MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

视网膜色素上皮细胞与光感受器 细胞在功能和发育上 整合 RPE的紊乱涉及到 某些视网膜疾病。 增强RPE的知识- 表达的基因和表达的调节 这些基因将提高我们对视网膜色素上皮的理解。 在 在过去的几年里，我们已经识别，克隆， 特征在于RPE特异性蛋白RPE 65，一种新的 保守的，发育调节的65 kDa蛋白。 使用 RPE 65的cDNA，我们已经分离并克隆了全长 人和小鼠RPE 65基因。 比较牛， 人类和部分小鼠推导的蛋白质序列揭示了 一种高度保守的分子， 物种之间的相似性。 为了研究转录 对这个RPE特异性基因的调控，我们做了启动子; 驱动细菌氯霉素的构建体 乙酰转移酶（CAT）基因，覆盖2.8个 人5'侧翼序列，并在瞬时 在培养的RPE细胞中进行转染测定以鉴定 监管区域。 小鼠5'侧翼区也具有 进行测序并与人类进行比较， 保守区块，包括已鉴定的转录 在推定的启动子区域的因子结合元件。 小鼠5'侧翼区将用于产生 用于启动子体内分析的转基因动物 功能 除了在转录水平上的调节， RPE 65的表达也在转录后调节， 翻译水平。 进一步表征 在3'非翻译区鉴定出不同的序列 (UTR)RPE 65 mRNA的稳定性和表达， RPE 65信息的翻译效率表明， 该效果可以是至少部分编码区序列 特异性，因为3' UTR序列影响 翻译效率对CAT表达无影响。

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.