REGULATION OF SODIUM/MYOINOSITOL COTRANSPORTER GENE

当面对高渗环境时，细胞通过积累相容的渗透剂来适应它，这减少了细胞内无机盐，并因此保护细胞免受高细胞内无机盐浓度的有害影响。 由于尿浓缩机制，肾髓质细胞暴露于极高水平的渗透压，通过积累肌醇（一种已知的相容性渗透剂）来保护自身。 这主要是通过上调转运蛋白，称为钠/肌醇协同转运蛋白（SMIT）。 先前的研究表明，转录是SMIT活性和Kwonet上调的主要步骤。在我的实验室中，J.A.等人鉴定了五个参与SMIT基因调控的假定顺式作用序列。 这些是分布在SMIT基因启动子的5 '侧翼区域中的55个酶对上的张力响应增强子（TonE）。 我们推测，在染色质中存在特殊的结构特征，允许所有的TonE与SMIT启动子相互作用。 为了验证我们的假设，我将准备一个100 kb的酵母人工染色体，其中含有人SMIT基因座-整个基因和60 kb的侧翼序列，其中含有所有的增强子。 利用同源重组，通过删除增强子并改变其位置来制备基因座的变体。然后，我将用这个构建体转染MDCK细胞系，以评估变化的影响。 我还将研究TonEs和SMIT启动子之间是否存在其他基因，并将研究它们在转染人SMIT YAC构建体及其变体的MDCK细胞中通过高渗性的可能调控。

When faced to a hypertonic environment, cells adapt to it by accumulating compatible osmolytes, which reduces intracellular inorganic salts and as a result protects the cell from the deleterious effects of the high intracellular inorganic salt concentration. Being exposed to variably high levels of osmolality due to the urinary concentrating mechanism, renal medullary cells protect themselves by accumulating myo-inositol, which is a known compatible osmolyte. This is primarily through the up-regulation of transporter protein, called sodium/myo-inositol cotransporter (SMIT). Previous studies have indicated that transcription is the primary step in the up-regulation of SMIT activity and Kwonet. al. in my lab identified five putative cis-acting sequences involved in the regulation of the SMIT gene. These are tonicity-responsive enhancers (TonEs) spread over 55 kilobase pairs in the 5'-flanking region of the SMIT gene promoter. We hypothesize that there are special structural features in the chromatin that allow all TonEs to interact with SMIT promoter. To test our hypothesis, I will prepare a 100 kb yeast artificial chromosome containing the human SMIT locus-the entire gene and 60 kb of flanking sequence containing all the enhancers. Taking advantage of homologous recombination, variations of the locus will be prepared by deleting the enhancers and altering their location. Then I will transfect MDCK cell line with this construct to assess the effects of the changes. I will also investigate whether there are other genes present between TonEs and SMIT promoter and will study their possible regulation by hypertonicity in MDCK cells transfected with human SMIT YAC construct and its variations.