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The Effect of Cell Mass on the Cell Cycle Timing and Duration of S-Phase in Fission Yeast

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The Effect of Cell Mass on the Cell Cycle Timing and Duration of S-Phase in Fission Yeast J. Cell Sci. 39, 215-233 (i979) 315 Printed in Great Britain © Company of Biologists Limited 1979 THE EFFECT OF CELL MASS ON THE CELL CYCLE TIMING AND DURATION OF S-PHASE IN FISSION YEAST KIM NASMYTH.f PAUL NURSE* AND R. S. S. FRASERJ • Department of Zoology, University of Edinburgh, West Mains Road, Edinburgh EHg 2JT, U.K. SUMMARY Two isotopic methods for measuring DNA replication in the fission yeast Schizosaccharo- myces pombe are described. The first is a method for measuring the total quantity of PFTJuracil incorporated into DNA after pulse labelling. The second is a means of detecting DNA replica- tion in single cells by autoradiography. Both of these techniques have been used to investigate the timing and duration of S-phase in a series of mutant strains whose cell mass at division varies over a 3-fold range. The results support the hypothesis that in S. pombe there are 2 dif- ferent controls over the timing of 5-phase: an attainment of a critical cell mass and a dependency upon the completion of the previous mitosis coupled with a short minimum time in G-i. Strains whose cell mass at birth is above this critical level initiate DNA replication almost immediately after septation, that is, very soon after the previous mitosis. Strains whose cell mass at birth is below the critical level do not initiate replication until the critical cell mass is attained. The duration of S-phase has been estimated from the proportion of cells whose nuclei are labelled after a pulse of given duration. 5-phase is short in S. pombe, lasting only about o-i of a cell cycle in wild type. Cell mass at S-phase does not have any consistent effect on this length. We have also investigated the degree of synchrony of S-phase initiation in daughter cells, and have found that, in a cell cycle 240 min long, their 5-phases are initiated within 1-2 min of each other. This result indicates that between sisters variability in the duration of the G1 phase is small compared with variability in the total cell cycle time, and argues against the hypothesis that the rate of cell cycle traverse is determined by a random transition in Gx. INTRODUCTION In eukaryotic cells DNA is replicated during a discrete period of the cell cycle known as the iS-phase. It is not yet clear what factors are most important in determining the timing and duration of 5-phase. In the fission yeast Schizosaccharomyces pombe we have proposed that the initiation of 5-phase is subject to 2 controls: (1) the necessity for the cell to attain a particular minimal cell mass; and (2) the dependency of DNA replication upon completion of the previous mitosis (Nurse, 1975; Nurse, Thuriaux & Nasmyth, 1976; Nurse & Thuriaux, 1977); which of these 2 controls was operative in a particular cell was determined by the mass of that cell at its previous cell division. If cells divided at a small mass, then the requirement to attain a particular minimal cell mass determined the cell cycle timing of S-phase. If cells divided at a large mass, • Request for reprints to Paul Nurse. f Present address: Department of Genetics, University of Washington, Seattle, Washington 98195, U.S.A. % Present address: National Vegetable Research Station, Wellesbourne, Warwick, U.K. 2i 6 K. Nasmyth, P. Nurse and R. S. S. Fraser then it was the dependency of DNA replication upon the completion of mitosis coupled with a short minimum time in G1 that determined the timing of 5-phase. This hypothesis can be tested by investigating the timing of 5-phase in mutant strains of S. pombe which undergo cell division at varying cell masses. Wild type and 4 mutant strains are available which vary in cell mass at division over a 3-fold range, with little change in generation time (Nurse, 1975; Thuriaux, Nurse & Carter, 1978; Nurse & Thuriaux, unpublished). In this paper we describe the use of these 5 strains to investigate the role cell mass plays in the cell cycle timing of 5-phase. We also examine the effect cell mass has on the duration of 5-phase, since some evidence suggests that the duration of S-phase is affected by cell mass (Callan, 1973; Blumen- thal, Kriegstein & Hogness, 1973; Taylor, 1977), Convenient methods for the isotopic estimation of DNA synthesis in bulk cultures and for autoradiography have not been previously available for 5. pombe. It is not possible to label DNA specifically with radioactive thymidine as fungi lack thymidine kinase activity (Grivell & Jackson, 1968). Methods are available in Saccharomyces cerivisiae for isotopic detection of DNA by labelling total nucleic acid with a less specific precursor such as uracil