MOLECULAR CLONING OF BRAIN MRNA IN ETHANOL DEPENDENCE

越来越多的证据表明酒精影响基因 大脑中的表达比以前想象的要多。 这是 特别是在慢性乙醇治疗期间，当基因 在中枢神经系统中的表达受神经化学物质的影响， 与耐受性和依赖性的发展相关的变化。 的 拟议的项目将研究乙醇成瘾的演变， 基因表达的初始产物的反应性的功能， 信使RNA（mRNA）。 “差异”的分子克隆技术 将使用“菌落杂交”和“消减文库杂交 鉴定和表征大鼠脑的互补DNA（cDNA）克隆， mRNA改变（诱导或抑制）作为乙醇依赖的结果， 戒断 通过重复给药，大鼠将呈现乙醇依赖性。 在5天的时间内胃内给予高剂量的乙醇。 互补DNA文库将使用分离自以下的mRNA构建： 对照组、酒精依赖组和酒精依赖组大鼠的脑组织 从乙醇中提取。 这些cDNA文库将进行以下处理： 重组杂交技术将揭示mRNA的cDNA克隆 其在乙醇依赖性和戒断中经历上调或下调。 差异表达的克隆将基于核酸序列进行表征。 酸序列和根据大小，组织分布和丰度 每个互补的mRNA。 将进行时程分析， 监测乙醇诱导的这些mRNA的变化，并选择克隆将 用于将mRNA定位于大鼠脑中的乙醇响应位点。 的 本建议的实验设计将提供一个公正的方法， cDNA克隆的发现，可用于研究基因表达， 从急性乙醇摄入过渡期间的中枢神经系统 酒精依赖和戒断。 差异化选择- 表达的cDNA克隆最终将用作分子探针， 乙醇中分子相关物数据库的扩充 成瘾 未来的研究探索乙醇诱导的机制 基因表达的变化（即，转录），神经解剖位点， 成瘾的分子基础（即，原位杂交）和研究 基因连锁和限制性片段长度多态性 这些cDNA克隆也将促进酒精中毒。

An increasing body of evidence suggests that alcohol affects gene expression in the brain more than had been previously thought. This is particularly tue during periods of chronic ethanol treatment, when gene expression in the central nervous system is subject to the neurochemical changes associated with the development of tolerance and dependence. The proposed project will examine the evolution of ethanol addiction as a function of the responsiveness of the initial product of gene expression, messenger RNA (mRNA). The molecular cloning techniques of "differential colony hybridization" and "subtracted library hybridization" will be used to identify and characterize complementary DNA (cDNA) clones of rat brain mRNA altered (induced or repressed) as a result of ethanol dependence an withdrawal. Rats will be rendered ethanol-dependent by repeated intragastric administration of high doses of ethanol over a 5 day period. Complementary DNA libraries will be constructed using mRNA isolated from the brains of control and ethanol-dependent rats as well as dependent rats withdrawn from ethanol. These cDNA libraries will be subjected to recombinant hybridization techniques which will reveal cDNA clones for mRNA which undergo up-or down-regulation in ethanol dependence an withdrawal. Differentially-expressed clones will be characterized based upon nucleic acid sequence and according to the size, tissue distribution and abundance of each complementary mRNA. A time-course analysis will be performed to monitor ethanol-induced changes of these mRNA, and selected clones will be used to localize mRNA to ethanol-responsive sites in rat brain. The experimental design of this proposal will provide an unbiased approach for the discovery of cDNA clones that can be used to study gene expression in the central nervous system during the transition from acute ethanol intake through ethanol dependence and withdrawal. Selected differentially- expressed cDNA clones will eventually be used as molecular probes for expansion of the data base of molecular correlates found in ethanol addiction. Future studies exploring the mechanisms of ethanol-induced changes in gene expression (i.e., transcription), neuro-anatomical loci for the molecular basis of addiction (i.e., in situ hybridization) and studies of the genetic linkages and restriction fragment length polymorphisms in alcoholism will also be facilitated by these cDNA clones.