ERROR PRONE DNA SYNTHESIS AND ONCOGENE MUTAGENESIS

容易出错的 DNA 合成和癌基因诱变

• 批准号: 3170358
• 负责人: BERNARD S. STRAUSS
• 金额: $ 16.54万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1990-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3170358
• 关键词: DNA repair; Escherichia coli; acetylaminofluorene; bacterial virus; carcinogenesis inhibitor; chemical carcinogen; fibroblasts; human tissue; lymphoblast; molecular site; mutagens; mutant; neoplasm /cancer genetics; radiation genetics; radiotracer; tissue /cell culture; ultraviolet radiation; xeroderma pigmentosum

Activation of the c-Ha-ras oncogene can occur by point mutation at a limited number of sites. c-Ha-ras-containing plasmids treated with chemical carcinogens yield transformants on introduction into recipient human cells. These observations suggest the following questions: a) Are the mutations the result of mispairing at a targeted site or does carcinogen treatment induce an error-prone condition in the cells?; b) Are segments of the c-Ha-ras oncogene particularly susceptible to mutation because of their sequence or does selection alone account for the frequency of change to a transformed state. We plan to study these questions utilizing an in vitro DNA system obtained from human cells and with plasmids containing c-Ha-ras. We propose: 1) to replicate such plasmids in single and double stranded form in vitro using purified prokaryotic and eukaryotic enzymes; 2) to develop mammalian cell extracts and partially purified systems which will replicate c-Ha-ras; 3) to apply previously developed methodology to the replication of carcinogen-reacted c-Ha-ras templates; 4) to utilize extracts prepared from carcinogen-reacted cells to see whether they promote adduct bypass more readily, as would be expected if an inducible system had developed; 5) to assay the reaction products for mutation by transfection of rodent cells to the transformed state; and 6) to study the role of deoxynucleoside triphosphate pool bias, as affected by treatment with carcinogen, to see whether it is possible to account for increases in mutability on the basis of pool effects. These in vitro studies should suggest answers to the questions asked and should also provide information useful in the design of plasmids for in vivo tests of mutability. The long term goal of these studies is the determination of how nucleotide sequence and the physiological state of the cell interact in the production of precarcinogenic mutagenic events.

c-Ha-ras癌基因的激活可以通过在一个位点的点突变而发生。 网站数量有限。 用以下物质处理的含c-Ha-ras质粒 化学致癌物进入受体后产生转化体 人体细胞 这些观察结果提出了以下问题：a） 突变是靶位点错配的结果， 致癌物质治疗会在细胞中诱发一种易出错的状态吗？B）是 c-Ha-ras癌基因的一段特别易受突变的影响 是因为它们的顺序还是选择本身就决定了 转变为一种转变的状态。 我们计划研究这些问题 利用从人细胞获得的体外DNA系统， 含有c-Ha-ras的质粒。 我们建议：1）复制这样的质粒 单链和双链形式体外培养， 真核生物酶; 2）开发哺乳动物细胞提取物， 纯化的系统，将复制c-Ha-ras; 3）应用于先前 开发了复制致癌物反应的c-Ha-ras的方法 模板; 4）利用从致癌物反应的细胞制备的提取物， 看看它们是否如预期的那样更容易促进加合物旁路 如果诱导系统已经开发; 5）测定反应产物， 通过转染啮齿动物细胞至转化状态而突变;以及6） 研究脱氧核苷三磷酸合并偏倚的作用， 用致癌物治疗，看看是否有可能解释 基于池效应的可变性增加。 这些体外 研究应提出问题的答案，还应 提供了在设计用于体内试验的质粒中有用的信息， 易变性 这些研究的长期目标是确定 核苷酸序列和细胞的生理状态如何相互作用， 致癌前的诱变事件的产生。