The aim of this SGER proposal is to isolate clones for cytoplasmic dynein chains from a budding yeast (S. cerevisiae) genomic library, with the long term aim of studying molecular aspects of dynein structure and function. Degenerate primers and PCR have generated a 300 bp probe that appears to hybridize with dynein cDNA. The project calls for the further characterization of the genomic clones that hybridize with the probe. A series of restriction maps are planned to determine whether these clones actually encode yeast dynein(s). The second major aim of the project is to generate a null mutation in diploid yeast cells to assess the role of the gene product in growth, cell division, and mating. The experiments are carefully-planned, well-controlled and provide alternative strategies in the event that the primary plan of attack is unsuccessful. %%% The significance of this work is potentially high because this work could alter our understanding of the molecular structure of this immense mechanochemical protein complex in ways that have been impossible thusfar. The risk in this project is fairly evident; it is not at all clear that budding yeast requires significant levels of dynein to survive. The functional studies involving the null mutant(s) may be totally equivocal. Another possibility is that null mutations for cytoplasmic dynein could be lethal. If the null mutants survive, but fail to develop properly, the role for a cytoplasmic dynein may be finally established. This is an important project that entails a high risk, but has a potentially significant payoff.
