ALCOHOL RESPONSE ELEMENTS IN HUMAN GABA/AR GENE CLUSTERS

乙醇对氨基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，急性暴露于 乙醇增强氨基丁酸A型受体(GABAAR) 中介反应，而慢性暴露改变了药理学和 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 切换受体亚单位mRNAs的表达，产生 异构体(S)，具有改变的药理和生理特性。 然而，受体功能改变的机制仍然存在。 未知但对我们理解乙醇耐受性和 依赖。在我们调查的过程中 人GABAAR亚单位基因启动子的鉴定及其功能， 我们发现乙醇可以刺激(大约20倍)阿尔法。 转基因大鼠新皮质原代培养中的启动子活性 位于启动子近端的63个碱基对 乙醇监管。β1启动子的活性因以下原因而降低 乙醇，这表明一个或多个顺式调节元件是 参与了细胞对乙醇的适应性反应。一个主要目标 这个项目的目的是检验我们的工作假设乙醇 与细胞表面受体相互作用并发挥基因组调节作用 通过细胞信号转导系统(S)，激活或 抑制与乙醇结合的转录因子 顺式元素。我们发现GABAAR基因多样性来自于 人类祖先的复制和随后的移位 α-α-β-伽马基因簇，在 染色体4，5和15。这是我们的假设 基因在簇内和簇间的进化关系预测 不同簇中相关基因的协同调控 用乙醇。为了检验上述假设，乙醇反应 α、β和GAMM亚单位基因启动子中的元件将是 利用原代培养的神经元细胞进行功能鉴定 利用包含各种缺失的荧光素酶报告构建体 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 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激活剂将用于 确定偶联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 对神经元适应性反应的理解(S) 酒精暴露，并，从长远来看，对诊断的发展 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.