Distinct controls of DNA replication and of nuclear division in the cell cycles
of the chlorococcal alga Scenedesmus quadricauda
V. ZACHLEDER* and I. SETLiK
Institute of Microbiology, Czechoslovak Academy of Sciences, Tfeboii, Czechoslovakia
* Author for correspondence
Summary
In the course of the cell cycle of Scenedesmus
quadricauda, the syntheses of RNA and total protein
occur in steps. Each step represents an approximate
doubling of the preceding amount of RNA or protein per cell. The increase in protein content per cell
runs parallel to, but with a constant delay behind,
the corresponding RNA steps. When protein synthesis is suppressed (e.g. by maintaining the cells in
the dark) after an RNA synthesis step has already
occurred the cells double their DNA content, but no
corresponding nuclear division occurs and uninuclear daughter cells with double the amount of DNA
may be formed. Under conditions of phosphorus or
nitrogen starvation RNA synthesis is stopped while
Introduction
The process of cell division by binary fission is thought to
consist of two interacting cycles. These have been termed
the "growth cycle" and the "DNA-division cycle"
(Mitchison, 1971, 1977). Most macromolecular syntheses
occur during the growth cycle, which results in the
increase of cell mass and the formation of cell structure.
The main events in the DNA-division cycle are: replication of DNA, nuclear division, and cytokinesis.
The coordination of the growth and DNA—division
cycle appears to be controlled by the achievement of a
critical cell size necessary for the initiation of DNA
replication (Nasmyth, 1979; Nasmyth et al. 1979).
Another cell size control is supposed to be a prerequisite
for the onset of nuclear division (Fantes & Nurse, 1977,
1978; Fantes et al. 1975). However, it is assumed that it is
not the cell size itself, but some other more specific
processes, which can be coupled or coordinated with the
increase of the cell size.
Syntheses of RNA and protein are the most important
features of the growth cycle and both processes are
considered to play a major role in the control of cell
reproductive processes (Alberghina & Sturani, 1981;
Johnston & Singer, 1978; Darzynkiewicz et al. 1979a,b;
Journal of Cell Science 91, 531-539 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
protein synthesis continues. In this case, the number of DNA replication rounds corresponds to the
reduced RNA content while the number of nuclear
divisions tends to follow the number of protein
synthesis steps until one genome per nucleus is
attained. These results indicate that with each
doubling of RNA content the cells become committed to DNA replication, while doubling of protein
content is required for the commitment to the
corresponding nuclear divisions.
Key words: DNA replication control, nuclear division
control, RNA synthesis, protein synthesis, Scenedesmus
quadricauda.
Popolo et al. 1982). A considerable effort has been made
to elucidate the nature of the signals that commit a cell to
DNA replication and to nuclear division, but the problem remains unsolved.
Chlorococcal algae are characterized by multiple
fission, i.e. 2" daughter cells are released at the end of the
cell cycle. Consequently, n DNA replications and n
nuclear divisions occur during one cell cycle. Temporal
uncoupling of DNA replications, and of nuclear and
cellular divisions leads to formation of multinucleate
cells, which often have polyploid nuclei, at least for a
certain part of the cell cycle. Therefore, a simple cellcycle model for cells dividing by binary fission seems to
be insufficient for the interpretation of the cell cycles of
chlorococcal algae.
In previous papers, we have tried to develop a more
appropriate model of the cell cycle corroborated by our
studies of the chlorococcal algae (Setlfk et al. 1972;
Zachleder et al. 1975; Zachleder & Setlfk, 1982; Setlfk &
Zachleder, 1984). In this paper, a study of the regulatory
relationships between the cell growth steps and the
corresponding reproductive steps (DNA replication and
nuclear division) in the chlorococcal alga Scenedesmus
quadricauda is presented.
531
Materials and methods
Organism
The chlorococcal alga Scenedesmus quadricauda (Turp.)
