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Fission yeast cells with a temperature-sensitive Orp1 protein, a component of the origin recognition complex, cannot perform DNA replication at the restrictive temperature. Seventy percent of orp1-4 cells arrest with a 1C DNA content, whereas 30% proceed to mitosis (`cut'). The arrest depends upon the checkpoint Rad proteins and, surprisingly, the Chk1 protein, which is thought to act only from late S phase. The arrested cells maintain a 1C DNA content, as judged by flow cytometry, and the early origin ars3001has not been initiated, as judged by 2D gel analysis. We show that in G1-arrested orp1-4 cells, Wee1 phosphorylates and inactivates Cdc2. Activation of Chk1 occurs earlier than Cdc2 phosphorylation, indicating a novel role for Chk1, namely to induce and/or maintain Cdc2 phosphorylation upon checkpoint activation in G1. We also show that commitment to cutting occurs already in early G1 phase.

In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression. These mutations define a single gene which we call ded1. We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species. Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex. When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins. We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G1 arrest and subsequent mating. Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis.

Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate.