Systematic Exploration of Essential Yeast Gene Functions with Temperature-sensitive Mutants

The budding yeast Saccharomyces cerevisiae is the most well characterized model organism for systematic analysis of fundamental eukaryotic processes. Approximately 19% of S. cerevisiae genes are considered essential. Essential genes tend to be more highly conserved from yeast to humans when compared to nonessential genes. The set of essential yeast genes spans diverse biological processes and while the primary role of most essential yeast genes has been characterized, the full breadth of function associated with essential genes has not been examined, due, at least in part, to the lack of adequate genetic reagents for their conditional and systematic perturbations. To systematically study yeast essential gene functions using synthetic genetic array analysis and to complement the current yeast deletion collection, I constructed a collection of temperature-sensitive yeast mutants consisting of 795 ts strains, covering 501 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. This is the largest collection of isogenic ts yeast mutants constructed to date. I confirmed the correct integration of over 99% of the ts alleles using PCR-based strategy and the identity of the ts allele by complementation of the ts phenotype with its cognate plasmid. The ts mutant collection was characterized by high-resolution profiling of the temperature sensitivity of each ts strain, distribution analysis of gene ontology molecular function and biological process, and comparison of ts allele strains to the strains carrying Tet-repressible alleles of essential genes. The results demonstrated that the ts collection is a powerful reagent for the systematic study of yeast essential gene functions and provides a valuable resource to complement the current yeast deletion collection. I validated and demonstrated the usefulness of the ts collection in a number of different ways. First, I carried out detailed temperature profiling of each mutant strain using liquid growth assays and found that ts mutants that define particular biological pathways often show highly similar profiles. Second, I showed that the ts mutant array can be used to screen compounds for suppression of growth defects and thus is useful for exploration of chemical-genetic interactions. Third, I demonstrated that the ts collection represents a key reagent set for genetic interaction analysis because essential genes tend to be highly connected hubs on the global genetic network. Fourth, I further validated the ts array as a key resource for quantitative phenotypic analysis by using a high-content screening protocol to score six different fluorescent markers, diagnostic for different subcellular compartments or structures, in hundreds of different mutants. Quantification of the marker behaviour at the single-cell level enabled integration of this data set to generate a morphological profile for each ts mutant to reveal both known and previously unappreciated functions for essential genes, including roles for cohesion and condensin genes in spindle disassembly.Ph