INACTIVATION OF GENE EXPRESSION BY DNA ALKYLATING AGENTS

拟议中的实验将确定分子基础， DNA烷化剂和化学致癌物甲烷磺酸乙酯(EMS)， 稳定和遗传地抑制催乳素(RPRL)基因的高表达 通过增加基因组中胞嘧啶残基的甲基化来提高频率 GH3大鼠垂体瘤细胞。生物化学和分子技术 将被用来检验EMS使DNA烷基化的假设 促进修饰DNA的“维持”甲基化。增加了 经EMS处理的GH3细胞DNA中的5-甲基胞嘧啶含量将 代谢标记法和DNA甲基化酶体外测定法 化验。哺乳动物DNA甲基酶的部分纯化制剂将是 用于测定不同烷基加合物对甲基接受性的影响 DNA的容量。烷基化的DNA也将被引入培养的 细胞通过基因转移技术和随后的甲基化 引入的DNA将通过限制性内切酶和 南方基因组杂交技术。结构和功能 RPRL基因失活的后果也将在 RPRL缺失、突变和野生型细胞。甲基化程度 将分析rPRL基因及其侧翼区的CpG序列 通过抗性酶消化和基因组Southern杂交技术。 还将确定停用是否会导致(1)降低 RPRL基因对DNase I消化的敏感性；(2)获得 5英尺侧翼序列中的过敏性部位，以及(3)减少 核前体RNA到细胞质前rPRL信使RNA的水平。 次黄嘌呤基因错义突变的HeLa细胞突变 磷酸核糖转移酶(HPRT)将被用来开发一种遗传模型 用于监控激活和失活的频率的系统 沉默的HPRT基因对多种化学致癌物的反应。

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.