J. Cell Sci. 33, 1-23 (i977)

Printed in Great Britain

EFFECTS OF HEAT SHOCK AND

CYCLOHEXIMIDE ON GROWTH AND DIVISION

OF THE FISSION YEAST,

SCHIZOSACCHAROMYCES POMBE

WITH AN APPENDIXESTIMATION OF DIVISION DELAY FOR S. POMBEFROM CELL PLATE INDEX CURVES

MARK M.POLANSHEK*Department of Zoology, University of Edinburgh, Edinburgh,Scotland, EHg jjfT

SUMMARYEffects of superoptimal temperature shock (HS) and the protein synthesis inhibitor, cyclo-

heximide (CH), on growth and division of the fission yeast, Schizosaccharomyces pontbe, werestudied. Experiments on asynchronous cultures have shown that a 15-min HS of 41 °C inhibitsRNA and protein synthesis and growth in cell length while delaying mitosis, division, andDNA synthesis. A 10-min CH pulse (100 /ig/ml) inhibits protein and RNA synthesis brieflywhile delaying mitosis, division, and DNA synthesis. These single heat or CH pulses partiallysynchronize mitosis, division, and DNA synthesis.

Experiments in which either 15-min HS or 10-min CH pulses were applied at different timesin selection-synchronized cultures have demonstrated several kinds and periods of sensitivityto these agents. During roughly the first two-thirds of the cell cycle (measured betweendivisions) mitosis, division, and DNA synthesis are delayed equally, delay increasing as a pulseis applied progressively later in the cycle. The magnitude of the delay from a heat shock isalways greater than that from a CH pulse, but for both agents there is a period during whichdelay is greater in magnitude than pulse length. The pattern of delay from cultures synchronizedby an induction method suggests that the period of increasing delay lies obligately within G2.

At 0-65 in the cycle the nature of the sensitivity to heat and CH changes. Between this transi-tion point and the formation of a cell plate, CH has no effects on the timing of mitosis or cellplate stage. However, CH can block the final splitting of the cell plate, leading to a permanentcell plate and the formation of transient 4-celled pseudo-filaments upon resumed growth.HS foOowing the transition point allows mitosis to be completed with normal timing to a stagein which daughter nuclei occupy terminal positions in the cell, but formation of the cell plateis delayed by about 30 min. Cells pulsed in the last third of the cycle may develop severalmorphological aberrations. The cell plate is sometimes oblique or positioned at one end of thecell, giving rise to daughter cells with 2 or no nuclei. Thus, it appears that some functionsrelated to normal positioning of the cell plate occur during the last third of the cycle.

Recovery from a heat shock applied prior to 065 in the cycle includes a period in which thereis increasing delay when a CH pulse is applied progressively later following the heat shock.However, applied together, CH plus HS produce roughly the amount of delay due to the HSalone. These facts are considered evidence for a common effect of both agents during the firsttwo-thirds of the cycle.

The results are discussed with reference to possible controls over several events of the cellcycle.

• Present address: McArdle Laboratory for Cancer Research, University of Wisconsin,Madison, Wisconsin, 53706, U.S.A._

2 M. M. Polanshek

INTRODUCTION

It is now known that many preparations for cell division occur and are completedduring cell cycles prior to a particular division in both prokaryotes and eukaryotes(Mitchison, 1971; Slater & Schaecter, 1974). Among these preparations are generalrequirements for some quantity of growth, expressed in terms of RNA and proteinsynthesis, as well as a doubling of DNA during the 5-period in eukaryotes. Morespecifically, the products of at least 32 genes are required for cell cycle traverse inbudding yeast (reviewed in Hartwell, 1974), and we suppose that these few dozenproducts are components of one or more sequences of events which lead to division(Mitchison, 1971; Hartwell, Culotti, Pringle & Reid, 1974).

A component of one sequence leading to division has been termed the 'heat-sensitive' function (see Swann, 1957). This function was described in detail for theciliate, Tetrahymena, and relevant literature has been reviewed extensively (Zeuthen,1964, 1974; Zeuthen & Rasmussen, 1971). The early observation was that a non-lethal shock of superoptimal temperature would delay or arrest division if it wasapplied during roughly the first three-quarters of the cell cycle, with the inferencethat a temperature-sensitive process required for division occurred throughout thisinterval. When sensitivity ceased, the required preparation was believed either to becomplete or became heat-stable. The point at which insensitivity is achieved has beencalled a 'critical point', 'point of no return', and 'transition point' (Mitchison, 1971)

The pattern of division delay prior to the transition point has received muchattention because it is the basis for theories of the control of cell division in which alimiting synthesis or accumulation of substance needed for division spans a consider-able portion of the cycle, and which has some properties of a clock (Rusch, Sach-senmaier, Behrens & Gruter, 1966; Sachsenmaier, Remy & Plattner-Schobel, 1972;Bradbury, Inglis, Matthews & Langan, 1974; Zeuthen, 1961, 1974; Winfree, 1975).In one paradigm, Tetrahymena, the key observation is that for a standard pulsetreatment, division delay becomes greater as a shock is applied later in the cycle.Further, division delay is greater than the duration of a shock. One effect of the latteris that division synchrony can be induced in an initially asynchronous culture becausecells must recover from a shock beginning a fixed time in the cell cycle before division.This phenomenon has become known as 'excess delay' or 'setback'; it implies thatdelay is greater than a given shock period without specifying a mechanism by whichthis comes about. However, the standard interpretation of setback due to heat shockis that heat discharges some cumulative preparations for division until cells reach thetransition point. Since the accumulation begins at a fixed point in cycle time beforedivision, a shock forces a population of cells to renegotiate division preparations to-gether, which in turn constrains cells to proceed through division in synchrony(Zeuthen, 1964).

The heat-sensitive preparation for division in Tetrahymena has not been unequivo-cally identified at the molecular level, but is generally believed to involve 'divisionproteins' (Zeuthen, 1961). Evidence for this interpretation includes studies in whichdivision delay with a pattern similar to that for heat shocks has been effected with

Heat and cycloheximide effects on fission yeast 3

inhibitors of protein synthesis or amino acid analogues, and for these agents a singletransition point has been found (Frankel, 1962, 1967a, b; Rasmussen & Zeuthen,1962; Hamburger, 1962).

Our interest in the phenomena outlined above was to see how similar were effectsof heat shock and inhibition of protein synthesis on a fission yeast, Schizosaccharomycespombe. It has been shown that this yeast can be synchronized by heat shocks, and thatdivision delay increases during the cell cycle for heat-synchronized cultures (Kram-hoft & Zeuthen, 1971). Also, in one strain of yeast, cycloheximide has been found todelay division (Herring, 1973). A difference between this and previous work is thatwe studied the effects of shocks on cultures of yeast synchronized by a selectionmethod, and we have completed more detailed analyses of effects on nuclear division,cell plate formation, and various aspects of normal growth. Included is evidence thatboth cycloheximide and heat affect more than one phase of the cell cycle, and evidenceconsistent with the interpretation that both agents affect some processes in common.

