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压来适应高渗环境，从而减少细胞内的无机盐，从而保护细胞免受高细胞内无机盐浓度的有害影响。由于尿液浓缩机制，肾髓质细胞暴露在不同的高渗透压下，通过积累肌醇来保护自己，肌醇是一种已知的相容渗透调节物质。这主要是通过上调转运蛋白，称为钠/肌醇共转运蛋白(SMIT)。以往的研究表明，转录是上调SMIT活性和KWUNET的主要步骤。艾尔在我的实验室里，我发现了五个与SMIT基因调控有关的假定顺式作用序列。这些是分布在SMIT基因启动子5‘侧翼区域的55kb碱基对的音调反应增强子(音调)。我们假设染色质中有特殊的结构特征，允许所有的音调与SMIT启动子相互作用。为了验证我们的假设，我将准备一个100kb的酵母人工染色体，其中包含人类SMIT基因座--整个基因和包含所有增强子的60kb的侧翼序列。利用同源重组的优势，通过删除增强子和改变它们的位置来准备基因座的变异。然后，我将此载体导入MDCK细胞系，以评估这些变化的效果。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结构及其变异的MDCK细胞中通过高渗性可能的调节。

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.