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.
OBJECTIVE
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.
Borst
D E
DE
Department of Anatomy, Physiology, and Genetics, Uniformed University of the Health Sciences, Rockville, MD, USA.
Boatright
J H
JH
Si
J S
JS
Stodulkova
E
E
Remaley
N
N
Pallansch
L A
LA
Nickerson
J M
JM
D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D018983:DNA Footprinting; D005136:Eye Proteins / Q000235:genetics*; D015870:Gene Expression; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012160:Retina / Q000166:cytology; D012177:Retinol-Binding Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099214:Retinol-Binding Proteins, Interstitial
D005136:Eye Proteins; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial