Interaction of an Overexpressed γ-Tubulin with Microtubules In Vivo and In Vitro

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γ-Tubulin is an ubiquitous MTOC(microtubule-organizing center)component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides(amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells(Vassilev et al., J. Cell Sci. 108 : 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports(Li and Joshi, J. Cell Biol. 131 : 207, 1995 ; Shu and Joshi, J. Cell Biol. 130 : 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.
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Interaction of an Overexpressed γ-Tubulin with Microtubules In Vivo and In Vitro

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