REGULATED EXPRESSION--CATECHOLAMINE BIOSYNTHESIS GENES

多巴胺的生物合成速率， 去甲肾上腺素和肾上腺素，可以通过许多调节 神经、激素和环境影响。 的目的 本提案中描述的实验是定义和 描述两个基因调控元件 儿茶酚胺生物合成酶，酪氨酸羟化酶（TH）， 起始酶和限速酶，以及多巴胺β- 羟化酶（DBH），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 由诱导刺激调节。 该项目的主要重点是 以确定刺激的个体效应器是否 每个基因的转录都是通过相互作用进行的， 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基因是由于共同的顺式作用的遗传调控位点 其由相似或相同的反式作用因子识别。 大鼠TH的cDNA和基因组克隆都已经可用， 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因子的转录 嗜铬细胞瘤细胞密度。 的5'侧翼序列。 TH基因连接到异源“报告”基因， 测定TH序列赋予诱导剂的能力， 对报告基因的反应。 所需序列 边界将被定义，距离和方向 将进一步分析这些遗传元件的特异性。 大鼠DBH的cDNA将通过免疫选择从一个 来源于肾上腺髓质肿瘤的cDNA文库。 的 cDNA将被用来分析DBH RNA转录物的水平 在已知诱导DBH酶活性的条件下， 组织培养和完整动物。 cDNA还将用于 分离DBH的基因组克隆， 对DBH的特异性和调节重要的元素 基因表达将被定义。 的调控元件 TH和DBH基因将通过DNA序列分析进行比较 也可以通过确定这些基因元件是否 具有并且能够完成类似的反式作用控制因素。

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.