Benatti L, Bonecchi L, Cozzi L, Sarmientos P
Department of Biotechnology, Farmitalia Carlo Erba, Nerviano, Italy.
J Clin Invest. 1993 Mar;91(3):1149-56. doi: 10.1172/JCI116274.
Endothelin-1, initially identified as potent vasoconstrictor secreted by vascular endothelial cells, was subsequently found to have many effects on both vascular and nonvascular tissues. We have identified from a human placenta cDNA library a clone (cDNA-2) which corresponds to a novel 5'-extended preproendothelin 1 (preproET-1) mRNA. Comparison with the known preproET-1 mRNA (cDNA-1), showed that the two molecules share the same coding sequence but differ in the 5'-untranslated region. Interestingly, cDNA-2 extends upstream of promoter regions previously shown to be essential for full preproET-1 expression. Primer extension and PCR analysis of human placenta RNA demonstrated the presence of additional transcription initiation sites located upstream of the previously identified preproET-1 CAP site. Moreover, the two mRNAs show different pattern of expression. To elucidate the mechanisms controlling the production of alternative transcripts we transfected COS-1 cells with a series of preproET-1 promoter deletion mutants. This analysis revealed that the human preproET-1 gene can be transcribed from a proximal and a distal promoter element which has hitherto been undetected. In addition, we demonstrate the presence of a region in the down-epithelial specific expression.
内皮素-1最初被鉴定为血管内皮细胞分泌的一种强力血管收缩剂，随后发现它对血管组织和非血管组织均有多种作用。我们从人胎盘cDNA文库中鉴定出一个克隆（cDNA-2），它对应一种新的5'-延伸型前内皮素原1（preproET-1）mRNA。与已知的preproET-1 mRNA（cDNA-1）比较表明，这两个分子具有相同的编码序列，但在5'-非翻译区有所不同。有趣的是，cDNA-2延伸至先前已证明对前内皮素原1的完整表达至关重要的启动子区域的上游。对人胎盘RNA进行引物延伸和PCR分析表明，在先前鉴定的preproET-1帽位点上游存在额外的转录起始位点。此外，这两种mRNA表现出不同的表达模式。为了阐明控制替代转录本产生的机制，我们用一系列preproET-1启动子缺失突变体转染了COS-1细胞。该分析表明，人preproET-1基因可从一个近端启动子元件和一个迄今未被检测到的远端启动子元件转录。此外，我们证明了在上皮细胞特异性表达下调区域的存在。
