J. Cell Sci. 33, 399-411 (1978)Printed in Great Britain © Company of Biologists Limited 1978

OXYGEN UPTAKE DURING THE CELL

CYCLE OF THE FISSION YEAST

SCHIZOSACCHAROMYCES POMBE

JAMES CREANORDepartment of Zoology, West Mains Road, Edinburgh EHg 3 JT, Scotland

SUMMARYOxygen uptake was measured in synchronous cultures of the fission yeast Schisosaccharomyces

pombe. The rate of oxygen uptake was found to increase in a step-wise manner at the beginningof the cycle and again in the middle of the cycle. The increases in rate were such that overall,oxygen uptake doubled in rate once per cell cycle.

Addition of inhibitors of DNA synthesis or nuclear division to a synchronous culture didnot affect the uptake of oxygen. In an induced synchronous culture, in which DNA synthesis,cell division, and nuclear division, but not 'growth' were synchronized, oxygen uptake in-creased continuously in rate and did not show the step-wise rises which were shown in theselection-synchronized culture.

These results were compared with previous measurements of oxygen uptake in yeast and anexplanation is suggested for the many different patterns which have been reported.

INTRODUCTION

Oxygen uptake has been examined frequently in yeast using a wide variety oftechniques to produce synchronous cultures. Most investigations have been carriedout in Saccharomyces cerevisiae and there is a wide variation in the patterns whichhave been reported for the uptake of oxygen in synchronous cultures of this species.For instance, there are reports of step-wise increases in the rate of oxygen uptakeduring the cell cycle (Scopes & Williamson, 1964; Greksak & Hanicova, 1973).Another investigation has shown that the rate increased continuously in the samestrain of this yeast (Cottrell & Avers, 1970). There are also numerous reports ofoscillations in the rate of oxygen uptake during the cell cycle of 5. cerevisiae (Nosoh &Takamiya, 1962; Kiienzi & Fiechter, 1969; von Meyenburg, 1969; Wiemken, Matile& Moor, 1970; Dharmalingam & Jayaraman, 1973). These will be discussed morefully later.

The situation in the fission yeast Schizosaccharomyces pombe is equally unclear and3 different investigations into the oxygen uptake of synchronous cultures of this yeasthave produced 3 different results. A step-wise increase in the rate of oxygen uptakehas been demonstrated (Osumi & Sando, 1969; Osumi, Masuzawa & Sando, 1968).Using similar techniques, Marchant (1971) showed 2 steps in the rate of oxygen uptakeper cycle. Poole & Lloyd (1973) have shown that in glucose-grown cells, oxygenuptake increased exponentially overall but rose to maxima twice per cycle; in glycerol-

400 jf. Creanor

grown, cells, these same workers showed that oxygen uptake increased in rate twiceper cycle, remaining constant in the intervening periods (Poole & Lloyd, 1974).

This paper describes an investigation into the oxygen uptake of the fission yeastSchizosacchqromyces pombe using the various techniques for the preparation ofsynchronous cultures and asynchronous control cultures which have been developedin this laboratory. It was shown in the preceding paper (Creanor, 1978) that theobserved pattern of C02 evolution in synchronous cultures of 5. pombe was notaffected by distorting the cell cycle using inhibitors of DNA synthesis and nucleardivision. It will be shown that the same situation applies to the uptake of oxygen inthe cell cycle of S. pombe.

METHODS

Growth and synchronization procedure

Schisosaccharomyces pombe, strain NCYC 132 (ATCC 24751) was grown in a minimalmedium EMM 2 (Mitchison, 1970). Synchronous cultures were prepared by the selectionmethod of Mitchison & Vincent (1965). A 4-I. culture, in exponential phase at 1-4 x io6 cells/ml, was harvested on a Whatman no. 50 filter paper, resuspended in 4 ml of medium andlayered on to 80 ml of either a 75-30 % lactose gradient or a 10-40 % sucrose gradient, madeup in EMM 2. The gradient was centrifuged for approximately 5 min at 500 g at room tem-perature. The top layer of cells was then removed and added to 100 ml warm medium to giveabout 5 x io6 cells/ml. The effect of the synchronization procedure on subsequent cell growthwas tested by proceeding through' the technique until after the gradient had been centrifuged.The gradient was then thoroughly mixed and a sample, which contained cells at all stages ofthe cell cycle, was inoculated into fresh medium at the same cell density as was used in thesynchronous cultures. A culture of this nature was judged to be asynchronous as measured bythe cell plate index.

