Genetic analysis of budding yeast has identified functions mediating entry into the cell cycle. A key event essential to spindle formation and other processes is the duplication of the spindle pole body (SPB) -- the sole locus for microtubule organization. We propose investigation of four genes controlling different aspects of SPB duplication: CDC31 is specifically required for duplication per se; mutations permit the SPB to double in size but not in number. Ensuing monopolar division often results in polyploidization. Homology of the CDC31 product with calmodulin-related proteins suggests that altered Ca++ levels regulate its activity. The cloned gene will permit production of new alleles useful for evaluating how Ca++ regulation is exerted. Others will help identify interacting functions by immunochemical assays and isolation of pseudorevertants. Two other genes, CDC4 and CDC34, mediate separation of duplicated SPBs and other G1 events. The derived product of CDC4 shares a striking periodic motif with Beta-transducin, suggesting functional homology with G-protein regulatory mechanisms. Mutations in CDC4 will establish the role of the homologous domain and facilitate isolation of pseudorevertants, which may clarify the key regulatory mechanisms of the CDC4 and CDC34 products. DNA sequencing of CDC34 will establish whether its product is homologous with other transducing proteins and will permit molecular analysis. The fourth gene is ESP1, which serves in negative regulation on the cycle of SPB duplication and separation. Molecular characterization of ESP1 and its product will further address the control of spindle pole behavior.
