CLONING, GENOMIC ORGANIZATION AND TRANSCRIPTION OF THE ENTAMOEBA-HISTOLYTICA ALPHA-TUBULIN-ENCODING GENE

We have isolated and characterizated an Entamoeba histolytica alpha-tu bulin (alpha Tub)-encoding gene (Eh alpha tub). A 700-bp DNA fragment was amplified by PCR using primers derived from consensus alpha- and b eta-Tub amino acid (aa) sequences from different organisms and E. hist olytica DNA as the template. These PCR fragments were used to screen b oth genomic DNA and cDNA libraries in order to isolate an Eh alpha tub structural gene. Two overlapping clones containing the complete alpha tub ORF (1392 bp) were isolated from the genome and cDNA libraries. T he deduced aa sequence of Eh alpha Tub has 55.5, 50 and 52% identity t o Plasmodium falciparum alpha Tub 2, Saccharomyces cerevisiae alpha Tu b 2 and human alpha Tub, respectively. Interestingly, the predicted Eh alpha Tub protein lacks a poly-acidic motif at its C terminus which i s involved in Tub polymerization and microtubule-associated protein bi nding in other organisms. This fact may indicate a difference in tubul e assembly in this organism and could provide a potential key for the development of therapeutic agents. According to Southern blot experime nts and the sequences of several clones, at least two non-adjacent cop ies of alpha tub are present in the E. histolytica genome. A 1.5-kb tr anscript corresponding to the alpha tub mRNA was detected in mRNA from asynchronous E. histolytica trophozoites.