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

项目描述（由申请人提供）：本项目将对人类NAT1和NAT2的最小启动子区域进行表征，并研究遗传多态性对人类乳腺细胞表达的影响。特异性目的1涉及通过5'-RLM-RACE识别人乳腺上皮细胞中NAT1和NAT2转录起始位点。Specific Aims 2和3将通过构建含有NAT1和AT2 5′-UTR区域的荧光素酶报告基因，进行定点诱变和缺失分析，鉴定NAT1和NAT2的功能启动子，并通过MCF-7和MCF-10A细胞系中的瞬时表达评估这些区域的转录作用。特异性目标4包括通过测定NAT1和NAT2 mRNA水平、蛋白质表达、内在蛋白质稳定性以及作为5'- utr和编码区基因型的N-和o -乙酰化活性的催化活性，研究正常人类乳腺组织中NAT1和NAT2的转录调控。特异性目的5涉及对人类乳腺组织中NAT1和NAT2的5'-UTR进行测序，以鉴定和表征NAT1和NAT2的5'-UTR多态性的影响。了解NAT1和NAT2的5'-UTR的转录调控将有助于进一步了解遗传多态性在环境致癌物致乳腺癌遗传易感性中的作用。

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.