Fission yeast cells tolerate the total absence of the cdc25 mitotic inducer in two cases, either in cdc2-3w or in wee1 genetic backgrounds. In the cdc2-3w cdc25Delta double mutant, the rate-limiting step leading to mitosis is reaching a critical size. However, the size control of this mutant operates in late G2, which is different from wild-type (WT) cells. This fact suggests that in WT the rate-limiting molecular process during the G2 timer is the Tyr15 dephosphorylation of cdc2, for which the cdc25 phosphatase (together with its back-up, pyp3) is dependent. In the wee1-50 cdc25Delta mutant, the population splits into different clusters, all lacking mitotic size control. This strain maintains size homeostasis by a novel method, which is random movement of the cells from one cluster to another in the successive generations. These cells should normally have a ‘minimal cycle’, a ‘timer’ with short G1 and G2 phases. However, very often the cells abort mitosis, possibly at an early event and return back to early G2, thus lengthening their cycles. The inability of these cells to start anaphase might be caused by the absence of the main mitotic regulators (wee1 and cdc25) and the improper regulation of their back-up copies (mik1 and pyp3, respectively).

An analysis was made of cell length and cycle time in time-lapse films of the fission yeast Schizosaccharomyces pombe using wild-type (WT) cells and those of various mutants. The more important conclusions about ‘size controls’ are: (1) there is a marker in G2 in WT cells provided by a rate change point (RCP) where the linear rate of length growth increases by approximately 30%. The period before this RCP is dependent on size and can be called a ‘sizer’. The period after the RCP is nearly independent of size and can be called a ‘timer’. The achievement of a critical threshold size is at or near the RCP which is on average at about 0.3 of the cycle (halfway through G2). This is much earlier than was previously believed. (2) The RCP is at about the time when H1 histone kinase activity and the B type cyclin cdc13 start to rise in preparation for mitosis. The RCP is also associated with other metabolic changes. (3) In wee1 mutants, the mitotic size control is replaced by a G1/S size control which is as strong as the mitotic control. As in WT cells, there is a sizer which precedes the RCP followed by a timer but the RCP is at about the G1/S boundary and has a larger increase (approximately 100%) in rate. (4) cdc25 is not an essential part of the size control at mitosis or at the G1/S boundary. (5) Three further situations have been examined in which the mitotic size control has been abolished. First, induction synchronisation by block and release of cdc2 and cdc10. In the largest oversize-cells which are produced, the RCP is pushed back to the beginning of the cycle. There is no sizer period but only a timer. Second, when both the antagonists wee1 and cdc25 are absent in the double mutant wee1-50 cdc25 delta. In this interesting situation there is apparently no mitotic size control and the cycle times are quantised. Third, in rum1 delta wee1-50 where the normal long G1 in wee1 is much reduced, there is probably no size control either in G1 or in G2 causing a continuous shortening of division length from cycle to cycle.

To contribute to a deeper understanding of M-phase control in eukaryotic cells, we have constructed a model based on the biochemistry of M-phase promoting factor (MPF) in Xenopus oocyte extracts, where there is evidence for two positive feedback loops (MPF stimulates its own production by activating Cdc25 and inhibiting Wee1) and a negative feedback loop (MPF stimulates its own destruction by indirectly activating the ubiquitin pathway that degrades its cyclin subunit). To uncover the full dynamical possibilities of the control system, we translate the regulatory network into a set of differential equations and study these equations by graphical techniques and computer simulation. The positive feedback loops in the model account for thresholds and time lags in cyclin-induced and MPF-induced activation of MPF, and the model can be fitted quantitatively to these experimental observations. The negative feedback loop is consistent with observed time lags in MPF-induced cyclin degradation. Furthermore, our model indicates that there are two possible mechanisms for autonomous oscillations. One is driven by the positive feedback loops, resulting in phosphorylation and abrupt dephosphorylation of the Cdc2 subunit at an inhibitory tyrosine residue. These oscillations are typical of oocyte extracts. The other type is driven by the negative feedback loop, involving rapid cyclin turnover and negligible phosphorylation of the tyrosine residue of Cdc2. The early mitotic cycles of intact embryos exhibit such characteristics. In addition, by assuming that unreplicated DNA interferes with M-phase initiation by activating the phosphatases that oppose MPF in the positive feedback loops, we can simulate the effect of addition of sperm nuclei to oocyte extracts, and the lengthening of cycle times at the mid-blastula transition of intact embryos.

CO2 production was followed by manometry in starved cell-free extracts of fission yeast stimulated by unstarved cell-free extracts from a synchronous culture. The degree of stimulus, measured by the lag time in CO2 production, varied markedly during the cell cycle, with a maximum for cells at about mitosis and a minimum for septated cells. Similar differences in lag time were found with unstarved extracts of cdc13.117 grown at 37 degrees C and 35 degrees C.

Oxygen consumption was measured with an oxygen electrode in synchronous cultures of S. pombe. There were changes during the cell cycle in the rate of oxygen uptake, which are most clearly shown as oscillations in acceleration curves (rate of the rate of uptake). Under various conditions of selection and induction synchrony the acceleration curves are similar to those found earlier for CO2 production. As with CO2 production, the oscillations continued after a block to the DNA-division cycle. There were, however, two differences between oxygen uptake and CO2 production. The oxygen oscillations were more marked and also were out of phase by half a cycle. The respiratory coefficient therefore changes through the cycle.

