REGULATED EXPRESSION OF CATECHOLAMINE BIOSYNTHESIS GENES

神经系统由成千上万种不同类型的细胞组成 表现出适当的细胞-细胞所必需的功能特异性 沟通。细胞的生化和生物物理特性是 这在很大程度上取决于对该细胞表达的基因的选择。这个 基因转录成信使核糖核酸是通过相互作用来调节的 具有遗传识别功能的核转录调控蛋白 该基因内部和周围的元素。这里面的实验 建议的目的是确定调节模式的因素 大鼠多巴胺β-羟基酶(DBH)基因转录。胸径 催化儿茶酚胺中多巴胺转化为去甲肾上腺素 生物合成途径，并在特定的脑核中表达， 交感神经节和肾上腺髓质。在前一次授予中 期间，我们在5‘-侧翼区域发现了一个调控元件 影响特定细胞类型转录的大鼠胸腺激素基因 图案。这个30个碱基的调控元件，我们将其命名为DB1， 在儿茶酚胺能细胞系中起转录增强剂的作用， 但在成纤维细胞或上皮细胞系中不存在。这里面的实验 格兰特提案将进一步描述DB1 DNA与蛋白质的相互作用， 包括(1)确定DB1的精确结合部位(S) DBH基因的结合因子，(2)DB1结合因子的纯化 亲和层析法分离DB1cDNA克隆(S) 然后进行序列分析，(4)确定组织分布 DB1RNA转录本的特征，以及(5)分子的特征 DB1结合络合物的组成。除了这些研究之外， 定义了DB1绑定活动后，我们确定了第二个绑定 位于胸径基因5‘侧翼区的位点 转录因子AP-2的识别序列。此绑定活动 在神经母细胞瘤的提取物中观察到，但在上皮细胞中没有观察到 细胞系。拟议的实验将进一步表征这种AP-2 如结合活性，使用生化和分子遗传学方法 类似于描述DB1的特征的那些。功能界别 AP-2结合位点的意义将通过确定 AP-2对胸腺激素基因转录的影响。的目标之一 这项提议中的实验是为了了解这些 胸径基因的转录调节因子也起着影响 其他神经元基因的转录，特别是那些共同参与转录的基因。 在儿茶酚胺能细胞中表达。为了解决这个问题，我们将 确定DB1和AP-2蛋白调节细胞周期的能力 转录酪氨酸羟基酶基因，它编码初始的 儿茶酚胺生物合成途径的酶。最后，我们会 描述其他未确定特征的区域的功能重要性 胸径基因，就基因表达的特异性而言。结果是 在本申请中提出的实验将提供一个 对控制基因选择的遗传事件的理解 特定的神经递质表型。

The nervous system is composed of thousands of diverse cell types which exhibit functional specificity which is essential for appropriate cell-cell communication. The biochemical and biophysical properties of a cell are largely a function of the selection of genes expressed in that cell. The transcription of a gene into mRNA is regulated through the interaction of nuclear transcriptional modulatory proteins with genetic recognition elements within and surrounding that gene. The experiments in this proposal are designed to identify the factors which modulate the pattern of transcription of the rat dopamine beta-hydroxylase (DBH) gene. DBH catalyzes the conversion of dopamine to norepinephrine in the catecholamine biosynthetic pathway, and is expressed in specified brain nuclei, the sympathetic ganglia and the adrenal medulla. In the previous granting period we identified a regulatory element on the 5'-flanking region of the rat DBH gene which influences transcription in a cell type specific pattern. this 30 base regulatory element, which we have named DB1, functions as an enhancer of transcription in catecholaminergic cell lines, but not in fibroblast or epithelial cell lines. The experiments in this grant proposal will further characterize this DB1 DNA-protein interaction, and include (1) a determination of the precise binding site(s) of the DB1 binding factor on the DBH gene, (2) purification of the DB1 binding factor via DNA-affinity chromotography, (3) isolation of DB1 cDNA clone(s) followed by sequence analysis, (4) determination of the tissue distribution of the DB1 RNA transcript, and (5) characterization of the molecular composition of the DB1 binding complex. In addition to the studies which have defined the DB1 binding activity, we have identified a second binding site on the 5'-flanking region of the DBH gene corresponding to the recognition sequence of transcription factor AP-2. This binding activity was observed in extracts derived from neuroblastoma, but not epithelial cell lines. The experiments proposed will further characterize this AP-2 like binding activity, using biochemical and molecular genetic approaches similar to those described for the characterization of DB1. The functional significance of the AP-2 binding site will be studied by determining the ability of AP-2 to influence DBH gene transcription. One of the goals of the experiments in this proposal is to understand whether these transcriptional modulators of the DBH gene also function to influence transcription of other neuronal genes, especially those which are co- expressed in catecholaminergic cells. To address this question, we will determine the ability of the DB1 and AP-2 proteins to modulate the transcription of the tyrosine hydroxylase gene, which encodes the initial enzyme of the catecholamine biosynthetic pathway. Finally, we will characterize the functional importance of other, uncharacterized regions of the DBH gene, with regard to specificity of gene expression. The results of the experiments proposed in this application will provide an understanding of the genetic events which control the selection of a specific neurotransmitter phenotype.