Genomic profiling of yeast resistance to AFB1, a P450-activated carcinogen

描述（由申请人提供）：环境因素估计是19%的癌症的原因。然而，人类对环境致癌物的易感性是高度可变的。低渗透等位基因可能会增加患特定环境相关癌症的风险。然而，流行病学研究受到其统计能力和对大量患者的要求的限制。由于许多DNA代谢和管家基因从酵母到人类都是保守的，因此对赋予致癌物抗性的酿酒酵母（出芽酵母）基因的高通量分析已经确定了赋予对环境致癌物抗性的人类基因。利用约5000个酵母单基因缺失的单倍体和二倍体菌株进行基因组表型分析，在确定对辐射和化学制剂具有抗性的基因方面取得了很大成功。然而，75%的环境因子本身并不致癌，但需要生物活化，如组织特异性细胞色素p450介导的代谢活化。我们之前成功地将人类CYP1A2和CYP1A1引入酵母并激活多种致癌物，包括黄曲霉毒素B1 (AFB1)，苯并[a]芘二氢二醇（BaP-DHD）和杂环胺（HA，食品致癌物）。该项目的目的是确定哪些酵母基因需要抵抗强致癌物AFB1，使用高通量系统生物学方法。我们将把表达人CYP1A2的质粒引入到约5000个二倍体纯合单缺失和杂合单缺失菌株中。在第一个目标中，我们将使用二倍体单缺失菌株对AFB1抗性进行基因组分析。赋予耐药性的基因将通过测量细胞生长的高通量敏感试验和使用高通量测序或微阵列的分子条形码来鉴定。考虑到AFB1是一种有效的重组原，在第二个目标中，我们将通过对一组单DNA修复突变体衍生的双突变体的高通量敏感性分析，确定赋予AFB1抗性的DNA修复和重组途径。因此，这些研究将确定参与介导致癌物抗性和敏感性的新基因，并为重组修复过程如何积极耐受DNA加合物提供见解。菌株收集将提供给科学界，并将成为表征环境因子遗传易感性的宝贵资源。该项目将成为系统和计算生物学的宝贵培训工具。

DESCRIPTION (provided by applicant): Environmental factors are estimated to be the cause of 19% of cancers. However, human susceptibility to environmental carcinogens is highly variable. Low penetrant alleles may increase the risk for particular environmental-associated cancers. Epidemiological studies, however, are limited by their statistical power and the requirement for large patient numbers. Because many DNA metabolism and housekeeping genes are conserved from yeast to man, high throughput analysis of Saccharomyces cerevisiae (budding yeast) genes that confer resistance to carcinogens have identified human genes that confer resistance to environmental carcinogens. Genomic phenotyping using the ~5,000 yeast single-gene deletion haploid and diploid strains have been highly successful in determining genes that confer resistance to radiation and chemical agents. However, 75% of environmental agents are not carcinogenic per se, but require bioactivation, such as tissue - specific cytochrome P450-mediated metabolic activation. We previously were successful in introducing human CYP1A2 and CYP1A1 into yeast and activating a variety of carcinogens, including aflatoxin B1 (AFB1), benzo[a]pyrene dihydrodiol (BaP-DHD), and heterocyclic amines (HA, food carcinogens). The aim of this project is to determine which yeast genes are required for resistance to the potent carcinogen, AFB1, using a high throughput systems biology approach. We will introduce plasmids that express human CYP1A2 into the ~5,000 diploid homozygous single-deletion and heterozygous single-deletion strains. In the first aim, we will profile the yeat genome using the diploid single deletion strains for resistance to AFB1. Genes that confer resistance will be identified by high-throughput sensitive assays to measure cell growth and by molecular bar codes using high throughput sequencing or microarrays. Considering that AFB1 is a potent recombinagen, in the second aim, we will identify DNA repair and recombination pathways that confer AFB1 resistance by high throughput sensitivity analysis of double mutants derived from a set of single DNA repair mutants. These studies will thus identify novel genes involved in mediating carcinogen resistance and sensitivity, and provide insights into how recombinational repair processes actively tolerate DNA adducts. The strain collections will be made available to the scientific community and will be a valuable resource for characterizing the genetic susceptibility to environmental agents. The project will be a valuable training tool in systems and computational biology.