Cloning and characterization of a divergent alpha-tubulin that is expressed specifically in dividing amebae of Naegleria gruberi.
A novel alpha-tubulin gene (alpha6) was cloned from a genomic library of Naegleria gruberi strain NB-1 and characterized. The open reading frame of alpha6 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 alpha6 showed considerable divergence (68.4% identity) when compared with previously cloned N. gruberi alpha-tubulin genes, which share about 97% identity in DNA sequences. The deduced aa sequence of alpha6-tubulin was 61.9% identical to that of alpha13-tubulin, which was cloned from the same strain, and showed similar identities to those of alpha-tubulins from other species (54 approximately 62%). These data showed that alpha6-tubulin is one of the most divergent alpha-tubulins so far known. Alpha6-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 alpha6-tubulin showed that alpha6-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 alpha-tubulin that is specifically utilized for spindle-fiber microtubules and distinct from the flagellar axonemal alpha-tubulins in N. gruberi, hence confirmed the multi-tubulin hypothesis in this organism.
Department of Biology and the Institute of Life Science and Biotechnology, College of Science, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, South Korea.
Sunglan
S
Chung
Jaeyoung
J
Cho
HyeonJoo
H
Cheon
Soonyoung
S
Paik
JooHun
J
Lee
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D014644:Genetic Variation; D008938:Mitosis / Q000235:genetics; D008969:Molecular Sequence Data; D009253:Naegleria / Q000235:genetics*; D010802:Phylogeny; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014404:Tubulin / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D016054:DNA, Protozoan; D020033:Protein Isoforms; D014404:Tubulin