The degree of synchrony was measured by the cell plate index which is the percentage ofcells in a population showing cell plates. This is roughly equivalent to the measurement ofmitotic index in other systems.

Measurement of oxygen uptake

Oxygen uptake was measured by a polarographic method which utilized a Clark oxygenelectrode. A Rank Brothers Oxygen Electrode (Rank Brothers, Bottisham, Cambridge) wasused to measure the oxygen uptake of cultures of S. pombe. A 3-ml sample containing at least5 x io6 cells/ml was placed in the reaction vessel. Air was bubbled through this sample for1 min and 2 min later, the top was placed down on to the reaction chamber leaving no air spaceand taking care that no air bubbles were trapped in the reaction vessel. The culture was stirredthroughout using a small magnetic follower. A Rikadenki 2 Pen Recorder, set at 1 mV fullscale deflexion, was used to measure the current flowing.

Two minutes after sealing the reaction chamber, the recorder was switched on and the fallin current measured, thus obtaining a measure of the oxygen consumption of the culture. Ingeneral, 6-10 min were required to establish the rate of oxygen uptake in a mid-exponentialculture. The sample was then discarded and the procedure was repeated using a further 3-mlsample from the experimental culture.

RESULTS

Oxygen uptake in asynchronous and synchronous cultures

Initially, oxygen uptake was measured in S. pombe using a manometric method.However, because of the low oxygen uptake rate of this yeast (De Deken, 1966)oxygen uptake could only be measured in very dense cultures and there were problems

0 2 uptake during cell cycle of S. pombe 401

about the changing environment during measurement of the rate. Because of the im-precise nature of these experiments, it was decided to concentrate on measurementsof the rate of oxygen uptake using the oxygen electrode.

Synchronous cultures were prepared using lactose gradients. The uptake of oxygenwas measured on 3-ml samples from the culture about once every 10 min. Rates ofoxygen uptake in 3 synchronous cultures are shown in Fig. 1. There were 2 periodsduring the cell cycle when the rate of oxygen uptake increased sharply; during the

40

30

2 0

1 1 0E

0-6 I-

° 04o

0-2 -

0-13

Time, h

Fig. 1. Rates of oxygen uptake in 3 synchronous cultures of S. pombe. Synchronouscultures were prepared using lactose gradients. Oxygen uptake was measured on 3-mlsamples removed from the culture about every 10 min. • , rate of oxygen uptake x 2;• , rate of oxygen uptake; A, rate of oxygen uptake X05 . Arrows indicate the cellplate peaks of that particular culture.

rest of the cycle the rate remained constant. The extent of the changes in the rate weresuch that overall, the rate of oxygen uptake doubled once each cell cycle.

Asynchronous control cultures were prepared by the method described above andthe rate of oxygen uptake was measured on 3-ml samples removed from the cultureat intervals. The results from 3 such experiments are shown in Fig. 2. While therewas some degree of fluctuation in the rate of oxygen uptake during the growth ofthese cultures, they all showed a fairly smooth increase with a doubling time equal

26-2

402 J. Creanor

to the doubling time of the culture (about 160 min). The sharp changes in the rate ofoxygen uptake which were shown in the synchronous cultures can therefore be con-sidered to be real cell cycle events.

The collected results from measuring the rate of oxygen uptake in 7 synchronouscultures are shown in the cell cycle map in Fig. 3. The results suggest that there is a

40

2 0

c"E

1 1 - iE

•a. 0-i

| 0 -

0-4

0-2

0-12 3

Time, h

Fig. 2. Rates of oxygen uptake in asynchronous control cultures of S. pombe. The con-trol cultures were prepared by shaking up lactose gradients and inoculating samplesof cells into fresh medium. Measurements of the rate of oxygen uptake were madeon 3-ml samples once every 10-15 min. • , rate of oxygen uptake X2; # , rate ofoxygen uptake; A, rate of oxygen uptake xo-5.

step in the rate of oxygen uptake at the beginning of the cycle (at 0-05) and againmid-way through the cycle (at 0-57). In some experiments, it was not possible to seea rate change at the start of the third cell cycle since the synchrony of the culture wasdeteriorating by this time.

