The cdc2 protein kinase of fission yeast plays a critical role in regulation of the cell cycle and appears to have a homolog in all eukaryotes. In fission yeast, the kinase acts at both the initiation of DNA replication and also independently at entry into mitosis. The experimental objectives fall broadly into four interconnected sections, each relating to some aspect of regulation of cell cycle by the cdc2 protein kinase. 1. The product of the cdc13gene, which was identified as an extragenic suppressor of cdc2 mutants, is a mitotic cyclin that exists in a complex with the cdc2 protein kinase. The specific role of the cyclin in regulating the kinase will be tested and the cdc2/cdc13 complex will also be purified to homogeneity from yeast. Purified material will be used for determination of the subunit structure of the kinase. 2. The signature of cyclins is their destruction at the metaphase/anaphase transition by a protease that appears to be remarkably tightly regulated. The cdc13gene product shares this property with all other cyclins and provides an opportunity to approach the mechanism of cyclin degradation genetically. We propose to isolate mutants of cdc13 that cannot be degraded, to identify the cyclin protease and to obtain protease deficient mutants. 3. The mcs mutants define six new genes that interest with cdc2 in mitotic control. Some of these are expected to encode potential substrates of the cdc2 or wee1 protein kinases. We have isolated the mcs genes. We propose to generate null-alleles of each and particularly to characterize the mcs gene products and define their interactions with these protein kinases. 4. cdc2 acts in both G2 and G2 phases of the yeast cell cycle. We propose to isolate mutant alleles of cdc2 that are defective for G1 but not G2 function and to isolate extragenic suppressors.
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