A short, highly active photoreceptor-specific enhancer/promoter region upstream of the human rhodopsin kinase gene.
OBJECTIVE
Rhodopsin kinase (Rk or GRK1) is a photoreceptor-specific enzyme that mediates adaptation of photoreceptors to light and protects these cells against light-induced injury. This study examined the transcriptional mechanisms that maintain physiologic levels of this essential enzyme in photoreceptors.


METHODS
The 2.0-kb region flanking the 5' end of the human Rk gene was isolated, mapped, and sequenced. The sequence was fused upstream of the luciferase gene and was tested for promoter activity in retinoblastoma cells by transient transfection. Transcriptionally active segments were identified by deletion and site-directed mutagenesis. Transgenic mice were generated that carried the immediate 5' flanking segment linked upstream of the enhancerless green fluorescent protein (GFP) gene. GFP expression was analyzed by RT-PCR, fluorescence microscopy, and immunohistochemistry.


RESULTS
Mapping and sequence analysis uncovered a TATA-less promoter with several recognizable elements concentrated proximally. A conserved putative homeodomain response element H1 and a GC- and a GA-rich motif were noted within a 0.11-kb region. Another putative homeodomain binding site, H2, and a stretch of C-rich repeats were present distally. Mutagenesis in conjunction with transient transfection in retinoblastoma cells identified the 0.11-kb region and H1 sequence as the key active enhancer-promoter sequences. The distal sequences were inhibitory. Transgenic mice that carried the 0.11-kb DNA segment with the GFP gene linked downstream showed GFP transcript, fluorescence, and immunoreactivity that were restricted to photoreceptors.


CONCLUSIONS
The experiments defined a short, highly active photoreceptor-specific enhancer-promoter region upstream of the Rk gene. The H1 element contributed substantially but not exclusively to the transcriptional activity of the region. The findings support a transcriptional basis for photoreceptor-specific expression of Rk.
Young
Joyce E
JE
Department of Ophthalmology and Biochemistry, State University of New York at Buffalo, New York 14215, USA.
Vogt
Todd
T
Gross
Kenneth W
KW
Khani
Shahrokh C
SC
D000818:Animals; D001483:Base Sequence; D004742:Enhancer Elements, Genetic; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D051606:G-Protein-Coupled Receptor Kinase 1; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection; D014407:Tumor Cells, Cultured
D005136:Eye Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496764:GRK1 protein, human; C496762:Grk1 protein, mouse