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.
Kube-Granderath
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Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, München/Germany.
Schliwa
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M
D000595:Amino Acid Sequence; D000656:Amoeba / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014404:Tubulin