Practice problem 4A Telomere position effects in budding yeast

4. Four genes in yeast are required for silencing of a reporter gene positioned near a telomere: RAP1, SIR2, SIR3, and SIR4. Rap1 protein binds to a specific DNA sequence found only at telomeres. Binding of Rap1 recruits Sir3 and Sir4, which can also bind to deacetylated histone H3. SIR2 encodes a histone deacetylase. Spreading is thought to occur by the action of Sir2 clearing a binding site for Sir3 and Sir4 proteins.

If ADE2 is used as the reporter gene, silencing can be visualized using a colony-sectoring assay that was described in class. If ADE2 is silenced in all cells, then pure red colonies form. If ADE2 is expressed all cells then white colonies form. When the reporter is positioned near the telomere a variegated colony phenotype is typically observed in otherwise wild-type cells (red and white sectors within a colony).


A. You want to construct a rap1 sir3 DOUBLE-MUTANT HAPLOID (both alleles exhibit complete loss of function for silencing). You mate a sir3 RAP1 haploid with a SIR3 rap1 haploid to generate a diploid. Following sporulation, you dissect tetrads and score the sectoring phenotype of each colony arising from a single spore. FILL IN THE CIRCLES below to represent the red/white phenotype you would observe among colonies in the tetrad that would allow you to unambiguously find the double mutant you seek. WRITE THE GENOTYPE under each spore clone and DRAW A BOX around the spore clone(s) exhibiting the double-mutant phenotype.

For your analysis, assume that sir3 and rap1 are recessive loss-of-function mutations and are not linked to one another. In addition, assume that the ADE2 reporter is present in both parent strains. (For the diagram: no color is white and whatever you fill in is red).

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