J. Cell Set. 55, 51-67(1982)
Printed in Great Britain © Company of Biologists Limited 1982
51
POLY(A)+ RNA POPULATIONS, POLYPEPTIDE
SYNTHESIS AND MACROMOLECULE
ACCUMULATION IN THE CELL CYCLE OF
THE EUKARYOTE
CHLORELLA
P. C. L. JOHN, C. A. LAMBE*, R. McGOOKINf,
B. ORR AND M. J. ROLLINS
Department of Botany, The Queen's University, Belfast BTy iNN, U.K.
SUMMARY
Synchronous cultures of Chlorella, that were obtained with minimum metabolic perturbation
by centrifugal selection, reveal that progress through the cell cycle requires no change in
the poly(A) + mRNA population, although changes do occur during nutritional adaptation.
Of the abundant soluble proteins, 93 % are synthesized continuously through the cell cycle
and those that are discontinuous show similar patterns in control cells. T h e synthesis of
proteins is compared with parallel studies of accumulation of enzyme activity and it is shown
that there is no discrepancy in their pattern of accumulation when both are studied under
the same culture conditions. The eukaryote cell cycle can allow stable relative rates of synthesis
of most proteins and balanced rates of accumulation of most enzyme activities. Macromolecule
classes differ in their rates of accumulation throughout the cell cycle: total RNA increases
linearly, poly(A) + RNA accumulation is restricted to Gt phase, but total protein accumulation
accelerates smoothly through Glt S and mitosis phases, pausing at cytokinesis. There is no
evidence that the cell cycle requires an extensive programme of differential enzyme synthesis.
The cycle can therefore proceed with minimum disturbance of metabolism required for
growth.
INTRODUCTION
Most reports of enzyme activity in the cell cycle have described discontinuous
patterns of increase. Such periodicity is particularly clear in synchronized populations
of algae such as Chlorella (John et al. 1973; Schmidt, 1974; Lorenzen & Hess,
1974), and has been the subject of numerous reports in synchronous cultures
of Saccharomyces cerevisiae (see reviews by Mitchison, 1969; Halvorson, Carter &
Tauro, 1971). Paradoxically, individual proteins in yeast have shown continuous
synthesis when non-synchronous cells were pulse-labelled and then segregated into
phases of the cycle (Elliott & McLaughlin, 1978). It has therefore been uncertain
whether there is a programme of biochemical development extending throughout
the cell cycle.
• Present address: Biochemistry Group, Engineering Science Division, Harwell, OXi 1 oRA,
U.K.
t Present address: Department of Botany, The King's Buildings, Mayfield Road, Edinburgh,
EH9 3JH, U.K.
52
P. C. L. John, C. A. Lambe, R. McGookin, B. Orr and M. J. Rollins
The present study rejected the use of synchronization by periodic illumination,
which is commonly used to study the cell cycle of unicellular algae (John et al.
1973; Lorenzen & Hess, 1974), because, although periodic illumination does yield
highly synchronous populations of cells, we have recently compared such cultures
with others in which cells experienced continuous illumination and a constant degree
of self-shading, and we have detected that synchronization that involves changes
in illumination initiates metabolic adaptations that are not part of the cell cycle.
Change in the amount of light available to individual cells causes fluctuations in
photosynthetic capacity, in levels of starch, in rates of respiration and in the accumulation of enzyme activities that occupy a whole cell cycle (John, Lambe, McGookin
& Orr, 1981). Similar distortions of enzyme accumulation caused by starvation
and by osmotic stress were noted when sucrose density-gradient centrifugation
was used to select small cells of Schizosaccharomyces pombe for synchronous culture
(Mitchison, 1977).
In the present study synchronous cultures were prepared by selecting small cells
by continuous-flow centrifugation from an asynchronous population that was growing
under constant environmental conditions. Care was taken that the selected cells
were cultured at the same turbidity as they were in the parent culture, since a change
in culture density would alter the amount of light reaching individual cells and so
alter the growth rate. The potential hazard of such changes in growth rate for the
study of enzyme synthesis is indicated by fluctuations in the rate of accumulation
of four enzymes in Chlorella, following a 40% change in culture density (John, Cole,
Keenan & Rollins, 1980).
MATERIALS AND METHODS
Cell culture
Chlorella strain 211-Sp was cultured in mineral salts medium under conditions of illumination and aeration that have been described previously (McCullough & John, 1972). For
turbidostat culture a control system similar to that of Myers & Clark (1944) was used. An
imbalance between a photocell receiving light through the culture and a reference photocell
allowed an inflow of fresh medium, which stabilized culture turbidity. Medium inflow Was
continually monitored by the automatic recording of liquid level in the medium reservoir.
