Genomic profiling of yeast resistance to heterocylic aromatic amines

摘要/概要 结直肠癌是美国癌症死亡的第二大原因。不到5%的结肠 癌症病例与已知的高渗透性遗传因素有关，而生活方式因素包括高渗透性饮食。 红肉消费在煮熟的红肉中发现的致癌物包括杂环芳香胺（哈斯）， 其在被P450酶和N-乙酰转移酶2激活后成为有效的DNA损伤剂 （NAT2）。然而，尚不清楚哪些个体最易受哈斯和流行病学研究的影响 由于抽样规模小，往往缺乏意义。由于许多DNA代谢和管家 基因从酵母到人是保守的，酿酒酵母的高通量分析（出芽 酵母）基因，赋予致癌物的抵抗力，已经确定了人类基因，赋予抵抗力， 环境致癌物。使用约5，000个酵母单基因缺失单倍体和 二倍体菌株在确定赋予对化学试剂的抗性的基因方面非常成功。我们 先前已成功地将人CYP 1A 2和CYP 1A 1引入酵母并激活多种 致癌物，包括黄曲霉毒素B1（AFB 1）、苯并[a]芘二氢二醇（BaP-DHD）和哈斯。的目的 该项目的目的是确定哪些酵母基因是抵抗强有力的HAA致癌物所必需的。我们将 将表达人CYP 1A 2和NAT 2的质粒导入约5，000个二倍体纯合单缺失 菌株在第一个目标中，我们将使用二倍体单缺失菌株进行抗性分析 到哈斯。赋予抗性的基因将通过高通量敏感性测定来鉴定，以测量细胞的抗性。 生长和使用高通量测序的分子条形码。考虑到CYP 1A 2主要是 在第二个目标中，我们将确定哪些结肠相关CYP可以 代谢活化哈斯。该项目产生的信息将帮助卫生保健提供者， 确定由于饮食致癌物而最有可能患结肠癌的个体。该项目将是一个有价值的 系统和细胞生物学研究生和本科生的培训工具。

Abstract/Summary Colorectal cancer is the second leading cause of cancer deaths in the United States. Fewer than 5% of colon cancer cases are linked to known high-penetrant genetic factors, while lifestyle factors include a diet high in red meat consumption. Carcinogens found in cooked red meat include heterocyclic aromatic amines (HAAs), which become potent DNA damaging agents after activation by P450 enzymes and N-acetyl transferase 2 (NAT2). However, it is unknown which individuals are most susceptible to HAAs and epidemiological studies often lack significance because of small sampling sizes. Since 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 chemical agents. 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 HAAs. The aim of this project is to determine which yeast genes are required for resistance to the potent HAA carcinogens. We will introduce plasmids that express human CYP1A2 and NAT2 into the ~5,000 diploid homozygous single-deletion strains. In the first aim, we will profile the yeast genome using the diploid single deletion strains for resistance to HAAs. 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. Considering that CYP1A2 is mostly expressed in the liver and not the colon, in the second aim, we will identify which colon-associated CYPs can metabolically activate HAAs. The information resulting from this project will aid health care providers in identifying individuals most at risk for colon cancer due to dietary carcinogens. The project will be a valuable training tool for graduate and undergraduate students in systems and cell biology.