Bre'b. strain Greifswald/lS was obtained from the Culture
Collection of Autotrophic Microorganisms kept at the Institute
of Botany, Tfebon, Czechoslovakia.
Culture conditions
The cultures were synchronized by alternating light (L) and
dark (D) periods (LD 14: 10h). The suspensions of synchronous cells were cultivated in plate-parallel vessels (2200 ml)
illuminated from one side by incandescent lamps. Irradiance at
the surface of the culture vessels was approximately 180 Wm~ 2
of photosynthetically active radiation (PhAR) (400-720 nm).
Carbon dioxide concentration in the aerating gas mixture was
maintained between 1-5 and 3-0% ( v /v). The inorganic nutrient solution was that described by Zachleder & Setlfk (1982).
The culture vessels were immersed in a water bath at a constant
temperature of 30°C. Continuously diluted cultures were used
for experiments. Details of culture equipment and conditions
were the same as those described by Doucha (1979).
The experiments were carried out with synchronized cultures
under conditions of different irradiances and illumination
regimes or under conditions of nitrogen or phosphorus
starvation.
Assessment of commitment cutves
Samples were taken from synchronous cultures at one- or twohourly intervals and were incubated under aerated conditions in
the dark at 30°C. At the end of the cell cycle in the master
population, the percentages of binuclear daughter cells, fourcelled, eight-celled daughter coenobia, and undivided mother
cells were estimated. Also the content of RNA, DNA, and
protein was assayed in the dark-incubated samples. The values
obtained by the assay of samples were plotted against the time of
sampling. The curves obtained are termed the 'commitment'
curves. Note that in our earlier papers we have used the term
'induction' instead of 'commitment' (Setlfk et al. 1972; Zachleder et al. 1975). The significance of the term'commitment'for
various reproduction and synthetic processes has been
explained elsewhere (Setlfk & Zachleder, 1984).
Cell counting
Cells were counted in the BUrker counting chamber (produced
by Meopta, Czechoslovakia).
Staining of nuclei
Nuclei were fluorochromed with Acridine Orange and observed
through a fluorescent microscope using the method described
by Zachleder et al. (1974).
Chemicals
All chemicals used for the analyses were of analytical grade.
DNA, RNA, casein, and albumin for calibration assays were
obtained from Serva, Heidelberg, FRG. Other chemicals were
supplied by Lachema, Prague, Czechoslovakia.
Total nucleic acid assay
The procedure of Wanka (1962) as modified by Lukavsky et al.
(1973) was used for the extraction of the total nucleic acids. The
samples were centrifuged in 10-ml centrifuge tubes, which also
served for storage of the samples. The pellet of algal cells was
stored under 1 ml of ethanol at —20°C.
The algae were extracted 5 times with 0 2 M-perchloric acid in
532
V. Zachleder and I. SetUk
50% ethanol for 50min at 20°C, and 3 times with an ethanolether mixture (3:1) at 70°C for lOmin. Such pre-extracted
samples can be stored in ethanol. Total nucleic acids were
extracted and hydrolysed by 0-5 M-perchloric acid at 60°C for
5 h. After hydrolysis, concentrated perchloric acid was added to
achieve a final concentration of 1 M-perchloric acid in the
sample. Absorbance of total nucleic acids in the supernatant was
read at 260 nm.
DNA assay
The light-activated reaction of diphenylamine with hydrolysed
DNA, as described by Decallonne & Weyns (1976), was used
with the following modification (Zachleder, 1986). The
diphenylamine reagent (4% diphenylamine in glacial acetic
acid, w/v) was mixed with the samples of total nucleic acid
extracts in a ratio of 1:1 and the mixture in the test tubes was
illuminated from two sides with fluorescent lamps (Tesla Z,
40 W). The incident radiation from each side was 20Wm~ .
After 6h of illumination at 40°C, the difference between the
absorbance at 600 nm and at 700 nm was estimated.
RNA content calculation
The RNA content was calculated as a difference between the
total nucleic acid and DNA content.