The extent of culture dilution up to each sample time was calculated, taking into account
periods when the culture was recovering its volume after sampling and periods when the
culture was overflowing (Herbert, Elsworth & Telling, 1956). Data presented in Figs. 1 and 2
have been corrected for culture dilution. They therefore provide a direct indication of growth
and show patterns of accumulation that would have been observed in batch culture, if effects
of increasing turbidity and increasing mutual shading could have been eliminated.
Cell selection
Small cells were selected from asynchronously dividing culture by continuous-flow centrifugation (Lloyd, John, Edwards & Chagla, 1975). A nylon rotor was made in the form of
a shallow cup of 90 mm diameter and 25 mm deep. The sides tapered inwards leaving an
opening of 76 mm and the inner face of the rotor contained 11 semi-circular pockets 6 mm
deep, into which the larger cells sedimented. Smaller cells passed out of the rotor, over the
lip of the rotor cup with the overflowing medium, and were collected in an enclosing dish,
from which they drained and were then aerated to await refractionation or inoculation into
culture.
mRNA and proteins in the cell cycle
53
Cell number
Cell density was determined using a Coulter Counter BZ.
Protein and DNA
Protein was estimated in extracts of broken cells after precipitation with 5 % (w/v) trichloracetic acid and DNA was estimated by diphenylamine reaction, as described previously
(McCullough & John, 1972).
RNA isolation and estimation
RNA was extracted in the presence of RNase inhibitors and was estimated by measuring
absorbance at 260 nm. Poly(A)+ RNA was purified by binding to oligo(dT)-cellulose and
determined by measuring absorbance at 260 nm. Both RNA fractions were prepared as
described previously (Lambe & John, 1979). Estimation of relative poly(A)+ RNA levels by
hybridization with [*H]poly(U) followed the procedure of Fraser & Carter (1976).
Cell-free protein synthesis
The in vitro protein synthesizing system isolated from wheat germ was employed, as
described by Roberts & Paterson (1973), with endogenous protein synthesis reduced below
levels detectable by autoradiography, by prior treatment with Ca1+-dependent nuclease
(Pelham & Jackson, 1976). The rate of protein synthesis was linear with time for 1 h and with
up to 1 fig of poly(A)+ RNA added. In the assays performed for Figs. 3 and 4, 0-5 fig poly(A)+
RNA was added and incubation was for 1 h.
Electrophoresis
One-dimensional electrophoresis in the presence of sodium lauryl sulphate was performed
in a 15 % (w/v) acrylamide gel according to the method of Laemmli (1970).
Two-dimensional separation, with isoelectric focusing followed by electrophoresis in the
presence of sodium lauryl sulphate, according to the method of O'Farrell (1975), was used to
fractionate 20 fig samples of extracted protein for the study of in vivo protein synthesis.
RESULTS
A major advantage of preparing synchronous cultures by cell selection is that
control experiments can be performed to test for the effects of synchronizing. Fig. 1
shows a control experiment in which asynchronously dividing cells, held at constant
turbidity and illumination, were at time o subject to continuous-flow centrifugation,
but at a reduced rotor speed so that cells at all stages of the cell cycle remained in
the supernatant fraction. After centrifugation the cells were transferred into a smaller
vessel, to mimic the treatment of selected cells, and were grown at the same constant
illumination as in the larger parent culture. The similarity of environment before
and after selection is reflected in the unaltered rates of increase in each class of
macromolecules and in cell number (Fig. 1). There is therefore no evidence that
changes in the rate of macromolecule accumulation are caused by the cell selection
procedure.
