INACTIVATION OF GENE EXPRESSION BY DNA ALKYLATING AGENTS

所提出的实验旨在了解 DNA烷化剂和化学物质的分子基础 致癌物甲基磺酸乙酯（EMS）可增强甲基化 胞嘧啶的CpG位点和失活表达的大鼠 催乳素（rPRL）基因在GH 3大鼠垂体瘤细胞中的表达。 为此目的，基因的转录活性将被抑制。 在野生型、缺陷型和回复突变型GH 3细胞中测定， 测量RNA聚合酶II分子的数量， rPRL基因转录通过核径流转录。 rPRL基因的初级转录物也将通过 北方印迹法。 rPRL mRNA的5'和3'末端将被 通过引物延伸和S1核酸酶消化映射到 确定是否相同的转录开始和终止 位点用于变体和回复突变体系。 细胞质 还将测量成熟rPRL消息的周转率 为了确定mRNA的稳定性是否受到影响， 缺陷细胞 为了确定rPRL缺陷细胞是否具有 一种转录因子表达缺失，瞬时表达 含有启动子和5'侧翼区的载体， rPRL和大鼠生长激素（rGH）基因将被用于检测 野生型和缺陷株系。 侧翼DNA区域 将检测含有顺式作用增强子位点的结合 从野生型和变体中提取的潜在调节因子 细胞核 大鼠rPRL基因中潜在的EMS靶序列， 经EMS修饰刺激酶促甲基化并灭活 将寻求rPRL表达。 5'侧翼的CpG位点 区域将被甲基化，并测定它们改变 rPRL启动子功能和基因组测序将用于 定位CpG位点，其甲基化与 基因在野生型和缺陷株系中的表达模式。 还将分析对DNA酶I消化高度敏感的位点， 与野生型和变体中基因的表达相关 留置权 哺乳动物DNA甲基化酶在CpG位点上的活性 将测量含有胞嘧啶5'和3'的N7乙基鸟嘌呤 以确定N7乙基加合物是否负责 体外刺激EMS处理的DNA甲基化酶活性 聚（dCdG）、聚（dC-dG）。 使用一组嵌套的 在GCG位点重叠的限制酶位点将用于 测量在任一G位点的N7乙基加合物是否刺激 酶甲基化的胞嘧啶的网站。

The proposed experiments are directed toward understanding the molecular basis by which the DNA alkylating agent and chemical carcinogen, ethly methanesulfonate (EMS), enhances methylation of cytosines in CpG sites and inactivates the expression of the rat prolactin (rPRL) gene in an GH3 rat pituitary tumor cells. Toward this end, the transcriptional activity of the gene will be assayed in wildtype, deficient and revertant GH3 cells by measuring the number of RNA polymerase II molecules engaged in rPRL gene transcription by nuclear runoff transcription. Primary transcripts of the rPRL gene will also be assayed by Northern blotting. The 5' and 3' ends of the rPRL mRNA will be mapped by primer extension and S1 nuclease digestion to determine whether the same transcription start and termination sites are used in variant and revertant lines. Cytoplasmic turnover rate of the mature rPRL message will also be measured to determine whether stability of the mRNA is affected in the deficient cells. To determine whether rPRLdeficient cells have lost the expression of a transacting factor, transient expression vectors containing the promotors and 5' flanking regions of the rPRL and rat growth hormone (rGH) gene will be used to transfect wildtype and deficient lines. Regions of flanking DNA containing cisacting enhancer sites will be assayed for binding potential regulatory factors extracted from wildtype and variant cell nuclei. Potential EMS target sequences in the rat rPRL gene which when modified by EMS stimulate enzymatic methylation and inactivates rPRL expression will be sought. CpG sites in the 5' flanking region will be methyulated and assayed for their ability to alter rPRL promotor function and genomic sequencing will be used to locate CpG sites whose methylation correlates with the expression pattern of the gene in wildtype and deficient lines. Sites hypersensitive to DNAse I digestion will also be analyzed to correlate with expression of the gene in wild-type and variant liens. The activity of mammalian DNA methylase on CpG sites containing N7ethylguanine 5' and 3' to cytosine will be measured to determine whether the N7ethyl adduct is responsible for in vitro stimulation of DNA methylase activity on EMS-treated poly(dCdG).poly(dC-dG). An in vivo assay using a nested set of restriction enzyme sites overlapping at a GCG site will be used to measure whether N7ethyl adducts at either G site stimulates enzymatic methylation of cytosine in the site.