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.