To show more clearly the inter-relationship of the rate changes in oxygen uptakebetween the different synchronous cultures, the first rate change in oxygen uptake ineach experiment is taken as time o. The rest of the rate changes during the 2 cellcycles are then calculated in cell cycle time from the previous rate change. Thesedata are shown in Table 1. It is obvious from these data that there is a fairly consistent

0 2 uptake during cell cycle of S. pombe 403

First cycle

$T T TTT T T T T T T TT

0-5 10

Second cycle

irTT TTT

I I I I I 1

10 1-5 20Fig. 3. Cell-cycle map for rate changes in oxygen uptake measured in synchronouscultures of S. pombe. Each triangle represents a single determination in a synchronousculture of the time at which the midpoint of the increase in oxygen uptake occurred.The rate changes were grouped into first, second, third and fourth rate changes in eachsynchronous culture. The large arrows denote the means for each group of rate changeswith the cross-bars representing 2 standard errors. First group: mean 0-58, S.E. 0-033.Second group: mean 1-08, S.E. 0030. Third group: mean 1-56, S.E. 0044. Fourthgroup: mean 2-03, S.E. 0-060.

Table 1. Time between successive rate changes in oxygen uptakeexpressed as fractions of the cell cycle

N o .

i

2

34567

1st rate change 2nd rate change(mid 1st cycle) (start 2nd cycle)

0 0570 0560 0-570 0520 0500 0-500 0-48

Mean 0.53S.E. 0-014

3rd rate change(mid 2nd cycle)

0 4 90-65°-430 4 1

0 4 70 3 6

O'430.460035

4th rate change(start 3rd cycle)

—

OS7—

0-400-460-67O'3S0.490-058

The first rate change in each experiment, during the middle of the first cell cycle, is takenas time o. The rest of the rate changes are calculated in cell cycle time from the previous ratechange.

26-3

404 y. Creanor

time interval between the individual rate changes and that this is very close to halfof a cell cycle. There is also no significant difference between the intervals from thefirst to the second rate changes and the differences in time between the later ratechanges.

0-6 r

0-4

0 2

0-1

<

w

7 30

20

10 g?

Time, h

Fig. 4. Oxygen uptake in a synchronous culture of S. pombe containing deoxyadeno-sine. A synchronous culture was prepared using a lactose gradient. At 40 min,deoxyadenosine was added to the culture at 2 mM. Oxygen, uptake ( • ) was measuredon i-ml samples every 10 min. Cell plate percentages ( • ) were measured at intervals.

Oxygen uptake in a synchronous culture containing deoxyadenosine

A synchronous culture was prepared using a lactose gradient as previously de-scribed. Forty minutes after preparation of the culture, deoxyadenosine was added tothe culture to give a concentration of 2 mM and oxygen uptake was measured aboutevery 10 min. The results, shown in Fig. 4, show that the rate of oxygen uptake in-creased in the same periodic manner as was seen in the normal synchronous cultures,with a step in the rate in the middle of the first cycle, a further step at the time of thecell plate peak, followed by further steps in what would normally be the second cell

02 uptake during cell cycle of S. pombe 405

cycle. The effect of adding deoxyadenosine to a synchronous culture is to inhibitDNA synthesis at the time of the first cell plate peak (Mitchison & Creanor, 1971)and therefore to delay nuclear division and cell division during the second cell cycle.So the conclusion from this experiment is that the oxygen uptake pattern which wasseen in a normal synchronous culture can occur in the absence of DNA synthesis.

Oxygen uptake in induced synchronous cultures

There are 2 other ways of preparing synchronous cultures of S. pombe apart fromthe gradient method. One involves the addition of deoxyadenosine, the other theaddition of hydroxyurea, both of which inhibit DNA synthesis in S. pombe for a time(Mitchison & Creanor, 1971). When added for a 3-h period to an exponentiallygrowing culture and removed at the end of this time by filtration, these inhibitorscause the cells to undergo a synchronous division 2 h later, followed by a second semi-synchronous division after 1-5 h. Although cell division and DNA synthesis aresynchronized in a culture of this nature, cells are not of uniform size as in a culturesynchronized by the selection method. Since all of the cells have been growing for atleast 1 generation time without dividing, the smallest cells in an induced synchronousculture at the cell plate peak are as large as the biggest cells in an exponentially growingculture.

