Sequence analysis of the mouse IRBP gene and cDNA

Abstract

Purpose. To understand the structure of the mouse interphotoreceptor retinoid-binding protein (IRBP) gene and to compare the predicted primary structure within each repeat of IRBP with its relatives. To compare the levels of expression of IRBP RNA in normal and knockout mice. Methods. The DNA sequence was determined by subcloning restriction fragments of the IRBP gene from 129/Sv P1 clones. Primers were designed to utilize the walking approach. Additional sequences were obtained by PCR amplification from genomic DNA and direct sequencing of products. Mouse retina RNA was subjected to reverse transcription coupled to PCR and the accumulation of double stranded DNA product was monitored with SYBR Green. The PCR primers flanked Intron C, to avoid the analysis of contaminating genomic DNA. Results. Altogether a contig was assembled with a final length of about 14.4kb. The mouse gene 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. The splice site boundaries closely match consensus sequences and the exons appear to be identically placed among the three species (bovine, human, and mouse). A region containing a repeated sequence of low complexity is located about 1.75 to 1.4kb upstream of the transcription start site. A second region containing another low complexity repeat is found in Intron C close to the end of Exon 3. A limited number of weak consensus polyadenylation signals in the 3' region suggest at least three different transcription terminators that apparently give rise to the previously known mouse IRBP mRNAs. The mRNA for IRBP was detected in normal and part of the mRNA was detected in the IRBP knockout mouse, consistent with previous observations. The level of the IRBP mRNA remnant was reduced about 10 fold in the knockout mouse, also consistent with the previously reported absence of Repeat 4 immunoreactivity. Conclusions. The strong conservation in intron-exon positions, gene structure, and protein sequence 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 detectable Repeat 4 protein in this mouse.