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 benz imidazole drugs. Trichomonas vaginalis, a flagellated protozoan respon sible for urogenital tract infections in humans, is highly sensitive t o certain benzimidazoles in vitro. As a first step towards defining th e roles of microtubules in this organism, the regulation of their expr ession, and the basis for their benzimidazole sensitivity, we have cha racterized the genes encoding T. vaginalis beta-tubulin. A combination of genomic DNA cloning using bacteriophage lambda and PCR amplificati on using conserved beta-tubulin gene primers was employed. Southern bl ots of DNA from two different T. vaginalis strains suggest there are 6 -7 beta-tubulin gene copies. Sequencing identified three distinct gene s: btub1, btub2, and btub3. Amplification of cDNA with gene-specific p rimers indicated that the relative expression of RNA transcripts was b tub1 > btub2 much greater than btub3. The promoter region from btub1 i ncludes a 15-bp repeat also found (with 1-bp difference) upstream of t he T. vaginalis ferredoxin gene. Primer extension suggests the 5' lead er of the mRNA transcribed from btub1 is only 10 nucleotides long, sim ilar to the lengths found in other anaerobic protozoa. In 152 residues examined by PCR, btub2 and btub3 differed by 1 and 12 amino acids, re spectively, from btub1 All three sequences, however, have diverged con siderably (20-24%) from beta-tubulins of other protozoa. T. vaginalis beta-tubulins include residues Tyr167 and Phe200, previously implicate d in resistance and sensitivity, respectively, to the benzimidazole de rivative benomyl.