Genetic polymorphisms in 5'-UTR of human NAT1 and NAT2

描述(申请人提供)：这个项目将表征人类NAT1和NAT2的最小启动子区域，并研究遗传多态对人类乳腺细胞表达的影响。具体目标1包括用5‘-RLM-RACE鉴定人乳腺上皮细胞中的NAT1和NAT2转录起始位点。特异的AIMS 2和3将通过构建含有Nat1和AT2的5‘-UTR区的荧光素酶报告基因，并进行定点突变和缺失分析，并通过在MCF-7和MCF-10A细胞中的瞬时表达来评估这些区域的转录效应，从而确定Nat1和Nat2的功能启动子。具体目标4包括通过测定NAT1和NAT2的mRNA水平、蛋白质表达、固有的蛋白质稳定性以及对N-和O-乙酰化活性的催化活性随5‘-非编码区和编码区基因的变化，来研究NAT1和NAT2在正常人类乳腺组织中的转录调控。特定目的5涉及对人类乳腺组织中NAT1和NAT2的5‘-UTR区进行测序，以确定和表征NAT1和NAT2的5’-UTR区的多态的影响。了解NAT1和NAT2 5‘-UTR中转录调控的知识将有助于更好地理解基因多态对环境致癌物导致乳腺癌的遗传易感性的作用。

DESCRIPTION (provided by applicant): This project will characterize the minimal promoter region of human NAT1 and NAT2 and study effects of genetic polymorphisms on expression in human mammary cells. Specific Aim 1 involves identifying the NAT1 and NAT2 transcriptional start site in human mammary epithelial cells by 5'-RLM-RACE. Specific Aims 2 and 3 will identify the functional promoter of NAT1 and NAT2 by construction of luciferase reporter genes containing 5'-UTR regions of NAT1 and AT2 and site-directed mutagenesis and deletion analysis, and assessing the transcriptional effects of these regions by transient expression in MCF-7 and MCF-10A cell lines. Specific Aim 4 involves investigating transcriptional regulation of NAT1 and NAT2 in normal human mammary tissues by determining NAT1 and NAT2 mRNA levels, protein expression, intrinsic protein stability, and catalytic activity for N- and O-acetylation activities as a function of 5'-UTR and coding region genotypes. Specific Aim 5 involves sequencing the 5'-UTR of NAT1 and NAT2 in human mammary tissues to identify and characterize effects of polymorphisms in the 5'-UTR of NAT1 and NAT2. Knowledge of transcriptional regulation in the 5'-UTR of NAT1 and NAT2 will allow for an increased understanding the role of genetic polymorphisms towards genetic predisposition to breast cancer from environmental carcinogens.