My laboratory presently is isolating and characterizing recombinant DNA molecules necessary for the study of the structure and expression of IRBP (Interphoto-receptor Retinoid- Binding Protein). We have cloned many different cDNAs (copies of the IRBP messenger RNA) from bovine retina that correspond to 4.8 kb of the IRBP mRNA. We have sequenced portions of all of these overlapping cDNA clones and have about 3000 bases in continuous sequence. The IRBP mRNA is long (7000 bases) and gives only one band on a Northern blot; however, we have evidence that suggests that there is sequence heterogeneity near the 3' end of the IRBP mRNA. Two authentic cDNA clones show a striking divergence in their sequences, yet sequences both 5' and 3' to the divergence are identical. Both sequences in the divergence hybridize to one gene clone. Alternative splicing of the IRBP gene primary transcript could explain the origin of the two types of cDNA clones. The cDNA sequences have been used to predict the amino acid sequence of the protein. These sequences have been helpful in the analysis of the uveitogenic peptides in IRBP. The entire gene for bovine IRBP has been cloned. Partial DNA sequence analysis of the gene clone has identified the authentic N-terminus, the putative initiator methionine codon and a putative signal peptide sequence of the IRBP polypeptide. A second different complete bovine IRBP gene has been identified. In human, the IRBP gene is on chromosome 10, as determined by in situ hybridization of our bovine cDNA probe to human chromosome squashes. This result was verified by isolating genomic clones from a human chromosome 10 specific library. We have screened a human retinal cell cDNA library with the bovine IRBP cDNA probe and have identified several large cDNA clones up to 4.5 kb in length for human IRBP.
