MECHANISMS OF CHEMICAL MUTAGENESIS IN MAMMALIAN CELLS

我们建议开展一项研究项目， 哺乳动物细胞 作为突变的目标，这些研究将 使用导入哺乳动物细胞的小细菌基因（gpt） 作为逆转录病毒穿梭载体的一部分，并整合到 前病毒形式的哺乳动物染色体DNA。 我们有 最近开发了这个系统，并证明了它的潜力， 提供哺乳动物细胞突变的信息， 具体的基础变化。 研究的目标是 阐明突变的分子机制， 哺乳动物细胞 拟议的研究将利用穿梭载体转化的Gpt+ 小鼠细胞和大量的Gpt+突变体， 已经被分离出来并以特定的碱基为特征 变化，以及其他待分离的转化株系和突变体。 主要的具体目标是：（1）确定 自发发生的或由于诱变而发生的突变 （2）通过化学或物理因素分析， 核苷酸库扰动和基于诱变剂诱导的 DNA的变化，（3）确定分子机制， 哺乳动物细胞中哪些突变基因可以恢复;（4）分离 哺乳动物细胞抑制突变体，（5）确定是否 GPT基因的突变谱是不同的， 受体物种或穿梭体的整合位点 载体，（6）检查哺乳动物细胞中的错配修复，（7） 将穿梭载体中的gpt基因导入小鼠并分析 Gpt+小鼠突变的分子机制。

We propose to carry out a program of research on mutation in mammalian cells. As the target for mutations, these studies will use a small bacterial gene (gpt) introduced into mammalian cells as part of a retroviral shuttle vector and integrated into mammalian chromosomal DNA in a proviral form. We have recently developed this system and demonstrated its potential for providing information on mutations in mammalian cells at the level of specific base changes. The goal of the research is the elucidation of the molecular mechanisms of mutation in mammalian cells. The proposed studies will utilize shuttle vector transformed Gpt+ mouse cells and the large number of Gpt+ mutants that we have already isolated and characterized in terms of specific base changes, and other transformed lines and mutants to be isolated. The major specific aims are: (1) determine the base changes in mutations occuring spontaneously or as the result of mutagenesis by chemical or physical agents, (2) analyze the relationshp between nucleotide pool perturbation and mutagen-induced based changes in DNA, (3) determine the molecular mechanisms by which mutant genes in mammalian cells can revert; (4) isolate mammalian cell suppressor mutants, (5) determine if the mutational spectra for the gpt gene are different depending upon the recipient species or the site of integration of the shuttle vector, (6) examine mismatch repair in mammalian cells, (7) introduce the gpt gene in a shuttle vector into mice and analyze the molecular mechanisms of mutation in Gpt+ mice.