The continuous-flow centrifugation procedure can be used to select a homogeneous
population of small daughter cells, which form a synchronously dividing culture
(Fig. 2) in which S phase is completed between 5 h and 10 h and subsequent release
of daughter cells occurs between 12 h and 18 h. Cell-cycle events are shown 5I1
54
P. C. L. John, C. A. Lambe, R. McGookin, B. On and M. J. Rollins
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Fig. i. The effect of cell selection procedures imposed at time o, on the increase in
cell number ( O ) ; the accumulation of DNA (A); total protein ( • ) ; total RNA ( • ) ;
and poly(A) + RNA (A) in synchronously dividing Chlorella. A zo-1 culture was
grown at 25 °C in a narrow trough-like vessel with sides 1200 mm x 236 mm and
a breadth of 70 mm. Illumination was provided by two banks of warm-white
fluorescent strip-lights giving i4O/<Einsteins M~ 2 S" 1 at 400-700 nm onto both sides
of the vessel. The cells in mineral salts medium, were agitated with sterile air
containing 0 5 % (v/v) CO 2 and were maintained at a constant density {Ax cm68 o nm =
o-i) by a turbidostat-controlled inflow of fresh medium at 25 °C. At time o the
cells were subject to the selection procedure (as described in Fig. 2 legend) with
the sole difference that rotor speed was reduced so that all cells were recovered in the
non-sedimented fraction. The cells were then transferred to a 1 -8-1 culture with the
same 70 mm breadth and at the same turbidity as the parent culture and growth was
continued under identical conditions. Samples for the estimation of RNA levels
were processed immediately. Cells were broken at o °C in buffer containing 2 %
sodium tri-isopropyl-naphthalenesulphonate (Lambe & John, 1979), by passage
through a French pressure cell, and the effluent from the cell was passed directly
through 400 mm of 1 mm diameter stainless steel tubing in an ice bath, to re-chill
the suspension quickly. RNA was immediately purified and precipitated with
ethanol. Total RNA was estimated by measuring absorbance at 260 nm and poly(A) +
RNA, in 20-fig samples of total RNA, was measured by hybridization with excess
[ 3 H]poly(U). Identical profiles were obtained when poly(A) + RNA was purified
from i-mg samples of total RNA by binding to oligo(dT)-cellulose, and then
measured by determining absorbance at 260 nm. TCA, trichloroacetic acid.
mRNA and proteins in the cell cycle
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Fig. 2. Growth of cells obtained by selection from an asynchronous population, which
were cultured without change in growth environment and analysed for increase in
cell number (O); for accumulation of DNA (A); total protein (D); total RNA ( • )
and poly(A)+ RNA (A). Cells in a zo-1 asynchronous culture, identical to that
described for Fig. i, were harvested using a Sharpies continuous-flow centrifuge
and the cells were suspended in I 1 of their previous growth medium and were
aerated. Small cells were selected by continuous-flow centrifugation. Culture was
pumped into the centre pi the rotor well at 120 ml min"1 and speed of rotation was
adjusted with a rheostat so that half of the cells were sedimented. The culture was
re-passed through the centrifuge until only 10 % of the cells remained in suspension
and those were returned to a 1-8 1 culture vessel under growth conditions identical
to those of the parent culture, as described for Fig. 1. The process of harvest and
cell selection occupies nearly 1 h and this is a period of growth interruption, since
the cells cannot photosynthesize. The selected cells were cultured, sampled and
analysed as described for Fig. 1.
earlier here than is usually reported in the synchronizing regime of periodic illumination. In the synchronizing regime it is usual to begin sampling at the start of
illumination, when daughter cells of the strain 2ii-8p have not yet ruptured the
mother cell wall (Atkinson, Gunning & John, 1972), but here the daughter cells
that were selected at time o have already escaped from the mother cell and are 5 h
later in the cell cycle.
56
P. C. L. John, C. A. Lambe, R. McGookin, B. On and M. J. Rollins
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The conventional procedure for synchronizing division in algae by periodic
illumination does not allow a discrimination between phenomena that are caused
by the recurring interruption of growth and those that are part of the cell cycle.
Therefore, the comparison that is possible here, between selected cells and control
cells, reveals for the first time changes in the rate of macromolecule accumulation
that are unambiguously part of the Chlorella cell cycle.
Protein accumulation accelerates throughout Glt S and mitosis phases, as is
revealed in the semilogarithmic Fig. 2 by the straight line describing the accumulation
of protein between o and 12 h. Calculations from the data in Fig. 2 reveal that
mRNA and proteins in the cell cycle
57
individual cells, in successive 3-h periods between o and 12 h, achieve the following
progressively larger mean increments of protein (in pg); 1*2, i-8, 2-7, 3-3. Accelerating
protein accumulation resumes in the subsequent cell cycle beginning at 15 h (Fig. 2).
However, when cytokinesis occurs there is an interruption of protein accumulation,
which is seen as a pause between 12 h and 15 h. This brief pause is invariably
observed in selected cells and also in synchronized cells (Forde & John, 1973). There
is evidence that the pause in accumulation is achieved by faster protein breakdown,
since rates of incorporation into protein are reduced very little compared with
earlier stages in the cycle. Relative to the preceding S and mitosis phases, rates of
incorporation during the cytokinesis pause, are 93% and 89%, respectively, when
measured with 36SO4 and pHJleucine, both supplied for 2-5 h period, and 95 %
when measured with [^SJmethionine supplied in 3-h pulses (labelled proteins are
described and illustrated in the legend to Fig. 5).
