MOLECULAR BASIS OF TYROSINE HYDROXYLASE REGULATION

酪氨酸羟化酶调节的分子基础

• 批准号: 6071934
• 负责人: Dona M Chikaraishi
• 金额: $ 5万
• 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 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6071934
• 关键词: adrenal glands; afferent nerve; animal genetic material tag; cholinergic receptors; enzyme induction /repression; gene deletion mutation; genetic enhancer element; genetic mapping; genetic promoter element; genetic transcription; genetically modified animals; laboratory mouse; molecular cloning; nucleic acid sequence; olfactory lobe; protein isoforms; site directed mutagenesis; stress proteins; sympathetic ganglion; synapses; tissue /cell culture; transcription factor; tyrosine 3 monooxygenase

In the nervous system, the selective expression of tyrosine hydroxylase (TH) leads to the development of catecholaminergic cells. TH is the rate- limiting enzyme in the synthesis of dopamine, norepinephrine and epinephrine, which are expressed in anatomically and functionally distinct locations within the central and peripheral nervous systems. This proposal seeks to define the regulatory elements that direct cell-specific expression and trans-synaptic induction of TH in cultured cells and transgenic mice. By mutational analyses of the 5' flanking region of the rat TH gene, two enhancer regions are required for full transcriptional activation in cultured cells: an AP1/E box region at -205 and an CRE site at -45. We propose to evaluate the role of these sites in transgenic mice using site- directed mutations. Since the CRE site has been implicated in mediating depolarization-induced transcription, the effect of CRE mutations will be assessed after trans-synaptic induction in vivo. Two paradigms will be investigated: tonic, activity-dependent expression in the dopaminergic periglomerular cells of the olfactory bulb and stress-dependent induction by cholinergic afferents in the adrenal and sympathetic ganglia. Using new CNS TH+ cell lines and deletion mapping in transgenic mice, we propose to identify sites responsible for TH expression in different TH+ cell types. Preliminary data from transgenic mice, suggest that multiple elements, both activators and repressors, may be involved. Fine mapping of the AP1/E box and the TH promoter regions is proposed in cultured cells. Using a panel of mutant AP1/E box sites assessed in the mapping experiments, proteins that bind transcriptionally active sites will be cloned from expression libraries and their relevance evaluated by several criteria.

在神经系统中，酪氨酸羟化酶的选择性表达 （Th）导致儿茶酚胺能细胞的发展。这是速率 - 在多巴胺，去甲肾上腺素和 肾上腺素，在解剖和功能上以不同的方式表达 中央和周围神经系统中的位置。这个建议 旨在定义直接细胞特异性的调节元素 培养细胞中Th的表达和反触发诱导 转基因小鼠。 通过对大鼠Th基因的5'侧翼区域的突变分析，两个 增强子区域是完全转录激活所必需的 培养的细胞：-205处的AP1/E盒区域和-45的CRE位点。我们 建议使用位点 - 定向突变。由于CRE站点与中介有关 去极化引起的转录，CRE突变的效果将是 在体内反式突触诱导后评估。两个范式将是 研究：多巴胺能中的补品，活性依赖性表达 嗅球的周围细胞和应力依赖性诱导 通过肾上腺和交感神经的胆碱能传入。 在转基因小鼠中使用新的CNS Th+细胞系和缺失映射，我们 建议识别负责在不同Th+中表达的位点 细胞类型。来自转基因小鼠的初步数据表明多个 可能涉及激活因子和阻遏物的元素。 AP1/E框的精细映射和TH启动子区域提出了 培养的细胞。使用一组突变的AP1/E盒站点 映射实验，结合转录活性位点的蛋白质 将从表达式库克隆及其相关性。 几个标准。