ALCOHOL RESPONSE ELEMENTS IN HUMAN GABA/AR GENE CLUSTERS

乙醇对氨基的受体有深刻的急性和慢性影响乙醇对氨基的受体有深刻的急性和慢性影响 中枢神经系统中的酸性神经递质 特别是，急性接触 乙醇增强γ-氨基丁酸A型受体（GABAAR） 介导的反应，而慢性暴露改变了药理学， GABAARs在动物模型和死后脑中的密度 酗酒者 有人提出，慢性乙醇可能会导致 受体亚基mRNA表达的开关， 具有改变的药理学和生理学特性的同种型。 然而，受体功能改变的机制仍然存在， 未知但对于我们了解乙醇耐受性至关重要， 依赖 在我们调查的过程中 人GABAAR亚基基因启动子的鉴定和功能， 我们发现，乙醇可以刺激（约20倍）α 1 在转染的原代大鼠新皮层培养物中的启动子活性，和 位于近端启动子中的63 bp区域对于 乙醇法规 β 1启动子的活性被以下物质降低： 乙醇，这表明一个或多个顺式调节元件， 参与了细胞对乙醇的适应性反应。 一个主要目标 这个项目的一部分将是测试我们的工作假设， 与细胞表面受体相互作用， 通过细胞信号转导系统，激活或 抑制转录因子结合乙醇受体 顺式元件 我们发现GABAAR基因的多样性来自于 人类祖先的复制和随后的易位 α-α-β-γ基因簇，在 4号5号和15号染色体 我们的假设是 簇内和簇间基因的进化关系预示着 不同簇中相关基因的协同调控 乙醇。 为了检验上述假设，乙醇响应 α、β和γ亚基基因启动子中的元件将被 使用转染的原代神经元培养物， 用含有各种缺失的荧光素酶报告基因构建体， 点突变 我们将比较乙醇的序列和功能 来自不同GABAAR亚基启动子的响应元件，以鉴定 保守的调控区域和共享的转录因子。 细胞内信使的抑制剂和活化剂将用于 确定耦合受体调节的细胞反应系统 到转录调控。 总的来说，这些实验将增加 对我们理解神经元对慢性疾病的适应性反应的重要性 酒精暴露，从长远来看， 和治疗剂。

Ethanol exerts profound acute and chronic effects onreceptors for amino acid neurotransmitters in the CNS. In particular, acute exposure to ethanol potentiates the ganna-aminobutyric acid type-A receptor (GABAAR) mediated response, while chronic exposure alters the pharmacology and density of GABAARs in animal models and inpost-mortem brain from alcoholics. It has been proposed that chronic ethanol may cause a switch in the expression of receptor subunit mRNAs, producing an isoform(s) with altered pharmacolgical and physiological properties. However, the mechanism by which receptor function is altered remains unknown yet crucial to our understanding of ethanol tolerance and dependence. During the course of our investigation into the identification and function of promoters for human GABAAR subunit genes, we discovered that ethanol can stimulate (by about 20-fold) alpha1 promoter activity in transfected primary rat neocortical cultures, and that a 63 bp region located in the proximal promoter is crucial for ethanol regulation. The activity of the beta1 promoter is reduced by ethanol, suggesting that one or more cis-regulatory elements are involved in the cell's adaptive response to ethanol. A major objective of this project will be to test our working hypothesis that ethanol interacts with cell surface receptors and exerts genomic regulatory effects through a cellular signal transduction system(s), activating or inhibiting transcription factors which bind to ethanol reposive cis-elements. We have discovered that GABAAR gene diversity arose fromm the duplication and subsequent translocation of an ancestral human alpha-alpha-beta-gamma gene cluster, spawning the clusters on chromosomes 4, 5 and 15. It is our hypothesis that the close evolutionary relationsip of genes within and across clusters predicts coordinate regulation regulation of related genes in different clusters by ethanol. To examine the above hypotheses, ethanol responsive elements in alpha, beta and gamm subunit gene promoters will be identified functionally, using primary neuronal cultures transfected with luciferase reporter constructs containing various deletions and point mutations. We will compare the sequence and fucntion of ehtanol responsive elements from different GABAAR subunit promoters to identify conserved regulatory regions and shared transcription factors. Inhibitors and activators of intracellular messengers will be used to determine the cellular response system that couples receptor modulation to transcriptional regulation. Collectively, these experiments will add to our understanding of the neuron's adaptive response(s) to chronic ethanol exposure, and, in the long-run to the development of diagnostiv and therapeutic agents.