Oxygen uptake was first measured in a culture induced to divide synchronouslyusing deoxyadenosine. The results, shown in Fig. 5 A, show that there was a con-tinuous increase in the rate of oxygen uptake during the pulse and afterwards whenthe culture was dividing synchronously. So in this culture, although DNA synthesis,nuclear division and cell division are occurring in a synchronous manner, the rate ofoxygen uptake is increasing continuously.

A similar result, shown in Fig. 5B, was obtained when a culture was synchronizedusing 11 rain hydroxyurea. Here again, although the cells were dividing and under-going DNA synthesis synchronously, oxygen uptake increased continuously and didnot show the periodic pattern which was seen in a normal synchronous culture. Thisexperiment also shows that the rate of oxygen uptake is declining after a 3-h pulse ofhydroxyurea. It has been noted before that growth is inhibited during and after apulse of 11 mM hydroxyurea (Mitchison & Creanor, 1971) and this is one of the reasonsfor choosing deoxyadenosine as a DNA-synthesis inhibitor in S. pombe. Nevertheless,this result with hydroxyurea confirms that obtained using deoxyadenosine.

Oxygen uptake in the presence of mitomycin C

One further experiment was carried out to confirm the independence of the oxygenuptake pattern from DNA synthesis, nuclear division and cell division. Mitomycin Cinhibits nuclear division in S. pombe (Robinson, 1972) and S. cerevisiae (Williamson& Scopes, 1962). While hydroxyurea and deoxyadenosine arrest cells in Gx, afternuclear division has occurred but before DNA synthesis, mitomycin C arrests cellsin G2.

Oxygen uptake was examined in an asynchronous culture which contained mito-mycin C. This experiment is similar to those described in Fig. 5 below involving

406 J. Creanor

0-4

cE

I 02

0)

ID

§• 01

O 008

006

30

20 |• Q .

"510 °

0 7 r

Fig. 5. Oxygen uptake in S. pombe synchronized by pulses of either deoxyadenosine(A) or hydroxyurea (B).

A. At time o, deoxyadenosine was added to a culture containing 3 x io6 cells/ml togive 2 min. Oxygen uptake ( • ) was measured at intervals on 3-ml samples of this cul-ture. At 3 h the culture was filtered and resuspended in fresh medium. Oxygen uptakemeasurements were continued and cell plate percentages ( • ) were measured in theperiod after the pulse.

B. Hydroxyurea was added to an exponential culture of S. pombe containing 2 x io6

cells/ml to give 11 mM. Three hours later (time o on graph) the culture was filteredand resuspended in fresh medium. Oxygen uptake (#) was measured on 3-mlsamples every 15 min. Cell plate percentages ( • ) were measured in the period after thepulse.

0 2 uptake during cell cycle of S. pombe 407

pulses of deoxyadenosine and hydroxyurea. In this case, however, cells which hadnot undergone nuclear division when the inhibitor was added were unable to do so,with the result that a short time after the addition of mitomycin C there was no furtherdivision in the culture. Rather than remove the inhibitor after 3 h as above, oxygenuptake was measured in the continuous presence of mitomycin C for a total of 6 h.It can be seen in Fig. 6 that the rate of oxygen uptake continued to increase throughoutthis treatment. This again suggests that an increase in the rate of oxygen uptake isindependent of nuclear division, DNA synthesis and cell division. It should be noted

08r-

- 0 6cE_ 0-4

2 02

D.D

0-1

20 r

i 10CD

O W _^-»r«-»0 1 2 3 4 5 6 7

Time, h

Fig. 6. Oxygen uptake in a culture of S. pombe containing mitomycin C. Oxygenuptake was measured on an exponential culture of 5 . pombe at 30-min intervals.After 120 min, mitomycin C was added (arrow) to the culture to give o-i mg/ml.Oxygen uptake ( • ) was measured for a further 4 h along with percentage cell plates( • ) •

that in this experiment, the increase in rate of oxygen uptake began to decline afterabout 2 h in the presence of the mitomycin C and that the inhibition of cell divisionwas not complete. It was shown in the previous paper that the evolution of CO2 wasalso inhibited eventually in the presence of mitomycin C and it is likely that both ofthese effects are caused by the presence of a number of damaged cells.

