Genomic profiling of yeast resistance to heterocylic aromatic amines

摘要/摘要 在美国，结直肠癌是癌症死亡的第二大原因。不到5%的冒号 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饮食中高含量的 红肉消费。在煮熟的红肉中发现的致癌物质包括杂环芳胺(HAAs)、 它们被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，目前尚不清楚哪些人最容易感染禽流感和流行病学研究。 由于样本量较小，通常缺乏意义。由于许多DNA新陈代谢和家务劳动 基因从酵母到人都是保守的，酿酒酵母(芽生)的高通量分析 酵母)对致癌物产生抗性的基因已经确定了人类对致癌物具有抗药性的基因 环境致癌物质。~5,000酵母单基因缺失单倍体与基因组表型 二倍体菌株在确定赋予化学试剂抗性的基因方面取得了极大的成功。我们 以前成功地将人的细胞色素P1A2和细胞色素P1A1导入酵母，并激活了多种 致癌物，包括黄曲霉毒素B1(AFB1)、苯并[a]芘二氢二醇(BaP-DHD)和HAAs。这样做的目的是 该项目是为了确定哪些酵母基因是抵抗强大的HAA致癌物所必需的。我们会 在~5,000个二倍体纯合性单缺失中引入表达人CYP1A2和Nat2的质粒 菌株。在第一个目标中，我们将使用二倍体单个缺失菌株来分析酵母基因组的抗性 敬哈斯。赋予抗性的基因将通过高通量、灵敏的分析来测量细胞 生长和使用高通量测序的分子条形码。考虑到CYP1A2主要是 表达在肝脏而不是结肠，在第二个目标中，我们将确定哪些与结肠相关的Cyps可以 新陈代谢激活哈斯。这个项目产生的信息将帮助医疗保健提供者在 确定因饮食致癌物而患结肠癌风险最高的个人。这个项目将是一个有价值的 为研究生和本科生提供系统和细胞生物学方面的培训工具。

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.