The Frequency of Survivorship in Heterozygous Diploids of Cdc13-1exo1Δ Mutants of S. <i>cerevisiae</i> Is One Survivor Cell in 72 Cells/Generation at 36&deg;C

Abstract

Telomeres cap ends of eukaryotic chromosomes prevent them from degradation and ensur e genomic stability. Cdc13 is an essential telomere recruitment and maintenance protein. A temperature - sensitive point mutation in cdc13 gene leads to telomere impairment, giv ing rise to cdc13-1 mutants that suffer lethality at enhanced temperatures. Deleting Exo1 gene from these mutants, however, leads to the emergence of temperature - tolerant mutants called sur vivors. Yeasts are known to exist as either diploids or haploids. These yeast genotypes generate survivors. The frequency of survivorship in the haploid genotype is one cell in 104 cells/generation at 36 &deg;C , however, the frequency at which they emerge in their diploid counterparts at the same temperature is not known. In this study, we investigated the frequency of Survivorship in heterozygous diploids of cdc13-1exo1 Δ mutants of S. cerevisiae at 36 °C . Diploids were constructed by mating haploid strains of opposite mating type cdc13-1 exo1:LEU strains with strains of cdc13-1 exo1:HIS. The crosses were 1296 × 3181, 2561 × 3182, 1296 × 3182 and 2561 × 3181. Genetic mark ers and phenotypic appearance were considered while mating the mutant cells. Using a stick, a smear of one haploid strain was made on each YEPD plate labelled C2, C8, C9, D1, D14, and D15. A smear of another opposite mating type was made on the previous strain. They were mixed and allowed to mate over night, before culturing on media lacking Luecine and Histidine (-L and -H). Survivors were generated by culturing these diploids at 36 &deg;C . Using SPSS 20.0 software for windows SPSS, 2011, the frequency was determined as one Survivor cell in 72 cells/generation, as their frequency of survivorship averaged 5.9 × 10 - 5 ± 0.04.