Although protein accumulation accelerates throughout the cell cycle, we consistently observed that total RNA accumulation is linear. Cells establish a rate of
RNA accumulation at the beginning of Gx phase and maintain the same rate throughout the cycle, including the period at cytokinesis when protein does not accumulate.
The two patterns are revealed in the semilogarithmic plot (Fig. 2), where protein
shows a straight line for accumulation up to 12 h and again after cytokinesis from
15 h, while for RNA there is a curve, with a rate change at 18 h as daughter cells
establish their rate of RNA increase early in Gx phase. In even stronger contrast
with protein accumulation is the increase in poly(A)+ RNA, which is restricted to
Gx phase. Fig. 2 shows the termination of poly(A)+ RNA accumulation at 3 h, as
cells commence S phase, and its resumption 9 h later in newly formed daughter
cells. The accumulation of protein, by contrast, continues to accelerate through 5
Fig. 3. Translation products of poly(A)+ RNA taken from cells that are adapting to
different nutritional conditions. Photosynthetically growing cells were allowed to
continue in autotrophic growth (lane 1), or were darkened for 6 h and provided
with no organic carbon source (lane 2), 10 mM-sodium acetate (lane 3), or 10 mMglucose (lane 4). Total RNA was immediately extracted from cells and precipitated,
as described for Fig. 1. Poly(A)+ RNA was purified using oligo(dT)-cellulose and
O'5-fig aliquots were translated into protein using a wheat-germ cell-free system
containing ["SJmethionine, in which endogenous protein synthesis was reduced to
levels not detectable by autoradiography by prior treatment with Cal+-dependent
nuclease. Proteins produced by in vitro synthesis were resolved by electrophoresis
under dissociating conditions (Laemmli, 1970) and were detected by autoradiography.
Cell-mass doubling times under these growth conditions were: lane 1, 8 h; lane 2,
infinity; lane 3, 60 h; and lane 4, 12 h; and although some mRNAs (O) remain at
similar translatable levels under each of these conditions, most mRNA levels can
be changed in response to metabolic state. Some ( < ) are present at high levels
only at higher growth rates, whether autotrophic or heterotrophic and are present
(in lanes 1 and 4), while others ( • ) are elevated in autotrophic growth and are at
high levels only in (lane 1), or are elevated in heterotrophic growth (O) and are
present in lanes 2, 3 and 4. Particular forms of heterotrophic growth influence
certain mRNAs, some (#) being elevated during glycolysis from glucose, others
such as isocitrate lyase (ICL) during glyconeogenesis from acetate, and others ( • )
are repressed by glucose.
3
CEL55
58
P. C. L. John, C. A. Lambe, R. McGookin, B. On and M. J. Rollins
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Cytokinesis
i
Fig. 4. Translation products of poly(A)+ RNA taken from cells in Glt S and cytokinesis phases. The synchronous culture, which was prepared by continuous-flow
centrifugal selection of small daughter cells and illustrated in Fig. 2, was sampled
in Gi phase at 3 h, in S phase at 6 h and in cytokinesis at 12 h. Total RNA was
immediately extracted and then poly(A)+ RNA was purified and translated into
protein as described for Fig. 3.
and mitosis phases, between 3 h and 12 h, when the level of mRNA does not
increase. Protein synthesis, measured by incorporation of ^SO^ (not shown) also
increases smoothly during this period of stationary level of messenger. Poly(A)+ mRNA
must therefore be present in excess of the amount required for protein synthesis.
To establish whether Chlorella cells have the facility to change the composition
mRNA and proteins in the cell cycle
59
of their mRNA population within the time-span of a cell cycle, cells previously
grown photosynthetically in batch culture were incubated for a period of less than
a third of a cell cycle, under a variety of nutritional conditions, and then their
poly(A)+ RNA was extracted and translated. Autoradiography of the protein products
seen in Fig. 3 reveals that, although some mRNAs are constitutive, Chlorella has
an ability to elevate or depress functional levels of many individual mRNAs. Some
messengers are present at high levels during faster growth, whether photosynthetic
or in heterotrophic growth on glucose. Particular modes of carbon nutrition also
influence individual mRNAs and some are specific for inorganic carbon fixation in
photosynthesis (Bassham, 1973), others for heterotrophic growth with either glucose
or acetate, and others are specific for particular forms of organic carbon: either
glucose, which allows glycolysis, or acetate, which requires glyconeogenesis (Syrett,
Bocks & Merrett, 1964), while some are specifically repressed by glucose.