DISCUSSION

Experiments measuring oxygen uptake during the normal cell cycle of synchronouscultures of S. pombe have shown that there are 2 stages during the cell cycle when therate of oxygen uptake increased. There was one change in the rate of oxygen uptake inthe middle of the cycle followed by a second rate change at the end of the cycle ornear the start of the next. During the intervening periods, the rate of oxygen uptake

408 J. Creanor

remained constant. Each change in the rate was an increase of about 50%, with theresult that overall, the rate doubled each cycle. Controls,.in the form of asynchronouscultures which had gone through the synchronization procedure, showed that thesechanges in the rate of oxygen uptake were real cell cycle events.

Most investigations into the oxygen uptake of yeast have been carried out inS. cerevisiae and a variety of techniques have been used in the preparation of syn-chronous cultures. There is also a wide variation in the patterns which have beenreported for the oxygen uptake in cultures of this species. The rate of oxygen uptakewas shown to increase in a series of sharp steps with the rate constant during theintervening periods in synchronous cultures of S. cerevisiae growing in a semi-definedmedium and synchronized by a feeding-starvation method (Scopes & Williamson,1964). A similar result was reported for the oxygen uptake of the same yeast syn-chronized on ficol gradients and growing in a complex medium (Greksak & Hanicova,1973). However, using the same yeast and the same methods, Cottrell & Avers (1970)reported a continuous increase in the uptake of oxygen in synchronous cultures.Another investigation, using a starvation method of synchronization and growing thecells in a semi-defined medium, produced a peak pattern, with a maximum in therate of oxygen uptake once per cycle (Dharmalingam & Jayaraman, 1973). Thispattern was shown with glucose as the carbon source but not with maltose. Kiienzi &Fiechter (1969) using a starvation method of synchronization, showed that oxygenuptake oscillated during the cell cycle with a maximum in rate occurring once percycle. Nosoh & Takamiya (1962), using an elaborate temperature-shock procedure toprepare synchronous cultures of S. cerevisiae, showed that the rate of oxygen uptakeoscillated during the cell cycle, with a maximum at the end of the GL period, vonMeyenburg (1969) has also shown oscillations in the rate of oxygen uptake in syn-chronous cultures prepared by limiting glucose and growing the cells in a semi-defined medium. Wiemken et al. (1970) grew cells in 3% glucose and synchronizedby a density gradient method. They found a maximum in the rate of oxygen uptakeat the time of bud initiation. Using a complex medium and synchronizing by equili-brium gradient centrifugation, these workers also found a maximum in the rate ofoxygen uptake at bud initiation (Wiemken et al. 1970).

Varying patterns also emerged from the various measurements of oxygen uptakein synchronous cultures of S. pombe. A stepwise increase in rate has been shown incultures synchronized by a starvation procedure (Oaumi & Sando, 1969; Osumi et al.1968). Using the same techniques, Marchant (1971) has shown that there were 2 stepsper cycle in the rate of oxygen uptake. Poole & Lloyd (1973) have shown that overallthe rate of oxygen uptake increased exponentially in glucose-grown synchronouscultures but rose to maxima at 2 points in the cycle; in glycerol-grown cells, on theother hand, the rate was shown to increase twice per cycle, remaining constant in theintervening periods (Poole & Lloyd, 1974).

The overall result is that step patterns, exponential patterns and oscillatory patternshave been reported. There is at present no way to account for these differences. Ithas been suggested that different synchronous cultures may have mitochondrialpopulations having different degrees of synchrony (Cottrell & Avers, 1970). The his-

0 2 uptake during cell cycle of S. pombe 409

tory of the cells prior to synchronization may be important (Kimball, Casperson,Svensson &• Carlson, 1959) and.it is very obvious that the history of many of thecultures discussed above was very different before synchronization. The methodsused to prepare each of the synchronous cultures discussed above may well affect thesubsequent growth of the culture. This effect might be investigated by examination ofcontrol cultures. When a selection method of synchronization is used, it is quitepossible to measure the effect of the procedure on an asynchronous population, e.g.by mixing up a gradient. But when an induction method of synchronization is used,obviously a control culture cannot be made since the effect of the treatment is to makeall of the cells in a population divide synchronously. Control cultures were not madein any of the selection methods described above. With the discovery of many syn-chronization-induced artifacts (for a review see Mitchison, 1976), testing the syn-chronization procedure is obviously of great importance.

