Transcriptional analysis of the EhPgp5 promoter of Entamoeba histolytica multidrug-resistant mutant.
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.
Pérez
D G
DG
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.
Gómez
C
C
López-Bayghen
E
E
Tannich
E
E
Orozco
E
E
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D022762:CCAAT-Enhancer-Binding Proteins; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000235:genetics*; D004748:Entamoeba histolytica / Q000235:genetics*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors