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

描述（由申请人提供）：本项目将表征人NAT 1和NAT 2的最小启动子区域，并研究遗传多态性对人乳腺细胞中表达的影响。 具体目标1涉及通过5 '-RLM-RACE鉴定人乳腺上皮细胞中的NAT 1和NAT 2转录起始位点。 具体目的2和3将通过构建含有NAT 1和AT 2的5 '-UTR区域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细胞系中瞬时表达来评估这些区域的转录效应，来鉴定NAT 1和NAT 2的功能性启动子。 具体目标4涉及通过测定NAT 1和NAT 2 mRNA水平、蛋白表达、内在蛋白稳定性以及作为5 '-UTR和编码区基因型的函数的N-和O-乙酰化活性的催化活性，研究正常人乳腺组织中NAT 1和NAT 2的转录调控。 具体目标5涉及对人乳腺组织中NAT 1和NAT 2的5 '-UTR进行测序，以鉴定和表征NAT 1和NAT 2的5'-UTR中多态性的影响。 NAT 1和NAT 2的5 '-UTR中的转录调控的知识将允许增加理解遗传多态性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.