Protein assay
The sediment remaining after the nucleic acid extraction was
used for protein content determination after Lowry et al.
(1951).
Measurement of irradiance
A non-selective device measuring photosynthetically active
radiation (400-720 nm) and calibrated in energy units as
described by Kubfn (1971) was used to measure radiation (in
Wm~ z ) at the surface of culture vessels (I,) and radiation
transmitted (I t ) through the suspensions. From these two
values the mean irradiance was calculated according to the
formula: I = (Ij-I t )/ln(I,/l t ).
Results
The course of mactvmolecular syntheses
RNA synthesis. In the course of the cell cycles of
Scenedesmus quadricauda, periods of fast RNA synthesis alternated with periods when this synthesis was
slowed down or even stopped. The curves recording the
increase in the amount of RNA per cell had, therefore, a
stepwise form (Fig. 1A). In a well-synchronized culture,
each step corresponded approximately to a doubling of
the previous RNA content in the cell.
As the synchrony of the cultures investigated was not
perfect, a certain fraction of the cells in the population
started a new wave of RNA synthesis before another
fraction had terminated the preceding one. Due to this
overlapping, the amounts of RNA during the periods of
its slow synthesis sometimes corresponded to RNA
doubling. At high growth rates, this overlapping tended
to smooth down the stepwise character of the RNA
increase in the population (Fig. 1A). Similar observations were made in the cases of protein and DNA
synthesis.
The rate of RNA synthesis increased with the growth
- 100
-50
0
10
15
Time (h)
Fig. 1. Course of RNA and DNA synthesis and their
committed values in synchronous cultures of Scenedesmus
quadricauda grown under optimal growth conditions (A) and
under conditions of slowed growth (B). I = 95 W m~ ,
D = 0-10h~'. The CO2 concentration in aerating mixture was
lowered to 0-5 %. Light and dark periods are indicated by
white and black strips at the top of each panel and are
separated by a vertical line. ( • ) RNA; (A) DNA;
(O) committed RNA; (A) committed DNA.
rate. The times required for successive doublings of
RNA in the population became shorter. Also the number
of waves, i.e. the number of RNA doublings in one cell
cycle, increased with the growth rate (compare Fig. 1A
and B).
Protein synthesis. The increase in protein content in
the course of the cell cycle had similar characteristics to
that of RNA. The curves recording the increase of
protein per cell run more or less in parallel to those for
RNA content but are delayed by an interval of several
hours (Fig. 2C).
DNA synthesis. DNA synthesis started later in the
cycle than RNA and protein synthesis. The replication
rounds were separated by a distinct time interval
(Fig. 1A). Similarly, as in the case of RNA and protein
synthesis, the separation of the steps on the curves
representing a doubling of DNA amount in the whole
population were less distinct at high growth rates
(Fig. 1A).
5
10
Time (h)
15
20
Fig. 2. Effect of darkening after different light intervals on
the course of RNA, DNA and protein synthesis and of
nuclear divisions in synchronous cultures of Scenedesmus
quadricauda. Culture conditions and labelling of light and
dark intervals are the same as described in Fig. 1A.
(O) RNA; (A) DNA; ( • ) protein; ( • ) nuclei.
The course of the committed macromolectilar syntheses
and of some cell cycle events
Subcultures were removed at one-hourly intervals from
the synchronously growing culture and were incubated in
the dark under the same temperature and aeration.
Analyses were performed on the samples taken from
these subcultures at the time at which the cell cycle in the
mother culture was terminated.
Committed RNA synthesis. RNA synthesis stopped
immediately upon darkening of the culture or a short time
thereafter (Fig. 2). In cells darkened early in the cell
cycle, no additional RNA synthesis occurred in the dark,
so that the committed values of RNA per cell were the
same as the actual values or lower because of a certain
amount of RNA degradation (Fig. 1A,B). In the cells
darkened later in the cell cycle, a commitment for cell
division was attained and in the released daughter cells,
the restoration of RNA synthesis occurred. The preceding decay in RNA amount per cell was more than
compensated for by this new RNA synthesis (Fig. 2B).
