The long term objective os this research plan is to identify the genetic and environmental factors involved in the regulation of cellular and subcellular concentrations of photoreceptor cell- specific gene products during development of the photoreceptor cells in normal mice and in mice with inherited retinal degeneration. The more immediate objectives include the generation of recombinant DNA probes corresponding to three photoreceptor specific proteins. They are: The alpha subunit of cyclic GMP phosphodiesterase (PDE); (2) 33k, a phosphoprotein which binds the transducin beta, gamma complex and is unique to photoreceptor cells; and (3) 23k, a protein which is presence in normal adult mouse retina and absent from the photoreceptorless retina of the adult rd mouse. The cDNA's will be isolated from mouse retinal cDNA expression libraries generated in this laboratory by screening with antibodies, cDNA's and synthetic oligonucleotides. The qualitative and quantitative characteristic of the specific gene products will be determined during the development of the retinas of normal mice and of those with inherited retinal degeneration (rd and rds), using Western, Northern, Southern, and nucleotide sequence analysis; immunocytochemistry and in situ hybridization techniques. The positional assignment of the genes for these proteins will be made using Southern analysis of restriction digests of somatic cell hybrid chromosome panels, in situ hybridization and recombinant inbred lines of mice. The subcellular localization of 33k, 37k (beta transducin) and 48k has been shown by immunocytochemistry to be transient and dependent on the lighting environment to which the animal is exposed. The mechanism by which light induces a shift in the specific staining for these proteins between the inner and the outer segments will be examined and characterized with respect to the light, the genotype of the animal, the rate and direction of movement, the specific protein, and the biochemical requirements. Site specific monoclonal antibodies against 48k will be used to visualize changes in epitope availability and/or actual protein movement. It is anticipated that the results and probes obtained will provide for the detailed examinations of the expression of the genes coding for the identified retinal specific proteins during normal retinal development and during the genetically programmed degeneration of photoreceptor cells that occurs in mice with hereditary.
