Pérez D G, Gómez C, López-Bayghen E, Tannich E, Orozco E
Department of Patología Experimental, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, CINVESTAV Instituto Politecnico Nacional AP 14-740, México 07300, D.F. México.
J Biol Chem. 1998 Mar 27;273(13):7285-92. doi: 10.1074/jbc.273.13.7285.
We report here the cloning and transcriptional characterization of the EhPgp5 multidrug resistance gene promoter isolated from the drug-resistant clone C2 of Entamoeba histolytica. The EhPgp5 promoter has the TATA-like motif at -31 base pairs; transcription initiates three nucleotides upstream from the ATG in trophozoites grown in 225 microM emetine (clone C2(225)), whereas in those grown without the drug (clone C2) a product with no open reading frame was detected. The promoter was active in transfected clone C2 trophozoites, its activity increased when trophozoites were cultured in 40 microM emetine, while it was turned off in the drug-sensitive clone A. The first -235 base pair kept full promoter activity, suggesting that it has important drug responsive elements. Gel shift assays detected the complex Ib in clone C2, which was augmented in clone C2(225). Competition experiments suggested that complex Ib may be constituted by HOX and AP-1 like factors in clone C2, whereas in clone C2(225), complex Ib was only competed by the HOX sequence. Complexes Ie, detected in clones A and C2 but not in C2(225), and Ia, present in all clones, were competed by the TATA box oligonucleotide. Our results suggest that proteins forming complexes Ib and Ie may be participating in the regulation of the EhPgp5 gene expression.
我们在此报告从溶组织内阿米巴耐药克隆C2中分离出的EhPgp5多药耐药基因启动子的克隆及转录特征。EhPgp5启动子在 -31个碱基对处有类似TATA的基序；在225微摩尔依米丁中生长的滋养体（克隆C2(225)）中，转录起始于ATG上游三个核苷酸处，而在无药物培养的滋养体（克隆C2）中，检测到一个没有开放阅读框的产物。该启动子在转染的克隆C2滋养体中具有活性，当滋养体在40微摩尔依米丁中培养时其活性增加，而在药物敏感克隆A中则关闭。首个 -235个碱基对保持了完整的启动子活性，表明它具有重要的药物反应元件。凝胶迁移实验在克隆C2中检测到复合物Ib，在克隆C2(225)中其增加。竞争实验表明，在克隆C2中复合物Ib可能由HOX和AP-1样因子组成，而在克隆C2(225)中，复合物Ib仅被HOX序列竞争。在克隆A和C2中检测到但在C2(225)中未检测到的复合物Ie，以及在所有克隆中都存在的复合物Ia，被TATA盒寡核苷酸竞争。我们的结果表明，形成复合物Ib和Ie的蛋白质可能参与了EhPgp5基因表达的调控。
