IDENTIFICATION OF GENE FOR ACTH-RECEPTOR BY CLONING

压力诱发疾病的生化基础，如 免疫抑制、生长抑制、神经元变性和骨骼 肌肉萎缩和神经精神疾病如健忘症， 阿尔茨海默氏病的病因知之甚少。 促肾上腺皮质激素 促肾上腺皮质激素（ACTH）及其受体是肾上腺皮质激素分泌的重要分子组成部分。 感知和调解的压力反应。 过去十五 多年来，我们已经开发了成功检测的工具， ACTH受体（ACTH-R）的表征和鉴定。 我们现在 建议从牛肾上腺皮质分离ACTH受体基因 互补DNA文库，并使用牛基因分离人基因 促肾上腺皮质激素R 这些目标将通过以下研究来实现 策略：a）开发ACTH的配体结合和功能测定， 用来自牛肾上腺皮质的mRNA显微注射的非洲爪蟾卵母细胞中的R; B） mRNA的大小分级以鉴定活性mRNA级分， 用于在含T7的载体中构建cDNA文库 启动子; c）通过免疫组化鉴定含有ACTH-R基因的亚克隆; 体外转录和RNA表达测定的组合 d）测定以下的核苷酸序列： 活跃的亚克隆 然后ACTH受体基因将在 几种已建立的细胞系。 ACTH可能的异质性 肾上腺皮质的三个区域和不同区域中的受体亚型 将通过北方分析原位研究大脑区域 使用针对以下的抗体进行细胞化和免疫细胞化学 对应于受体序列的合成肽。 强效 将使用过表达ACTH的细胞系开发拮抗剂- 受体基因

The biochemical basis for stress induced diseases, such as immunosuppression, inhibition of growth, neuronal degeneration and skeletal muscle atrophy and for neuropsychiatric disorders such as amnesia and Alzeheimer's disease are poorly understood. Adrenocorticotropic hormone (ACTH) and its receptor are essential molecular components in the perception and mediation of the stress response. In the past fifteen years, we have developed the tools for the successful detection, characterization and identification of ACTH receptors (ACTH-R). We now propose to isolate the ACTH-receptor gene from bovine adrenal cortical complimentary-DNA library and use the bovine gene to isolate the human gene for ACTH-R. These goals will be attained by the following research strategy: a) development of ligand binding and functional assays for ACTH- R in Xenopus oocytes microinjected with mRNA from bovine adrenal cortex; b) size fractionation of mRNA to identify the active mRNA fraction which will be used for the construction of cDNA library in a vector containing T7 promoter; c) identification of the ACTH-R gene containing sub-clones by the combination of in vitro transcription and assay of expression of RNA microinjected into oocytes; d) determination of the nucleotide sequence of the active sub-clone. The ACTH receptor gene will then be expressed in several established cell lines. The possible heterogeneity of ACTH receptor subtypes in the three zones of adrenal cortex and in the various regions of the brain will be studied by Northern analysis, in situ hydribization and immunocytochemistry using antibodies raised against synthetic peptides corresponding to the receptor sequence. Potent antagonists will be developed using cell lines over expressing ACTH- receptor gene.