MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

视网膜色素上皮细胞（RPE）在视网膜色素变性中起着关键作用。 外视网膜的发育和功能。 我们有兴趣 RPE特定机制，在监管和功能层面。 这将有助于我们更好地了解正常和 疾病状态，并为我们提供进一步分析RPE的工具。 为此，我们一直在研究 RPE 65，一个新的保守的发育调节的65 kDa RPE特异性 蛋白 我们已经克隆了RPE 65的全长人类和小鼠基因。 牛、人和部分小鼠推导蛋白的比较 序列揭示了一种高度保守的分子， 物种之间的相似性。 我们构建了启动子/报告基因结构， 通过使用与氯霉素连接的人5'侧翼序列， 乙酰转移酶（CAT）基因，并测试这些在瞬时转染 在原代RPE细胞培养物中进行测定以鉴定转录 监管区域。 我们发现人RPE 65基因5'侧翼 区域驱动报告基因的RPE特异性表达。 这是 正在进一步分析。 小鼠5'侧翼区也已被克隆。 测序并与人类进行比较，揭示了保守的区块， 包括鉴定的转录因子结合元件， 推定的启动子区。 我们已经用 小鼠RPE 65基因启动子/报告基因构建体，用于体内分析 启动子功能 这些动物目前正在进行分析。 到 进一步探索该基因在RPE中的作用，以及可能的 为了研究RPE 65对神经视网膜的影响，我们正在产生RPE 65敲除小鼠。 小鼠基因组序列已被用于构建靶向 用于小鼠胚胎干细胞电穿孔的载体。 除了在转录水平上的调节，RPE 65的表达 在翻译水平上也受到转录后调控。 我们已经确定了3'非翻译区（UTR）的不同序列， RPE 65 mRNA的稳定性和效率， 翻译RPE 65报文。 进一步分析表明， 作用可以是至少部分编码区序列特异性的，因为 影响翻译效率3 ′ UTR序列对 翻译一封外国邮件。 这可能产生的影响是 被研究。

The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. This will help us gain a better understanding of the RPE in normal and disease states and provide us with tools for further analysis of the RPE. To this end, we have been studying the function and gene regulation of RPE65, a novel conserved developmentally regulated 65 kDa RPE-specific protein. We have 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 between species. We have made promoter/reporter constructs by using human 5' flanking sequence linked to the chloramphenicol acetyltransferase (CAT) gene and tested these in transient transfection assays in primary RPE cell cultures to identify transcriptional regulatory regions. We found that the human RPE65 gene 5' flanking region drives RPE-specific expression of the reporter gene. This is being further analyzed. The mouse 5' flanking region has also been sequenced and compared with the human, revealing conserved blocks, including identified transcriptional factor-binding elements in the putative promoter region. We have generated transgenic animals using mouse RPE65 gene promoter/reporter constructs for in vivo analysis of promoter function. These animals are currently being analyzed. To further explore the role of this gene in the RPE as well as possible effects on the neural retina, we are generating an RPE65 knockout mouse. The mouse genomic sequences have been used to construct a targeting vector for electroporation of mouse embryonic stem cells. In addition to regulation at the transcriptional level, RPE65 expression is also regulated post-transcriptionally at the level of translation. We have 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. Further analysis suggests that the effect may be at least partially coding-region sequence specific because the 3' UTR sequence affecting translational efficiency had no effect on translation of a foreign message. Possible implications of this are being studied.