IDENTIFICATION OF GENE FOR ACTH-RECEPTOR BY CLONING

应激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障碍，如健忘症和 人们对阿尔泽海默氏症知之甚少。促肾上腺皮质激素 促肾上腺皮质激素(ACTH)及其受体是血管紧张素转换酶的重要分子成分。 对应激反应的感知和调节。在过去的15年里 多年来，我们已经开发了成功检测的工具， 促肾上腺皮质激素受体的特性和鉴定。我们现在 从牛肾上腺皮质分离ACTH受体基因的建议 免费DNA文库，并用牛基因分离人类基因 对于ACTH-R。这些目标将通过以下研究来实现 策略：a)发展ACTH-的配基结合和功能分析 在显微注射牛肾上腺皮质mRNA的非洲爪哇卵母细胞中；b) 信使核糖核酸的大小分级以鉴定活性信使核糖核酸组分 用于构建含T7的载体中的cDNA文库 启动子；c)含亚克隆的ACTH-R基因 体外转录与RNA表达检测相结合 显微注射入卵母细胞；d)测定 活动子克隆。ACTH受体基因随后将在 几个成熟的细胞系。促肾上腺皮质激素可能的异质性 肾上腺皮质三区和不同脑区的受体亚型 大脑的区域将通过原位的Northern分析进行研究 利用抗肿瘤抗体进行氢化反应和免疫细胞化学 与受体序列相对应的合成肽。强有力的 将利用高表达ACTH-1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.