Coordination of growth and division during the cell cycle of fission yeast.
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Alcohol Dehydrogenase↗
Biomedical subjects
J M Mitchison
Publications and source records attributed to J M Mitchison.
The fission yeast, Schizosaccharomyces pombe.
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Cell Division↗
CO2 production after induction synchrony of the fission yeast Schizosaccharomyces pombe: the origin and nature of entrainment.
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)
Biological Clocks↗
Changes in the rate of oxygen consumption in synchronous cultures of the fission yeast Schizosaccharomyces pombe.
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.
Carbon Dioxide↗
Continued DNA synthesis after a mitotic block in the double mutant cut1 cdc11 of the fission yeast Schizosaccharomyces pombe.
DNA synthesis is normally dependent on a cell having previously gone through mitosis. Hirano et al. (1986), however, found that DNA synthesis continued at the restrictive temperature in the double mutant cut1 cdc11 of Schizosaccharomyces pombe even though mitosis was blocked in some of the cells. We have confirmed this result with bulk DNA assays of asynchronous cultures. Synchronous cultures of a diploid double mutant at the restrictive temperature showed two peaks of incorporation with an interval between them that was approximately the same as the doubling time in cell length. Flow cytometry showed that the cells had increased their DNA content from 4C (the diploid value) to about 16C after 7h. The cytological appearance at this time was mixed, with uninucleate, binucleate and dead cells, but fluorescence measurements on single cells indicated that about half the population had single nuclei with about the 16C value and had therefore gone through two rounds of DNA synthesis without mitosis.
Cell Nucleus↗
Observations on ultracentrifuging wild-type and mutant (cdc2.33) cells of Schizosaccharomyces pombe.
Ultracentrifuging (400,000 g for 4-6 h at 4 degrees C) living wild-type cells of the fission yeast Schizosaccharomyces pombe moves the nucleus towards the ends of the cells but scarcely affects their viability. However, in the long cells produced by growing the mutant cdc2.33 for 4-6 h at the restrictive temperature (36.5 degrees C), ultracentrifuging (as above) gives an intense fluorescence with DAPI in about half of the cytoplasm in about 80% of the cells. This is probably nuclear DNA that has moved into the cytoplasm, both because of the DAPI stain and because it is removed by DNase treatment. These cells ultimately divide and are viable, and we suggest that the extended cytoplasmic DNA returns to the nucleus.
Cell Nucleus↗
Nucleoside diphosphokinase, an enzyme with step changes in activity during the cell cycle of the fission yeast Schizosaccharomyces pombe. II. Dissociation of the steps from the DNA-division cycle after induction synchronization.
Synchrony was induced in cultures of the mitotic mutant cdc2.33 of Schizosaccharomyces pombe by shifting up an asynchronous culture to the restrictive temperature for a period of 3.5-4.5 h and then shifting down to the permissive temperature. The resulting synchronous divisions had short cycle times, down to 50% of the normal cycle. The oscillatory control of nucleoside diphosphokinase activity was also synchronized by the shift-down and the activity rose in a step pattern. Unlike the situation in the normal cycle, this step pattern was dissociated from the shortened cell cycle and had a longer period and different phase relations. It may be that the normal entrainment or coupling between the cell cycle and the activity control fails if the cell cycle is too short. The period of the activity control (equal to the protein doubling time at the restrictive temperature) appears to be temperature-compensated.
Cell Cycle↗
The first transition point of the mutant cdc2.33 in the fission yeast Schizosaccharomyces pombe.
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.
Cell Cycle↗
Periodic cell cycle changes in the rate of CO2 production in the fission yeast Schizosaccharomyces pombe persist after a block to protein synthesis.
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.
Carbon Dioxide↗
Change in the rate of CO2 production in synchronous cultures of the fission yeast Schizosaccharomyces pombe: a periodic cell cycle event that persists after the DNA-division cycle has been blocked.
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.
Carbon Dioxide↗
Nucleoside diphosphokinase, an enzyme with step changes in activity during the cell cycle of the fission yeast Schizosaccharomyces pombe. I. Persistence of steps after a block to the DNA-division cycle.
In confirmation of earlier results, nucleoside diphosphokinase is shown to be a 'step' enzyme in Schizosaccharomyces pombe with a sharp doubling in activity at the beginning of the cell cycle. These doubling steps occur at the same time in the cycle in the smaller cells of the mutant wee1.6. An important result is that the activity steps persist with normal cell cycle timing after a block to the DNA-division cycle imposed by the cycle mutants cdc2.33 and cdc2.33wee1.6. This is clear proof that oscillatory controls of some cell cycle events can persist after the main periodic events of the DNA-division cycle have been abolished.
Cell Cycle↗
Growth in cell length in the fission yeast Schizosaccharomyces pombe.