Chlorella cells can therefore alter their population of mRNAs when they are
synthesizing the different enzymes required for different metabolic patterns (Syrett,
Merrett & Bocks, 1963; Bassham, 1973). To test whether similar changes in mRNA
are required for progress through the cell cycle, samples were taken from the
synchronous cells analysed in Fig. 2. Exactly the same method of analysis showed
no comparable change in the levels of individual mRNAs. Samples taken in G1
phase at 3 h, in S phase at 6 h and in cytokinesis at 12 h all show very similar mRNA
populations (Fig. 4). The sample at 3 h was taken at the end of a period of poly(A)+
RNA accumulation when the proportion of mRNA in total RNA was maximal, while
the sample at 12 h was taken after a 9 h period during which there was no increase
in mRNA level when the proportion of mRNA in total RNA was at a minimum.
The similarity between these samples indicates that all the abundant translatable
mRNA species follow the same periodic pattern of accumulation. We have not
tested directly whether proteins showing the same electrophoretic mobility when
coded by different samples of poly(A)+ RNA are the same proteins, but we consider
it unlikely that messengers are consistently replaced throughout the cell cycle with
others that code for different proteins with identical mobilities that occur in identical
amounts.
To investigate whether changing populations of protein are synthesized throughout
the cell cycle, successive 200-ml samples were removed from a synchronous culture
identical to that described in the legend to Fig. 2 and the cells were pulse-labelled
under the same growth conditions for one seventh of a cycle (3 h) with [35S]methionine.
The cells were smashed at o °C in a French pressure cell and the proteins were
divided into an insoluble pelleted fraction and a post-ribosomal soluble fraction.
Proteins in these fractions were subjected to one-dimensional electrophoresis'under
dissociating conditions by the method of Laemmli (1970) and were detected by
autoradiography. Differences in radioactivity of less than twofold are not clearly
detected by visual inspection of autoradiographs (Lutkenhaus, Moore, Masters &
Donachie, 1979), and with this reservation more than 96% of the proteins in the
insoluble fraction and 94% of the proteins in the soluble fraction were synthesized
throughout the cell cycle. There is no evidence of periodic cessations of synthesis.
3-2
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P. C. L. John, C. A. Lambe, R. McGookin, B. Orr and M. J. Rollins
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The absence of periodicity is not a failure of resolution since the same procedures,
when applied to cells in periodic illumination, showed periodic synthesis in the
majority of proteins throughout the cell cycle (John et al. 1981).
To test further whether periodicity of synthesis may have been masked by the
co-migration of proteins in one-dimensional electrophoresis, proteins in the soluble
fraction were subject to two-dimensional electrophoresis, by the method of O'Farrell
X
ecu lar weigh
CD
CD
mRNA and proteins in the cell cycle
61
(1975). Examples of the more abundant proteins that were resolved are shown in
Fig. 5. In the present study exposure of autoradiographs was terminated before
the fainter spots had developed, to prevent fogging of the central area. Even with
the improved resolution provided by two-dimensional separation, 93% of the
quantitatively predominant proteins were seen to have been synthesized in all
phases of the cell cycle. We think it unlikely that there is concealed periodic synthesis
within this abundant class of proteins since, to replace one periodically synthesized
protein by another without visible effect would require them to have identical
iso-electric pH values and identical molecular weights, to be present in identical
amounts and to be synthesized at inverse rates.
The periodic synthesis, which is noted here for 7% of the soluble proteins in
selected cells, may be a normal part of the cell cycle that has been made evident
in the population because of their synchrony, but may alternatively be part of
recovery from the selection procedure. If selection did cause their periodic synthesis
then non-synchronous cells would show the same periodicity if subjected to the
same stress. The control asynchronous cells described in the legend to Fig. 1 were
therefore pulse-labelled, exactly as the selected cells were, for the 24 h period after
mock selection. In these cells the majority of proteins were synthesized continuously
but each of the proteins found to be periodically synthesized in synchronous culture
was also periodically synchronized in the control asynchronous culture. Some
proteins were synthesized preferentially soon after mock selection, synthesis of others
was initially depressed but recovered at 9 h and 12 h and some showed accelerations
of synthesis as late as 18 h. Therefore, perturbations affecting a minority of proteins
persisted for the whole of a period equal to one cell cycle after selection.
Fig. 5- Two-dimensional electrophoresis of pulse-labelled soluble proteins, taken
from synchronously dividing cells (Fig. 2) growing under constant illumination
and turbidity, which were selected at time o by continuous-flow centrifugal selection
from an asynchronous population growing under the same constant conditions.
