PURPOSE: 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.
PURPOSE: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.
Authors: Cristy A Ku; Vince A Chiodo; Sanford L Boye; Andrew F X Goldberg; Tiansen Li; William W Hauswirth; Visvanathan Ramamurthy Journal: Hum Mol Genet Date: 2011-08-31 Impact factor: 6.150
Authors: Basil S Pawlyk; Oleg V Bulgakov; Xiaoqing Liu; Xiaoyun Xu; Michael Adamian; Xun Sun; Shahrokh C Khani; Eliot L Berson; Michael A Sandberg; Tiansen Li Journal: Hum Gene Ther Date: 2010-08 Impact factor: 5.695
Authors: Cristy A Ku; Vince A Chiodo; Sanford L Boye; Abigail Hayes; Andrew F X Goldberg; William W Hauswirth; Visvanathan Ramamurthy Journal: Hum Mol Genet Date: 2014-09-30 Impact factor: 6.150
Authors: Hanna Västinsalo; Reetta Jalkanen; Astra Dinculescu; Juha Isosomppi; Scott Geller; John G Flannery; William W Hauswirth; Eeva-Marja Sankila Journal: Eur J Hum Genet Date: 2010-08-18 Impact factor: 4.246
Authors: Tiffany Whitcomb; Keisuke Sakurai; Bruce M Brown; Joyce E Young; Lowell Sheflin; Cynthia Dlugos; Cheryl M Craft; Vladimir J Kefalov; Shahrokh C Khani Journal: Invest Ophthalmol Vis Sci Date: 2009-10-15 Impact factor: 4.799
Authors: X Sun; B Pawlyk; X Xu; X Liu; O V Bulgakov; M Adamian; M A Sandberg; S C Khani; M-H Tan; A J Smith; R R Ali; T Li Journal: Gene Ther Date: 2009-08-27 Impact factor: 5.250