GENOMIC CLONING OF THE HUMAN COCAINE RECEPTOR

这项工作的长期目标是了解 参与行为和生理的分子机制 与可卡因使用有关的症状。 的第一步 剖析这一系列复杂的分子事件，我建议： 1)开发小鼠细胞系，其表达编码 可卡因受体 2)分离编码可卡因受体的DNA片段， 表达所述受体的转染的人基因的细胞系。 3)定义这个基因的边界，准备一个详细的限制性内切酶图谱， 并对基因进行测序， 基因的分子结构，以及 受体的 这将通过修改这些方法来实现 成功地选择了基因重组人类运输机， 小鼠成纤维细胞中的5-羟色胺，以及 Cos细胞中的去甲肾上腺素。 我建议制备小鼠细胞系， 表达可卡因敏感的多巴胺转运蛋白（即，可卡因受体） 通过分离商业上含有的大块人类DNA， 可用的粘粒文库。 稳定转化的细胞系将被 筛选可卡因敏感性3 H-多巴胺转运的特异性摄取 这些细胞。 人类的DNA将从细胞中被拯救出来 特异性表达可卡因受体，然后系统地 使用限制性内切酶消化缩短。 缩短的片段 将人类DNA引入Cos细胞，以识别 最短的人类DNA片段，含有编码多巴胺的基因 传送器 一旦这个短的DNA片段在手， 该基因将使用RNase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.