CELL SPECIFIC TYROSINE HYDROXYLASE GENE TRANSCRIPTION

细胞特异性酪氨酸羟化酶基因转录

• 批准号: 3302796
• 负责人: WILLIAM W MORGAN
• 金额: $ 11.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302796
• 关键词: DNA binding protein; affinity chromatography; antibody; cell differentiation; chimeric proteins; complementary DNA; embryogenesis; fusion gene; gene expression; genetic regulatory element; genetic transcription; genetically modified animals; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rabbit; molecular cloning; neurons; northern blottings; nucleic acid sequence; polymerase chain reaction; protein purification; site directed mutagenesis; tissue /cell culture; transcription factor; transfection; tyrosine 3 monooxygenase

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines, major neurotransmitters-hormones released by adrenal medullary cells and by life-supporting neurons. TH activity increases with enhanced catecholamine biosynthesis, in part by elevated expression of TH mRNA. The TH gene is only expressed in cells which release catecholamines suggesting a regulation by precise cell-specific molecular mechanisms. These include interactions between several segments of DNA (cis-elements) located in the upstream or 5' flanking region of the TH gene and nuclear proteins (trans-acting factors) which bind in a sequence specific fashion to these cis-elements. Our data suggest that one element is an AP-1-like sequence in the 5' DNA which interacts in a cellspecific manner with protein(s) found only in nuclear extracts from THexpressing cell lines,' i.e., PC-12 cells. The first specific aim is to isolate and characterize the protein which recognizes this AP-1-like sequence. in the second specific aim, antibodies to this protein will be Used for immunohistochemical studies in mouse embryos in combination with PCR and in situ hybridization to determine the temporal and spatial onset of expression of the gene which encodes this trans-acting factor. By comparing these data, with the first appearance of immunoreactive TH in cells, insight will be gained into the role of the AP-1-like binding protein in the embryologic commitment to the expression of the catecholaminergic phenotype. The last specific aim will employ the transgenic mouse model to investigate the role of the AP-1-like cis-element as well as additional cis-elements located upstream of the TH coding sequence in the cell-specific expression of the TH gene in the whole animal. Ultimately, these results may suggest novel approaches to treat diseases, e.g., Parkinson's Disease, a sinister malady ultimately leading to an almost complete loss of voluntary locomotor control.

酪氨酸羟化酶（Tyrosine hydroxylase，TH）是生物合成的限速酶 肾上腺释放的主要神经递质--激素--儿茶酚胺 和维持生命的神经元。 TH活性随 增强儿茶酚胺的生物合成，部分通过TH的表达升高 mRNA。 TH基因仅在释放儿茶酚胺的细胞中表达 表明通过精确的细胞特异性分子机制进行调节。 这些包括几个DNA片段（顺式元件）之间的相互作用。 位于TH基因的上游或5'侧翼区， 以序列特异性方式结合的蛋白质（反式作用因子） 这些顺式元件。 我们的数据表明，一种元素是AP-1样的 在5' DNA中的序列，其以细胞特异性方式与 仅在TH表达细胞系的核提取物中发现的蛋白质， 也就是说，PC-12细胞 第一个具体目标是分离和表征 识别AP-1样序列的蛋白质。在第二 针对特定目的，该蛋白的抗体将用于 小鼠胚胎中的免疫组织化学研究结合PCR和 原位杂交以确定时间和空间起始 编码该反式作用因子的基因的表达。 通过 比较这些数据，与免疫反应性TH的第一次出现， 细胞，洞察将获得AP-1样结合的作用， 蛋白质的胚胎承诺的表达， 儿茶酚胺能表型 最后一个具体目标将采用 研究AP-1样顺式元件作用的转基因小鼠模型 以及位于TH编码上游的另外的顺式元件 序列在整个TH基因的细胞特异性表达中 动物 最终，这些结果可能会提出新的治疗方法， 疾病，例如，帕金森氏病，一种最终导致 几乎完全失去自主运动控制