MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

视网膜色素上皮细胞和光感受器

Retinal pigment epithelium (RPE) cells and photoreceptor cells re functionally and developmentally closely integrated. Derangements of the RPE are involved in certain retinal diseases. An enhanced knowledge of RPE- expressed genes and of the regulation of expression of these genes will improve our understanding of the RPE. In previous years, we have identified, cloned, and characterized an RPE-specific protein, RPE65, a novel conserved, developmentally regulated 65 kDa protein. Using the cDNA for RPE65, we have isolated and cloned full-length human and mouse genes for RPE65. Comparison of the bovine, human, and partial mouse deduced protein sequences reveals a highly conserved molecule with about 99 percent similarity among species. To study the transcriptional regulation of this RPE-specific gene, we have made promote; constructs driving the bacterial chloramphenicol acetyltransferase (CAT) gene, covering 2.8 kilobases of human 5' flanking sequence, and tested them in transient transfection assays in cultured RPE cells to identify regulatory regions. The mouse 5' flanking region also has been sequenced and compared with the human, revealing conserved blocks, including identified transcriptional factor-binding elements in the putative promoter region. Mouse 5' flanking regions will be used to generate transgenic animals for in vivo analysis of promoter function. In addition to regulation at the transcriptional level, RPE65 expression is also regulated posttranscriptionally at the level of translation. Further characterization of the identified distinct sequences in the 3' untranslated region (UTR) of the RPE65 mRNA that control the stability and the efficiency of translation of the RPE65 message reveals that the effect may be at least partially coding-region-sequence specific because the 3' UTR sequence affecting translational efficiency had no effect on CAT expression.