MOLECULAR GENETICS OF LENS CONNEXIN50 (MP70)

晶状体 CONNEXIN50 (MP70) 的分子遗传学

• 批准号: 6179048
• 负责人: ROBERT L CHURCH
• 金额: $ 26.89万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179048
• 关键词: DNA footprinting; animal genetic material tag; cell cell interaction; electrophysiology; fiber cell; gap junctions; gel mobility shift assay; gene mutation; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; laboratory mouse; lens proteins; membrane channels; membrane proteins; molecular cloning; nucleic acid sequence; nucleoproteins; protein binding; protein biosynthesis; protein structure function; tissue /cell culture; transcription factor; transfection

DESCRIPTION: Maintenance of lens transparency depends in part on intercellular communication between lens fiber cells and epithelial cells. This communication is primarily mediated by gap junctions, specialized plasma membrane structures which consist of cell-to-cell channels. A major component of these channels is MP70, a gap junctional lens intrinsic membrane protein (connexin or Cx), which plays a critical role in the maintenance of lens transparency. Aspects of the structure, topology, and function, of MP70 are already being investigated. However, to date, the mechanisms of the MP70 gene's regulation have not yet been investigated. Thus the overall goal of this project is to understand the gene regulation of the lens fiber cell membrane protein MP70/Cx50 in both the normal and cataractous state. To achieve this goal, the PI proposes to investigate the following topics: a) to identify the mouse MP70 gene (Gja8) transcription initiation site(s) by performing primer extension assay and S1-nuclease mapping; b) to determine the region(s) responsible for the transcriptional activity of the Gja8 gene, using 5'-upstream sequencing from the Gja8 gene and CAT reporter gene constructs in transient transfection assays; c) to characterize protein:DNA interactions associated with the Gja8 gene basal promoter region, using gel mobility shift and DNA footprinting procedures; d) To search for regulatory elements in the Gja8 gene intron and then perform transient transfection assays on the identified elements to discover their effect on promoter activity; e) To use transgenic mice and transfected cell lines in order to study the lesions known in the Gja8 gene which cause heritable cataract. These studies will shed light on the control of lens gap junction protein MP70 synthesis and its relationship with lens health, and disorders such as cataracts.

描述：透镜透明度的保持部分取决于 透镜纤维细胞和上皮细胞之间的细胞间通讯。 这种通讯主要由缝隙连接介导， 由细胞间通道组成的质膜结构。 一个主要 这些通道的一个组成部分是MP 70，一个间隙连接透镜固有的 膜蛋白（连接蛋白或Cx），在细胞膜中发挥着关键作用 保持透镜的透明度。 结构、拓扑结构和 MP 70的功能已经在研究中。 然而，迄今为止， MP 70基因的调控机制尚未研究。 因此，本项目的总体目标是了解基因调控 透镜纤维细胞膜蛋白MP 70/Cx 50在正常和 白内障状态 为实现这一目标，PI建议调查以下主题： a）鉴定小鼠MP 70基因（Gja 8）转录起始位点， 通过进行引物延伸测定和S1-核酸酶作图; B） 确定负责转录活性的区域， Gja 8基因，使用Gja 8基因和CAT报告基因的5 '上游测序 瞬时转染测定中的基因构建体; c）表征 与Gja 8基因基础启动子相关的蛋白质：DNA相互作用 区域，使用凝胶迁移率变动和DNA足迹法; d） 在Gja 8基因内含子中寻找调控元件，然后进行 对鉴定的元件进行瞬时转染测定，以发现它们的 对启动子活性影响; e）使用转基因小鼠和转染的细胞 为了研究Gja 8基因中已知的病变， 遗传性白内障。 这些研究将为透镜间隙连接蛋白的调控提供新的思路 MP 70合成及其与透镜健康的关系，以及诸如 白内障