Cloning of the promoters for the β-cell ATP-sensitive K-channel subunits Kir6.2 and SUR1

被引:38
作者
Ashfield, R [1 ]
Ashcroft, SJH [1 ]
机构
[1] John Radcliffe Hosp, Nuffield Dept Clin Biochem, Oxford OX3 9DU, England
基金
英国惠康基金;
关键词
D O I
10.2337/diabetes.47.8.1274
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
The P-cell ATP-sensitive potassium channel (K-ATP channel), which regulates insulin secretion, is composed of two types of subunits: 1) a sulfonylurea receptor (SUR1) and 2) an inwardly rectifying potassium channel(Kir6.2). We have isolated clones containing 5'-flanking DNA for both genes by hybridization screening of a human genomic library. Sequencing of over one kilobase of each upstream region has revealed that the putative promoters are G + C rich, with no TATA box. Several E-boxes and potential Spl sites are present in both promoters, and the Kir6.2 upstream region contains an Alu repeat. Using a luciferase reporter gene in transient transfection assays, me demonstrate that the upstream DNA contains promoters that are active in the p-cen lines HIT T15 and MIN6. The promoters are completely inactive in the fibroblast cell line COS7 but show some activity in HepG2 (liver) and HEK293 (epithelial) cell Lines. Deletion analysis suggests that a short (173-base pair [bp]) fragment of SUR1 5'-flanking sequence is sufficient for maximal promoter activity. In contrast, over 900 bp of Kir6.2 5' sequence are required for similar high level expression, and deletion of the Alu repeat results in an increase in promoter activity.
引用
收藏
页码:1274 / 1280
页数:7
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