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.