MATERIALS AND METHODS

The fission yeast, Schizosaccharomyces pombe Lindner, strain 927b", was obtained from Pro-fessor U. Leupold, University of Bern, and was grown in Edinburgh Minimal Medium 2(EMM2, Mitchison, 1970). The doubling time for cell numbers in exponential growth(0-5-8 x io8 cells/ml) at the normal culture temperature of 32 °C ia 140 min.

Synchronous cultures were usually prepared by the selection method of Mitchison & Vincent(1965). Typically, cultures were harvested at 1-4 x i o ' cells/ml, yielding synchronous culturesof O'S-2'5 x 10' cells/ml at time zero. The selection procedure is described in detail in Mitchison(1970).

The induction method of Mitchison & Creanor (1971), using 3'-deoxyadenosine (Adr,Sigma) to block DNA synthesis, was used for preparing synchronous cultures in severalexperiments. The induction conditions for this strain of yeast are a period of 4 h in 3 mM Adrin EMM2.

Division synchrony was usually monitored by estimating the cell plate index, which behaveslike a mitotic index (Mitchison, 1970).

Cell numbers were determined with a Coulter Counter Model B following light sonicationto break up cell pairs and clumps of cells (see Mitchison, 1970, for details).

Nuclear division figures can be seen in cells stained with Giemsa following acid hydrolysisto remove RNA (Mitchison, 1970). The percentage of binucleate cells is based upon scoring400 cells/sample. Our convention is to score as 'binucleate' all cells which show any stagebetween the initially swollen, oval nucleus and that in which cells contain 2 nuclei but in whicha cell plate is not yet visible (stages 1-4 in Fig. 8).

Culture increase in dry mass was followed by measuring the absorbance at 595 nm (Am,6)(Mitchison, 1970).

DNA was estimated colorimetrically using the diphenylamine reaction following Schneiderextraction, with 2'-deoxyadenosine as standard (Bostock, 1970). RNA was estimated from theabsorbance at 260 nm of appropriate dilutions of the hot perchloric acid extract from theSchneider extraction (Munro & Fleck, 1966).

Relative rates of protein synthesis were estimated by incorporation of L-[4,5-'H]leucine(Amersham) into acid-precipitable material during 10-min pulses of label. Typically, 1 ml ofculture was added to 1 /iCi [°H]leucine, 30 fig carrier L-leucine dissolved in 50 fi\ EMM2.Incorporation was stopped after 10 min by addition of 1 ml of ice-cold 10% (w/v) trichloro-acetic acid (TCA). Samples were collected on Whatman GF/A filters, and assayed for radio-activity with a liquid scintillation counter.

Cell lengths were measured with a calibrated graticule and x 100 objective on cells dried onto a slide, negatively stained with 10 % (v/v) India ink, and positively stained with 0-25 % (w/v)crystal violet (Mitchison, 1970).

4 M. M. Polanshek

Heat shocks were applied by shifting a flask of cells from a water bath at 32 °C to anotherat 41 °C. The pulse durations shown on graphs refer to the times at which transfers betweenbaths were made. The shock temperature is taken from experiments by Harnden (1957) andKramhoft & Zeuthen (1971). For most experiments, cultures of 10—25 Iri^ were shocked in50-100 ml conical flasks to ensure a large surface area for heating and cooling, and the changesin temperature took approximately 1 min. In larger cultures required for DNA determinations(500 ml culture in a 3-I. flask), heating and cooling took as much as 5 min, and in some casesthe temperature rose to only 405 °C. However, the effect of shocks on cell number increase issimilar for both large and small culture volumes, so the speed of heating and the maximumtemperature reached are probably not critical within a few minutes or a degree.

Cycloheximide (CH, Sigma) solutions in EMM2 were always made up on the day of use.The effective concentration of 100 /ig/ml was chosen after experiments showed that this wasthe minimum concentration which produced maximum delay for io-min pulses of the drugapplied at about mid-cycle in cultures synchronized by selection (Polanshek, 1973). Pulses ofinhibitor were given by adding the drug solution to cultures in the volume ratio, 5 ml drug: 95 mlculture. CH was washed out of cultures by collecting cells at room temperature (20 °C) on0-45 grade Oxoid membrane filters with suction, rinsing with EMM2 at 32 °C, and resuspendingcells in fresh 32 °C EMM2. Controls, in which a pulse of medium was applied with identicalwashing, showed that none of the parameters measured in this study were affected appreciablyby the pulsing procedure alone.

RESULTS

Effects of heat shock and cycloheximide on protein synthesis

In this study, effects of heat shock and an inhibitor of eukaryotic protein synthesis(Siegel & Sisler, 1965; Cooper, Banthorpe & Wilkie, 1967; Rao & Grollman, 1967;Baliga & Munro, 1971) have been compared. An assumption is that inhibitionsresulting from thermal stress can be understood by comparison with the effects of aninhibitor whose mechanism of action is reasonably clear. Thus, we need to knowwhether CH does in fact inhibit protein synthesis in this yeast, and how such inhibitioncompares with that produced by elevated temperature.

Fig. 1 shows effects of 41 °C heating and 100/ig/mI CH on incorporation of pH]-leucine into TCA-precipitable material during io-min pulses of label. CH cuts in-corporation to 5-10% of the control rate of incorporation within 2 min, while heatingto 41 °C takes some 20 min to inhibit incorporation maximally. The maximum inhibi-tion of pHJleucine incorporation by HS is slightly less than that achieved with CH.The inhibition is reversible in either case when released by lowering the temperatureor washing out the drug. The lag which precedes recovery to the control rate ofincorporation increases with increasing pulse duration (Polanshek, 1973). For thislatter reason, and in order to fit many pulses into the cell cycle, short pulses of10-20 min for CH or 15 min for HS were used. Experiments in which pulses wereapplied at different times in selection-synchronized cultures showed that the kineticsand degree of inhibition of pH]leucine incorporation and the kinetics of recoveryfrom either treatment match the data in Fig. 1, and do not vary during the cell cycle.

Effects of heat and cycloheximide on asynchronous growth

By analogy with systems such as that of Tetrahymena, one expects that any dis-turbance which releases a setback reaction will cause partial synchronization of divisionin an initially asynchronous culture of cells (see Introduction). Figs. 2 and 3 show the

Heat and cycloheximide effects on fission yeast 5

effects of single heat or cycloheximide pulses respectively on asynchronous cultures ofS. pombe. Prior to either pulse, growth is characteristic of asynchronous cultures ofyeast. Following either pulse, cell number increase is depressed up to the partiallysynchronous division, which is reflected in the cell plate index, percentage binucleate

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Fig. 1. Effect of 100 fig/ml cycloheximide (A) or 41 CC heat shock (B) on L-fH]leucineincorporation into acid-precipitable material during 10-min pulses of label. Details ofthe pulsing procedure are in Materials and methods. Pulses began at the times indi-cated by the symbols. Controls, • ; continuous treatment with either CH (A) or heat(B) beginning at time = o, OI recovery from a 10-min CH pulse (A) or a 15-min heatshock (B), • .

cells, and increases in DNA and cell numbers. Growth then returns to normal. Forthe CH-treated culture, the burst in division comes about 70 min after the end of thepulse, while a similar burst follows the heat shock by roughly 90 min.

