The broad long-term objective of this project is to determine the cellular functions of cytoplasmic dynein by studying its role in yeast as a model for its role in cells of vertebrates and other higher organisms. More specifically, it is intended (i) to utilize the yeast DNA probe obtained from the gene encoding dynein to characterize the gene by determining whether it is a single or a multiple copy gene, obtaining its restriction map and base sequence at a fragment that encodes the hydrolytic site, and mapping it to its physical position on the chromosome, (ii) to localize the yeast cytoplasmic dynein during various stages of the cell cycle, (iii) to study the phenotypes of mutated forms of the gene in order to understand the general role of yeast cytoplasmic dynein, and the specific importance of different regions on the protein, (iv) to study the possible interaction between dynein and the other motor proteins of the cytoskeleton, (v) to study the relationship between dynein expression and the various mechanisms regulating the cell cycle. For the first goal, a genomic library of Saccharomyces cerevisiae will be screened with our probe in order to construct a plasmid that will carry the whole yeast dynein gene including its promoter, which is expected to contain more than 14 kb, based on the size of dynein from higher cells. This cloned gene will serve for later experiments of genetic manipulations. The localization of the yeast cytoplasmic dynein will be probed with methods of immunofluorescence microscopy using epitope-tagged antibodies. The variations in the cellular distribution of dynein at different stages of the cell cycle will clarify the processes in which dynein is involved. The extensive molecular genetic tools available in yeast will be exploited to study the phenotypic behavior of cells in which the dynein gene was entirely or conditionally disrupted. These targeted mutations will be induced, utilizing our sequenced yeast probe and methods that are widely used in yeast for these purposes. In observing the phenotypic behavior of conditional site-directed point mutations, special attention will be given to the effect on cellular processes that involve motility such as chromosome movement during cell division or nuclear migration during conjugation. The results of this study are expected to elucidate the involvement of cytoplasmic dynein in mitosis and meiosis and so to contribute to understanding of the health problems associated with improper chromosome segregation in higher organisms.
