Essential genes About 1000 of the 6100 ORF are essential genes Test for essential gene: –Gene disruption in diploid –Sporulation and tetrad dissection –2 viable 2 dead spores Many genes would be essential in nature that are dispensable on laboratory rich media –eg. carbon source –Temperature –Salts...

The genetic and physical map of chromosome III  Both strands contain about the same number of ORFs  Often several ORFs on one strand not interrupted by ORFs on the other strand  Very few overlapping ORFs on the same strand  No overlap of divergently transcribed ORFs  Close shared promoters of divergently transcribed ORFs  Most DNA is ORF  Few and small introns  Genes close to the centromer

 Dispersed tRNAs (270 / genome)  Ty elements and there remnants are 5’ to tRNAs  Dispersed snRNAs and snoRNAs  3kb / cM (200x less than in humans)  rRNA on Chr XII, no recombination, nucleolus remains associated with the chromosome during meiosis  Moderate suppression of recombination around centromeres  Genome 4300 cM -> 45 x 2 crossovers per meiosis chromosome III (cont.)

Genetic nomenclature 3 letter name with digit, eg. CDC33, CMD1 Italic = genes Uppercase = wild-type Loss of function, cdc33 cdc33-1, known allele Cdc33 protein, Cdc33p Phenotypes TS +, ts - arg2∆, arg2::LEU2 Dominant / recessive ORF designation YCR49w

whole genome duplication 100 mio years ago (polyploidy; Ohno’s hypothesis, shark)  Tetraploid -> diploid + many deletions and reciprocal translocations  55 duplicate regions, 13% of all ORFs, 50% of the genome !  functions in anaerobiosis ? The genome duplication

Genetic elements of yeast