Imagawa S, Goldberg M A, Doweiko J, Bunn H F
Laboratory of the Howard Hughes Medical Institute, Boston, MA.
Blood. 1991 Jan 15;77(2):278-85.
Because the human hepatoma cell line Hep3B produces erythropoietin (Epo) in a regulated fashion, it can be used to investigate the cis-acting regulatory elements of the Epo gene. Comparison of primate and mouse sequences shows strong homology not only in the coding sequence but also within the 5' flanking region, the first intron, and the 3' flanking region. These portions of the Epo gene were inserted 5' and 3' to a reporter gene, human growth hormone (GH). 5A is a 1,192-base pair (bp) HindIII-Xbal fragment that extends from 378 bp 5' to the cap site through the first intron. To obviate the problem of false initiation of translation from the Epo ATG start codon, this site was changed to TAG by site-directed mutagenesis. 3A is a 255-bp Accl-BglII fragment that extends 67 bp upstream from the Epo termination codon and covers most of the 3' noncoding region of homology. The plasmid DNAs were transfected by electroporation into Hep3B cells with RSVCAT as an internal standard to correct for transfection efficiency. One aliquot of cells was exposed to 50 mumol/L CoCl2 or to 1% O2. At the end of the incubations, GH and Epo were measured in the cell media and the cell pellet was assayed for CAT. Production of GH was stimulated 1.7-fold by cobalt or hypoxia. Furthermore, addition of 3A to the GH gene, irrespective of orientation, stimulated GH production 2.6-fold with CoCl2 and 2.3-fold with hypoxia. Stable cell lines were produced by cotransfection of the above constructions, along with the selectable marker pSV-Neo. In two clones, exposure to hypoxia resulted in much more marked (16-fold) induction of GH. Stimulus of both GH and Epo production by hypoxia was partially abrogated by carbon monoxide. These results demonstrate the presence of promoter and enhancer elements within the human Epo gene that are appropriately responsive to hypoxia and cobalt.
由于人肝癌细胞系Hep3B以一种受调控的方式产生促红细胞生成素（Epo），它可用于研究Epo基因的顺式作用调控元件。灵长类和小鼠序列的比较显示，不仅在编码序列中，而且在5'侧翼区域、第一个内含子和3'侧翼区域内都有很强的同源性。Epo基因的这些部分被插入到报告基因人生长激素（GH）的5'和3'端。5A是一个1192碱基对（bp）的HindIII-Xbal片段，从5'端距帽位点378 bp处开始，穿过第一个内含子。为了避免从Epo的ATG起始密码子错误起始翻译的问题，通过定点诱变将该位点改为TAG。3A是一个255 bp的AccI-BglII片段，从Epo终止密码子上游67 bp处开始，覆盖了大部分3'同源非编码区。通过电穿孔将质粒DNA转染到Hep3B细胞中，以RSVCAT作为内标来校正转染效率。将一份细胞暴露于50 μmol/L的CoCl2或1%的O2中。培养结束时，在细胞培养基中测量GH和Epo，并对细胞沉淀进行CAT检测。钴或缺氧刺激GH的产生增加了1.7倍。此外，无论方向如何，将3A添加到GH基因中，在CoCl2作用下刺激GH产生增加2.6倍，在缺氧条件下增加2.3倍。通过共转染上述构建体以及选择标记pSV-Neo产生稳定细胞系。在两个克隆中，暴露于缺氧条件下导致GH的诱导更为显著（16倍）。缺氧对GH和Epo产生的刺激被一氧化碳部分消除。这些结果表明，人Epo基因中存在启动子和增强子元件，它们对缺氧和钴有适当的反应。
