As a step in the characterization of the microtubule system of Dietyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium α- and β-tubulins, as well as the Dictyostelium α-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single α- and β-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete α-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete β-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 α-tubulin displays the greatest sequence divergence yet described. Single α- and β-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 α- and β-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the α- and β-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium α- and β-tubulins. These antisera recognize one α- and two β-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of cell science|
|State||Published - Jan 1 1993|
ASJC Scopus subject areas
- Cell Biology