The TATA-box binding protein of Entamoeba histolytica: cloning of the gene and location of the protein by immunofluorescence and confocal microscopy

Program of Molecular Biomedicine Department of Experimental Pathology, CINVESTAV, IPN, A.P. 14–740, Mexico 07300 DF, Mexico CICATA, IPN, Legaria 694, Mexico 11590 DF, Mexico Author for correspondence: Esther Orozco. Tel: +52 5 747 7000 ext. 5642. Fax: +52 5 747 7108. e-mail: esther@mail.cinvestav.mx ABSTRACT Summary: A 309 bp DNA fragment from Entamoeba histolytica was amplified by PCR using primers derived from the Acanthamoeba castellanii consensus TATA-box binding protein amino acid sequence. The amplified fragment was used to isolate cDNA and genomic DNA clones containing an ORF encoding the complete E. histolytica TATA-box binding protein ( Ehtbp, 702 bp, 234 aa, molecular mass 26 kDa). The EhTBP functional domain showed 55% sequence identity to that of Homo sapiens, 54% to A. castellanii and 37% to Plasmodium falciparum TBPs. In Southern blot experiments we detected a single Ehtbp band, which was transcribed as a 1.3 kb mRNA containing a 420 nt 5' untranslated region. However, the probe hybridized with the 0.8 and 1.5 Mb chromosomes, suggesting that this sequence is diploid. In situ PCR assays showed two signals in 95% of trophozoites, one located in the nucleus and another in EhkO, the novel DNA-containing organelle recently reported. The recombinant E. histolytica TATA-box binding protein was expressed in Escherichia coli. Antibodies against it recognized two proteins of 26 and 29 kDa in E. histolytica nuclear extracts. Confocal microscopy immunofluorescence analysis located the protein in both the nucleus and EhkO. Keywords: Entamoeba histolytica , TATA-box binding protein, tbp gene expression The EMBL accession number for the sequence reported in this paper is Z48307 .