MOLECULAR GENETICS OF LENS CONNEXIN50 (MP70)

描述：维持镜片透明度在一定程度上取决于 晶状体纤维细胞和上皮细胞之间的细胞间通讯。 这种交流主要是通过缝隙连接来调节的， 由细胞间通道组成的质膜结构。一位少校 这些通道的成分是MP70，一种固有的缝隙连接透镜 膜蛋白(连接蛋白或CX)，它在 保持镜头的透明度。结构、拓扑和 MP70的功能已经在研究中。然而，到目前为止， MP70基因的调控机制尚未被研究。 因此，本项目的总体目标是了解基因调控。 正常人和正常人晶状体纤维细胞膜蛋白MP70/CX50的表达 白内障状态。 为实现这一目标，公安部建议调查以下主题： A)确定小鼠MP70基因(Gja8)转录起始点(S) 通过进行引物延伸分析和S1-核酸酶作图； 确定负责转录活动的区域(S) Gja8基因，使用Gja8基因和CAT报告基因的5‘上游测序 瞬时转染法中的基因构建；c)表征 蛋白质：与Gja8基因基本启动子相关的DNA相互作用 区域，使用凝胶迁移率转移和DNA足迹程序；d) 搜索Gja8基因内含子中的调控元件，然后执行 对已鉴定的元素进行瞬时转染分析，以发现其 对启动子活性的影响；e)使用转基因小鼠和转基因细胞 为了研究Gja8基因中已知的导致 遗传性白内障。 这些研究将有助于控制晶状体缝隙连接蛋白。 MP70合成及其与晶状体健康的关系，以及 白内障。

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.