Genetic Control of Nutrition Starvation in Yeast

本提案将利用酿酒酵母和白色念珠菌的分子遗传学来确定多倍体和基因复制在基因表达中的作用。基因组是通过现有遗传物质的复制而进化的，要么是整个基因组，要么是单个基因；然而，这种重复会导致异常生长。许多肿瘤含有很大比例的高倍体细胞，对一些肿瘤来说，高倍体预示着良好的预后，预示着对抗有丝分裂药物的敏感性增加。实验旨在利用全基因组敲除文库鉴定多倍体形成和多倍体细胞在固定期存活所需的所有酵母菌基因。基因沉默在多倍体中控制gone表达中的作用将通过全基因组染色质免疫沉淀来确定。FLO基因是一个高度重复的基因家族，具有一个表达位点（FL011）和许多沉默的亚端粒成员，该基因沉默和变异的机制将被阐明。这些实验将确定染色质因子、营养、突变和重组在FLO基因集合沉默和解除沉默中的作用。实验还旨在揭示非端粒基因FL011在“开”和“关”状态之间切换的表观遗传学机制。基因组宽筛选将确定基因表达变异的普遍性。由于FLO基因是真菌的细胞表面粘附素，它们的转换能力可能是病原体的关键毒力因素。沉默和去沉默对人类健康的重要性将在人类真菌病原体白色念珠菌中确定，这是一种专性二倍体。念珠菌对抗真菌剂氟康唑的耐药性在隐性erg3突变的杂合菌株中是不稳定的。我们将确定二倍体和组蛋白去乙酰化酶在产生高水平耐药性中的作用，并将通过这种沉默机制筛选念珠菌基因组中导致氟康唑耐药性的其他基因。这些念珠菌研究可以确定提高当前抗真菌治疗有效性的因素。使用DNA微阵列的实验旨在识别酵母菌用来感知其密度的化合物。提出的实验将导致对低细胞密度下增殖能力的更深入理解，这是真菌病原体更现实的情况。

This proposal will use the molecular genetics of Saccharomyces cerevisiae and Candida albicans to determine the role of polyploidy and gene 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.