ERROR PRONE DNA SYNTHESIS AND ONCOGENE MUTAGENESIS

C-Ha-ras癌基因的激活可通过点突变发生在 网站数量有限。含c-Ha-ras基因的质粒的处理 化学致癌物在进入受体后产生转化子 人类细胞。这些观察结果提出了以下问题：a)是 突变是靶点错配的结果，或者 致癌物治疗在细胞中导致容易出错的情况？；b)是 C-Ha-ras癌基因突变易感性片段 是因为它们的序列，还是只有选择才能解释频率 转变为一种转变的状态。我们计划研究这些问题 利用从人类细胞获得的体外DNA系统 含有c-Ha-ras的质粒。我们建议：1)复制这种质粒 用纯化的原核细胞和 真核酶；2)开发哺乳动物细胞提取物和部分 将复制c-Ha-ras的纯化系统；3)以前应用 建立致癌物反应c-Ha-ras复制的方法学 模板；4)利用从致癌物反应细胞中制备的提取物来 看看他们是否像预期的那样更容易地促进内收旁路 如果已开发出可诱导系统；5)检测反应产物 通过将啮齿动物细胞转移到转化状态而发生突变；以及6) 研究脱氧核苷三磷酸偏倚的作用，其影响因素 用致癌物质治疗，看看是否有可能解释 在集合效应的基础上增加了可变性。这些是体外培养的 研究应该对所提出的问题提出答案，而且还应该 为设计用于体内试验的质粒提供有用的信息 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 致癌前诱变事件的产生。

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.