Identification of the RLBP1 gene promoter.
Identification of the RLBP1 gene promoter.
复制标题
RLBP1 基因启动子的鉴定。
DOI：
10.1167/iovs.06-1523
复制
发表时间：
2007
影响因子：
4.4
通讯作者：
Howard,EricW
中科院分区：
文献类型：
--
作者：
Vogel,JohnS;Bullen,ElizabethC;Teygong,CrystalL;Howard,EricW
目的。细胞视黄醛结合蛋白是由视黄醛结合蛋白1基因转录而来的一种36 kDa的水溶性蛋白，含有316个氨基酸，存在于视网膜色素上皮和视网膜Müler细胞中。它被认为在视觉周期中发挥着关键作用，因为它是异甲基水解酶反应中11-顺式视黄醇的受体。这里的目的是评估该基因的功能启动子。用体外培养的人源性RPE细胞(ARPE-19和D407)进行5‘RACE分析、启动子-报告基因分析和外显子特异的半定量聚合酶链式反应(PCR)，分析RLBP1基因侧翼的5’端序列。此外，在电子计算机中对小鼠、牛和猪的RLBP1基因进行了评估，以确定可能与人类基因相似的近端启动子/外显子1序列。5‘RACE分析表明，在RLBP1基因中存在一个先前未描述的外显子。对GenBank人类EST数据库的分析证实了这一点，发现存在18个与外显子1匹配的序列。外显子特异性聚合酶链式反应显示大多数在ARPE-19细胞中表达的CRALBP转录本同时含有外显子1和最终外显子，这表明CRALBP的主要启动子存在新发现的外显子1的5‘端。最后，启动子-报告构建揭示了启动子功能所需的最小序列，并表明与先前描述的RLBP1启动子相比，启动子活性显著更高。这里的发现表明，RPE细胞中的CRALBP转录本除了新描述的启动子外，还含有一个非编码外显子，根据定义，还有一个额外的内含子。这一发现为从机制上理解RLBP1高度细胞类型特异性表达奠定了基础。
purpose. Cellular retinaldehyde-binding protein (CRALBP), transcribed from the RLBP1 gene, is a 36-kDa water-soluble protein with 316 amino acids found in the retinal pigment epithelium (RPE) and in retinal Müller cells. It is thought to play a critical role in the visual cycle by functioning as an acceptor of 11-cis-retinol from the isomerohydrolase reaction. The goal here was to evaluate the functional promoter of this gene.methods. 5′ RACE analysis, promoter-reporter assays, and semiquantitative PCR with exon-specific primers were performed using human-derived RPE cells (ARPE-19 and D407) in culture to evaluate the 5′ sequence flanking the RLBP1 gene. In addition, the murine, bovine, and porcine RLBP1 genes were evaluated in silico to identify likely proximal promoter/exon 1 sequences similar to the human gene.results. 5′ RACE analysis revealed the presence of a previously undescribed exon in the RLBP1 gene. This was confirmed by analysis of the GenBank Human EST database, which revealed the presence of 18 sequences matching exon 1. Exon-specific PCR revealed that most CRALBP transcripts expressed in ARPE-19 cells contain both exon 1 and the final exon, suggesting that the primary promoter of CRALBP exists 5′ of the newly identified exon 1. Highly homologous sequences in the murine, bovine, and porcine genes were also identified. Finally, promoter-reporter constructs revealed a minimal sequence necessary for promoter function and indicated significantly greater promoter activity compared with previously described RLBP1 promoters.conclusions. The findings presented here suggest that CRALBP transcripts in RPE cells contain a noncoding exon in addition to a newly described promoter and, by definition, an additional intron. This finding sets the stage for a mechanistic understanding of the high degree of cell type–specific expression of RLBP1.