Molecular cloning and characterization of chicken interphotoreceptor retinoid-binding protein

Purpose: Previous studies have suggested that the retina of birds does not express an interphotoreceptor retinoid-binding protein In view of the importance of the avian retina to retinal molecular and cell biology, we re-evaluated this question. Methods: The soluble fraction of the interphotoreceptor matrix and neural retina were obtained from dark-adapted chickens. Western blot analysis utilized an antiserum directed against the fourth module of Xenopus IRBP. Phylogenetic alignment of the nucleic acid sequence of rat, goldfish, zebrafish and Xenopus IRBPs was used to identify conserved regions. These regions were used to design RT-PCR oligonucleotide primers. cDNA screening was carried out following standard protocols Results: Western blot analysis showed an immunoreactive 140 kDa band in the matrix extract. RT-PCR amplified a 1 3 kb fragment. Of known IRBPs, the chicken cDNA fragment had the highest identity of 67.8%. The RT-PCR generated cDNA was used to isolate two partial length cDNAs (maximum length = 2.4 kb) from a chicken retinal cDNA library. Genomic screens are now in progress to determine the full-length sequence and isolate the promoter region. Conclusions: IRBP appears to be a major component of the chicken interphotoreceptor matrix Isolation of the chicken IRBP gene will allow phylogenetic studies of the protein's structure and use of the avian retina in molecular and cellular studies of IRBP expression and function. EY09412 (F.G.-F.).