RECEPTOR CDNA EXPRESSION CLONING USING LIGAND AUTORADIOGRAPHIC SCREENING

了解识别滥用药物的受体分子， 药物对神经递质的影响是确定 药物滥用的分子机制知之甚少 许多这些分子，因为它们已经被证明非常难以 通过常规方法纯化。实验室继续 致力于建立一种直接克隆这些分子的方法， 它们的配体结合特性，并利用这种方法。 在过去的一年里，这些工人取得了实质性进展， 这个结局 以前，克隆的β肾上腺素能受体cDNA被用于 记录并优化β肾上腺素能受体结合的表达 COS细胞中。 在今年完成的研究中， 回收技术和电穿孔已经导致了 质粒存在于非常少量的细胞中， 频率. 这导致有记录的富集高达300倍， 一步 工人们在采用这种方法以 可卡因和五氯酚受体的作用本年度目标 包括使用该技术尝试基于两种基因克隆受体 cDNA回收和亚分级文库以及cDNA文库的适应性改造 聚合酶链反应，以帮助恢复非常少量的 质粒存在。 这一做法继续为以下方面提供了希望： 允许直接克隆基因的关键行动的几类 滥用物质。

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.