Hammond-Kosack M C, Kilpatrick M W, Docherty K
Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, U.K.
J Mol Endocrinol. 1992 Dec;9(3):221-5. doi: 10.1677/jme.0.0090221.
An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human beta-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived beta-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehyde-reactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat beta-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.
人类胰岛素基因上游360 bp处的高变区内存在一种改变的DNA结构。本研究的目的是确定这种结构在体内胰岛素基因中是否存在，以及其存在是否与胰岛素基因的表达有关。然而，由于没有可用于此类研究的克隆人β细胞系，因此将人类胰岛素基因转染到大鼠胰岛素瘤衍生的β细胞系中，并筛选出几个表达人类胰岛素的克隆。用DNA结构探针溴乙醛对其中一个这样的细胞系进行体内处理，然后提取染色体DNA。用TaqI消化，随后用S1核酸酶在溴乙醛反应位点进行切割，将DNA进行琼脂糖凝胶电泳，通过Southern印迹分析检测胰岛素基因片段。从大鼠β细胞系分离的人类胰岛素基因中，溴乙醛产生了2500、1700和800 bp的亚片段，而在不表达的HeLa细胞系中的人类胰岛素基因对溴乙醛无反应。这些结果表明，体内人类胰岛素基因的胰岛素基因连锁多态区内可能存在一种改变的结构，并且这种结构可能在胰岛素基因的表达中发挥作用。