The cylindrical cells of Schizosaccharomyces pombe grow in length by extension at the ends and not the middle. At the beginning of the cell cycle, growth is restricted to the 'old end', which existed in the previous cycle. Later on, the 'new end', formed from the septum, starts to grow at a point in the cycle that we have called NETO ('new end take-off'). Fluorescence microscopy on cells stained with Calcofluor has been used to study NETO in size mutants, in blocked cdc mutants and with different growth temperatures and media. In wild-type cells (strain 972) NETO happens at 0.34 of the cycle with a cell length of 9.5 microns. With size mutants that are smaller at division, NETO takes place at the same size (9.0-9.5 microns) but this is not achieved until later in the cycle. Another control operates in larger size mutants since NETO occurs at the same stage of the cycle (about 0.32) as in wild type but at a larger cell size. This control is probably a requirement to have completed an event in early G2, since most cdc mutant cells blocked before this point in the cycle do not show NETO whereas most of those blocked in late G2 do show it. We conclude that NETO only happens if: (1) the cell length is greater than a critical value of 9.0-9.5 microns; and (2) the cell has traversed the first 0.3-0.35 of the cycle and passed early G2. NETO is delayed in poor media, in which cell size is also reduced. Temperature has little effect on NETO under steady-state conditions, but there is a transient delay for some hours after a temperature shift. NETO is later in another wild-type strain, 132. Time-lapse photomicrography was used to follow the rates of length growth in single cells. Wild-type cells showed two linear segments during the first 75% of the cycle. There was a rate-change point (RCP), coincident with NETO, where the rate of total length extension increased by 35%. This increase was not due simply to the start of new-end growth, since old-end growth slowed down in some cells at the RCP. cdc 11.123 is a mutant in which septation and division is blocked at 35 degrees C but nuclear division continues.(ABSTRACT TRUNCATED AT 400 WORDS)
Cell Cycle↗
Protein synthesis and its relation to the DNA-division cycle in the fission yeast Schizosaccharomyces pombe.
The rate of protein synthesis has been measured with pulse labels of [3H]tryptophan in synchronous and asynchronous cultures of cdc mutants of Schizosaccharomyces pombe shifted up to the restrictive temperature. The cell cycle related fluctuations in rate that occur in normal synchronous cultures vanish when nuclear division is blocked in synchronous cultures of cdc2 and cdc10. But they persist in cdc11 where nuclear division continues and cleavage is stopped. We conclude that nuclear division affects the rate of synthesis and that this effect is inhibitory and probably persists for the last 40% of the cycle. When nuclear division has been blocked, the rate of synthesis continues to increase until a plateau is reached where the rate remains constant. Three size mutants of cdc2 reach the plateau at the same average protein content per cell although their initial protein contents vary over a threefold range. Comparison of these results with those from cdc10 leads to the tentative conclusion that the plateau starts when the cells reach a critical protein/DNA ratio.
Ascomycota↗
Absence of step changes in activity of certain enzymes during the cell cycle of budding and fission yeasts in synchronous cultures.
Synchronous cultures prepared by selection from an elutriating rotor were used to measure activity changes during the cell cycle of the following enzymes: acid phosphatase in Schizosaccharomyces pombe and Saccharomyces cerevisiae, alpha-glucosidase in S. cerevisiae and beta-galactosidase in Kluyveromyces lactis. There was no sign of step rises in activity in acid phosphatase but there were indications in S. cerevisiae of the linear pattern with rate doublings once per cycle that had been found previously in S. pombe. There was also no sign of step rises in the other two enzymes, in contrast to earlier results using different techniques. Asynchronous control cultures showed little or no perturbations after the first hour.
Acid Phosphatase↗
Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.
A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.
Ascomycota↗
Anaesthetics delay and accelerate division in the fission yeast Schizosaccharomyces pombe.
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Ascomycota↗
The effect of 8-hydroxyquinoline on enzyme synthesis in the fission yeast Schizosaccharomyces pombe.
The effect of 8-hydroxyquinoline, a rapid inhibitor of RNA synthesis, was followed on the activity of a number of enzymes in cultures of the fission yeast Schizosaccharomyces pombe. Two types of effect were found. In the first the activity continued to rise for a period and then remained constant. This occurred with alkaline phosphatase, basal and derepressed acid phosphatase, hexokinase, and derepressed sucrase and maltase at low cell density. It is consistent with control being exercised by an unstable mRNA or by an unstable stimulator of translocation. In the second the activity increased above the control values for several hours. This occurred with basal sucrase and maltase, and suggests a stable mRNA and an unstable inhibitor of translation. The extent of 'superproduction' of sucrase varied with cell density and with growth medium and this may be due to differences in the degree of translational inhibition. The possiblilty of a stable mRNA has interesting implications for the control of enzyme synthesis through the cell cycle.
Acid Phosphatase↗
Germination of Schizosaccharomyces pombe spores separated by zonal centrifugation.
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Ascomycota↗