REGULATED EXPRESSION--CATECHOLAMINE BIOSYNTHESIS GENES

调控表达--儿茶酚胺生物合成基因

• 批准号: 3466401
• 负责人: Elaine J. Lewis
• 金额: $ 9.75万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466401
• 关键词: catecholamines; complementary DNA; dopamine; dopamine beta monooxygenase; enzyme mechanism; epinephrine; gene expression; genetic mapping; genetic regulation; genetic transcription; laboratory rabbit; laboratory rat; molecular cloning; norepinephrine; nucleic acid sequence; reporter genes; tissue /cell culture; transcription factor; tyrosine 3 monooxygenase

The rate of biosynthesis of the catecholamines dopamine, norepinephrine and epinephrine, can be modulated by numerous neuronal, hormonal and environmental influences. The aim of the experiments described in this proposal is to define and characterize the genetic regulatory elements for two catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH), the initial and rate-limiting enzyme, and dopamine beta- hydroxylase (DBH), the third enzyme of the pathway. In many tissues, including the adrenal medulla, sympathetic ganglia and locus ceruleus, TH and DBH are co-expressed and coordinately regulated by induction stimuli. The major focus of this project is to determine whether individual effectors which stimulate transcription from each gene do so through interactions at common or unique sites on that gene, and also whether the coordinated expression and modulation of activity of the TH and DBH genes is due to shared cis-acting genetic regulatory sites which are recognized by similar or identical trans-acting factors. Both cDNA and genomic clones for rat TH are already available, and will be used to define and characterize the regulatory elements of the TH gene required for stimulation of TH transcription by cyclic AMP, epidermal growth factor and cell density in pheochromocytoma cells. The 5' flanking sequences of the TH gene are ligated to a heterologous "reporter" gene and the TH sequences are assayed for the ability to confer inducer responsiveness onto the reporter gene. The required sequence boundaries will be defined, and the distance and orientation specifities of these genetic elements will be further analyzed. A cDNA for rat DBH will be isolated by immunoselection from a cDNA library derived from a tumor of the adrenal medulla. The cDNA will be used to assay the levels of DBH RNA transcripts under conditions known to induce DBH enzyme activity in both tissue culture and intact animals. The cDNA will also be used to isolate genomic clones for DBH, and the genetic regulatory elements important for the specificity and modulation of DBH gene expression will be defined. The regulatory elements of the TH and DBH genes will be compared by DNA sequence analysis and also by determining whether these genetic elements interact with, and can complete for, similar trans-acting control factors.

儿茶酚胺多巴胺的生物合成速率， 去甲肾上腺素和肾上腺素，可以通过多种调节 神经元、荷尔蒙和环境的影响。 该组织的目标是 本提案中描述的实验是为了定义和 表征两个基因调控元件 儿茶酚胺生物合成酶、酪氨酸羟化酶（TH）、 初始酶和限速酶，以及多巴胺β- 羟化酶（DBH），该途径的第三种酶。 在许多 组织，包括肾上腺髓质、交感神经节和 蓝斑、TH 和 DBH 共表达且协调 受诱导刺激调节。 该项目的主要重点是 确定是否刺激个体效应器 每个基因的转录是通过相互作用来实现的 该基因上的共同或独特位点，以及是否 TH 和 TH 活性的协调表达和调节 DBH 基因是由于共享顺式作用基因调控位点 它们被相似或相同的反式作用因子识别。 大鼠 TH 的 cDNA 和基因组克隆均已可用， 并将用于定义和表征监管 刺激 TH 所需的 TH 基因元件 环腺苷酸、表皮生长因子和细胞的转录 嗜铬细胞瘤细胞的密度。 5'侧翼序列 TH 基因与异源“报告”基因连接， 分析 TH 序列赋予诱导物的能力 对报告基因的反应。 所需的顺序 将定义边界，距离和方向 这些遗传因素的特殊性将得到进一步分析。 大鼠 DBH 的 cDNA 将通过免疫选择从 源自肾上腺髓质肿瘤的 cDNA 文库。 这 cDNA 将用于测定 DBH RNA 转录物的水平 在已知可诱导 DBH 酶活性的条件下 组织培养和完整的动物。 该 cDNA 还将用于 分离 DBH 的基因组克隆，以及遗传调控 对 DBH 的特异性和调节很重要的元素 基因表达将被定义。 监管要素 TH和DBH基因将通过DNA序列分析进行比较 并通过确定这些遗传元件是否相互作用 具有类似的反式作用控制因素，并且可以完成类似的反式作用控制因素。