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JM Nickerson, E Gross, WY He, GI Liou, RK Shuler; The Mouse IRBP Gene and Quantitative Measure of Its mRNA Levels . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1435.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To assess structure-function relationships, we determined the sequence of the mouse interphotoreceptor retinoid binding protein (IRBP) gene and compared the predicted primary structure of mouse IRBP with other mammals. To compare normal and IRBP knockout mice, we quantified the amounts of IRBP-derived RNA. Methods: The DNA sequence was determined by subcloning restriction fragments of the IRBP gene from 129/Sv P1 clones. Primers were obtained by the walking approach. Additional sequences were obtained utilizing PCR amplification from genomic DNA and direct sequencing. Mouse retina RNA was subjected to reverse transcription coupled to PCR and the accumulation of double stranded DNA was quantified with SYBR Green. The PCR primers flanked Intron C, to avoid the analysis of contaminating genomic DNA. Results: A single contig was assembled with a final length of about 14.2 kb demonstrating a single IRBP gene in the mouse. The structure is similar to the pattern of exons and introns in the bovine and human genes, with a long first exon encoding most of the protein. Splice site boundaries closely match consensus sequences and the exons appear to be identically placed among the three species (bovine, human, and mouse). Several tandem and interspersed repetitive elements were found in the mouse IRBP gene sequence. Some are common to the human and bovine genes. Polyadenylation signals in the 3' untranslated region suggest different transcription terminators that give rise to two mouse IRBP mRNAs detected on northern blots. These were verified by analysis of several IRBP cDNA sequences. The mRNA for IRBP was detected in normal mouse retina and part of the mRNA from Exons 3 and 4 was detected in the IRBP knockout mouse. The level of the remnant of IRBP mRNA was reduced about 10 fold in the knockout mouse relative to normal IRBP mRNA. Conclusion: The strong conservation in intron-exon positions, gene structure, and protein sequence in among mammals supports an important biological role for these signals and for the IRBP protein in vision. Low levels of aberrant IRBP mRNA in the knockout mouse are consistent with no immunologically detected Repeat 4 protein in this mouse.
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