Increases in the A69S and RNA/ml are hardly affected by CH, but A^g increase ismarkedly inhibited following a heat shock. This observation indicates a decrease inthe accumulation of dry mass by heat-shocked cells. Consistent with this interpretation

6 M. M. Polanshek

is the fact that cells do not appear to grow in length for 30-45 min following a heatshock when viewed in time-lapse films (Polanshek, unpublished). Since the cell wallmaterial must be a large fraction of the total cell mass, it is probably inhibition of thisaspect of growth which accounts for the depression in the A ^ following heat shock.

Fig. 2. Effect of a single 15-min, 41 °C heat shock (bar, 1—1) on growth of an asyn-chronous culture of yeast. The curve for the percentage binucleate cells (dashedline) is based upon other experiments because mitotic cells were not scored in thiscase. DNA, A ; RNA, • ; cell number, A J Absorbance695 Tim, • ; cell plate index, O-

A slower increase in dry mass is also reflected in measurements of cell length at thetimes of the cell plate peaks in Figs. 2 and 3. Since fission yeast are roughly cylindrical,a measurement of length is proportional to cell volume, so the histograms presentedin Fig. 4 can be read for the trend in either property. Cells at division are roughlythe same length on average for heat-shocked or control cultures, but the distributionof lengths is somewhat broader in the treated culture, with some rather long dividingcells. Indeed, the size distribution of dividing cells in the shocked culture is ratherlike the distribution of sizes for random cells in the control population. This suggeststhat all cells of the population contributed proportionately to the decrease in drymass seen in Fig. 2.

There are some similarities in the effects of HS and CH on cell length at division.Fig. 4E-F shows the length distributions for control and CH-treated cells at the cellplate peak in Fig. 3. The length distributions are again somewhat broader in thetreated culture than in the control. In contrast with the HS effects, the mean celllengths are markedly greater in the CH-treated culture than in the control. This fact,

Heat and cycloheximide effects on fission yeast 7

together with the minor depression in the \ K following the CH pulse (Fig. 3),implies that culture growth measured so broadly has been little affected by the CHpulse, and cells have managed somewhat more growth in size and mass than is usualat the time of division.

Summing up, the data in Fig. 4 suggest that cell lengths are not standardized whencell division is partially synchronized as a result of HS or a CH pulse. Thus, alignmentof division bears no direct relation to cell volume (see Faed, 1959; Mitchison &Creanor, 1971).

Fig. 3. Effect of a single 10-min, 100 jUg/ml cycloheximide pulse on growth of an asyn-chronous culture of yeast. The position of the pulse is indicated by the bar (h-1). RNA,• ; DNA, A ; cell number, A ; AbsorbanceM6 „„, • ; cell plate index, O ; binucleatecells, • .

Division delay in synchronous cultures

Since cell division is partially synchronized as a result of a single pulse of eitherheat or cycloheximide, we expect that division delay due to a standard shock willincrease as the shock is applied progressively later in the cycle (Mitchison, 1971;Kxamhoft & Zeuthen, 1971). Fig. 5 shows the results of experiments in which eithera heat shock or CH pulse was applied at several times during the cell cycle in culturessynchronized by selection. Pulses given before about 0-40 in the cycle result in singlecell plate peaks occurring somewhat later than the controls. Later pulses split syn-chronous cultures into 2 groups of cells which can be described from their respectivecell plate peaks. The earlier peak is interpreted to include cells which have escaped thedelaying effects of a pulse because they are past a transition point for division delaywhich is late in G2. The later peak represents cells which were sensitive to a treatment

8 M. M. Polanshek

because they were positioned in the cycle prior to the transition point at the time of thepulse. The escape peak increases as the peak of the delayed population declines, therebeing first few and finally all cells in the escape peak. The transition point for divisiondelay occurs at about 0-65 in the cycle from the fact that a pulse applied at this timesplits a synchronous culture into 2 numerically equal groups of cells.

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Fig. 4. A—H. Length distributions and mean lengths for cells at the times of the cell platepeaks in Figs. 2 (A-D) and 3 (E-H). 100 cells were sized for each histogram from randomfields of cells stained with crystal violet on an India ink background (see Materialsand methods), A, E, controls, cells with cell plates only; B, F, controls, random cells;C, G, cells with cell plates only, after HS (c) or a CH pulse (G) ; D, H, random cells, afterHS (D) or a CH pulse (H). Figures given are mean length ± standard deviation.

From a series of pulsing experiments a curve of division delay as a function of theposition of a pulse in the cell cycle can be drawn. Delay is estimated as described inthe Appendix. Fig. 6A,B gives delay curves for heat shocks and CH pulses, respectively.For both agents there is a period of increasing delay over a large fraction of the cycle.In the case of CH pulses, delay is zero or a few minutes for escape peak cells, but cellspast the transition point for increasing delay are still delayed roughly 30 min by aheat shock. While delay is greater in magnitude and begins earlier for heat shock, thetransition point for the phase of increasing delay is approximately 0-65 in the cyclefor either agent. For at least part of the period of increasing delay, both heat and CHproduce excess delay (see Introduction).

When cell plate formation is delayed during the major period of increasing delay,division as measured by increase in cell numbers is also delayed, as is DNA synthesis

Heat and cycloheocimide effects on fission yeast 9

(Polanshek, 1973, and see Figs. 2 and 3). However, the earliest event which we canshow to be delayed is nuclear division. This process is detectable in Giemsa-stainedpreparations by about 0-75 in the cycle, as estimated from the peak of dividing nuclearfigures which occurs roughly 15 min before the peak in the cell plate index. Fig. 7shows control and shocked synchronous cultures in which mitosis was followed. Forthe CH-treated culture (D), nuclear division is delayed along with cell plate formation,

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Fig. 5. Effect of a 15-min, 41 °C heat shock or a 10-min, ioo/ig/ml CH pulse on the cellplate index in selection-synchronized cultures of yeast. Shocks, indicated by bars ( M ) ,were applied at 3 different times between the first and second divisions followingselection. The position of each shock is given as a fraction of a cell cycle betweendivisions, assuming that the times of half-doubling in cell numbers occur 15 min(o-io of the cycle) after the mean times of the cell plate peaks for control cultures.Mean times for cell plate peaks are indicated by arrows, and were determined asdescribed in the Appendix.

but remains in the same relative position prior to the cell plate peak as in the control (c).When there are delayed and escape peaks of cell plates, as shown, the population issplit with respect to the time of nuclear division as well, suggesting that after thetransition point for increasing delay, nuclear division proceeds with normal tuning,and cells enter into cell plate stage as in untreated cultures. This is confirmed forcultures left in CH, as cells continue to enter cell plate stage for roughly 45 min

IO M. M. Polanshek

when cycloheximide is present, and mitotic figures decrease to zero (Polanshek, un-published).

