Microbiology (1999), 145, 33-40 Printed in Great Britain The TATA-box binding protein of Entarnoeba histolytica: cloning of the gene and location of the protein by immunofluorescence and confocal microscopy J ua n P. Lu n a-Ar ias, If3 Rosa u ra Hernandez-Rivas, Guadalupe de Di~s-Bravo,~ Job Garcia’/ Leobardo Mendoza3 and Esther O ~ O Z C O * ~ ~ Author for correspondence: Esther Orozco. Tel: +52 5 747 7000 ext. 5642. Fax: +52 5 747 7108. e-mail: esther@mail.cinvestav.mx l,* Program of Molecular Biomedicinel, and Department of Experimental Pathologyz, CINVESTAV, IPN, A.P. 14-740, Mexico 07300 DF, Mexico 3 CICATA, IPN, Legaria 694, Mexico 11 590 DF, Mexico 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 Em 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 INTRODUCTION Entamoeba histolytica is a protozoon responsible for human amoebiasis. Knowing its DNA structure and its mechanisms for regulating gene transcription is a step toward understanding virulence. E. histolytica has an A+T rich genome (67 mol%) (Sanchez et al., 1994; Tannich & Horstmann, 1992) and its codon usage is unusual, with 85 O / O A + U in the third codon position (Bruchhaus et al., 1993). Putative TATA boxes with TATTAAA, TATAAA (Sanchez et al., 1994) and TATTTAAA sequences (Bruchhaus et al., 1993; Li et al., 1992) have been located 27 to 50 bp upstream from the ATG start codon. The ATTCA and ATCA se- quences have been described as consensus transcription start sites (Bruchhaus et al., 1993).Remarkably, protein- encoding gene transcription is insensitive in uitro to 1 mg a-amanitin ml-’, suggesting that the RNA poly- merase differs from those of other eukaryotic organisms (Lioutas & Tannich, 1995), but little is known about the transcription machinery. Studies of promoter structure and function began recently with the characterization of the hg12 and hgl5 gene promoters, which encode the slightly different 170 kDa heavy subunits of the galactose-inhibitable lectin (Bug et al., 1995; Purdy et al., 1996; Singh et al., 1997). The TATA box, the CCAAT box (hg12) and the GAAC (hg15) were identified as regu1ators for full promoter activity in vivo. We have studied factors involved in expression of the EhPgp2 and EhPgp5 genes, both responsible for the multidrug resistance phenotype ....................................................... ‘............I ................................................................. . .......... Abbreviations: TAFE, transverse alternating field electrophoresis; TBP, TATA-box binding protein; UTR, untranslated region. The EMBL accession number for the sequence reported in this paper is 248307. 0002-2676 0 1999 SGM 33