Yildiz N, Diedrich T, Knepel W
Department of Biochemical Pharmacology, University of Göttingen, Germany.
Exp Clin Endocrinol Diabetes. 1996;104(3):218-27. doi: 10.1055/s-0029-1211446.
In a subset of patients with non-insulin-dependent diabetes mellitus an 8-base pair (bp) repeat was found from -322 to -315 in the 5'-flanking region of the insulin gene. This 8-bp repeat is inserted into a sequence that is highly homologous to a sequence motif, called PISCES (pancreatic islet cell-specific enhancer sequences), found within cell-specific enhancer elements of the rat insulin I (Ins-E1, from -332 to -285), rat glucagon (Glu-G3) and rat somatostatin (SMS-UE) genes. The PISCES motif confers pancreatic islet-specific activity and is recognized by an islet-specific transcription factor (PISCES-BP). The consequences on functional activity and on protein binding of the 8-bp repeat sequence in the human insulin promoter was investigated. When fused to a reporter gene and transiently transfected into an insulin-producing islet cell line, the 8-bp repeat decreased basal transcriptional activity of the human insulin promoter (from -366 to +42) whereas the induction of promoter activity by cAMP was unaffected. The isolated rat Ins-E1 element was sufficient to confer basal transcriptional activity to a minimal promoter; the corresponding fragments of the normal and variant human insulin genes (from -329 to -288), however, were not. Using nuclear extracts in an electrophoretic mobility shift assay, it was found that PISCES-BP recognizes rat Ins-E1, but PISCES-BP binding to the corresponding normal and variant human insulin promoter fragments was not detectable and weak, respectively. However, a nuclear protein was found that binds to the variant but not normal human sequence. These data suggest that the 8-bp repeat in the variant human insulin promoter found in patients with non-insulin-dependent diabetes mellitus allows the binding of a nuclear protein that interferes with promoter function.
在一部分非胰岛素依赖型糖尿病患者中，胰岛素基因5′侧翼区-322至-315处发现了一个8碱基对（bp）的重复序列。这个8 bp的重复序列插入到了一个与序列基序高度同源的序列中，该序列基序被称为双鱼座（PISCES，胰岛细胞特异性增强子序列），存在于大鼠胰岛素I（Ins-E1，从-332至-285）、大鼠胰高血糖素（Glu-G3）和大鼠生长抑素（SMS-UE）基因的细胞特异性增强子元件内。双鱼座基序赋予胰岛特异性活性，并被一种胰岛特异性转录因子（PISCES-BP）识别。研究了非胰岛素依赖型糖尿病患者中人类胰岛素启动子8 bp重复序列对功能活性和蛋白质结合的影响。当与报告基因融合并瞬时转染到胰岛素产生胰岛细胞系中时，8 bp重复序列降低了人类胰岛素启动子（从-366至+42）的基础转录活性，而cAMP对启动子活性的诱导不受影响。分离的大鼠Ins-E1元件足以赋予最小启动子基础转录活性；然而，正常和变异人类胰岛素基因的相应片段（从-329至-288）却不能。在电泳迁移率变动分析中使用核提取物，发现PISCES-BP识别大鼠Ins-E1，但未检测到PISCES-BP与相应正常和变异人类胰岛素启动子片段的结合，且结合较弱。然而，发现一种核蛋白可与变异而非正常人类序列结合。这些数据表明，在非胰岛素依赖型糖尿病患者中发现的变异人类胰岛素启动子中的8 bp重复序列允许一种干扰启动子功能的核蛋白结合。
