Yeast_suppressors

Many genes are essential only in particular genetic backgrounds. The ability to survive and proliferate without an otherwise required gene implies existence of variants that complement its function. Such natural suppressors are potentially important for human health, as they point to ways that an organism can circumvent deleterious mutations, yet remain rare and poorly characterized. We have comprehensively assessed the ability of eight different wild strains to suppress 1742 temperature-sensitive knockout alleles of essential yeast genes. To do so, we created diploid hybrids of the reference strain carrying a temperature-sensitive (TS) allele and a wild strain, and generated a large number of haploid meiotic progeny selected for the TS allele. We then tested for lack of fitness defects in restrictive temperature (30C) compared to permissive one (23C), indicating suppression of the essential gene deletion. In parallel, we generated 1500 alleles of these strains that are similarly able to bypass the essentiality by a de novo mutation, without crossing to the wild strain. Importantly, all meiotic progeny that survive at the restrictive temperature carry a complement of alleles that together suppress essentiality, while other alleles segregate randomly. Thus, we can map the elusive natural suppressors by sequencing the surviving pool of progeny, and testing for allele frequency deviations from the expected 50%. We propose to sequence two biological replicates of 96 wild strain/TS allele combinations to identify the suppressor mutations, and 1500 suppressor strains to identify the de novo changes that have led to suppression.