Systematic Genetic Analysis with Conditional Estradiol-Regulated Gene Expression Alleles in Yeast

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The sophisticated molecular genetic methods available with yeast, makes this organism an ideal model system for studying fundamental cellular functions. The proposed project involves the construction of a new genome-wide collection of yeast mutants, in which each one of its ~6000 different genes is placed under the control of an inducible promoter, which means we can turn the expression of the gene on or off, simply by including or removing an inducer from the growth medium. This new mutant collection, which we refer to as the Z3 collection, has numerous applications and thus it will fuel research in numerous different labs around the world. We are particularly interested in this new mutant collection because it will allow us to track how cells die when particular genes are turned off. Interestingly, only ~1000 yeast genes are essential, such that the cell will not grow or divide when they are turned off or deleted, the other ~5000 genes are nonessential and the cell remains alive even when they are deleted or shut off. We think most genes are nonessential because cells are buffered from genetic and environmental perturbations and thus they have evolved backup genes and pathways to compensate for defects in a single gene. In another project, our group has tested all possible yeast double mutants for a lethal phenotype. While we now know many gene pairs work together to control cellular life, at a mechanistic level, we do not know exactly why the cell depends on both genes. We will use the new Z3 collection to investigate the terminal phenotype of a select set of yeast essential genes and lethal double mutants. We have assembled a number of diagnostic markers that allow us to follow the function of pathways and cellular compartments. We anticipate that different terminal phenotype trajectories will provide us with new knowledge of how genes and their corresponding pathways are connected to one another and thus will provide us with valuable new insights on how cells work.