The picture for heat-shocked cultures is more complex. First, nuclear division isdelayed together with cell plate formation during the period of increasing delay. Aswith CH, there is a single peak in both the percentage binucleate cells and the cell plate

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Fig. 6 A, B. Division delay in yeast cultures synchronized by selection as a function ofthe position of a is-min, 41 °C heat shock (A) or 10-min, 100 fig/ml cycloheximidepulse (B) in the cell cycle. Shocks were applied between the time of selection and thesecond cell plate peak. 00 and 1 -o in the cycle are the times of half-doubling in cellnumbers, so the data are plotted for a cell cycle running between divisions, not betweencell plate peaks. Filled circles represent cells delayed in the major period of sensi-tivity to heat or CH (see text), while open circles represent 'escape peak' cellswhich are past a transition point for division delay at 0-65 in the cycle (arrows). Thecurves are drawn by eye. 'Excess delay' is total delay minus the length of a pulsetreatment or shock (see Introduction).

index early in the cycle, while later pulses split the population. Fig. 7 shows data forsuch a population split into escape and delayed fractions, and it is clear that there arealso 2 peaks of binucleate cells. We have noted that cell plate formation is delayedroughly 30 min following the transition point for increasing delay. However, nucleardivision occurs at the normal time compared with the control for those cells which

I IHeat and cycloheximide effects on fission yeast

form the escape peak of the cell plate index curve. This means that nuclear divisionand cell plate formation have been separated by about 30 min longer than in the normaltemporal sequence.

Transition points and periods of delay

We can reconstruct the events which occur following the first transition point insomewhat greater detail from slides stained for mitotic nuclei. Such a reconstruction isgiven in Fig. 8. Representative morphologies of cells at various times in the cell cyclewere traced from photographs, and represent the normal course of growth, mitosis,

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Fig. 7. Effect of heat shock or cycloheximide on mitosis. Cell plate index, O ; binucleatecells, 0 . A, control for heat-shocked culture (B); C, control for CH-pulsed culture(D). Positions of pulses are shown by bars (h-i). Arrows indicate mean times for theseveral peaks. For both controls (A, C), the mean time of the % binucleate peak is about15 min before that for the cell plate index. In (D), a CH pulse has split both the % bi-nucleate and cell plate index curves into 2 peaks. However, the % binucleate peakremains 10-15 min prior to the cell plate index peak for each of the 2 sub-populations.

In (B), the heat shock has split the culture into 2 major cell plate peaks and 2 %binucleate peaks. The second % binucleate peak represents cells which form cell platesat the second major cell plate peak (3), and these experience severe division delay;nevertheless, the % binucleate peak is about 15 min prior to the cell plate peak. Thefirst peak of binucleate cells represents cells which form cell plates at the first majorcell plate peak (2), but the time between these peaks is roughly 40 min, or 25-30 minmore than in controls. Thus, mitosis has been separated from cell plate stage by 25-30 min extra for these escape peak cells. The cell plate peak at (J) in (B) represents asmall fraction of the culture which was at or near the end of mitosis at the time of theheat shock, and which formed cell plates during and after the shock.

12 M. M. Polanshek

and division (see also McCully & Robinow, 1971; Johnson, Yoo & Calleja, 1974).The total cell cycle is taken to be the 140 min characteristic of growth in EMM2 at32 °C, and o-o and i-o in the cycle are the times of division denned operationally fromcell number increase measured with a Coulter Counter after sonication (Mitchison,1970).

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Delay of mitosis, cell plateformation, division, DNAsynthesis.Delay Increases as pulse Isjpplcd progressively later

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Fig. 8. Summary of heat and cycloheximide effects during the cell cycle. The cycle ofapproximately 140 min is plotted in fractions of a cycle between divisions, and is alsobroken into the conventional periods of S, G%, M, Gx (Howard & Pelc, 1953). Outlinesof cells with nuclei were traced from photographs, and those above the cell cycle maprepresent the normal course of growth, mitosis, and division. Effects of heat andcycloheximide pulses are summarized for periods of sensitivity indicated by the widthof the boxes and the arrowheads. Cells with aberrant morphologies were also tracedfrom photographs.

The evidence for a major period of increasing delay due to pulse treatments hasbeen presented earlier. Cultures are observed to be split into either of 2 populationswhen pulses are applied late in the cycle, and this is the main evidence that a transitionpoint exists at 0-65 and behaves as a point. That is, cells very suddenly become in-sensitive to increasing division delay, and do not gradually lose their sensitivity overa measureable fraction of the cycle. However, for CH there must be a second transi-tion point during cell plate stage. The evidence for this is that the cell plate index roughlydoubles during the first 30-45 min of continuous exposure to CH in asynchronous cul-tures, or for approximately the amount of time between the first transition point and cellplate formation (Polanshek, 1973). Further, a normal preparation for cell plate comple-tion must fail during this part of the cycle because most of the cells which accumulatewith cell plates in the presence of the drug do not separate at these cell plates, but re-main attached during subsequent growth when the drug is removed. If a culture remainsin CH, the cell plates which form thicken and become visibly double and roughlyelliptical, with the 2 sides of the cell plate connected at the circumference of the cell.

Heat and cycloheximide effects on fission yeast 13

The impression is that a cell plate may be completed in the presence of CH, butsplitting at the margins cannot be accomplished. The result of resumed growth ofcells with double cell plates is the production of 4-celled filaments in the configurationdrawn in Fig. 8. These filaments are transient since daughter cells at either end splitoff normally, though not usually in synchrony.

Heat shocks produce more complex effects during the cycle, and induce more variedaberrant cell types than do CH pulses. Shocks applied before the transition point at0-65 delay the events of division which follow the transition point, but mitosis, cellplate formation, DNA synthesis, and division are then completed in normal sequenceand timing. Following this first transition point, mitosis, once begun, seems to occurnormally at the shock temperature until stage 4, Fig. 8. During a shock, the per-centage of cells in early stages of nuclear division declines as there is a reciprocal in-crease in the percentage of cells in stage 4 (Polanshek, 1973). Once collected at thisstage, the nuclei remain at the extreme ends of the cell even though a cell plate formsroughly 30 min after the heat shock has ended. Normally, the cell plate would appearimmediately after, or even during, stage 4. Thus, there is apparently a block late innuclear division, and this can be considered a second transition point for thermalshock.

Between the first and second heat transition points, shocks delay cell plate formationroughly 30 min. Shocks during this period also result in a number of aberrant morpho-logies, shown in Fig. 8. These configurations may be interpreted as mistakes in theplacement of the cell plate such that nuclei either are not sorted into different cells,or are sorted into cells of markedly unequal size. It is not known whether small seg-ments containing single nuclei are viable cells, but some of the small segments appearhighly refractile, and are probably dead.

