MOLECULAR CLONING OF BRAIN MRNA IN ETHANOL DEPENDENCE

越来越多的证据表明酒精会影响基因 在大脑中的表达比之前想象的要多。这是 尤其是在慢性乙醇治疗期间，当基因 中枢神经系统的表达受神经化学物质的影响 与宽容和依赖的发展相关的变化。这个 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(信使核糖核酸)。“差异”的分子克隆技术 采用“菌落杂交”和“消减文库杂交” 大鼠脑内互补DNA(CDNA)克隆的鉴定与鉴定 酒精依赖引起的mRNA改变(诱导或抑制)和 戒烟。反复饮酒会使大鼠出现酒精依赖 连续5天灌胃高剂量乙醇。 互补的DNA文库将用从 正常大鼠、酒精依赖大鼠和依赖大鼠的脑组织 从乙醇中提取。这些cDNA文库将受到 重组杂交技术将揭示mRNA的cDNA克隆 它们在酒精依赖和戒断过程中会经历上调或下调。 差异表达的克隆将根据核糖核酸进行表征 酸序列，并根据大小、组织分布和丰度 每个互补的信使核糖核酸。将执行时间进程分析以 监测乙醇诱导的这些mRNA的变化，选定的克隆将被 用于将信使核糖核酸定位于大鼠脑内对酒精有反应的部位。这个 这项建议的实验设计将提供一种公正的方法 可用于基因表达研究的基因克隆的发现 急性酒精摄入过渡过程中的中枢神经系统 通过酒精依赖和戒断。差异化地选择- 表达的cdna克隆最终将作为分子探针用于 乙醇中分子相关数据库的扩展 上瘾。未来研究探索乙醇诱导的机制 基因表达(即转录)、神经解剖学基因座的变化 成瘾的分子基础(即原位杂交)及其研究 的遗传连锁和限制性片段长度多态 这些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will be constructed using mRNA isolated from the brains of control and ethanol-dependent rats as well as dependent rats withdrawn from ethanol.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.