Two investigations into the uptake of oxygen during the cell cycle of S. pombe haveshown changes in rate, either steps or peaks, twice per cycle occurring at the sametime as the steps in oxygen uptake which I have reported here (Marchant, 1971;Poole & Lloyd, 1973). The difference between these and other results for S. pombecould well be accounted for by differences in the medium and in the synchronizationtechnique. It was shown that the pattern of CO2 evolution in synchronous culturesof S. pombe differed when the cultures were grown in complete medium or minimalmedium. This situation could exist in the case of oxygen uptake.

Because respiration is much more efficient than fermentation in the production ofenergy, the low level of oxygen uptake in S. pombe is producing half of the energyof the cell (Hamburger, Kramhoft, Nissen & Zeuthen, 1977). So it is likely thatoxygen uptake would be strongly influenced by the condition of a culture both duringthe growth of the synchronous culture and, equally, during the growth of the culturebefore synchronization. Until it is possible to find out what brings about changes inoxygen uptake during the cell cycle and how this unknown factor is influenced indifferent growth conditions, then there is no satisfactory explanation for the widelyvarying results obtained by measuring oxygen uptake in synchronous cultures.

A pattern has been established for the rate of oxygen uptake in synchronous culturesof S. pombe. This pattern has been shown to persist in synchronous cultures in whichDNA synthesis, nuclear division and cell division have all been inhibited but not incultures synchronized only with respect to DNA synthesis, nuclear division and celldivision. Poole (1977) has shown a more-or-less similar result with oxygen uptakeincreasing continuously after synchronization of 5. pombe using deoxyadenosine butwith some minor fluctuations in the rate. This is similar to the situation shown for theevolution of CO2 in synchronous cultures of 5. pombe (Creanor, 1978) and is the secondcase where a rate-change pattern has been shown to persist after a DNA/cell divisionblock. The rate changes in oxygen uptake during the cell cycle of S. pombe are there-fore further support for the concept of a Growth Cycle, the existence of which hasbeen postulated in this yeast (Mitchison, 1971).

The rate change in oxygen uptake at the end of the cell cycle is associated with DNAsynthesis and cell division, but although there is a temporal connexion, the results

410 J. Creanor

with the inhibitors suggest that there is no causal connexion. The rate change inoxygen uptake in the middle of the cycle is not associated with any particular cellcycle event and there seems no apparent reason for it to occur at this point. Thesituation with C02 evolution is different in that there was only one rate change percycle, associated temporally, but not causally, with nuclear division. So while thereare many measurements of oxygen uptake and C02 evolution during the cell cycle ofyeast, the most novel feature in this work is the persistence of both in an unchangedmanner when the most prominent features of the cell cycle had been inhibited (i.e.nuclear division, cell division and DNA synthesis).

I should like to express my gratitude to Professor J. M. Mitchison and Dr P. A. G. Wilsonfor advice and criticism during the course of this work and preparation of this manuscript.

REFERENCES

COTTRELL, S. F. & AVERS, C. J. (1970). Evidence of mitochondrial synchrony in synchronouscultures of yeast. Biochem. biophys. Res. Cominun. 38, 750-757.

CREANOR, J. (1978). Carbon dioxide evolution during the cell cycle of the fission yeast Schizo-saccharomyces pombe. J. Cell Sci. 33, 385-397.

DE DEKEN, R. H. (1966). The Crabtree effect and its relation to the petite mutation. J. gen.Microbiol. 44, 149-167.

DHARMALINGAM, K. & JAYARAMAN, J. (1973). Mitochondriogenesis in synchronous cultures ofyeast. 1. Oscillatory pattern of respiration. Archs Biochem. Biophys. 157, 197-202.

GREKSAK, M. & HANICOVA, M. (1973). Activities of succinate dehydrogenase and cytochromeoxidase in synchronous aerobic cultures of aerobically and anaerobically grown yeast.Biologia (Bratislava) 28, 425-433.

