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INACTIVATION OF GENE EXPRESSION BY DNA ALKYLATING AGENTS

DNA 烷基化剂使基因表达失活

基本信息

批准号：
3171805
负责人：
Robert Ivarie
金额：
$ 10.89万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-07-01 至 1991-06-30
项目状态：
已结题

项目摘要

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.

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区域将测定含有顺式作用增强子位点的细胞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。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

甲磺酸乙酯对聚 (dC-dG).聚 (dC-dG) 的乙基化可在体外刺激哺乳动物 DNA 甲基转移酶的活性。

DOI：
10.1073/pnas.82.4.1045
发表时间：
1985
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Farrance,IK;Ivarie,R
通讯作者：
Ivarie,R

Prolactin-deficient GH3B3 cells are defective in the utilization of the endogenous prolactin promoter yet are fully competent to initiate transcription from a transfected prolactin promoter.

催乳素缺陷的 GH3B3 细胞在利用内源催乳素启动子方面存在缺陷，但完全有能力从转染的催乳素启动子启动转录。