The upper separation shows proteins that are labelled in Gx phase between o and
3 h, and the lower shows proteins labelled in S phase between 6 and 9 h. Successive
samples of culture were pulse-labelled during growth in parallel with the parent
culture, by incubation for 3 h with P'SJmethionine at 0-5 fid and 0-83 nmol per
ml. After labelling the cells were immediately harvested, washed and suspended in
50 mM-Tris-HCl, 5 mM-MgClt (pH 6-8) at o °C and broken in a French pressure
cell. Part of the total extract was immediately subject to centrifugation at 100 000 g
for 90 min and the proteins remaining in the supernatant constituted the soluble
fraction. Proteins were then frozen and stored for subsequent one-dimensional and
two-dimensional electrophoresis. Cell wall debris was removed from the total and
pelleted protein fractions by centrifugation at 5000 g before application to the gel.
The illustration shows the more abundantly labelled proteins resolved by twodimensional electrophoresis from the soluble fraction. Proteins were arbitrarily
assigned a number for systematic comparison of proteins in different culture samples.
Arrows indicate proteins that were preferentially synthesized relative to the other
proteins during the labelling period. The same proteins showed preferential synthesis, at the same time after centrifugation, in control cells that were centrifuged
but not size-selected and so remained asynchronous. For 93 % of the more than
5<x> proteins that were reproducibly resolved, there was no detectable change in
the rates of synthesis throughout the cell cycle.
62
P. C. L. John, C. A. Lambt, R. McGookin, B. Orr and M. J. Rollins
The simplest explanation for the periodicity seen in the small minority of proteins
in synchronously dividing cells after selection, is that their periodicity is part of the
same response to metabolic disturbance as is seen in control cells. From these data,
we cannot exclude the possibility that some of these proteins might also show
periodic synthesis in response to progress throughout the cell cycle. However, this
possibility can be eliminated by a similar analysis in cells that were made synchronous
by periodic illumination but then transferred to constant conditions in a turbidostat
for study. In this procedure known metabolic perturbations subside during the first
cell cycle (John et al. 1981) and protein synthesis can be studied by pulse-labelling
during the second cell cycle. This method has the limitation that no control is
available to test for possible perturbations persisting into the second cycle, but it
has proved a valuable adjunct to the procedure of cell selection for the present study
because all of the proteins that were synthesized periodically following selection by
continuous flow centrifugation were found to establish continuous rates of synthesis
through the second synchronous division in the turbidostat (M. Rollins & P. C. L.
John, unpublished observations). Therefore, we conclude that no abundant soluble
protein can be demonstrated to show periodic synthesis in the cell cycle.
DISCUSSION
Observations of macromolecule accumulation that have been made in synchronous
cultures of Chlorella prepared by centrifugal selection are significant for two reasons.
Firstly, because the cells were grown throughout at constant turbidity and so did
not suffer the reductions in photosynthesis and in growth rate that occur in batch
culture when increasing cell density causes increasing mutual shading. Secondly,
because synchrony was obtained by a means that allowed the performance of control
experiments to test for the effect of synchronizing procedures. Such controls are
not possible for the conventional procedure of synchronizing with repeated cycles
of growth interruption by darkening. When the effect of a dark period is tested on
asynchronous cells, significant synchrony is produced and it is therefore uncertain
whether subsequent periodicity in metabolism is due to perturbation or is a cell
cycle phenomenon made apparent by the synchrony. In the present study the
conditions of growth and procedures of cell selection were shown not to cause
fluctuations in macromolecule accumulation (Fig. 1). Synchronous cultures, under
the same conditions, revealed different patterns of increase throughout the cell cycle
in several classes of macromolecule.
Protein accumulation accelerates smoothly throughout the cell cycle up to cytokinesis but there is then a pause (Fig. 2), as has been observed in batch culture
(Forde & John, 1973). Our detection of continued protein synthesis by incorporation
of radioactive precursor indicates that the pause is achieved by an acceleration of
protein breakdown. There are parallels with this pause in growth at the time of
division, since in mammalian cells there is commonly a slowing of growth at mitosis
due to reduced protein synthesis (Prescott & Bender, 1962), and in S. pombe there
is a cessation of volume increase, but not of protein synthesis, during cytokinesis
mRNA and proteins in the cell cycle
63
(Mitchison, 1963); but in no case is it clear what contribution such pauses make to
cell division.