Effect of cycloheximide on recovery from heat shock

In view of the similarities in the pattern and period of sensitivity of cells to divisiondelay during the first two-thirds of the cell cycle, we may suppose that both heat andCH affect common processes leading toward division; evidence that division delayfrom the 2 treatments applied together is not equal to the sum of the delays for eithertreatment alone would be consistent with this idea. The reasoning is that eithertreatment should discharge the same preparation for division so that the total delayfrom a combined treatment should be equal to that for the single treatment whichalone produces the longer delay. One experiment in which this argument was testedis shown in Fig. 9. Here the shocks were sequential, with the heat shock first. Otherexperiments gave similar results when both treatments were applied coincidentally.It is clear that delay is not additive in this example, since an additional CH pulseadded only a few minutes to the delay induced by the heat shock, though CH aloneproduced 22 min of delay.

Because of this last result, experiments were done to see whether there was a phaseof increasing delay when CH pulses were applied at different times following a singleheat shock. The assumption was that if heat shock effects included CH-blocked events,then a potential for increasing delay from CH pulses would be part of the process of

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M. M. Polanshek

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10

Control

39 mm

46 min

22 mm

1 2 3Time, h

Fig. g. Effect of combined 15-min, 41 °C heat shock and 10-min, ioo/*g/ml cyclo-heximide pulse on division. Pulses were applied as shown by bars (l—l), and positionsof cell plate peak mean times are indicated by arrows. Pulses were applied betweenselection of cells and the first synchronous division. The division delays given on figureswere estimated as described in the Appendix.

00 05

Mid-point of CH pulse

1 0

Fig. 10. Division delay for cycloheximide pulses applied at different times followinga heat shock in cultures synchronized by selection. The position in the cycle of the 15-min, 41 °C heat shock is indicated by the bar (1—l); 10-min, 100 /tg/ml CH pulsesapplied at times shown by points resulted in delayed (#) and 'escape peak' popula-tions of cells (O)- All pulses were applied prior to the first division following selection.Delay due to the heat shock has been subtracted from the total delay to determine netCH delay. Arrow indicates time of CH transition point.

Heat and cycloheximide effects on fission yeast 15

recovery from heat shock. Three such experiments are summarized in the divisiondelay curve in Fig. 10. The predicted phase of increasing delay is apparent, but themagnitude of the delay is less than that found for CH pulses alone (Fig. 6B). Thismay reflect the fact that cultures are delayed progressively less by shocks subsequent

20

10

0

20

"a.

s° 0

30

20

10

- A d r 1 2 3Time, h

Fig. n.Effectof a is-min,4i CC heat shock on the cell plate index following synchroni-zation of a culture with 3'-deoxyadenosine (Adr). Heat shocks were applied at the timesindicated by bars (l—i). Approximate mean times for cell plate peaks are shown byarrows.

to the first one for either CH or heat, eventually dividing despite a series of severalshocks (Polanshek, 1973). There is evidence in Fig. 10 for a transition point for CHdelay, since populations are again found to be split into escape peaks and delayedpeaks by the CH pulse. The CH transition point, based upon these few experiments,has been delayed by roughly the 20 min maximum division delay produced by CH.

i 6 M. M. Polanshek

Division delay in cultures synchronized by induction

All of the experiments described so far were performed with either log-phase,asynchronous cultures of yeast, or with cultures synchronized by selection of small,young cells. One of the more powerful methods for studying cell cycle events is todisturb the cycle to see which events and temporal spacings can be altered, and whichremain obligatory. Thus, the effects of shocks on cultures synchronized by a secondmethod are of interest.

- 30

20 c1

10 |-o

40

c

i>; 30

•S

3 2 0

10

r>

i i i i i i i i I

/

/

• /° °/ */ °/ *

//

- - 1 0

- A d r 30 60 90 120 150Mid-point of heat shock, mm

Fig. 12. Summary of division delay for is-min,4i "C heat shocks applied to cultures ofS.pombe synchronized with a 3'-deoxyadenosine block (see Fig. 14). Delay was esti-mated for the first cell plate peak after removal of the block. Solid circles are delayedpeaks, open circles indicate 'escape peaks' (see Fig. 6 for similar data on culturessynchronized by selection).

The synchrony procedure chosen for comparison is that of Mitchison & Creanor(1971) in which an asynchronous culture is treated with 3'-deoxyadenosine (Adr) toblock DNA synthesis. This leads to division synchrony upon release from the block,and a long Gx period is created while G2 becomes short compared with the G2 seenin a culture synchronized by selection. A characteristic of this synchronization pro-cedure is the production of oversized cells which go through 2 abnormally closelyspaced divisions some time after release from Adr. It has been argued that the reasonthat the 2 divisions are so close together in time is that division proteins accumulateduring Adr treatment, allowing 2 rapid division cycles once DNA synthesis has caughtup with the level of division proteins (Mitchison & Creanor, 1971). We have assumedthat division delay resulting from heat shock assays for the same preparations fordivision under conditions of Adr synchrony as were measured in cultures synchron-ized by selection. Division delay has therefore been measured for pulses appliedbetween the end of the Adr treatment and the first synchronous division.

An experiment of the latter sort is shown in Fig. 11, and a series of such experi-ments is summarized in Fig. 12. Two facts are clear. First, division delay has a differentpattern here than for cultures synchronized by selection in that there is a period in thecycle when a heat shock produces no division delay. Delay of the first division beginsto increase shortly after DNA synthesis has resumed (see fig. 2 in Mitchison &

Heat and cycloheximide effects on fission yeast 17

Creanor, 1971). This suggests that heat-sensitive division preparations do notstart until at least the beginning of 5-phase. If the latter is correct, sensitivityto increasing division delay belongs in the G2 phase of the the cell cycle, with possibleoverlap back into S-phase; this is close to what is found for cells synchronized byselection.

A second point is that the time between the 2 synchronous divisions is not increasedwhen the first division is delayed. This suggests that heat-sensitive preparations forthe second division are not executed prior to the first division. An implication is thatdivision proteins have not been accumulated and sequestered during the time thatcells were in Adr, as has been suggested (Mitchison & Creanor, 1971).

DISCUSSION

Various factors and processes have been identified as determinants of cell divisionin eukaryotes (see Mitchison, 1971, and Introduction). In this paper we have con-centrated on processes which limit division when they are disrupted by heat shock orthe protein synthesis inhibitor, cycloheximide; as a result of the disruption, cellsexperience division delay. The paradigm is a system in which cultures of the ciliate,Tetrahymena, can be brought into division synchrony by a series of heat shocks(Zeuthen, 1974). Such synchronization has been explained using division delay data,and theories of the control of cell division derived from these data relate broadly toideas about possible initiators of mitosis (Mitchison, 1971). Our interest was to seehow closely parallel are the responses of a fission yeast, Schizosaccharomyces pombe,and the ciliate to heat shock and inhibition of protein synthesis.

