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We investigated the presence and the function of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations in 26 Japanese subjects with type 2 diabetes. The subjects were between 20 and 39 years of age on diagnosis and had diabetic first-degree relatives. Two different frameshift mutations were found in 2 subjects (8 %). One novel mutation, T539fsdelC (deletion of C in codon 539 for Thr), is predicted to generate a protein of normal 539 residues at the N-terminus followed by an abnormal 119 amino acid protein. The mutation, P291fsinsC (insertion of C in codon 291 for Pro) should lead to production of a truncated protein of 315 amino acids. Transfection reporter assay using MIN6 and HepG2 cells revealed both mutations to have null function in the transactivation of reporter gene expression. When transfected with wild-type gene, these mutations behaved as dominant-negative regulators in both cells. An equimolar amount of T539fsdelC reduced wild-type activity by approximately 80% in MIN6 cells, while the same concentration of P291fsinsc reduced it by 30%. The sequences responsible for the transactivation activity of HNF-1alpha are confined largely to amino acids 547-628, so that the T539fsdelC mutation, which affects this entire region, replacing amino acids 540-631 with an abnormal 119 amino acid protein, may acquire a potent dominant-negative function.  相似文献   

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研究谷氨酰半胱氨酸合成酶催化亚单位(GCLC)基因上游调控序列中2个AHR/ARNT元件的功能,从而了解γ-谷氨酰半胱氨酸合成酶(γ-GCS)基因转录调节特征.分别构建缺失2个位点AHR/ARNT元件的GCLC基因上游近端序列的萤光素酶报道基因载体以及含有2个AHR/ARNT元件核心序列的萤光素酶报道基因载体.转染大鼠支气管上皮细胞(RTE),比较检测野生与缺失报道载体的基因转录调控效率;利用电泳迁移率变动实验(EMSA)和超级迁移率变动实验检测AHR/ARNT元件与AHR以及ARNT因子的特异性结合;通过转染AHR因子真核表达质粒进一步确定AHR/ARNT元件与AHR结合在GCLC基因表达中的最终作用.结果显示,相比其野生序列,缺失AHR/ARNT元件(-1 090~-1 085)和双缺失AHR/ARNT元件(-1 090~-1 085,-215~-210)的GCLC上游调控序列报道载体在RTE显著提高萤光素酶表达(均P<0.05),而缺失AHR/ARNT元件(-215~-210)则未见显著影响(P>0.05); 独立AHR/ARNT元件(-1 090~-1 085)具有转录促进作用(P<0.05)而独立AHR/ARNT元件(-215~-210)无明显影响(P>0.05).转染CMV2-AHR能够抑制野生型和缺失型报道载体的萤光素酶表达(P<0.05).EMSA证实GCLC基因上游调控区域的2个AHR/ARNT元件均有核蛋白结合,并且超级迁移率变动实验显示结合的蛋白主要含有转录因子AHR以及ARNT.因此,2个AHR/ARNT元件均可以与异源二聚体AHR/ARNT结合,AHR/ARNT元件(-1 090~-1 085)是GCLC基因中重要的抑制元件.  相似文献   

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