Ahn Y H, Kim J W, Han G S, Lee B G, Kim Y S
Institute of Genetic Science, Yonsei University College of Medicine, Seoul, Korea.
Arch Biochem Biophys. 1995 Nov 10;323(2):387-96. doi: 10.1006/abbi.1995.0059.
Four overlapping lambda genomic clones encoding rat pancreatic beta-cell/liver type glucose transporter (GLUT2) have been isolated and characterized. The gene is about 35 kb long and contains 14 exons and 13 introns. Contrary to the exon 1 of the human or mouse counterpart, the rat GLUT2 gene has three additional noncoding exons which were identified by 5'-RACE and all four were designated exon 1a, 1b, 1c, and 1d. The intron sequences bordering the splice site junctions generally follow the GT/AG rule except for one intron which begins with GC. The exon sequences determined from genomic DNA sequencing showed some differences when compared to the published rat GLUT2 cDNA. Transcription initiation site was determined by primer extension and located 661 bp upstream of the ATG translation initiation codon. Several potential binding sites for transcription factors such as C/EBP, Sp1, AP1, HNF-5, and UPE were observed and they may be responsible for the regulation of GLUT2 gene expression. The promoter region of rat GLUT2 showed little homology when compared with those of human or mouse. However, striking sequence identity (84%) was found when the adjacent intron regions flanking exon 1c were compared with the -970/-721 region of the mouse GLUT2 promoter. A series of deleted mutant constructs of the putative promoter region linked to the CAT reporter gene showed promoter activity in the primary hepatocyte culture. The region containing -4542/+240 showed the highest CAT activity and further deletion of the region showed gradual decrease in CAT activity.
已分离并鉴定出四个重叠的λ基因组克隆，它们编码大鼠胰腺β细胞/肝脏型葡萄糖转运蛋白（GLUT2）。该基因约35kb长，包含14个外显子和13个内含子。与人类或小鼠对应物的外显子1不同，大鼠GLUT2基因有三个额外的非编码外显子，通过5'-RACE鉴定，所有四个分别命名为外显子1a、1b、1c和1d。除了一个以GC开头的内含子外，与剪接位点连接相邻的内含子序列通常遵循GT/AG规则。从基因组DNA测序确定的外显子序列与已发表的大鼠GLUT2 cDNA相比显示出一些差异。通过引物延伸确定转录起始位点，其位于ATG翻译起始密码子上游661bp处。观察到几个转录因子如C/EBP、Sp1、AP1、HNF-5和UPE的潜在结合位点，它们可能负责GLUT2基因表达的调控。与人类或小鼠的相比，大鼠GLUT2的启动子区域显示出很少的同源性。然而，当将外显子1c侧翼的相邻内含子区域与小鼠GLUT2启动子的-970/-721区域进行比较时，发现了惊人的序列同一性（84%）。一系列与CAT报告基因相连的假定启动子区域的缺失突变体构建体在原代肝细胞培养中显示出启动子活性。包含-4542/+240的区域显示出最高的CAT活性，该区域的进一步缺失显示CAT活性逐渐降低。