In the study most comparable to this one, Kramhoft & Zeuthen (1971) found aperiod of increasing delay for heat shocks in the cell cycle of yeast previously synchron-ized by heat shocks. The important points for comparison are the facts that in culturessynchronized either by selection (this study) or heat shock, increasing division delayand a transition point ending this period of sensitivity can be demonstrated. Addition-ally, delay due to a pulse is greater than pulse length during much of the cycle. Theperiod over which there is increasing delay is comparable in length, and the maximumdelay of 30-40 min for 15-min heat shocks is roughly the same as we find for the firstof 3 periods of sensitivity described here (Fig. 8).

Having confirmed that heat shocks produce a pattern of increasing excess delay inyeast, the question was whether pulses of CH produce a similar pattern, as would beexpected from a 'division protein' explanation of division delay (see Introduction).The period of increasing delay which we have demonstrated for CH pulses duringthe first two-thirds of the cycle fits this expectation. Also, delay from heat shock andfrom CH is not additive, and recovery from heat shock includes a potential for in-creasing division delay which is revealed when CH pulses are applied following a heatshock. These facts are consistent with the interpretation that both agents dischargea common preparation for division, as in the Tetrahymena division protein scheme(Zeuthen, 1974). However, heat shock and CH inhibit both protein synthesis and othermacromolecular syntheses. Additionally, the difference in the magnitude of division

18 M.M. Polanshek

delay produced by the 2 agents suggests that heat shock is either more potent thanCH while affecting comparable processes, or that, among its effects on cells, heat shockaffects processes which are also CH-sensitive. A similar problem in interpretationoccurs with ^-fluorophenylalanine-induced delay in Tetrahymena, where this drugproduces greater delay than heat shock early in the cycle (Zeuthen, 1964; Mitchison,1971). The fact that heat severely inhibits growth of fission yeast in length and in drymass suggests that recovery from heat shock includes more than simply resupply ofdivision proteins. To a first approximation, heat shock delay during the first two-thirdsof the cycle can be accounted for as the sum of a variable increasing delay equal tothat produced by CH alone and a constant delay roughly equal to the duration of aheat shock. The constant delay is perhaps reflected in the reduced rate of growth incell length following heat shock. Thus, heat delay may be due to an effect on particularsyntheses required for cell cycle traverse (division proteins?) and to a more generalsuppression of cellular metabolism.

Although it has been argued that division proteins are required for structures suchas the mitotic apparatus (Zeuthen & Williams, 1969; Mitchison, 1971), other datasuggest that a pattern of increasing delay with a transition point is not characteristicof one homologous sensitive process in various systems, but may rather relate tocommon and evolutionarily old patterns of cellular metabolism. For example, inChinese hamster cells treated during Gx with pulses of either cycloheximide or puro-mycin, initiation of DNA synthesis is delayed with a pattern of increasing excessdelay up to a transition point 55 min prior to 5-phase (Schneiderman, Dewey &Highfield, 1971; Highfield & Dewey, 1972). Further, it is hard to find an analogy witheukaryotic structural elements in Escherichia coli, where increasing division delayoccurs due to heat shocks and p-fluorophenylalanine pulses (Smith & Pardee, 1970).It may be that the accumulation of substances (initiators?) to threshold levels is thecommon mechanism which may or may not be sensitive to heat shock and to inter-ruption of protein synthesis; there is evidence for initiators of DNA synthesis inbacteria and mammalian cells (Donachie, Jones & Teather, 1973; Highfield & Dewey,1972), and of mitotic initiators in the eukaryotic slime mould, Physarum (Sachsen-maier et al. 1972; Bradbury et al. 1974). Alternatively, interruption of the normal cellcycle may require that a whole programme of biochemical synthesis be repeated, as inthe ' quantal control' idea developed to explain the recapitulation of enzyme synthesiswhich occurs following disruption of an aggregation stage in Dictyostelium (Newell,Franke & Sussman, 1972). There is some evidence that cells repeat sequences of eventsfrom the cell cycle in which they were delayed: Tetrahymena reconstructs an oralapparatus following heat shock, and fission yeast repeats a period of susceptibility tocycloheximide division delay.

A second period of heat-sensitivity for fission yeast covers the period of mitosis.During this period, cycloheximide has no division-delaying effects, and we concludethat abrupt and nearly complete inhibition of protein synthesis does not interferewith mitosis once it has begun. While mitosis proceeds at the higher temperature,formation of the cell plate is delayed. From the work of Johnston et al. (1974), we canpropose that heat interferes with the formation near the end of mitosis of the annular

Heat and cycloheximide effects on fission yeast 19

rudiment, the template from which centripetal growth of the septum occurs. Ifformation of the annular rudiment were disrupted by heat shock, nuclear division mightbe completed on time though cell plate formation could be delayed. This would notaccount for the fact that nuclei remain at the extreme ends of the mother cell when ashock occurs during mitosis. It is, however, well established that spindles and similarstructures are disrupted by high temperatures (Marsland, 1970). If daughter nuclei arepulled toward the middle of the presumptive daughter cells by the spindle, they mightnot suffer their normal final positioning at high temperatures.

We have presented some evidence that the position of the cell plate can be affectedduring this second phase of heat sensitivity, before there is any trace of a septum. Itis possible that sites at which a septum may form are integrated into the membrane orwall at regular intervals, as in bacteria and budding yeast (Donachie et al. 1973; Cabib& Farkas, 1971). If heat activates such sites, perhaps at random and possibly prema-turely, non-central cell plates could be formed. The usually strict relationship of acentral plane of fission perpendicular to the mitotic apparatus breaks down in thesecircumstances.

The last section of the cell cycle in which there is a distinct effect of inhibitors ona particular event is the cell plate stage. Heat shock can fix a cell in this stage for anindefinite period; cycloheximide has clear, sequential effects on cell plate formation,In the presence of CH, cell plates form as usual, but the septum fails to split at itscircumference. The central scar plug layers of the cell plate apparently split apart,leading to an elliptical double cell plate with daughter cells attached at the annularrudiment. It is likely that autolysin-like enzymes are required to break the old wall atthe annular rudiment after the cell plate is completed (Johnson et al. 1974). A tenta-tive conclusion is that CH interferes with such autolytic functions, possibly by blockingthe synthesis of the enzymes. Once autolysis has been blocked, the fault is not rectifiedin most of the cells which have suffered the blockage. This suggests that autolyticenzymes may be available only briefly once each cycle, and that they must be availableat a specific stage in cell plate maturation. It is also possible that the peripheral wallis not a suitable substrate for lytic enzymes once the cell plate has thickened excessively.

