INACTIVATION OF GENE EXPRESSION BY DNA ALKYLATING AGENTS

DNA 烷基化剂使基因表达失活

• 批准号: 3171802
• 负责人: Robert Ivarie
• 金额: $ 7.96万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 1986-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3171802
• 关键词: HeLa cells; alkylating agents; chemical carcinogen; chemical carcinogenesis; clone cells; gene expression; gene frequency; genetic manipulation; methylation; prolactin; radiotracer

The proposed experiments will determine the molecular basis by which the DNA alkylating agent and chemical carcinogen, ethyl methanesulfonate (EMS), stably and heritably inactivates prolactin (rPRL) gene expression at high frequency by increasing the methylation of cytosine residues in the genome of GH3 rat pituitary tumor cells. Biochemical and molecular techniques will be utilized to test the hypothesis that alkylation of DNA by EMS promotes "maintenance" methylation of the modified DNA. Increases in the 5-methylceytosine content in DNA from GH3 cells treated with EMS will be assayed by metabolic labelling techniques and by in vitro DNA methylase assays. Partially purified preparations of mammalian DNA methylase will be used to measure the effect of various alkyl adducts on the methyl-accepting capacity of DNA. Alkylated DNAs will also be introduced into cultured cells by gene transfer techniques and subsequent methylation of the introduced DNA will be assayed by restriction enzyme digestions and Southern genomic blotting techniques. The structural and functional consequences of rPRL gene inactivation will also be assayed in the rPRL-deficient, revertant and wild type cells. The extent of methylation of CpG sequences in the rPRL gene and its flanking regions will be assayed by restiction enzyme digestions and genomic Southern blotting techniques. It will also be determined whether inactivation results in (1) a reduced sensitivity of the rPRL gene to digestion by DNAse I; (2) an acquisition of hypersensitive sites in 5 feet flanking sequences, and (3) a decrease in the level of nuclear precursor RNAs to cytoplasmic pre-rPRL messenger RNA. A HeLa cell mutant with a missense mutation in the gene for hypoxanthine phosphoribosyl transferase (HPRT) will be used to develop a model genetic system for monitoring the frequency of activation and inactivation of a silent HPRT gene in response to a variety of chemical carcinogens.

拟议的实验将确定分子基础， DNA烷化剂和化学致癌物，甲基磺酸乙酯（EMS）， 稳定地和遗传性地使催乳素（rPRL）基因表达在高水平失活， 通过增加基因组中胞嘧啶残基的甲基化 GH 3大鼠垂体瘤细胞。 生物化学和分子技术 将被用来测试的假设，烷基化DNA的EMS 促进修饰的DNA的“维持”甲基化。 增加 5-来自用EMS处理的GH 3细胞的DNA中的甲基胞嘧啶含量将是 通过代谢标记技术和体外DNA甲基化酶测定 测定。 部分纯化的哺乳动物DNA甲基化酶制剂将被用于 用于测量各种烷基加合物对甲基接受的影响 DNA的能力。 烷基化DNA也将被引入培养的 通过基因转移技术和随后的细胞甲基化 引入的DNA将通过限制性内切酶进行分析， Southern基因组印迹技术。 结构和功能 rPRL基因失活的后果也将在 rPRL缺陷型、回复突变型和野生型细胞。 甲基化的程度 将分析rPRL基因及其侧翼区中的CpG序列 用限制酶消化法和基因组Southern印迹技术。 还将确定失活是否导致（1） rPRL基因对DNA酶I消化的敏感性;（2）获得 5足侧翼序列中的超敏位点，和（3） 细胞核前体RNA与细胞质前体rPRL信使RNA的水平。 一种次黄嘌呤基因错义突变的HeLa细胞突变体 磷酸核糖转移酶（HPRT）将用于开发模型遗传学 一种用于监控一个或多个传感器的激活和失活频率的系统， 沉默HPRT基因对多种化学致癌物的反应。