Structural characterization and comparison of promoter activity of mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) gene 5′ flanking regions in WERI, Y79, chick retina cells, and transgenic mice
Structural characterization and comparison of promoter activity of mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) gene 5′ flanking regions in WERI, Y79, chick retina cells, and transgenic mice
复制标题
DOI：
10.1076/ceyr.23.1.20.5419
复制
发表时间：
2001-07-01
影响因子：
2
通讯作者：
Nickerson, JM
中科院分区：
文献类型：
--
作者：
Borst, DE;Boatright, JH;Nickerson, JM
目的.为了确定小鼠和牛的感光细胞间类维生素A结合蛋白（IRBP）5'侧翼区的序列，以及这些5'侧翼区是否包含多种细胞类型中的功能性IRBP启动子活性，采用定量和统计分析。我们对牛和小鼠IRBP基因的5'侧翼区进行了测序，并将这些序列与人类基因序列进行了比较。为了测试该区域的功能活性，我们在四种不同的细胞类型中使用相同的DNA构建体p1783。迁移率变化，DNA酶足迹，和西南印迹被用来确定核蛋白复合物结合IRBP 5'侧翼区。牛、人和小鼠IRBP基因的5' Ranking区域在紧邻转录起始点的区域（长度约350个碱基）以及转录起始位点上游约1.25至1.50kb的220个碱基序列上表现出序列相似性。两种不同的统计方法表明IRBP 5'侧翼区在四种不同的细胞类型中具有启动子活性。通过迁移率改变、DNA酶I保护实验和西南印迹法，在-300位鉴定了一个约45个碱基的区域，该区域特异性结合牛视网膜和Y 79细胞核的蛋白质。特异性DNA结合事件是IRBP启动子活性的重要组成部分。保守的序列远上游的转录开始表明，未知的生理过程仍有待了解IRBP转录调控。
Purpose. To determine the sequences of the mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5' flanking regions contain functional IRBP promoter activity in multiple cell types using both quantitative and statistical analyses.Methods. We sequenced the bovine and mouse 5' flanking regions of the IRBP gene and compared these sequences to the human gene sequence. To test for functional activity of this region, we used the same DNA construct, p1783, in four different cell types. Mobility shift, DNase footprints, and southwestern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region.Results. The 5' Ranking regions of the bovine, human, and mouse IRBP genes exhibit sequence similarity in regions immediately adjacent to the start of transcription (roughly 350 bases in length) and also over a 220 base sequence about 1.25 to 1.50kb upstream of the transcription start site. Two different statistical approaches showed that the IRBP 5' flanking region possesses promoter activity in four different cell types. By using mobility shift, DNase I-protection experiments, and southwestern blotting, a region of about 45 bases at position -300 was identified that specifically binds a protein from the nuclei of bovine retina and Y79 cells.Conclusions. Specific DNA binding events are an essential part of IRBP promoter activity. The conservation of sequences far upstream of the transcription start suggest that unknown physiological processes remain to be understood in IRBP transcriptional regulation.