MOLECULAR MECHANISMS OF CHEMICAL TOXICITY

拟议研究的长期目标是阐明 代表性环境化学毒素产生的DNA变化 与DNA发生反应。 众所周知，毒性的一个组成部分 一些化学物质与DNA发生反应， 形成的加合物的化学性质。 关于这一点，我们所知甚少。 对DNA结构和功能的影响， 导致诱变和致癌的DNA改变的性质。 在 拟议的工作，成年大鼠肝脏（ARL）上皮细胞的遗传毒性 将进行研究，因为这些品系来自特定的体内 组织并保留生物激活化学基因毒素的实质能力。 在这些增殖的细胞系中，DNA的变化导致了细胞中的突变。 次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT）基因座和 这些突变的功能后果将通过各种研究， 手段 （a）大鼠HGPRT cDNA的克隆 基因及其通过DNA序列分析的表征（B） 化学诱导的ARL突变体的生化表征， Southern分析将用于区分基因组的性质， 由ARL细胞中的遗传毒性化学物质诱导的改变;（c）高度 聚合酶链式反应（PCR）将被应用于 用化学方法检测大鼠HGPRT基因中的单碱基替换， 诱导的HGPRT突变体;（d）北方印迹，RNA酶保护分析 核径流转录测定和体外翻译测定将被 用于探讨突变对转录、加工、 （e）HGPRT mRNA的总体半衰期的阐明 基于杂交研究的大鼠HGPRT基因的组织 从而允许区分位于不同位置的功能重要的序列 X染色体上的相关序列，可能发生在常染色体 染色体 这项研究除了记录 化学诱导的DNA变化，将有助于理解 老鼠基因组的组织，一个重要的物种， 实验毒理学，以及人类基因的变化 遗传病

The long term objective of the proposed research is to elucidate the changes in DNA produced by representative environmental chemical toxins that react with DNA. It is well established that a component of toxicity of some chemicals is reaction with DNA and a great deal is known about the chemical nature of the adducts formed. Less is known about the consequences to the structure and function of DNA or about the molecular nature of alterations in DNA leading to mutagenesis and carcinogenesis. In the proposed work, genotoxicity in adult rat liver (ARL) epithelial cells will be studied because these lines are derived from a specific in vivo tissue and retain a substantial ability to bioactivate chemical genotoxins. In these proliferating lines, the DNA changes leading to mutation in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus and the functional consequences of such mutations will be studied by a variety of means. These include the following: (a) Cloning of cDNA of the rat HGPRT gene and its characterization by DNA sequence analysis (b) In addition to the biochemical characterization of chemically induced ARL mutants, Southern analyses will be used to differentiate the nature of genomic alterations induced by genotoxic chemicals in ARL cells; (c) A highly precise technique, the polymerase chain reaction (PCR) will be adapted to detect single base substitutions in the rat HGPRT gene in chemically induced HGPRT mutants; (d) Northern blotting, RNase Protection analysis nuclear runoff transcription assay and in vitro translation assay will be used to explore the influence of mutation on the transcription, processing and half life of the HGPRT mRNA; (e) Elucidation of the overall organization of the rat HGPRT gene on the basis of hybridization studies and thus permit the distinction of functionally important sequences located on the X chromosome from related sequences that may occur on autosomal chromosomes. The proposed research, in addition to documenting the nature of chemically induced changes in DNA, will contribute to an understanding of the organization of the genome in the rat, an important species in experimental toxicology, and of changes in a gene involved in a human genetic disorder.