Transcriptional Analysis of the EhPgp5 Promoter of Entamoeba histolytica Multidrug-resistant Mutant

Abstract

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 M 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 M 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.The MDR 1 phenotype in Entamoeba histolytica seems to be a consequence of an increased expression of the P-glycoprotein, although the protein has not been detected (1). However, E. histolytica drug-resistant mutants overexpress mdr-like genes (EhPgp) (2, 3) and common characteristics are shared among the MDR phenotype of this parasite, Plasmodium falciparum, Leishmania tarentolae, and mammalian cells (Refs. 4 -6 and reviewed in Ref. 7). As in some mammalian transformed cells, where only certain mdr genes are expressed, three out of the four EhPgp genes are transcribed in the drug-resistant trophozoites (clone C2) (3, 4). While the EhPgp1 and EhPgp6 genes are transcribed in clone C2 grown in the absence of the drug, the amount of EhPgp5 transcript increases according to the emetine concentration (3, 4).The overproduction of the P-glycoprotein has been proposed to be mediated mainly by transcription, gene amplification, or both (8). However, the amplification alone may not be sufficient to activate the expression of a gene that is normally turned off or transcribed at very low levels (9). Increased mdr gene transcription precedes gene amplification in several mouse cell lines (10), and in human breast cancer and neuroblastoma cell lines (11,12), supporting that the regulation of the mdr genes is principally controlled at the transcriptional level.As occurs in the EhPgp5 gene, the expression of the mouse mdr1b gene is induced by the presence of the drug in the medium. The functional analysis of the mdr1b promoter demonstrated that three nuclear protein binding sequences from Ϫ82 to Ϫ59, from Ϫ123 to Ϫ101, and from Ϫ272 to Ϫ249, participate in the mdr1b gene regulation (13). In addition, DNase I footprinting experiments sho...

Transcriptional Silencing of an Amoebapore Gene inEntamoeba histolytica: Molecular Analysis and Effect on Pathogenicity

“…The results obtained with plasmid psAP-2, which was constructed to contain only the 5Ј flanking segment (470 bp) without the coding or 3Ј flanking sequences, clearly demonstrated that the same silencing of ap-a could be achieved by introducing this 5Ј flanking element alone. Preliminary sequence analysis of the 470 bp of the 5Ј flanking region indicated that it contains promoter motifs with a TATA-like box similar to those which have been previously found in numerous genes of E. histolytica (14,39,40). The 470-bp flanking segment of ap-a also contains at its 5Ј distal end a 120-bp region which appears to be present in additional sequences of the E. histolytica genome and which encodes an unidentified transcript.…”

Identification of Two Entamoeba histolyticaSequence-specific URE4 Enhancer-binding Proteins with Homology to the RNA-binding Motif RRM

“…The 5Ј-flanking regions of a handful of E. histolytica genes have been studied via truncation and mutational analysis (8,(12)(13)(14). In the hgl2 promoter, a CCAAT-like motif was identified that decreased reporter gene expression when mutated, but the existence of proteins recognizing this element has not been demonstrated (3).…”

Promoter Analysis of Palindromic Transcription Units in the Ribosomal DNA Circle of Entamoeba histolytica

“…End-labeled DNA fragments (0.5 to 1 ng, up to 20,000 cpm) were incubated with 15 g of nuclear extract, 1 g of poly(dI-dC), and 10% glycerol in the presence of DNA-protein binding buffer (12 mM HEPES [pH 7.9],150 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 4 mM Tris-HCl [pH 7.9], 1 mM spermidine, and 1.5 mM MgCl 2 ) for 15 min at room temperature (13). The bound and unbound complexes were separated on 7% nondenaturing polyacrylamide gels in 0.5ϫ TBE (44.5 mM Tris-HCl [pH 7.9], 44.5 mM boric acid, and 1 mM EDTA) at room temperature (ϳ25°C) and 200 V for 4 h. After electrophoresis, the gel was dried and visualized by autoradiography.…”