INTERACTION OF AN OVEREXPRESSED GAMMA-TUBULIN WITH MICROTUBULES IN-VIVO AND IN-VITRO

gamma-ubulin is an ubiquitous MTOC (microtubule-organizing center) com ponent essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis o f nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubutin clone, epitope-tag ged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various s izes. Although one of the foci was identified as the centrosome, the r est of the dots were not associated with any other centrosomal compone nts tested so far. The pattern of microtubule organization was not aff ected by induction of such gamma-tubulin-containing dots in transfecte d cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon remova l of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. U sing the monomeric form of Chlamydomonas gamma-tubulin purified from i nsect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), inter action between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubul in monomers in vitro were associated with more gold particles conjugat ed with gamma-tubulin than in controls where no exogenous gamma-tubuli n was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gam ma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles wer e seen along the microtubule length. These results contradict the prev ious 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 differ ence in the level of protein expression in transfected cells.