Two major experimental objectives are described in this proposal: (1) to complete the characterization and analyses of the promoter of the human cGMP PDE6A gene, and (2) to develop an in vitro cell system capable of expressing intact, function PDE6 enzymes. The first objective is a direct continuation of the research conducted during the previous grant period. The PI identified and characterized a short 177 bp sequence upstream of the transcription start point of the human PDE6A gene that is capable of directing expression of PDE6A to retina, but at subnormal levels. He has also carried out preliminary sequence analyses of approximately 4kb of the upstream promoter region. To complete the characterization of the human PDE6A promoter, experiments will be carried out to analyzed extended regions of the PDE6A promoter in an attempt to identify enhance and silence elements in the PDE6A promoter region that may be key to regulation of the levels of expression of PDE6A. Constructs containing longer segments of the upstream region will be cloned into a reporter vector that will be used to generate transgenic mice. Transient transfection analyses of the activity of these promoters will also be carried out in Y-79 cells. Analyses of transgene expression in the mice and in the in vitro cell systems will be used to determine the regulatory characteristics of these upstream regions. Sequences within the PDEGA promoter region that bind nuclear regulatory proteins will be identified using DNase I footprinting and electrophoretic mobility shift assays (EMSA). A major focus of these analyses will be to characterize PDE6A transactivation by the recently described photoreceptor-specific transcription factor, Crx. Preliminary studies by the Pi show that the proximal promoter of PDE6A, which contains three potential Crx binding sites, is robustly activated by Crx. Characterization of the importance of the Crx binding sites for stimulation of the PDEGA promoter will include DNase I footprinting, EMSAs, and mutagenesis studies. Initial studies will focus on the characterization of PDE enzyme intrinsic to Y-79 and WERI Rb cells. Preliminary analyses of these cells show that the transcripts encoding the subunits of both rod and cone cGMP PDE are present in these cells. Initially, PDE enzymes intrinsic to the RB cells will be purified and the cyclic nucleotide preference, kinetic analysis and drug inhibition profiles of each will be determined. The Rb cells will then be co-transduced with recombinant adenoviral constructs containing the full-length cDNA clones encoding the various subunits of either rod or cone cGMP PDE. Western analyses and immunocytochemistry will be used tot monitor expression of the transgenes. The biochemical properties of the expressed PDE enzyme will be compared to those of native PDE6 using DEAE chromatography and size fractionation. Ultimately, experiments will be carried out to examine the functional consequences of PDE6 mutations that have been linked to human RP.
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