With increasing length of the light period the amount of
newly synthesized RNA increased (Fig. 2C) and committed amounts became increasingly higher than the actual
ones (Figs 1, 3).
Committed protein synthesis. Protein synthesis is
slowed down upon darkening the culture and stops
Contmls of DNA replications and mitosis
533
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Time (h)
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Fig. 3. Variation in content of committed RNA, DNA and
protein per cell and in a number of committed nuclei in
synchronous cultures of Scenedesinus quadricauda.
I = 80Wm- 2 , D = 0-08h-1. (•JRNA; (A) DNA;
(•) protein; (O) nuclei.
shortly thereafter (Figs 2, 3). In general, the relative
increase of committed protein synthesis was lower than
the committed RNA synthesis (Figs 2C, 3).
Committed DNA replication. The timing as well as the
rate of DNA replication did not seem to be affected by
light deprivation of the cells. However, the number of
DNA replication rounds in the dark was strictly controlled by the duration of the light period. The potential
of the cells to replicate DNA in darkened subcultures
increased in steps, i.e. for each DNA replication round a
few extra hours of growth in light were required (Figs 1,
2). The steps of the committed DNA replications were
usually well separated. The last replication round in
rapidly growing cultures occurred in newly released
daughter cells just at the end of the dark period. Such
cells entered the next light period with two genomes
(Figs 1, 3).
Committed nuclear and cellular division. The cells
increased their potential for nuclear division as the length
of the light period was increased. A certain period of
growth in the light was required to attain the ability to
trigger the next mitosis. Under optimal growth conditions, three successive nuclear divisions were committed during one cell cycle of Scenedesmus. It is characteristic for Scenedesmus that the first nuclear division is not
followed by cellular division under the growth conditions
used and cells committed to this nuclear division become
binuclear in the dark but do not divide. The commitment
for the second and third nuclear division is attained
simultaneously with the commitment for the corresponding cellular division and four- or eight-celled daughter
coenobia with uninuclear cells are released during the
dark periods. At high growth rates at the maximum
length of the light period, part of the cells in the
population are committed to the fourth nuclear division.
534
V. Zachleder and I. Setlfk
10
15
Time (h)
Fig. 4. Increase in a number of committed genomes, nuclei
and variation in a number of committed genomes per
committed nucleus in a synchronous culture of Scenedesmus
quadncauda under conditions of slowed growth. Culture
conditions were the same as described in Fig. IB.
(A) Committed genomes; (O) committed nuclei;
( + ) committed genomes per committed nucleus.
This division occurs at the end of the dark period in
newly released daughter cells and belongs, therefore, to
the next cell cycle. Under such conditions, binuclear
daughter cells enter the next light period.
The requirement for distinct periods of light to achieve
commitment to DNA replication and nuclear division
It can be seen in Fig. 4 that replication of DNA was
committed earlier than the corresponding nuclear division. This became manifested in cells darkened immediately after the commitment to DNA replication had been
attained: the corresponding step of nuclear division did
not occur and daughter cells released from such mother
cells had nuclei with a doubled DNA content. This is
shown by the maxima on the curve showing DNA
content per nucleus in darkened cells (Fig. 4). The
minima on the curve demonstrate, however, that a small
increase of the light interval makes nuclei competent to
divide and to reduce their DNA content to that corresponding to one genome.
