This is a revised application to investigate the regulation of the renin gene promoter in a renin-expressing cell line, As4.1. The applicant will attempt to complete delineation of cis-acting sites in the 5' FS of the murine Ren-1c gene that appear to be critical for driving high level cell-specific expression of reporter constructs. This will be accomplished utilizing transient transfection assays in the renin-expressing AS4.1 cells, as well as the non-renin-expressing Ltk- cells. To ensure that the elements identified are germane to regulation in vivo, key elements will be tested for function in transgenic mice. Conventional assays of DNA-protein interactions will be used to refine the limits of the individual regulatory sites and will serve as a basis for the identification, purification and cloning of regulatory transcription factors which bind to these sites. Such proteins, once in hand, will be characterized for relevant functional domains and their developmental expression patterns will be examined under normal and pathophysiological conditions to determine whether these patterns are compatible, from a chronological and topographical standpoint, with regulation of renin gene transcription.
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