Structural characterization and comparison of promoter activity of mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) gene 5 ' flanking regions in WERI, Y79, chick retina cells, and transgenic mice

Purpose. To determine the sequences of the mouse and bovine interphotorecep tor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5 ' flanking regions contain functional IRBP promoter activity in multiple ce ll types using both quantitative and statistical analyses. Methods. We sequenced the bovine and mouse 5' flanking regions of the IRBP gene and compared these sequences to the human gene sequence. To test for f unctional activity of this region, we used the same DNA construct, p1783, i n four different cell types. Mobility shift, DNase footprints, and southwes tern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region. Results. The 5' Ranking regions of the bovine, human, and mouse IRBP genes exhibit sequence similarity in regions immediately adjacent to the start of transcription (roughly 350 bases in length) and also over a 220 base seque nce about 1.25 to 1.50kb upstream of the transcription start site. Two diff erent statistical approaches showed that the IRBP 5' flanking region posses ses promoter activity in four different cell types. By using mobility shift , DNase I-protection experiments, and southwestern blotting, a region of ab out 45 bases at position -300 was identified that specifically binds a prot ein from the nuclei of bovine retina and Y79 cells. Conclusions. Specific DNA binding events are an essential part of IRBP prom oter activity. The conservation of sequences far upstream of the transcript ion start suggest that unknown physiological processes remain to be underst ood in IRBP transcriptional regulation.