The several periods of sensitivity which we have described in the cell cycle of fissionyeast have all been related to interference with processes obviously associated withcell division. These periods are separated from one another by transition points.A transition point traditionally has been defined as a single time in the cell cycle atwhich sensitivity, usually measured as blockage of division or mitosis, ceases for aparticular agent or class of agents, such as inhibitors of protein synthesis (Mitchison,1971). However, multiple transition points for division can be found in many systemswhen such points are defined as times at which division sensitivity changes abruptly.For example, there are 2 periods of susceptibUity to ultraviolet-induced division delayin fission yeast, with sharp transition points between them (Gill, 1965). In Tetra-hymena, there is a heat transition point at the end of the period of increasing divisiondelay, but a second one at a late fission stage, since fission is disrupted by heat shockapplied while it is occurring (Zeuthen, 1964).

Transition points may identify times of change in the physiology of cells, especially

20 M. M. Polanshek

when agents block some events very rapidly. Nevertheless, a transition point for energymetabolism in sea-urchin eggs was shown by Epel (1963) to be artifactual when in-hibition of respiration was incomplete, as in early studies (see Swann, 1957). It ispossible that such a problem might occur when using inhibitors of protein synthesis.For example, Chinese hamster cells can move from G2 to mitosis when small amountsof leucine are added to leucine-deficient medium, though these cells are blocked inG1 with the same amount of leucine (Everhart & Prescott, 1972). In gut epithelium ofthe rat, cells move from G2 into mitosis when there is an 80 % reduction in proteinsynthesis (Verbin, Liang, Saez, Diluiso, Goldblatt & Farber, 1971). These examplessuggest that proteins required for cell cycle traverse are made even if the total capacityfor protein synthesis is well below normal. This must also be true for S. pombe,because cells will go through one or two division cycles when deprived of a nitrogensource (Faed, 1959, cited in Mazia, 1961), and cells make progress toward divisionwhich can be negated by CH following a heat shock, when the rate of protein synthesisis reduced.

I would like to thank Professor J. M. Mitchison and Drs R. G. Burns and J. Creanor fortheir help and advice during the course of this study, and the many people - especially (inaddition to the above) Drs R. S. S. Fraser, M. E. Rogers, H. C. Bennet-Clark, R. A. Kille,Miss P. Aitchison, Miss C.Wilson, Mr D. Cremer, and Mr N. McKay - who made theZoology Department at Edinburgh such a friendly place during my three years there.

It is a pleasure also to acknowledge the influence and inspired teaching of Professor DanielMazia of the University of California, Berkeley, who directed me first to Professoi Mitchison'scourse, thence to Edinburgh.

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BOSTOCK, C. J. (1970). DNA synthesis in the fission yeast, Schizosaccharomycespombe. Expl CellRes. 60, 16-26.

BRADBURY, E. M., INCUS, R. J., MATTHEWS, H. R. & LANGAN, T. A. (1974)- Molecular basisof control of mitotic cell division in eukaryotes. Nature, Lond. 249, 553-556.

CABIB, E. & FARKAS, V. (1971). The control of morphogenesis: an enzymatic mechanism forthe initiation of septum formation in yeast. Proc. natn. Acad. Sci. U.S.A. 68, 2052-2056.

COOPER, D., BANTHORPE, D. V. & WILKIE, D. (1967). Modified ribosomes conferring resistanceto cycloheximide in mutants of Saccharomyces cerevisiae. J. molec. Biol. 26, 347—350.

DONACHLE, W. D., JONES, N. C. & TEATHER, R. (1973). The bacterial cell cycle. In Society forGeneral Microbiology Symposium No. 23 (ed. J. M. Ashworth & J. E. Smith), pp. 9-44.New York and London: Cambridge University Press.

EPEL, D. (1963). The effects of carbon monoxide inhibition on ATP level and the rate ofmitosis in the sea urchin egg. J. Cell Biol. 17, 315-319.

EVERHART, L. P. & PRESCOTT, D. M. (1972). Reversible arrest of Chinese hamster cells in G±by partial deprivation of leucine. Expl Cell Res. 75, 170-174.

FAED, M. J. W. (1959). Division and Growth Relationships in Single Cells. Ph.D. Thesis, Uni-versity of Edinburgh.

FRANKEL, J. (1962). The effects of heat, cold, and ^>-fluorophenylalanine on morphogenesisin synchronized Tetrahymena pyriformis GL. C. r. Trav. Lab. Carlsberg 33, 1-52.

FRANKEL, J. (1967a). Studies on the maintenance of development in Tetrahymena pyriformisGL-C. I. An analysis of the mechanism of resorption of developing oral structures. J. exp.Zool. 164, 435-460.

FRANKEL, J. (19676). Studies on the maintenance of oral development in Tetrahymena pyri-formis GL-C. II. The relationship of protein synthesis to cell division and oral organelledevelopment. J. Cell Biol. 34, 841-858.

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GILL, B. F. (1965). The Effects of Ultraviolet Radiation during the Cell Cycle. Ph.D. Thesis,University of Edinburgh.

HAMBURGER, K. (1962). Division delays induced by metabolic inhibitors in synchronized cellsof Tetrahymena pyriformis. C. r. Trav. Lab. Carlsberg 32, 359-370.

HARNDEN, D. G. (1957). Synchronous Cell Division. Ph.D. Thesis, University of Edinburgh.HARTWELL, L. H. (1974). Saccharomyces cerevisiae cell cycle. Bad. Rev. 38, 164-198.HARTWELL, L. H., CULOTTI, J., PRENGLE, J. R. & REID, B. J. (1974). Genetic control of the cell

division cycle in yeast. A model to account for the order of cell cycle events is deducedfrom the phenotypes of yeast mutants. Science, N.Y. 183, 46-51.

HERRING, A. J. (1973). A Study of Induced Delay in the Division of the Yeast, Schizosaccharo-myces pombe. Ph.D. Thesis, University of Edinburgh.

HIGHFIELD, D. P. & DEWEY, W. C. (1972). Inhibition of DNA synthesis in synchronizedChinese hamster cells treated during G^ or early S phase with cycloheximide or puromycin.Expl Cell Res. 75, 314-320.

HOWARD, A. & PELC, S. R. (1953). Synthesis of desoxyribonucleic acid in normal and irradiatedcells and its relation to chromosome breakage. Heredity, hand. (Suppl.) 6, 261-273.

JOHNSON, B. F., YOO, B. Y. & CALLEJA, G. B. (1974). Cell division in yeasts. II. Templatecontrol of cell plate biogenesis in Schizosaccharomyces pombe. In Cell Cycle Controls (ed.G. M. Padilla, I. L. Cameron & A. Zimmerman), pp. 153-166. New York and London:Academic Press.

KRAMHOFT, B. & ZEUTHEN, E. (1971). Synchronization of cell division in the fission yeast,Schizosaccharomyces pombe, using heat shocks. C. r. Trav. Lab. Carlsberg 38, 351-368.

MCCULLY, E. K. & ROBINOW, C. F. (1971). Mitosis in the fission yeast Schizosaccharomycespombe: a comparative study with light and electron microscopy. J. Cell Sci. 9, 475—508.