or adenine, and then eliminating the radioactivity due to the RNA (Williamson, 1965; Hartwell, 1967; Hatzfield, 1973). In this paper we describe modifications of these methods that will work in S. pombe. The method for estimating DNA synthesis in bulk cultures has been used in synchronous cultures to determine the timing of 5-phase, whilst the autoradiographic method has been used to determine the duration of 5-phase. With some of the strains the autoradiographic method could also be used to determine the timing of S-phase and the variability in the length of the G1 phase in sister cells. Our results support the hypothesis that the initiation of »S-phase is subject to the 2 controls described above, and that there is a cross-over from one control to the other at a particular cell mass at division. We also found that the duration of 5-phase is short, being only about o-i of a cell cycle, and showed no consistent relationship with varying cell mass at 5-phase. Variability in the length of G1 of sister cells was found to be very small compared with the sister-sister variation in total cell cycle time, suggesting that Gx duration and 5-phase initiation are under precise control. MATERIALS AND METHODS Strains, media and growth conditions Wild type 972 and4 mutant strains wee 1.1, wee 1.50, toeez.i and cdc 2.M35 were used (Nurse, 1975; Thuriaux et al. 1978; Nurse & Thuriaux unpublished). Cells were routinely grown at 25 °C with shaking in a minimal medium described by Nurse et al. (1976). Preparation of synchronous cultures Synchronous cultures were prepared by selecting slowly sedimenting small cells after centrifugation in lactose gradients. The cells of the parent asynchronous culture were collected by nitration, suspended in a small volume of medium and centrifuged in 7 -5-30% lactose gradients (made up in medium). Small synchronous cultures, as used in DNA labelling experi- ments, were prepared using tubes as described by Mitchison & Carter (1975). Large synchronous Cell cycle timing and duration of S-phase 217 cultures, as used for measuring bulk DNA, were prepared using a zonal rotor (MSE type A), as described by Mitchison & Carter (1975). In both cases, a fraction of small cells was collected after centrifugation and innoculated into fresh medium. Measurement of cell number, DNA and protein content of cells These were performed as described by Nurse et al. (1976). DNA labelling and its measurement DNA was labelled by the addition of [6-3H]uracil (20 Ci/mmol, Amersham) to cell cultures, followed by incubation for varying periods of time. Pulse labels were terminated by transferring samples to a CC^/ethanol bath followed by storage at — 20 °C. Incorporation of radioactivity into DNA was measured by making up samples to 05 M NaOH, 10 mM ethylenediaminetetra- acetic acid, 100/ig/ml calf thymus DNA and incubating at 40 °C for 90 min. Samples were then cooled to o °C and acidified by addition of 60 % perchloric acid (PCA). Precipitation was allowed to proceed for at least 1 h at o °C, after which the samples were centrifuged at 5000g for 25 min, and the pellet washed once in 05 M PCA containing 100/tg/ml uracil and twice in 70% ethanol, ci M NaCl (both at o °C). The pellets were then dried under vacuum and hydrolysed in 0-5 ml 05 M PCA for 30 min at 70 CC. The hydrolysate was centrifuged for 1 min at 5000 g and 0-4 ml of the supernatant was added to 10 ml of scintillant containing 60% toluene, 40 % methoxyethanol, 05 % butyl PBD. The samples were counted in a Packard Tricarb scintillation spectrometer. Chromatography of HC1 digests of the PCA hydrolysate (Fig. i, p. 218) showed that no radioactivity remained as uracil and that at least 90 % of the radioactivity was in the form of cytosine and thymine. At least 90 % of the radioactivity of the PCA hydrolysate was shown to be DNase digestible when the final pellet was incubated in 50 mM 2(iV-morpholino)ethane sul- phonic acid, 25 mM MgClj buffer pH 70 containing ioo/tg/ml DNase. Under conditions of long-term labelling about 1 % of the total incorporated radioactivity was in the form of DNA. The visualization of S-phase by DNA autoradiography Cells were grown in minimal medium and labelled for 10-40 min in 50 /iCi/ml [8-3H]guano- sine (10 Ci/mmol, Amersham). Pulse labels were terminated by chilling the cells to o °C and adding an equal volume of cold (o °C) 0-5 M PCA. The cells could be left at this stage for several hours if required. Normally, cells were left at 0° for 5 min, collected by centrifugation, resuspended in 05 M PCA, and left at o °C for 15-30 min. The cells were then collected by centrifugation, washed twice in distilled water, suspended in 0-3 M sodium chloride, 0-03 M sodium citrate buffer pH 70 containing 200/fg/ml boiled RNase A (Sigma), and incubated at 37 °C for 70 min (with intermittent mixing).
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