The present data show that there is no obligate correspondence between the
patterns of total RNA and protein accumulation through the cell cycle, although
previous reports have shown that both total RNA and several enzyme activities
accumulate linearly in S. pombe (Fraser & Moreno, 1976) and both total RNA and
the rate of protein synthesis increase exponentially in 5. cerevisiae (Elliott & McLaughlin, 1978). We consistently observe in Chlorella that protein accumulation
accelerates through Glt S and M phases, but in the same cells total RNA accumulation
is linear and extends into cytokinesis. Although different patterns of accumulation
for RNA and protein have not previously been reported, those seen in Chlorella are
consistent with efficient utilization of ribosomal RNA through the cell cycle, since
an increase in the rate of protein synthesis in proportion to the linearly increasing
RNA could readily account for the accelerating accumulation of protein.
It is also revealed that the eukaryote cell cycle does not universally employ control
of protein accumulation by controlling the level of poly(A)+ mRNA. Correlations
that are consistent with this possibility have been observed in S. pombe, where a rise
in poly(A)+ RNA levels early in G* correlates with faster accumulation of several
enzymes (Fraser & Moreno, 1976), and in S. cerevisiae, where exponentially increasing
poly(A)+ RNA levels (Elliott & McLaughlin, 1979) correlate with exponentially
increasing protein synthesis (Elliott & McLaughlin, 1978). However, in Chlorella,
poly(A)+ RNA accumulation is restricted to Gx phase (Fig. 2) and, although its
level is constant through late Glt S and mitosis phases, the rate of protein accumulation
continues to accelerate; therefore, at the end of its periodic accumulation poly(A)+
RNA is present in excess.
An unsolved question about the cell cycle concerns the mechanisms by which
events leading to division are initiated. Comparison with other developmental processes
suggests that the accelerated synthesis of particular proteins could provide the
molecular switches (Tuchman, Alton & Lodish, 1974; Linn & Losick, 1976). Some
hypotheses of enzyme synthesis in the cell cycle, such as linear reading (Halvorson
et al. 1971) and oscillatory repression (Donachie & Masters, 1969), account for a
programme of periodic synthesis in a majority of proteins throughout the cell cycle.
Periodic synthesis of proteins that might control division could therefore be part of
a general programme. This possibility is not supported by our evidence of continuous
synthesis of individual proteins (Fig. 5). Our evidence confirms a similar finding in
S. cerevisiae (Elliott & McLaughlin, 1978) and extends that evidence by showing
that mRNA populations can remain stable throughout the cell cycle (Fig. 4). The
correlation between organisms that divide by such different mechanisms as budding
and multiple fission, suggests that continuous synthesis of the majority of proteins
is a common feature of the cell cycle.
Before the metabolic background to cell-cycle events can be understood, however,
it is necessary to resolve the discrepancy between the numerous reports of periodic
increase in enzyme activity (see reviews by Mitchison, 1969; Halvorson et al. 1971),
and the evidence for continuous synthesis of proteins. Elliott & McLaughlin (1978)
64
P. C. L. John, C. A. Lambe, R. McGookin, B. On and M. J. Rollins
suggested that the discrepancy might be due to change in activation state; that is,
changes in catalytic activity without a proportionate change in the amount of enzyme
protein. This possibility would allow the cell cycle to include numerous changes in
metabolic activity, in spite of continuous synthesis of proteins, and therefore it
could contain numerous potential mechanisms for the initiation of cell cycle events.
However, if this view is correct, sophisticated controls of enzyme activation state would
be required (Mitchison, 1981) on a scale for which there has not been any evidence
in cells that are simply growing and dividing.
The data presented here suggest a simpler explanation for the discrepancy between
the periodic increase in enzyme activity and the continuous synthesis of protein. We
have studied the accumulation of enzyme activity in the Chlorella cell cycle under
a variety of conditions. Therefore patterns of protein synthesis can be compared
with increase in enzyme activity under similar conditions in a way that is not yet
possible in S. cerevisiae.
Under the conditions of growth in batch culture with intermittent illumination,
which readily induce synchronous division in unicellular algae, most enzyme
activities are seen to increase discontinuously, e.g. in Chlamydomonas (Kates &
Jones, 1967) and in Chlorella (John et al. 1973; Schmidt, 1974; Lorenzen & Hess,
1974). In the strain 2ii-8p, which was employed here, only glutamate oxaloacetate
transaminase and nitrate reductase activities increased in proportion to total protein
under synchronizing conditions (P. C. L. John, F. Haldane & B. Taylor, unpublished
observations), while the following nine enzymes each increased in a single step at
different times in the cycle; aconitase, cytochrome oxidase, phosphoenolpyruvate
carboxylase, succinate dehydrogenase, ribulose-i,5-bisphosphate carboxylase (Forde
& John, 1973), ribulose-5-phosphate kinase (John et al. 1981), isocitric dehydrogenase, fumarase and citrate synthase (John et al. 1980). Under the same synchronizing
conditions, pulse-labelling with ^SOa and with ^HJleucine revealed that different
populations of proteins were also being synthesized at different times in the cell
cycle (John et al. 1981). Similar changes in protein synthesis have been noted in
Chlamydomonas under synchronizing conditions (Howell, Posakony & Hill, 1977).
