Unusual distribution of gamma-tubulin in the giant fresh water amoeba Reticulomyxa filosa.

We have isolated a cDNA encoding gamma-tubulin of Reticulomyxa. The deduced amino acid sequence revealed a high homology to known gamma-tubulin sequences from other organisms, underlining the hypothesis of conserved functions for gamma-tubulin in the cell. After introduction of restriction sites by site-directed mutagenesis and polymerase chain reaction (PCR), we cloned the cDNA into a bacterial expression vector. Recombinant gamma-tubulin was expressed in considerable amounts. Polyclonal antibodies raised against the recombinant material recognized in Western blots the bacterially expressed gamma-tubulin and in the amoeba crude extract a single band representing most likely the amoebal gamma-tubulin. In immunofluorescence studies the antibody showed a diffuse staining in the cytoplasm and no enrichment at centrosome-like structures. Because the assembly of centrosome-independent microtubules can be observed in the far extensions of the amoeba, the data suggest that in this amoeba gamma-tubulin might be involved in the centrosome-independent assembly of microtubules.