When exploring gene function, one valuable approach is to create mutations in a gene of interest and score the resulting mutant strains for phenotypes. Creating experimentally useful mutant strains is easy to do on a large scale for nonessential genes, but it is much more problematic for essential genes. The investigators propose to explore a new application of shuttle mutagenesis that should generate large numbers of hypomorphic (partial loss of function) alleles for essential yeast genes. The investigators have prepared a collection of yeast strains containing transposon insertions at known locations. These insertions are large (approximately 6 kb) and often generate null mutations, but sometimes result in hypomorphic mutations. They can be recombined by site-specific recombination to form short (279 bp) in- frame insertion elements that often result in a hypomorphic mutation. The investigators plan to use both large and small insertion mutations to generate a large collection of yeast strains containing hypomorphic mutations in essential genes. They will sporulate approximately 30,000 yeast strains containing the large insertion mutations to identify those that fall in yeast genes essential for cell growth. The investigators will also identify such insertions by means of data base searches. Diploid strains containing the small in-frame insertion mutation will be derived, and haploid segregants will be examined for viability and phenotypic effects. These studies should determine the feasibility of generating a large set of yeast strains containing hypomorphic mutations in essential genes. In the process they will produce a collection of approximately 800 yeast strains carrying potentially hypomorphic alleles of essential genes. These strains will be available for direct use by other researchers. Since the insertion alleles analyzed will also be available on plasmids, other researchers may introduce them into their own strain background, further enhancing the utility of the collection.
