RECEPTOR CDNA EXPRESSION CLONING USING LIGAND AUTORADIOGRAPHIC SCREENING

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Understanding the receptor molecules that recognize abused drugs and the neurotransmitters impacted by drugs is an important step in determining the molecular mechanisms underlying drug abuse. Little is known about many of these molecules, because they have proven very difficult to purify through conventional approaches. The laboratory has continued to work to establish a method for directly cloning these molecules based on their ligand binding properties, and to exploit this approach. Over the past year, these workers have made substantial progress toward this end. Previously, a cloned beta adrenergic receptor cDNA was used to document and optimize expression of the beta adrenergic receptor binding site in COS cells. In studies ompleted during this year, improved DNA recovery techniques and electroporation have resulted in recoveries of the plasmid present in very small number of cells with a reasonable frequency. This results in documented enrichments of up to 300-fold in a single step. The workers have progressed in adapting the method for use with the cocaine and PCP receptors. Goals for the current year include using this technique to attempt receptor cloning based on both cDNA recovery and subfractionating libraries and adaptation of the polymerase chain reaction to aid in recovery of the very small numbers of plasmids present. This approach continues to provide promise for allowing direct cloning of genes key to the action of several classes of abused substances.