hKv4.3基因5'非翻译区序列S160功能分析

hKv4.3基因是形成瞬时外向钾电流Ito的主要分子基础, 它在心脏和神经细胞中大量表达, 但在其它组织中则未见大量表达. 为了研究hKv4.3表达在基因水平的调节, 将hKv4.3基因的5'非翻译区的一段序列(+2~+160, 称之为S160)克隆到报告质粒中, 进行瞬时表达. 发现S160对hKv4.3基因的启动子和SV40的启动子都有强烈的抑制作用, 没有方向特异性, 但却有位置特异性. 经删除突变分析, 在S160片段中发现了一个抑制元件S(GAGGGGTTAA), 它位于hKv4.3基因中转录起始位点下游20-30 bp处. 在此基础上, 用RT-PCR方法对mRNA进行定量分析, 初步确认这个抑制元件对蛋白表达的抑制过程是在翻译水平上.     

Development of an efficient endothelial cell specific vector using promoter and 5' untranslated sequences from the human preproendothelin-1 gene

We report here, that a vector constructed based on ppET-1 gene promoter and 5' untranslated region induced a high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxia-mimic conditions due to a natural hypoxia responsive element within the promoter region. A naked DNA vector that confers endothelial cell specific gene expression as well as efficient levels of gene expression was constructed with an endothelial cell specific naked DNA vector, pETlong, by using the full length promoter of the preproendothelin-1 gene and the entire 5' untranslated region upstream from the start codon. Inclusion of the entire 5' untranslated region in pETlong increased gene expression 2.96 fold as compared with that from pETshort, which contains only the promoter sequences. Reporter gene expression from pETlong was 7.9 fold higher as compared with that from CMV-driven promoter based vector in calf pulmonary endothelial cells. However, in nonendothelial COS cells, luciferase activity from pETlong was only 0.3 fold as compared with that of CMV-based vector. Similar results were observed in other nonendothelial cells. These results demonstrate that the pETlong drives gene expression in endothelial cells with high efficacy and specificity. We have examined hypoxia responsiveness of pETlong as the promoter region of the preproendothelin-1 gene contains hypoxia responsive elements. The activity of the pETlong vector was increased 1.6 fold under hypoxia-mimic conditions using cobalt chloride. The high levels of hypoxia-inducible expression in endothelial cells relative to the low levels of background expression in other cells shows that pETlong could be a useful tool for vascular targeting of vascular disease and cancer gene therapy.     

hKv4.3最小功能启动子区与上游抑制元件分析

通过克隆hKv4.3的启动子区和相关的上游调控元件,对hKv4.3基因在转录水平的调控进行了分析.对启动子区5'端一系列的删除突变分析证明,hKv4.3基因的最小功能启动子区是位于转录起始位点附近的-156～+2bp序列.经序列分析发现,这个启动子缺乏典型的TATA-box,却存在另外3个元件,即E-box(CANNTG),CArG-box[CC(A/T)₆GG]和CACC-box(GGTGC),其中CArG-box对该启动子活性起关键作用.同时在启动子区找到一个未见报道的大小为10bp的抑制子T,删除抑制子T,则启动子活性增加1倍以上.