The long term objectives of the research program are to define the structure, expression, and spatial and temporal regulation of genes which encode the molecules essential to flow of information through the visual transduction pathway. The focus of this current proposal is on the gene encoding a large subunit, alpha, of rod photoreceptor cGMP phosphodiesterase (PDE), an essential component of mammalian phototransduction. The sequence of the human PDE alpha-subunit gene will be determined by polymerase chain reaction (PCR) of genomic DNA and direct sequencing, and by isolation and sequence analysis of genomic clones. Primer extension in conjunction with the ribonuclease protection assay or S1 nuclease mapping will be used to identify the transcriptional start point and putative core promoter region of the mouse alpha gene. Direct demonstration of promoter activity and specificity will be obtained in transgenic mice by directed expression of the reporter gene, Beta-galactosidase. As a first step towards promoter characterization, the sequences of mouse and human PDE alpha- and Beta-subunit genes upstream regulatory regions will be determined. Comparison of the upstream sequences may identify potentially important cis regulatory elements that will be confirmed for the mouse alpha-subunit gene by DNA footprinting, and gel retardation analysis. Studies will then be initiated to identify and characterize specific transcription factors in mouse retina that interact with the mouse PDE-alpha-subunit promoter. The proposed studies will provide the foundation necessary to understand the regulation of genes whose functions are essential for rod cell phototransduction and cyclic nucleotide metabolism. The information gained can be used to initiate candidate gene studies to assess the potential for a defective PDE alpha gene to be causal of any form of hereditary retinal degeneration. Additionally, new photoreceptor specific genes encoding transcriptional regulators may be identified. Thus, these studies should have direct impact on understanding the molecular basis of certain forms of human hereditary retinopathies.
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