My laboratory is presently isolating and characterizing recombinant DNA molecules necessary for the study of the structure and expression of IRBP (Interphotoreceptor Retinoid-Binding Protein). Clones and partial sequences from bovine IRBP have been obtained. We have cloned and completely sequenced six overlapping cDNA clones that constitute about 3500 bases of the 7000 base long bovine IRBP mRNA. The IRBP mRNA is unusually long especially given that the polypeptide encoding region of the mRNA should only require about 3600 bases. There is only one band on a Northern blot suggesting that there is only one IRBP mRNA species. The clones were used as probes for in situ hybridizations to frozen sections of bovine and monkey retinas. In large, only the rod cell perikarya were labelled with the probe. This showed us that the gene is regulated and expressed only in the rod cell, and it defined the site of synthesis of the IRBP polypeptide as the rod cell. Part of th gene for bovine IRBP has been cloned. Partial DNA sequence analysis revealed a perfect match of 75 bases of the cDNA and genomic clones. The sequences diverged at a consensus splice acceptor site in the gene clone, indicating an intron-exon boundary. We have obtained a putative genomic clone for the human gene, but it remains to be proven (by DNA sequencing) that this clone is authentic. We have begun to characterize the IRBP gene by analysis of southern genomic blots. We have shown that the gene is present in only one copy per haploid genome in a variety of species. In human, the gene is not on the X-chromosome, since hybridizing bands from male and female DNA samples are the same intensity. We are now in the process of mapping the precise chromosome.