There is therefore a correlation between accumulation of enzyme activity and protein
synthesis. However synchronizing conditions result in periodic starvation when cells
are darkened, and there are consequent changes in metabolism (John et al. 1981)
that affect enzyme synthesis.
To minimize environmental influence, the last six enzymes listed above have
been measured in synchronously dividing cells, which either have been transferred
to continuous illumination in a turbidostat and allowed to reach a balanced metabolic
state (John et al. 1981) or have been made synchronous without exposure to periodic
environmental changes by use of centrifugal selection, as in the present study (John
et al. 1980). Each of the enzymes then increased exactly in proportion with total
protein accumulation. In addition, glycollate dehydrogenase also increased with
constant specific activity throughout the cell cycle in turbidostat culture. In comparison with the number of proteins that can be resolved, far fewer enzyme activities
have been studied, but the evidence is consistent and we are not aware of any enzyme
mRNA and proteins in the cell cycle
65
activity involved in general metabolism that increases out of proportion with cell
growth in the cell cycle of Chlorella in the absence of environmental stimulus. There
is again, therefore, a correlation between enzyme activity and the continuous synthesis
of individual proteins that is described in this paper. Only if the continuous synthesis
of individual proteins, which prevails during the cell cycle under conditions of
balanced growth, is compared with the periodic increase in enzymes, which results
when the environment changes the processes of metabolism throughout the cell
cycle, is there an apparent discrepancy.
The periodic increases in enzyme activity, which are frequently observed in
synchronous cultures of yeasts, may also have resulted from the use of starvation or
sucrose density-gradient centrifugation to obtain synchrony. From the use of suitable
controls with S. pombe, Mitchison's group have shown (Mitchison, 1977) that of
the 19 enzymes that have been studied only one shows a periodic increase in activity
that is due to progress through the cell cycle, but similar controls for the effects of
selection have not yet been reported for S. cerevisiae. A few enzymes have been
studied in S. cerevisiae using zonal centrifugation to age-fractionate an entire cell
population (Carter & Halvorson, 1973; Sebastian, Carter & Halvorson, 1973), but
it is difficult to evaluate data from the unusually large and small cells at either end
of the population distribution. The discrepancy in 5. cerevisiae between individual
protein synthesis and the increase in activity may also be only an apparent one.
Therefore, unless periodic increases in activity can be shown to be a common feature
of the cell cycle in the absence of metabolic perturbation, it is unnecessary to consider
periodic enzyme activation to be a frequent occurrence for the enzymes of general
metabolism. The possibility remains that activation of a few key enzymes (Mitchelson,
Chambers, Bradbury & Matthews, 1978) is important in the cell cycle.
We cannot conclude that every protein is synthesized continuously throughout
the cell cycle. Many proteins could not be measured in the present study and these
included any proteins that did not contain methionine, the large number that were
insufficiently abundant to be detected by autoradiography, those with isoelectric
pH values too extreme to focus with the majority of proteins, and the messenger
population studies also excluded any mRNAs that lacked the usual poly(A) tail. For
example, histone synthesis was not detected in this study for these last two reasons,
but these basic proteins are synthesized periodically at the time of DNA replication
(Marks, Paik & Borun, 1973). Therefore, the possibility remains that a minority
of proteins is synthesized periodically and may drive the cell cycle.
We conclude that" a simplified view of the cell cycle is now appropriate, in which
a period of balanced growth, not involving an extensive programme of biochemical
differentiation, precedes an eventual commitment to divide. This model is consistent
with evidence that the period of cell growth prior to DNA replication can be readily
shortened (Tyson, Garcia-Herdugo & Sachsenmaier, 1979) or extended in response
to changes in cell size (Hartwell & Unger, 1977; Nurse & Thuriaux, 1977), nutrition
(Lorincz & Carter, 1979) or hormone supply (Stiles, Cochran & Scher, 1981).
We thank the Science Research Council for support.
66
P. C. L. John, C. A. Lambe, R. McGookin, B. On and M. J. Rollins
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{Received 12 October 1981)