Earlier work has shown that there is a periodic change in the rate of production of CO2 during the cell cycle of fission yeast and that this periodicity persists after a block to the DNA-division cycle and also after a block to protein synthesis. It appears that there is a periodic control or ‘oscillator’ affecting CO2 production that is normally closely entrained to the cell cycle, but which can ‘free-run’ after a block. In this paper, we examine what events in the DNA-division cycle can generate the entrainment signals and what is the nature of such signals. In the first set of experiments, CO2 production was measured by manometry during induction synchrony produced by blocking the DNA-division cycle in an asynchronous culture for a period and then releasing the block. Synchronous cell division occurs after the release with cell cycles shorter than normal. After release from a block imposed by shifting up the mutant cdc2.33 to the restrictive temperature, oscillations in CO2 production started rapidly and remained closely entrained to the division cycles (with slightly different patterns and timing from those after selection synchrony). This showed that there was an entrainment signal but did not show whether it came from start, the S period or mitosis. A similar experiment with cdc10.129 showed that an early signal came from either start or the S period, as did an experiment with release from N-starvation. The results with cdc25.22 were similar to those with cdc2.33. After a block with hydroxyurea, there was entrainment but with no signs of the early signal that occurred with cdc10. This showed that the early signal came from start and not from the S period. In a second set of double-block experiments, the first block was followed by a second different block. With cdc25.22 followed by MBC (an inhibitor of nuclear division) the cells passed through a narrow window of the cell cycle between the transition point of cdc25.22 and mitosis. This was sufficient to start the oscillations, showing that an entrainment signal could be generated at about the time of mitosis. The results from using hydroxyurea followed by cdc2.33 showed no genuine oscillations, confirming the conclusion from the single hydroxyurea block. The results from using hydroxyurea followed by cdc10.129 confirmed the existence of a mitotic signal.(ABSTRACT TRUNCATED AT 400 WORDS)

We show that the first of the two transition points of cdc2.33, a mutant of Schizosaccharomyces pombe, exists in exponential phase cells. Using flow cytometry and a double-block experiment, we have measured the position of this transition point both in the single mutant and in the double mutant cdc2.33 wee1.6. In the single mutant, this point is in early G1. In the double mutant, however, this point is only delayed slightly, if at all, despite much larger delays in the S period and in the transition point of cdc10, another ‘start’ mutant. There is therefore a significant dissociation in the timing of what are thought to be two start events, and the first one appears not to be subject to a size control and to be associated with the completion of mitosis rather than the G1/S boundary.

The effect of CO 2 removal on the cell cycle phases of Schizosaccharomyces pombe has been examinedin minimal, aspartate-containing and complete medium. The removal of CO 2 shortened the G 2 phase of the cell cycle and arrested the cells in G 1 phase in minimal medium. The G 1 block caused by CO 2 deprivation was demonstrated by transition-point and flow-cytometry analyses. The slow-down of anapleurotic CO 2 fixation might be responsible for this effect, as aspartic acid could abolish the G 1 block. The shortening of G 2 phase in the wild-type cells was observed in every medium irrespective of whether the growth rate was changed or not. The experiments in which growth rate was not changed by CO 2 shift-down suggest that this CO 2 effect can be independent from its action on CO 2 -fixing steps in metabolism. Therefore we propose that CO 2 inhibits mitosis infission yeast and we explain the proportionality between growth rate and cell size at mitosis found by Fantes & Nurse by this CO 2 inhibition. The larger CO 2 production in fast-growing cells leads to a higher CO 2 concentration, which could exerta stronger inhibition of mitosis. A wee mutant, which has lost its mitotic size control, also shows the G 1 block after CO 2 deprivation, but its mitosis is insensitive to CO 2 . Comparing the respiration of wee and wild-type cells we conclude that CO 2 inhibits the citric acid cycle in the wild type. The consequence of these results in the regulation of fission yeast cell cycle is discussed.

CO2 production has been followed by manometry in synchronous and asynchronous control cultures of Schizosaccharomyces pombe prepared by elutriation from the same initial culture. Earlier results showed a periodic change in the rate of production, which took place once per cell cycle. These changes were most clearly shown as oscillations in the difference between values of the second differential (acceleration) for the synchronous and asynchronous cultures. This paper shows that the oscillations continue for at least three cycles in the presence of cycloheximide (with and without chloramphenicol). Protein synthesis is virtually absent and there is no cell division. The control of this metabolic oscillation is therefore not directly dependent on translation. The period of the oscillation under these conditions is about 60% of the normal cycle time.

CO2 production has been followed by manometry in synchronous and asynchronous cultures of Schizosaccharomyces pombe prepared by elutriation from the same initial culture. The rate of production follows a linear pattern in synchronous cultures with a rate change once per cycle at the time of cell division. This pattern is most clearly shown in oscillations of the difference between values of the second differential (acceleration) for the synchronous and asynchronous cultures. The association between the rate change and the time of division is maintained during growth speeded up in rich medium and slowed down in poor medium and at lower temperature. It is also maintained after a shift-up in temperature. Results with wee mutants suggest that the association is with the S period rather than division itself. The rate and acceleration of CO2 production are approximately proportional to cell size (protein content) in asynchronous cultures. When synchronous cultures of the temperature-sensitive mutants cdc2.33 and cdc2.33 wee1.6 are shifted up to the restrictive temperature, the DNA-division cycle is blocked. The oscillatory pattern of CO2 production, however, continues for one to two cycles until the acceleration reaches a constant value, after which the oscillations are undetectable. This point is reached later in the double mutant and there is a phase difference in the oscillations compared to those in the single mutant. With both blocked mutants the ‘free-running’ oscillations are about 15% shorter than the normal cycle time. There are well-known examples of such oscillations in eggs but they are rare in growing systems.