Three clones for b-IRBP were isolated by anti b-IRBP screening of two bovine retina libraries in the expression vector λgt11. The cDNA inserts were then used as hybridization probes to screen and isolate three more clones in a bovine retina library in the non-expression vector λgt10. The six overlapping clones generated a b-IRBP cDNA sequence of 3400 nucleotides. An open reading frame encoded the complete amino acid sequences of 8 of the 35 b-IRBP tryptic peptides purified in the present study. One tentative glycosylation site was identified. The coding region was followed by TAG translation terminating codon and an untranslated stretch of about 1700 nucleotides that ended in a sequence containing a presumptive AATAAA polyadenylation signal that was 18 nucleotides upstream from a 10 nucleotide oligo(A) tract. The coding region for b-IRBP would be expected to be 3300 bp long, but Northern blot hybridization experiments performed with bovine retina polyadenylated RNA and probes containing part of the coding region established that the mRNA for b-IRBP consisted of a major species of about 6300 bp, and a minor species of 5200 bp. In vitro translation of bovine retina polyadenylated RNA in a rabbit reticulocyte lysate system yielded an immunoreactive protein that was comparable in size with nonglycosylated mature IRBP, showing that it is not synthesized from a large precursor, and supporting our finding that the mRNA contains an extensive non-coding region.
- In vitro translation
- Interstitial retinol-binding protein
- Northern blots
ASJC Scopus subject areas
- Sensory Systems