MOLECULAR MECHANISMS OF CHEMICAL TOXICITY

拟议研究的长期目标是阐明 典型环境化学毒素产生的DNA的变化 与DNA发生反应的物质。众所周知，毒性的一种成分 一些化学物质是与DNA发生反应，人们对这种化学物质有很多了解 形成的加合物的化学性质。人们对此知之甚少 对DNA结构和功能的影响或对分子的影响 导致突变和致癌的DNA改变的性质。在……里面 建议的工作，成年大鼠肝(ARL)上皮细胞的遗传毒性 将被研究，因为这些品系是从体内特定的 组织，并保持生物激活化学遗传毒素的能力。 在这些增殖系中，DNA的变化导致了 次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HGPRT)基因座 这些突变的功能后果将通过各种不同的 意思是。这些工作包括：(A)大鼠HGPRT基因的克隆 基因及其DNA序列分析特征(B) 化学诱变ARL突变体的生化特性， Southern分析将用于区分基因组的性质 遗传毒性化学物质引起的ARL细胞的改变；(C)高度 精密技术，聚合酶链式反应(PCR)将适用于 化学方法检测大鼠HGPRT基因中的单碱基替换 诱导HGPRT突变体；(D)Northern杂交、核糖核酸酶保护分析 核径流转录试验和体外翻译试验将 用来探讨突变对转录、加工的影响 和HGPRT mRNA的半衰期；(E)阐明 基于杂交研究的大鼠HGPRT基因的组织 并因此允许区分位于 在常染色体上可能出现的相关序列的X染色体上 染色体。拟议的研究，除了记录自然 化学诱导的DNA变化，将有助于理解 在老鼠体内的基因组的组织，一个重要的物种 实验毒理学，以及人类相关基因的变化 遗传性疾病。

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.