The B-type cyclins of S. cerevisiae are diversified with respect to time of expression during the cell cycle as well as biological function. We replaced the early-expressed CLB5 coding sequence with the late-expressed CLB2 coding sequence, at the CLB5 locus. CLB5::CLB2 exhibited almost no rescue of clb5-specific replication defects, although it could rescue clb1 clb2 lethality, and in synchronized cells Clb2p-associated kinase activity from CLB5::CLB2 rose early in the cell cycle, similar to that of Clb5p. Mutagenesis of a potential substrate-targeting domain of CLB5 reduced biological activity without reducing Clb5p-associated kinase activity. Thus, Clb5p may have targeting domains required for CLB5-specific biological activity.

Cyclical inactivation of B-type cyclins has been proposed to be required for alternating DNA replication and mitosis. Destruction box-dependent Clb5p degradation is strongly increased in mitotic cells, and constitutive overexpression of Clb5p lacking the destruction box resulted in rapid accumulation of inviable cells, frequently multiply budded, with DNA contents ranging from unreplicated to apparently fully replicated. Loss of viability correlated with retention of nuclear Clb5p at the time of nuclear division. CLB2-⌬db overexpression that was quantitatively comparable to CLB5-⌬db overexpression with respect to Clb protein production and Clbassociated kinase activity resulted in a distinct phenotype: reversible mitotic arrest with uniformly replicated DNA. Simultaneous overexpression of CLB2-⌬db and CLB5-⌬db overexpressers similarly resulted in a uniform arrest with replicated DNA, and this arrest was significantly more reversible than that observed with CLB5-⌬db overexpression alone. These results suggest that Clb2p and not Clb5p can efficiently block mitotic completion. We speculate that CLB5-⌬db overexpression may be lethal, because persistence of high nuclear Clb5p-associated kinase throughout mitosis leads to failure to load origins of replication, thus preventing DNA replication in the succeeding cell cycle.

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14.

LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detailed characterization of mutants carrying the lytJ-J allele showed that this mutation was recessive and pleiotropic, affecting both mitotic and meiotic functions. At the nonpermissive temperature of 37°C, lyti haploid strains budded at a distal position (instead of an axial one, as in wild-type haploid strains) and underwent autolysis when the buds were almost the size of the mother cells. These mitotic alterations in cell stability and budding topology were dependent on growth and protein synthesis. Autolysis was prevented by inhibiting DNA synthesis (with hydroxyurea) or by blocking the assembly of microtubules (with benomyl), suggesting that loss of cell viability must occur at a fixed mitotic cycle stage after DNA synthesis and mitotic spindle assembly. On the other hand, lyti-Jllyti-l diploids failed to sporulate at both 24 and 37°C. Taking into account these characteristics, the lyti mutant could be considered a cdc-like mutant. By genetic transformation of an appropriate lyti strain with a genomic library, ligated to the multicopy vector YEpl3, we isolated a gene capable of complementing mitotic alterations but not the meiotic defect. This was the sporulation-specific gene SP012, which is expressed under the control of the locus ML4T in meiosis and is also expressed in the mitotic cycle (V. Parkes and L. H. Johnston, Nucleic Acids Res. 20:5617-5623, 1992). A significant level of SP012 mRNA can be detected when this gene is inserted in a multicopy plasmid.The isolation of fungal mutants displaying a thermosensitive autolytic phenotype is a general strategy to identify genes controlling cell wall integrity and cell viability. Mutants of this type have been described from Saccharomyces cerevisiae (6), Candida albicans (25), Schizosaccharomyces pombe (29), and Aspergillus nidulans (3). The characteristic shared by these mutant strains is loss of viability when grown at nonpermissive temperatures, leading to the release of the intracellular content. The wide variety of autolytic mutants that can be isolated reflects the complexity of functions involved in cell wall integrity and cell viability. However, some insight into the defect of the corresponding mutant can be obtained by analyzing complementation of the phenotypic defect by osmotic stabilization with sorbitol, which can be precisely determined by means of flow cytometry (10). Some mutants are osmotic remedial, suggesting that the phenotypic defect must be primarily located in the cell wall (25,36). Others are minimally complemented or not complemented at all by the osmotic stabilizer, an indication that the primary alteration probably does not concern cell wall functions, although eventually the cells release their intracellular content.One aspect of the autolytic mutants that has not been examined in detail is the relationship that they may have with the cell cycle. In some S. cerevisiae autolytic mutants, cell lysis occurs at a particular stage of the cell cy...