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 interphotoreceptor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5' flanking regions contain functional IRBP promoter activity in multiple cell 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 functional activity of this region, we used the same DNA construct, p1783, in four different cell types. Mobility shift, DNase footprints, and southwestern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region.

RESULTS

The 5' flanking 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 sequence about 1.25 to 1.50 kb upstream of the transcription start site. Two different statistical approaches showed that the IRBP 5' flanking region possesses promoter activity in four different cell types. By using mobility shift, DNase I-protection experiments, and southwestern blotting, a region of about 45 bases at position -300 was identified that specifically binds a protein from the nuclei of bovine retina and Y79 cells.

CONCLUSIONS

Specific DNA binding events are an essential part of IRBP promoter activity. The conservation of sequences far upstream of the transcription start suggest that unknown physiological processes remain to be understood in IRBP transcriptional regulation.