REGULATED EXPRESSION OF CATECHOLAMINE BIOSYNTHESIS GENES

神经系统由成千上万种不同的细胞组成， 表现出对适当的细胞-细胞 通信 细胞的生物化学和生物物理特性是 这主要取决于细胞中表达的基因的选择。 的 基因转录成mRNA是通过以下相互作用来调节的： 具有遗传识别的核转录调节蛋白 基因内部和周围的元素。 这个实验 建议的目的是确定调整模式的因素， 大鼠多巴胺β-羟化酶（DBH）基因的转录。 胸径 催化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。 在过去的授予 在此期间，我们鉴定出了一个调控元件的5 '侧翼区， 影响细胞类型特异性转录的大鼠DBH基因 格局 这个由30个碱基组成的调控元件，我们将其命名为DB 1， 在儿茶酚胺能细胞系中起转录增强子的作用， 但不在成纤维细胞或上皮细胞系中。 这个实验 拨款提案将进一步表征这种DB 1 DNA-蛋白质相互作用， 并包括（1）确定DB 1的精确结合位点 DBH基因上的结合因子，（2）DB 1结合因子的纯化 通过DNA亲和层析，（3）分离DB 1 cDNA克隆 然后进行序列分析，（4）确定组织分布 DB 1 RNA转录物的分子特征，以及（5）DB 1 RNA转录物的分子特征。 DB 1结合复合物的组成。 除了研究， 在定义了DB 1的结合活性之后，我们确定了第二种结合 在DBH基因的5 '-侧翼区上对应于 转录因子AP-2的识别序列。 这种结合活性 在神经母细胞瘤的提取物中观察到，而在上皮细胞瘤的提取物中未观察到 细胞系 提出的实验将进一步表征这种AP-2 像结合活性，使用生物化学和分子遗传学方法， 类似于DB 1的表征所描述的那些。 功能 AP-2结合位点的重要性将通过测定 AP-2影响DBH基因转录的能力。 的目标之一 这项提议中的实验是为了了解这些 DBH基因的转录调节因子也起影响 其他神经元基因的转录，特别是那些与 在儿茶酚胺能细胞中表达。 为了解决这个问题，我们将 确定DB 1和AP-2蛋白调节 酪氨酸羟化酶基因的转录，该基因编码起始的 儿茶酚胺生物合成途径的酶。 最后我们将 表征其他未表征区域的功能重要性 DBH基因，就基因表达的特异性而言。 结果 在本申请中提出的实验将提供一个 了解控制选择的遗传事件， 特异性神经递质表型。

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.