Cloning, genomic organization and transcription of the Entamoeba histolytica alpha-tubulin-encoding gene

Abstract

We have isolated and characterized an Entamoeba histolytica alpha-tubulin (alpha Tub)-encoding gene (Eh alpha tub). A 700-bp DNA fragment was amplified by PCR using primers derived from consensus alpha- and beta-Tub amino acid (aa) sequences from different organisms and E. histolytica DNA as the template. These PCR fragments were used to screen both 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. The deduced aa sequence of Eh alpha Tub has 55.5, 50 and 52% identity to Plasmodium falciparum alpha Tub 2, Saccharomyces cerevisiae alpha Tub 2 and human alpha Tub, respectively. Interestingly, the predicted Eh alpha Tub protein lacks a poly-acidic motif at its C terminus which is involved in Tub polymerization and microtubule-associated protein binding in other organisms. This fact may indicate a difference in tubule assembly in this organism and could provide a potential key for the development of therapeutic agents. According to Southern blot experiments and the sequences of several clones, at least two non-adjacent copies of alpha tub are present in the E. histolytica genome. A 1.5-kb transcript corresponding to the alpha tub mRNA was detected in mRNA from asynchronous E. histolytica trophozoites.