The purpose of the research is to determine what fraction of the DNA sequences of the yeast Saccharomyces cerevisiae are required for cell viability. The experimental approach is to construct in vitro gene disruptions of random segments of yeast DNA cloned on recombinant plasmids. These disrupted sequences will be transformed into a diploid yeast strain, creating a gene disruption on one of the two homologous chromosomes. The diploid strain will then be induced to undergo meiosis. Gene disruptions that result in recessive lethals will meiotically segregate two live spores to two dead spores. The fraction of random gene disruptions resulting in haploid lethality should be directly related to the fraction of yeast DNA sequences required for cell viability. In strains containing haploid-viable gene disruptions, attempts will be made to correlate other phenotypes to the disruption (such as effects on cell growth rate, temperature sensitivity, auxotrophic requirements, mating or sporulation defects and mutagen sensitivity). The transcriptional activity of the disrupted sequences will also be monitored. In addition to information about the fraction of yeast DNA sequence required for cell viability, the Southern analysis of the various insertions that are part of the study will give a good estimate of the fraction of yeast DNA sequences that are repeated (as well as the repetition frequency of each family of repeats). The yeast strains containing haploid-viable insertions will be used in subsequent studies of the effects of flanking DNA sequences on gene expression and recombination.
NIH ExPorter
NIH ExPorter