BETA-TUBULIN GENES OF TRICHOMONAS-VAGINALIS

Microtubules, formed by polymerization of alpha and beta-tubulins, are major structural components of the mitotic spindle, cytoskeleton, and flagella, and are also an important target for the antiparasitic benzimidazole drugs. Trichomonas vaginalis, a flagellated protozoan responsible for urogenital tract infections in humans, is highly sensitive to certain benzimidazoles in vitro. As a first step towards defining the roles of microtubules in this organism, the regulation of their expression, and the basis for their benzimidazole sensitivity, we have characterized the genes encoding T. vaginalis beta-tubulin. A combination of genomic DNA cloning using bacteriophage lambda and PCR amplification using conserved beta-tubulin gene primers was employed. Southern blots of DNA from two different T. vaginalis strains suggest there are 6-7 beta-tubulin gene copies. Sequencing identified three distinct genes: btub1, btub2, and btub3. Amplification of cDNA with gene-specific primers indicated that the relative expression of RNA transcripts was btub1 > btub2 much greater than btub3. The promoter region from btub1 includes a 15-bp repeat also found (with 1-bp difference) upstream of the T. vaginalis ferredoxin gene. Primer extension suggests the 5' leader of the mRNA transcribed from btub1 is only 10 nucleotides long, similar to the lengths found in other anaerobic protozoa. In 152 residues examined by PCR, btub2 and btub3 differed by 1 and 12 amino acids, respectively, from btub1 All three sequences, however, have diverged considerably (20-24%) from beta-tubulins of other protozoa. T. vaginalis beta-tubulins include residues Tyr167 and Phe200, previously implicated in resistance and sensitivity, respectively, to the benzimidazole derivative benomyl.