Bovine interstitial retinol-binding protein (IRBP)-isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA

Gregory I Liou, S. L. Fong, W. G. Beattie, R. G. Cook, J. Leone, R. A. Landers, R. A. Alvarez, C. Wang, Y. Li, C. D.B. Bridges

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1645-1647
Number of pages3
JournalVision Research
Volume26
Issue number10
DOIs
StatePublished - Jan 1 1986

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Protein Sequence Analysis
Protein Biosynthesis
Complementary DNA
Clone Cells
Retina
Messenger RNA
Nucleotides
RNA Probes
Polyadenylation
Reticulocytes
interstitial retinol-binding protein
In Vitro Techniques
Glycosylation
Codon
Northern Blotting
Open Reading Frames
Amino Acid Sequence
Rabbits
Peptides
Proteins

Keywords

  • In vitro translation
  • Interstitial retinol-binding protein
  • Northern blots
  • cDNA
  • mRNA

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Bovine interstitial retinol-binding protein (IRBP)-isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA. / Liou, Gregory I; Fong, S. L.; Beattie, W. G.; Cook, R. G.; Leone, J.; Landers, R. A.; Alvarez, R. A.; Wang, C.; Li, Y.; Bridges, C. D.B.

In: Vision Research, Vol. 26, No. 10, 01.01.1986, p. 1645-1647.

Research output: Contribution to journalArticle

Liou, GI, Fong, SL, Beattie, WG, Cook, RG, Leone, J, Landers, RA, Alvarez, RA, Wang, C, Li, Y & Bridges, CDB 1986, 'Bovine interstitial retinol-binding protein (IRBP)-isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA', Vision Research, vol. 26, no. 10, pp. 1645-1647. https://doi.org/10.1016/0042-6989(86)90052-0
Liou, Gregory I ; Fong, S. L. ; Beattie, W. G. ; Cook, R. G. ; Leone, J. ; Landers, R. A. ; Alvarez, R. A. ; Wang, C. ; Li, Y. ; Bridges, C. D.B. / Bovine interstitial retinol-binding protein (IRBP)-isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA. In: Vision Research. 1986 ; Vol. 26, No. 10. pp. 1645-1647.
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AB - 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.

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