MARSLAND, D. (1970). Pressure-temperature studies on the mechanisms of cell division. InHigh Pressure Effects on Cellular Processes (ed. A. M. Zimmerman), pp. 259—312. New Yorkand London: Academic Press.

MAZIA, D. (1961). Mitosis and the physiology of cell division. In The Cell, vol. 3 (ed. J. Brachet& A. E. Mirsky), pp. 77-412. New York and London: Academic Press.

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. New York and London: CambiidgeUniversity Press.

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 cell cultures by sedi-mentation. Nature, Lond. 205, 987-989.

MUNRO, H. N. & FLECK, A. (1966). The determination of nucleic acids. Meth. biochem. Anal.14. 113-176-

NEWELL, P. C, FRANKE, J. & SUSSMAN, M. (1972). Regulation of four functionally relatedenzymes during shifts in the developmental program of Dictyostelium discoideum. J. molec.Biol. 63, 373-382.

POLANSHEK, M. M. (1973). Control ofCell Division in Schizosaccharomyces pombe. Ph.D. Thesis,University of Edinburgh.

RAO, S. S. & GROLLMAN, A. P. (1967). Cycloheximide resistance in yeast: A property of the60S ribosomal subunit. Biochem. biophys. Res. Commun. 29, 696—704.

RASMUSSEN, L. & ZEUTHEN, E. (1962). Cell division and protein synthesis in Tetrahymena asstudied with ^-fluorophenylalanine. C. r. Trav. Lab. Carlsberg 32, 333-358.

RUSCH, H. P., SACHSENMAIER, W., BEHRENS, K. & GRUTER, V. (1966). Synchronization ofmitosis by the fusion of the plasmodia of Physarum polycephalum J. Cell Biol. 31, 204-209.

SACHSENMAIER, W., REMY, U. & PLATTNER-SCHOBEL, R. (1972). Initiation of synchronousmitosis in Physarum polycephalum. A model of the control of cell division in Eukariots.Expl Cell Res. 73, 41-48.

SCHNEIDERMAN, M. H., DEWEY, W. C. & HIGHFIELD, D. P. (1971). Inhibition of DNA syn-thesis in synchronized Chinese hamster cells treated in Gt with cycloheximide. Expl Cell Res.67. 147-155-

22 M. M. Polanshek

SIEGEL, M. R. & SISLER, H. D. (1965). Site of action of cycloheximide in cells of Saccharomycespastorianus. III. Further studies on the mechanism of action and the mechanism of resistancein Saccharomyces species. Biochim. biophys. Acta 103, 558-567.

SLATER, M. & SCHAECTER, M. (1974). Control of cell division in bacteria. Bad. Rev. 38, 199-221.SMITH, H. S. & PARDEE, A. B. (1970). Accumulation of a protein required for division during

the cell cycle of Escherichia colt. J. Bad. 101, 901-909.SWANN, M. M. (1957). The control of cell division: A review. I. General mechanisms. Cancer

Res. 17, 727-757-VERBIN, R. S., LIANG, H., SAEZ, L. M., DILUISO, G., GOLDBLATT, P. J. & FARBER, E. (1971).

Effects of inhibitors of protein synthesis on structure and function of the crypts of the smallintestine. Expl Cell Res. 65, 81-93.

WINFREE, A. T. (1975). Unclocklike behaviour of biological clocks. Nature, Lond. 253, 315-319.ZEUTHEN, E. (1961). Cell division and protein synthesis. In Biological Structure and Function

(ed. T. W. Goodwin & O. Lindberg), pp. 537-548. New York and London: Academic Press.ZEUTHEN, E. (1964). The temperature-induced division synchrony in Tetrahymena. In Syn-

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[Received 25 May 1976)

APPENDIX: ESTIMATION OF DIVISION DELAY FOR S. POMBE

FROM CELL PLATE INDEX CURVES

In cultures of yeast synchronized by selection (Mitchison & Vincent, 1965), the cellplate index curve is generally symmetrical (Fig. 13 A). The curve broadens and themaximum becomes smaller at succeeding cell plate peaks as division synchrony wanes.Assuming that all cells take, on average, the same amount of time to complete cell platestage, the mean time of mid-cell plate stage for the population can be estimated byfinding the time at which the area under the cell plate peak is halved; for a normalcurve this mean time will be the time of the maximum value of the cell plate index.

When an agent is applied which causes division delay, the cell plate index curve isdisplaced to the right. For heat shock or cycloheximide, pulses early in the cycle dis-place the whole cell plate peak, and the mean time of mid-cell plate stage can be esti-mated as for a control culture (Fig. 13 A). Then the mean time for the control cell platepeak is subtracted from that for the treated culture, giving the magnitude of divisiondelay.

Pulses applied relatively late in the cell cycle split the cell plate index curve into 2(or more) peaks. With a split culture it is inaccurate to compare the time of the cellplate peak for either sub-population with the mean time for the whole population. Wehave used a graphical method for estimating division delay by comparing the time ofthe cell plate mean for each cell plate peak in a treated culture with the mean for thecorresponding fraction of the control population. With this method one eliminates an

Heat and cycloheximide effects on fission yeast 23

artifactual phase of increasing division delay for escape peak cells which is generatedwhen the times of the escape peaks are compared with the time of the control cell platepeak. One also eliminates negative values of delay for escape peak cells.

Time, h

Fig. 13. A and B are cell plate index curves from control and cycloheximide-pulsedcultures, respectively, and were taken from Fig. 5 in the main text. Arrows mark theestimated mean times for mid-cell plate stage for the various cell plate peaks, and thebar (l—l) shows the position of the cycloheximide pulse.

To estimate the mean time of mid-cell plate stage for each peak, we first smooth inthe curves, as in (c) and (D). The shapes of the curves for the pulsed culture will beassymetric as drawn in (D) if the pulse split the culture at a transition point or very shorttransition interval. The control population is assumed to be the union of the 2 popula-tions found for the pulsed culture. If the CH pulse has not altered the duration of cellplate stage for treated cells, then the areas under the two cell plate peak3 in D shouldsum to give the area under the control curve in c. The 2 cell plate peaks in D arefitted under the control curve in c by aligning the left hand edge of the left peak (finestippling) with the left hand edge of the control, and the right hand edge of the rightpeak (large stipples) with the right hand edge of the control. Note that the sum of the2 stippled areas is approximately equal to the area under the control. We can nowestimate when the cells in each peak of the pulsed culture would have divided in thecontrol. The mean time of mid-cell plate stage is estimated by finding the time whichsplits the area under each peak in half; these times are found for both peaks in thepulsed culture and for the same peaks fitted under the control in c. Subtracting the timesfound for the peaks under the control curve from the times found for the peaks in D,we estimate that the first peak in D is displaced 3-5 min from the position of the samefraction of the control population. Similarly, the second peak in D is delayed 18-20 mincompared with the corresponding fraction of the control.

The precision of this method of estimating division delay is indicated by the spreadof division delay values in Fig. 6 of the main text.