HAMBURGER, K., KRAMHOFT, B., NISSEN, S. B. & ZEUTHEN, E. (1977). Linear increase inglycolytic activity through the cell cycle of Schizosaccharomyces pombe. J. Cell Sci. 24, 69—79.

KIMBALL, R. F., CASPERSSON, T. O., SVENSSON, G. & CARLSON, L. (1959). Quantitative studieson Paramecium aurelia. 1. Growth in total dry weight measured by the scanning interferencemicroscope and X-ray absorption methods. Expl Cell Res. 17, 160-172.

KUENZI, M. T. & FIECHTER, A. (1969). Changes in carbohydrate composition during the bud-ding cycle of Saccharomyces cerevisiae. Arch. Mikrobioh 64, 396-407.

MARCHANT, R. (1971). The initiation of cell wall synthesis in parasynchronous cultures ofSchizosaccharomyces pomle. Arch. Mikrobiol. 78, 205-213.

MEYENBURG, H. K. VON (1969). Energetics of the budding cycle of Saccharomyces cerevisiaeduring glucose-limited growth. Arch. Mikrobiol. 66, 289-303.

MITCHISON, J. M. (1970). Physiological and cytological methods for Schizosaccharomycespombe. In Methods in Cell Physiology, vol. 4 (ed. D. M. Prescott), pp. 131-165. New Yorkand London: Academic Press.

MITCHISON, J. M. (1971). The Biology of the Cell Cycle. London: Cambridge University Press.MITCHISON, J. M. (1976). Enzyme synthesis during the cell cycle. In Cell Differentiation in

Micro-organisms, Plants and Animals (ed. L. Nover & K. Mothes), pp. 377-401. Jena:Fischer.

MITCHISON, J. M. & CREANOR, J. (1971). Induction synchrony in the fission yeast Schizo-saccharomyces pombe. Expl Cell Res. 67, 368-374.

MITCHISON, J. M. & VINCENT, W. S. (1965). Preparation of synchronous cultures by sedimen-tation. Nature, Lond. 205, 987-989.

NOSOH, Y. & TAKAMIYA, A. (1962). Synchronisation of the budding cycle in yeast cells, andthe effect of carbon monoxide and nitrogen deficiency on the synchrony. PI. Cell Physiol.(Tokyo) 3) 53-66.

OSUMI, M., MASUZAWA, E. & SANDO, N. (1968). Mitochondrial formation in synchronouscultures oi Schizosaccharomyces pombe. Jap. Women's Univ. J. 15, 33-41.

OSUMI, M. & SANDO, N. (1969). Division of yeast mitochondria in synchronous cultures.J. Electron Microsc, Chiba Cy 18, 47-56.

0 2 uptake during cell cycle of S. pombe 411

POOLE, R. K. (1977). Development of respiratory activity during the cell cycle of Schizo-saccharomyces pombe 972 h~: respiratory oscillations and heat dissipation in cultures syn-chronised with 2'-deoxyadenosine. J. gen. Microbiol. 103, 19-27.

POOLE, R. K. & LLOYD, D. (1973). Respiratory oscillations and heat evolution in synchronouslydividing cultures of the fission yeast Schizosaccharomyces pombe 972 h~. J. gen. Microbiol.77, 209-220.

POOLE, R. K. & LLOYD, D. (1974). Changes in respiratory activities during the cell cycle ofthe fission yeast Schizosaccharomyces pombe 972 h~ growing in the presence of glycerol.Biochem. J. 144, 141-148.

ROBINSON, A. A. (1972). The Relationship of Enzymes to the Cell Cycle with Particular Referenceto Schizosaccharomyces pombe. Ph.D. Thesis, University of Edinburgh.

SCOPES, A. W. & WILLIAMSON, D. H. (1964). The growth and oxygen uptake of synchronouslydividing cultures of Saccharomyces cerevisiae. Expl Cell Res. 35, 361-371.

WIEMKEN, A., MATILE, P. & MOOR, H. (1970). Vacuolar dynamics in synchronously buddingyeast. Arch. Mikrobioi. 70, 89-103.

WILLIAMSON, D. H. & SCOPES, A. W. (1962). Cell division and the synthesis of macromoleculesin synchronously dividing cultures of Saccharomyces cerevisiae. Proc. int. Union physiol. Sci.». 758.

(Received 27 January 1978)