CELL SPECIFIC TYROSINE HYDROXYLASE GENE TRANSCRIPTION

细胞特异性酪氨酸羟化酶基因转录

• 批准号: 3302797
• 负责人: WILLIAM W MORGAN
• 金额: $ 12.61万
• 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/3302797
• 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; protein sequence; 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(TH)是生物合成中的限速酶。 儿茶酚胺，主要的神经递质-由肾上腺释放的激素 骨髓细胞和维持生命的神经元。TH活性随 增强儿茶酚胺的生物合成，部分是通过上调TH的表达 MRNA.TH基因只在释放儿茶酚胺的细胞中表达 暗示了一种精确的细胞特异性分子机制的调节。 这包括DNA的几个片段之间的相互作用(顺式元件) 位于TH基因的上游或5‘侧翼区和核 以序列特异性方式结合的蛋白质(反式作用因子) 这些顺式元素。我们的数据表明，有一种成分是类似AP-1的 5‘DNA中以细胞特异性方式与之相互作用的序列 蛋白质(S)只存在于表达Th的细胞系的核提取液中， 即PC-12细胞。第一个具体目标是分离和鉴定 识别这种类似AP-1序列的蛋白质。在第二个 特定的目的，这种蛋白的抗体将用于 小鼠胚胎免疫组织化学结合聚合酶链式反应和免疫组织化学研究 用原位杂交法确定慢性阻塞性肺疾病发病的时间和空间 编码这种反式作用因子的基因的表达。通过 将这些数据与第一次出现免疫反应的TH 细胞，将深入了解AP-1样结合的作用 蛋白质在胚胎学中的承诺表达 儿茶酚胺能表型。最后一个特定目标将使用 转基因小鼠模型研究AP-1样顺式元件的作用 以及位于TH编码上游的附加顺式元素 TH基因全细胞特异性表达的序列分析 动物。最终，这些结果可能会提出新的治疗方法。 疾病，例如帕金森氏症，一种最终导致 几乎完全丧失了自主运动控制。