GENOMIC CLONING OF THE HUMAN COCAINE RECEPTOR

这项工作的长期目标是发展对 涉及行为和生理的分子机制(S) 与可卡因使用有关的表现。作为第一步， 剖析这一系列复杂的分子事件，我建议： 1)建立表达编码人类基因的小鼠细胞系 可卡因受体。 2)从其中一个中分离出编码可卡因受体的DNA片段 表达该受体的转基因人类基因的细胞系。 3)确定该基因的边界，准备详细的限制图， 并对该基因进行测序，以此作为深入研究 基因的分子结构，以及基因的分子结构 受体。 这将通过修改方法来实现 成功地选择在基因上重建人类转运蛋白 小鼠成纤维细胞中的5-羟色胺，以及人类转运蛋白 Cos细胞中的去甲肾上腺素。我建议准备一种小鼠细胞系 表达可卡因敏感的多巴胺转运体(即可卡因受体) 通过将包含在商业上的一种 可用的COSMID库。稳定转化的细胞系将是 对可卡因敏感的~3H-多巴胺转运的特异性摄取筛查 进入这些细胞。人类的DNA将从细胞中被拯救出来 专门表达可卡因受体，然后系统地 用限制性内切酶消化缩短。缩短的片断 将人类DNA的一部分导入Cos细胞，以鉴定 含有编码多巴胺基因的最短人类DNA片段 传送器。一旦这个短小的DNA片段在手中， 该基因将使用核糖核酸酶保护分析进行定位，该基因将 使用双脱氧测序技术进行测序。

The long range goal of this work is to develop an understanding of the molecular mechanism(s) involved in the behavioral and physical manifestations associated with cocaine usage. As a first step in dissecting this complex series of molecular events I propose to: 1) develop mouse cell lines which express the human gene encoding the cocaine receptor. 2) isolate the DNA fragment encoding the cocaine receptor form one of these cell lines expressing the transfected human gene for the receptor. 3) define the boundaries of this gene, prepare a detailed restriction map, and sequence the gene as a step toward thoroughly characterizing the molecular structure of the gene, and the molecular structure of the receptor. This will be accomplished using a modification of the approaches successfully chosen to genetically reconstitute the human transporter for serotonin in mouse fibroblasts, and the human transporter for norepinephrine in Cos cells. I propose to prepare mouse cell lines which express the cocaine sensitive dopamine transporter (ie., cocaine receptor) by transfecting large pieces of human DNA contained in a commercially available cosmid library. The stably transformed cell lines will be screened for specific uptake of cocaine sensitive 3H-dopamine transport into these cells. The human DNA will be rescued from the cells specifically expressing the cocaine receptor, and then systematically shortened using restriction endonuclease digestion. The shortened pieces of human DNA will be introduced into Cos cells in order to identify the shortest human DNA fragment containing the gene encoding the dopamine transporter. Once this short DNA fragment is in hand, the boundaries of the gene will be mapped using RNase protection analyses, and the gene will be sequenced using dideoxysequencing technology.