Part of the proposed project will be the completion of ongoing studies on the retinol-binding glycoprotein of the interphotoreceptor matrix. This proteins is known as interstitial retinol-binding protein (IRBP). It is secreted by the neural retina. The main thrust of the project is to initiate and develop a thorough investigation of the molecular biology of interstitial retinol-, cytosol retinol-, cytosol retinoic acid-and cytosol retinal-binding proteins. The goal of this work is to eludicate the regulation of the genes coding for these proteins in humans and rats, and to establish whether hereditary retinal dystrophies in the RCS and Wag-Rij rat models and human retinitis pigmentosa are related to abnormalities in these genes or their regulation. Such changes could affect the utilization of retinoids in the visual cycle, could limit the supply of retinol to the retina, or its removal during strong bleaching, and could affect the delivery of retinoids to the nuclei of retinal cells, where some aspects of normal gene expression are believed to be influenced by retinoids. It is proposed to isolate the mRNA coding for retinoid-binding protein (RBP's). The cDNA's will be cloned with appropriate expression vectors (e.g., bacteriophages Lamdagt11 and M13; pBR322 plasmids pBR322, pUC8). The cDNA probes will be used to detect and quantitate mRNA in various tissues and in normal rats and rats with hereditary retinal dystrophies. The cDNA probes will be used to screen the human genomic library to identify genes that code for RBP's and for in situ hybridization to determine mRNA distribution. Finally, the cDNA probes will be used to identify DNA polymorphisms in humans (including patients with retinitis pigmentosa). In this way, we hope to determine whether RP is linked with a mutation in the genes for RBP's, either in the coding segments or in flanking regions that may have a regulatory role. This work will also lay the groundwork for further research aimed at studying other proteins of the retina in relation to disease, such as the enzymes regulating cyclic nucleotide metabolism.
