Cloning and characterization of a divergent α-tubulin that is expressed specifically in dividing amebae of Naegleria gruberi

A novel α-tubulin gene (α6) was cloned from a genomic library of Naegleria gruberi strain NB-1 and characterized. The open reading frame of α6 contained 1359 nucleotides encoding a protein of 452 amino acids (aa) with a calculated molecular weight of 50.5 kDa. The nucleotide sequence of the open reading frame of α6 showed considerable divergence (68.4% identity) when compared with previously cloned N. gruberi α-tubulin genes, which share about 97% identity in DNA sequences. The deduced aa sequence of α6-tubulin was 61.9% identical to that of α13-tubulin, which was cloned from the same strain, and showed similar identities to those of α-tubulins from other species (54∼62%). These data showed that α6-tubulin is one of the most divergent α-tubulins so far known. α6-tubulin was found to be expressed in actively growing cells and repressed quickly when these cells were induced to differentiate. Immunostaining with an antibody against α6-tubulin showed that α6-tubulin is present in the nuclei and mitotic spindle-fibers but absent in flagellar axonemes or cytoskeletal microtubules. These data finally established the presence of an α-tubulin that is specifically utilized for spindle-fiber microtubules and distinct from the flagellar axonemal α-tubulins in N. gruberi, hence confirmed the multi-tubulin hypothesis in this organism.