The long term objective of this research plan is to identify and characterize the genetic and environmental factors involved in the regulation of cellular and subcellular concentrations of photoreceptor cell-specific gene products during the development and maintenance of vision. More immediately, the function and expression of the mouse gene for the retinal calcium sensor, recoverin, will be analyzed. The importance of specific recoverin peptides in the binding to rhodopsin kinase will be assessed using a coupled in vitro transcription/translation system in combination with recoverin molecules modified by expression/mutagenesis techniques. The temporal and spatial pattern of expression of recoverin in rods, cones, some bipolar cells and a rare population of ganglion cells is unique among the proteins participating in phototransduction. The extracellular signals, cis-acting elements and transcription factors involved in the regulation of this expression are unknown. An objective of the proposed research is to identify the genetic regulatory elements responsible for the expression of the recoverin gene and to assess the functional status of its transcription response elements detected by dideoxy nucleotide sequence analysis. The 5'end of the mouse gene will be sequenced, specific DNA protein binding motifs identified and their on/off status will be evaluated by immunocytochemical and Western analysis of the recoverin protein in postnatal retinal cells grown in a chemically defined medium to which the hormone or growth factor is added. The recoverin promoter will be further analyzed by in vitro transfection of Y79 retinoblastoma cells with a eukaryotic cell vector containing defined genomic sequences of the 5'end of the recoverin gene coupled to the bacterial B-galactosidase reporter gene. Conditions for successful transfection of the primary cell cultures will also be sought. Transgenic mice will be used to confirm the transfection data and to characterize the recoverin gene's ability to dictate the temporal, tissue- and cell- specific expression of the reporter gene. Following through on the initial strategy developed for this project, the 55kDa photoreceptor cell-specific protein, originally identified with recoverin and phosducin, will be cloned, sequenced and its expression characterized. The intron/exon boundaries of its gene will be determined and its chromosomal location assigned. The results from these studies will further our understanding of phototransduction, vision, hereditary blindness and blindness produced as a remote effect of cancer. Such knowledge may lead to methods of intervention to preserve and/or restore vision in retinas otherwise programmed for photoreceptor cell degeneration.
