Isolation and functional studies of D. discoideum cyclin B gene during growth and development

Cyclin B plays an essential role i n cell cycle regulation by complexing with Cdc2 protein kinase to direct cells into mitosis. A cyclin B gene (cycB) has been isolated from D. discoideum. The cyclin box region of the protein encoded by cycB has a high degree of sequence identity with B-type cyclins of other species. During celldivision synchronized growth, levels of cyclin B mRNA and protein were found to oscillate during the cell cycle with maximum accumulation occurring prior to and during cell division. Northern blot analysis showed that the cycB mRNA levels peaked twice during development, once during early aggregation (3 h) and again at the tipped aggregate stage (12 h). In contrast, the levels of cyclin B protein and histone H I kinase activity of the Cdc2-immunoprecipitate increased only during early development and then gradually decreased as development continued. The N-terminal region of the cyclin B protein has the conserved protein degradation motif, typical of cyclin B proteins. The 5' portion of the D. discoideum cycB gene was deleted and the truncated gene was cloned downstream from the regulatable discoidin promoter. Overexpression of this truncated cyclin B gene elevated the histone H I kinase activity of the Cdc2-immunoprecipitates, suggesting that the kinase activity was limited by the amount of the cyclin B i n the cells. The growth of these cyclin B overexpressing mutants was arrested during mitosis. These mitotically arrested cells formed very small fruiting bodies when allowed to undergo development. A protein that eluted together with the histidine tagged cyclin B fusion protein from a Ni-resin affinity column was identified as Cdc2, indicating a direct physical interaction between cyclin B and Cdc2. Indirect immunofluorescence staining using the cyclin B antibody revealed that D. discoideum cyclin B localized mainly in the nucleus. This is consistent with the finding that D. discoideum cyclin B is lacking the presumptive cytoplasmic signal sequence found in most of the B-type cyclins from multicellular organisms.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat