The highly divergent alpha- and beta-tubulins from Dictyostelium discoideum are encoded by single genes.
As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.
Triviños-Lagos
L
L
Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, IL 60611.
Ohmachi
T
T
Albrightson
C
C
Burns
R G
RG
Ennis
H L
HL
Chisholm
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014404:Tubulin / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D018076:DNA, Complementary; D004271:DNA, Fungal; D016054:DNA, Protozoan; D005656:Fungal Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D014404:Tubulin