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

A 309 bp DNA fragment from Entamoeba histolytica was amplified by PCR using primers derived from the Acanthamoeba castellanii consensus TATA-box bindi ng protein amino acid sequence. The amplified fragment was used to isolate cDNA and genomic DNA clones containing an ORF encoding the complete E. hist olytica TATA-box binding protein (Ehtbp, 702 bp, 234 aa, molecular mass 26 kDa). The EhTBP functional domain showed 55% sequence identity to that of H omo sapiens, 54% to A. castellanii and 37% to Plasmodium falciparum TBPs. I n Southern blot experiments we detected a single Ehtbp band, which was tran scribed as a 1.3 kb mRNA containing a 420 nt 5' untranslated region. Howeve r, the probe hybridized with the 0.8 and 1.5 Mb chromosomes, suggesting tha t 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 DN A-containing organelle recently reported. The recombinant E. histolytica TA TA-box binding protein was expressed in Escherichia coli. Antibodies agains t it recognized two proteins of 26 and 29 kDa in E. histolytica nuclear ext racts. Confocal microscopy immunofluorescence analysis located the protein in both the nucleus and EhkO.