MOLECULAR BASIS OF TYROSINE HYDROXYLASE REGULATION

酪氨酸羟化酶调节的分子基础

• 批准号: 2764095
• 负责人: Dona M Chikaraishi
• 金额: $ 40.1万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2764095
• 关键词: DNA binding protein; active sites; cAMP response element binding protein; enzyme activity; enzyme induction /repression; epitope mapping; gene deletion mutation; gene targeting; genetic enhancer element; genetic mapping; genetic promoter element; genetically modified animals; laboratory mouse; neurogenetics; reporter genes; site directed mutagenesis; stainings; tissue /cell culture; transcription factor; transfection; tyrosine 3 monooxygenase

DESCRIPTION: (from applicant's abstract): Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of the catecholamine neurotransmitters, dopamine, norepinephrine, and epinephrine, which are essential during development. TH 20 anatomically and functionally discrete nuclei in the mammalian CNS and peripheral nervous system, several of which sustain activity-dependent induction of TH. We have identified regulatory sites that direct TH expression in cell culture: principal among these are the cAMP response element (CRE) and the AP1 site. We wi11 mutate the TH CRE and AP I sites in a TH-driven reporter to the consensus DNA binding site for Ga14, a yeast transcription factor absent in mammalian cells. Expression from the mutated enhancers will be assessed in transgenic mice to determine if the CRE or API sites are important in vivo, for cell specific and trans- synaptic induction. Although many transcription factors composed of the transactivation domain of various transcription factors fused to a GaI4 DNA binding domain will be used in cultured cells and in transgenic mice to rescue basal and induced expression from the mutated CRE site. We seek to develop particle-mediated transfection into brain slices as an alternative to promoter mapping in transgemic mice. Reporter constructs will be biolistically transfected into acutely prepared where TH neurons retain their local physiological and anatomical connections. By co-staining for the endogenous TH gene, specificity at the single cell level can be assessed. We have identified a 4.5 KB 5' flanking region that directs expression to all TH CNS and PNS groups in transgenic mice. We hypothesize that discrete elements in this region direct cell-specific expression. Promoter mutations will be used to direct expression of an epitope- tagged TH transgene. Those lines lacking expression in discrete TH cells will be bred to a TH knockout mouse to restore catecholamines to all the but the ablated regions, creating place-specific knockouts. This aim will not only map putative cell-specific elements in vivo, but provide new animal models for Parkinson's disease, dysautonomia, affected disorders and drug abuse.

描述：（来自申请人的摘要）： 酪氨酸羟化酶（Th）是合成中的速率限制酶 儿茶酚胺神经递质，多巴胺，去甲肾上腺素和 肾上腺素，在发育过程中至关重要。 Th 20从解剖学上 在哺乳动物中枢神经系统和周围的功能上离散的核 神经系统，其中一些维持活动依赖性诱导 th。 我们已经确定了直接在细胞中表达TH表达的调节位点 文化：其中的主要是营地响应要素（CRE）和 AP1网站。我们将th驱动器中的th cre和ap i变为th驱动 酵母Ga14共有DNA结合位点的记者DNA结合位点 哺乳动物细胞中不存在转录因子。来自 将在转基因小鼠中评估突变的增强子，以确定是否是否 CRE或API位点在体内很重要，对于细胞特异性和反式 突触诱导。尽管许多转录因子由 融合到GAI4的各种转录因子的反式激活结构域 DNA结合结构域将用于培养细胞和转基因小鼠 从突变的CRE位点营救基础和诱导表达。 我们试图将粒子介导的转染转化为脑切片 在过度小鼠中的启动子映射的替代方法。记者 构建体将被生物息染为急性准备 神经元保留其局部生理和解剖联系。 通过为内源性TH基因染色，单个特异性 可以评估细胞水平。 我们已经确定了一个4.5 kb 5'的侧翼区域，该区域指导表达 转基因小鼠中的所有THS和PNS组。我们假设这一点 该区域直接细胞特异性表达中的离散元素。 启动子突变将用于指导表位表达 标记的TH转基因。那些在离散TH细胞中缺乏表达的线 将繁殖到淘汰鼠标，以将儿茶酚胺恢复到所有 但是融入的地区，创造了特定地点的淘汰赛。这个目标 不仅会在体内绘制假定的细胞特异性元素，还将提供 帕金森氏病的新动物模型，Dysautonomia受影响 疾病和吸毒。