HALOGENATED AROMATIC HYDROCARBONS

卤代芳烃

• 批准号: 3483754
• 负责人: ALAN P. POLAND
• 金额: $ 26.49万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-09-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3483754
• 关键词: DNA binding protein; affinity chromatography; alleles; antireceptor antibody; biochemical evolution; chemical carcinogen; chlorine; complementary DNA; dioxins; drug metabolism; drug receptors; environmental toxicology; enzyme induction /repression; enzyme mechanism; gel mobility shift assay; gene expression; halobiphenyl /halotriphenyl compound; laboratory mouse; molecular cloning; monoclonal antibody; protein structure function; radioimmunoassay; receptor binding; species difference; tissue /cell culture

The goal of this proposal is to characterize the Ah receptor - a soluble protein present in many tissues of vertebrate species and a presumed member of the erbA superfamily of receptors, which mediates the pleiotropic effects of halogenated aromatic hydrocarbons such as 2,3,7,8- tetrachlorodibenzo-p-dioxin. 1) We propose to clone and sequence the gene for the Ah receptor from cDNA libraries by using degenerate oligonucleotide probes and rabbit polyclonal antibodies to a synthetic peptide that corresponds to the N-terminal sequence of the receptor. We plan to analyze the structure-function analysis of this protein using mutagenesis and an expression assay. 2) We propose to characterize the four allelic variants of the Ah receptor among inbred mouse strains: a) in vitro by their binding kinetics K-D, B-MAX and ease of activation and b) in vivo by genetic analysis of the differences in functional phenotypes especially Ah(b-1)/Ah(d) vs. Ah(b-2)/Ah(d) and correlation of this with quantitative nuclear extract-gel retardation experiments. 3) We propose to expand on our photoaffinity labeling and immunologic methodology; a) to screen lower vertebrate and invertebrate species for the presence of the Ah receptor, b) to prepare new monoclonal and polyclonal antibodies to various regions of the Ah receptor, c) to prepare an immunoaffinity column to aid in rapid purification, and d) to examine murine tissues for the cellular localization of the Ah receptor using immunohistochemistry.

该提案的目标是表征 Ah 受体 - 一种可溶性受体 存在于脊椎动物许多组织中的蛋白质和推测的 erbA 受体超家族成员，介导 2、3、7、8-等卤代芳烃的多效性 四氯二苯并-对-二恶英。 1）我们建议克隆并测序该基因 使用简并从 cDNA 文库中获取 Ah 受体 寡核苷酸探针和兔多克隆抗体合成 对应于受体 N 端序列的肽。 我们 计划使用该蛋白质的结构-功能分析 诱变和表达测定。 2）我们建议描述 近交系小鼠品系中 Ah 受体的四种等位基因变体：a) 体外通过其结合动力学 K-D、B-MAX 和激活的难易程度 b) 通过基因分析体内功能差异 表型，尤其是 Ah(b-1)/Ah(d) 与 Ah(b-2)/Ah(d) 以及相关性 这是通过定量核提取物-凝胶延迟实验进行的。 3） 我们建议扩大我们的光亲和标记和免疫学 方法论； a) 筛选低等脊椎动物和无脊椎动物物种 Ah 受体的存在，b) 制备新的单克隆和 针对 Ah 受体各个区域的多克隆抗体，c) 准备免疫亲和柱以帮助快速纯化，并且 d) 检查小鼠组织中 Ah 受体的细胞定位 使用免疫组织化学。