The 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. Specifically the subproject for an MBRS student will attempt to determine the number of cytoplasmic dynein genes found in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Genomic DNA of both species will be screened with degenerate PCR primers derived from very highly conserved regions of sequence bridging the ATP-binding site of dynein in other organisms in order to amplify probes for the dynein genes in these two species from libraries of their genomic DNA. Preliminary indications are that S. cerevisiae contains only a single gene for cytoplasmic dynein, whereas sea urchins contain two that have only about 50% homology and presumably serve different functions in the cell. Since the nuclear division of S. pombe, and particularly the organization of its centromeres, resembles that of higher organisms more closely than that of S. cerevisiae, it will be of special interest to determine whether S. pombe possesses a second cytoplasmic dynein gene homologous to that of higher organisms. If only a single gene is found in S. pombe, then the project will be extended to other animals and plants in order to determine at what stage of evolution the second cytoplasmic dynein gene evolved. Correlation of the appearance of this second gene with the greater complexity of all functions in higher eukaryotes will be helpful in suggesting possible functions for the cytoplasmic dynein genes. These results will contribute to the overall project and help elucidate the involvement of cytoplasmic dynein in mitosis, meiosis and vesicle transport, and so contribute to our understanding of human health problems associated with improper chromosome segregation or secretory functions.
