CHOLINE ACETYLTRANSFERASE

描述（改编自申请人摘要）： 本项目的长期目标是了解 胆碱能基因位点及其基因产物胆碱的调节 乙酰转移酶（ChAT）和囊泡乙酰胆碱转运蛋白（VAChT）。 为实现这一目标，提出了三个具体目标。第一个目标是 旨在阐明细胞特异性表达的机制， 胆碱能基因位点这个基因座的独特之处在于它包含了 ChAT和VAChT在相同的转录方向。我们将测试 假设有上游和下游调控元件， 指导ChAT和/或VAChT的细胞特异性表达的基因。我们将 研究这些调节元件在“双重”背景下的活性， 报告基因”，其测量来自同一基因的ChAT和VAChT转录。 构建体第二个具体目标将检验以下假设： 转录因子REST到DNA受神经元特异性同种型调节 REST的第一个版本是REST 4。REST 4被认为是 胆碱能基因位点以及一组神经元特异性基因， 含有神经元限制性沉默元件NRSE/RE 1。在第三个具体 目的我们建议使用最近开发的系统研究VAChT功能。 我们有一个突变的PC 12细胞系，它缺乏ChAT和VAChT，但 含有突触囊泡，可以用ChAT和/或VAChT重建 cDNA。这提供了一个独特的机会，研究VAChT的功能， 不存在内源性VAChT的干扰背景活性。我们将使用 该系统研究囊泡填充的调节机制， 研究三聚体G蛋白对VAChT功能的调控，并研究一种VAChT 一个突变体，它似乎作为一个显性负抑制内源性 VAChT活性。

DESCRIPTION (Adapted from applicant's abstract): The long range goal of this project is to develop an understanding of the regulation of the cholinergic gene locus and its gene products, choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To achieve this goal three specific aims are proposed. The first aim is directed at elucidating the mechanism of cell specific expression of the cholinergic gene locus. This locus is unique in that it contains the genes for both ChAT and VAChT in the same transcriptional orientation. We will test the hypothesis that there are both upstream and downstream regulatory elements in the gene which direct cell specific expression of ChAT and/or VAChT. We will study the activity of these regulatory elements in the context of a "double reporter gene" which measures both ChAT and VAChT transcription from the same construct. The second specific aim will test the hypothesis that the binding of the transcription factor REST to DNA is regulated by a neuron specific isoform of REST called REST4. REST4 is proposed to be a key regulator of the cholinergic gene locus as well as of a group of neuron specific genes which contain the neuron restrictive silencer element NRSE/RE1. In the third specific aim we propose to use a recently developed system for studying VAChT function. We have a mutant PC12 cell line which is devoid of both ChAT and VAChT, but contains synaptic vesicles, and can be reconstituted with ChAT and/or VAChT cDNAs. This provides a unique opportunity to study VAChT function in the absence of interfering background activity from endogenous VAChT. We will use this system to study the mechanism by which vesicle filling is regulated, to study trimeric G protein regulation of VAChT function, and to study a VAChT mutant which appears to act as a dominant negative by inhibiting endogenous VAChT activity.