This proposal will use the molecular genetics of Saccharomyces cerevisiae and Candida atbicans to determine the role of polyploidy and gone duplications in gene expression. Genomes have evolved by duplication of existing genetic material, either whole genomes or individual genes; however, such duplications can lead to abnormal growth. Many tumors contain a large proportion of cells that are hyperploid, and, for some, increased ploidy carries a good prognosis, predicting an increased sensitivity to anti-mitotic drugs. Experiments are designed to identify all the Saccharomyces genes required for polyploid formation and for the survival of polyploid cells in stationary phase using whole genome knockout libraries. The role of gene silencing in the control of gone expression in polyploids will be determined by whole- genome chromatin immunoprecipitation. The mechanism of gene silencing and variegation will be elucidated for the FLO genes, a highly duplicated gone family with one expressed locus (FL011) and many silent subtelomeric members. These experiments will determine the role of chromatin factors, nutrition, mutation, and recombination in silencing and desilencing the ensemble of FLO genes. Experiments are also designed to reveal the mechanism by which the non-telomeric gene FL011 switches epigenetically between the """"""""on"""""""" and """"""""off"""""""" states. A genome wide screen will identify the prevalence of variegation in gene expression. As FLO genes are the cell surface adhesins of fungi, their ability to switch could be critical virulence factors in pathogens. The importance of silencing and desilencing for human health will be determined in the human fungal pathogen, Candida albicans, an obligate diploid. Candida's resistance to the antifungal agent, fluconazole, is unstable in strains heterozygous for the recessive erg3 mutation. We will determine the role of diploidy and the histone deactylases in generating high level drug resistance and will screen the Candida genome for other genes that lead to fluconazole resistance by this silencing mechanism. These Candida studies could identify the factors that will improve the effectiveness of current antifungal therapies. Experiments using DNA microarrays are designed to identify compounds used by Saccharomyces to sense its density. The experiments proposed will lead to a deeper understanding of the ability to proliferate at low cell densities, a more realistic scenario for fungal pathogens.
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