Evidence that synthesis of a critical amount of RNA is
a prerequisite for commitment to replicate DNA
The curve illustrating the variance of the RNA to DNA
ratio in a synchronous culture had a bell-shaped form
modulated by two or three maxima. At the end of the cell
cycle, the ratio of RNA to DNA dropped to a certain
minimal value which varied between 10 and 12 (see the
ends of the curves in Fig. 5A). This minimal value was
always found in the freshly released daughter cells
irrespective of the growth conditions under which their
mother cells had been grown (Figs 3, 5, 6). Similarly,
10
15
Time (h)
20
Fig. 6. Effect of different growth rates on variation in RNA
and DNA ratio during the cell cycle of Scenedesmus
quadncauda. Values of the mean irradiance and dilution rate
for individual curves: curve 1, 108 Wm , 0-11 h ; curve 2,
90 W m , 0-08 h ~ ' ; curve 3, 60 W m , 0-045 h
10
Time (h)
Fig. 5. Variation in the ratio of RNA to DNA in
synchronous cultures of Scenedesmus quadncauda grown for
different time intervals in light. Culture conditions are the
same as described in Fig. 1A. A. Open symbols indicate the
values found in light-grown cells and closed symbols in cells
deprived of light. The time at which the populations were
put into the dark or illuminated again are marked by arrows.
B. Deconvolution of composite curve of RNA to DNA ratio
(curve 4) found in light-growing culture (see A) into three
simple curves (curves 1, 2, 3). The maxima on these curves
are assumed to signal the attainment of a critical amount of
RNA and minima the termination of a replication round.
when RNA synthesis was stopped by nitrogen starvation
the number of DNA replications became correspondingly
reduced to attain this minimal value (Fig. 7). It indicated
that the maximal amount of DNA synthesized in a cell is
dictated by the amount of RNA synthesized some time
before the replication of DNA was initiated (Figs 3, 7).
Consequently, in a fast-growing culture, the commitment
to the second DNA replication round had been attained
before the first DNA replication round was terminated.
The value of the RNA to DNA ratio then exceeded for
some time the expected double value of the minimal one
(Fig. 6). The resulting curve recording variance in RNA
to DNA ratio during the cell cycle is assumed to be
composed of several simple distribution curves (Fig. 5B).
This assumption was confirmed by the experiment in
10
15
Time (h)
Fig. 7. Variation in RNA and DNA content per cell in
control, light-deprived and nitrogen-starved synchronous
cultures of Scenedesmus quadncauda. Culture conditions
and labelling of light and dark period in control culture are
the same as in Fig. 1A. Times of light deprivation and of
nitrate withdrawal are marked by arrows. ( • ) RNA;
(O) RNA, light-deprived; (3) RNA, nitrate-starved;
(A) DNA; (A) DNA, light-deprived; (A) DNA, nitratestarved.
which the population of cells were deprived of light just at
the times when maxima in the ratio of RNA to DNA were
reached. The ensuing DNA replication reduced the ratio
of RNA to DNA to the minimal value (about 10) (Figs 2,
5A).
If the darkened cells were again illuminated after the
Controls of DNA replications and mitosis
535
10
10
15
Time (h)
15
Time (h)
Fig. 8. Variation in RNA, protein and DNA content per cell
in control and phosphate-starved cultures of Scenedesmus
quadncauda. Culture conditions are the same as described in
Fig. 1A. ( • ) RNA; (O) RNA, phosphate-starved;
( • ) protein; (D) protein, phosphate-starved; (A) DNA;
(A) DNA, phosphate-starved.
first replication round had been terminated, two wellseparated peaks recording the attaining threshold level of
RNA for the first and the second DNA replication round
were observed (Fig. 5A). In the same culture growing in
continuous light, these two curves overlapped (Fig. 5B,
curves 1, 2) and consequently only small bumps on the
resulting curve of RNA to DNA ratio could be observed
(Fig. 5B, curve 4; Fig. 6).
Under conditions of continuous light, consecutive
reproductive sequences were more or less overlapping
and, consequently, two or three waves of RNA synthesis
could occur before the first committed replication round
was triggered. In these cases, the ratio of RNA to DNA
could reach higher values than twice the initial value
(Fig. 6).
