THE ROLE OF GAMMA-TUBULIN IN MITOTIC SPINDLE FORMATION AND CELL-CYCLEPROGRESSION IN ASPERGILLUS-NIDULANS

gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent resu lts suggest that this may not be the case. To clarify the role of gamm a-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a gamma-tubuli n disruption and observe the phenotypes caused by the disruption in th e first cell cycle after germination. Our results indicate that gamma- tubulin is absolutely required for the assembly of mitotic spindle mic rotubules, a finding that supports the hypothesis that gamma-tubulin i s involved in spindle microtubule nucleation. In the absence of functi onal gamma-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses withou t nuclear division. Our results indicate that gamma-tubulin is not ess ential for progression from G(1) to G(2), for entry into mitosis nor f or spindle pole body replication. It is also not required for reactivi ty of spindle pole bodies with the MPM-2 antibody which recognizes a p hosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule asse mbly but may play a role in the formation of normal cytoplasmic microt ubule arrays.