BINDING PROTEINS FOR RETINOIDS IN OCULAR TISSUES

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.