Evidence that pmtein synthesis is not sufficient to
commit cells to replicate DNA
A synchronous population of daughter cells was grown
from the beginning of the light period in a phosphate-free
medium. Under these conditions, RNA synthesis was
stopped early in the cell cycle, while protein synthesis
continued nearly to the end of the cell cycle (Fig. 8). The
number of DNA replications in this population attained a
value resulting again in the minimal ratio of RNA to
DNA content (about 10). However, the ratio of protein
to DNA increased to a higher value than in the control
culture and remained more than 60 % higher at the end of
536
V. Zachleder and I. Seth'k
20
25
Fig. 9. Variation in protein content per cell and number of
committed nuclei in control, light-deprived and nitrogenstarved synchronous cultures of Scenedesmus quadncauda.
Culture conditions and labelling of light and dark period in
control culture are the same as described in Fig. 1A. Times
of light deprivation and of nitrate withdrawal are marked by
arrows. ( • ) Protein; (D) protein, light-depnved;
(U) protein, nitrate-starved; ( • ) committed nuclei;
(O) committed nuclei, light-deprived; (3) committed nuclei,
nitrate-starved.
the cell cycle when all committed DNA replications had
been terminated (Fig. 8).
Evidence that synthesis of a critical amount ofptvtein
is a prerequisite for attaining the commitment to
individual nuclear division
The fact that daughter cells often had two genomes but
remained uninuclear also indicated that distinct controls
were involved in attaining commitments to DNA replication and to nuclear division. It is important that in this
situation RNA synthesis in the dark continued one step
further than protein synthesis (Fig. 2). Under conditions
of nutrient starvation, when RNA synthesis was stopped
while protein synthesis continued, the number of committed nuclei was always found to be proportional to the
content of protein (Figs 2, 9). This result indicated that
protein, and not RNA, amount was a prerequisite for
attaining commitment to divide nuclei.
Discussion
We have shown that the cells of Scenedesmus quadncauda require a longer period of growth for commitment
to nuclear division than for commitment to DNA replication. We suggest that the two commitments are controlled by two separate and distinct processes linked
directly to the growth rate of the cell. Ribosomal rRNA
and/or protein syntheses were considered to represent
these growth processes controlling the onset of reproductive processes during the cell cycle. However, three
contradictory interpretations can be found in the recent
literature concerning the control of reproductive processes:
(1) RNA and not protein accumulation to a certain
threshold value is a prerequisite for the cell to enter DNA
replication. This conclusion came firstly from experiments by Lieberman et al. (1963) and Baserga et al.
(1965), who prevented DNA replication by actinomycin
A, the specific inhibitor of rRNA synthesis. Recently, a
large number of papers have been published to confirm
that only cells with an rRNA amount above a certain
threshold level are able to initiate DNA replication
(Johnston & Singer, 1978; Darzynkiewicz et al. 1979a,b,
1980; Adam et al. 1983; Seuwen & Adam, 1983; Fujikawa-Yamamoto, 1982, 1983). Furthermore, the inhibition of rRNA synthesis led to arrest of budding yeast at
"start" even if bulk protein synthesis had not been
affected (Johnston & Singer, 1978; Singer & Johnston,
1979, 1981; Bedard et al. 1980). The present findings in
Scenedesmus quadricauda are quite consistent with all
the above papers.
(2) Protein and not RNA accumulation to a certain
threshold value is a prerequisite for the cell to enter both
DNA replication and mitosis. Alberghina and her coworkers (Alberghina, 1975; Alberghina & Sturani, 1981;
Popolo et al. 1982; Martegani & Alberghina, 1984)
suggested a model in which a critical level of protein and
not rRNA is required for entry into S phase. Alberghina's
concept is not, however, supported by other authors
(Johnston & Singer, 1978; Singer & Johnston, 1981;
Bedard et al. 1980) and it is also in discrepancy with the
present results.
These authors used O-phenanthroline and 8-hydroxyquinoline to stop rRNA synthesis. Popolo et al. (1982)
criticized their interpretation because the cell cycle arrest
might thus be due to a lack of synthesis of specific
proteins on newly synthesized mRNA. However, Popolo
et al. (1982) based their hypothesis on a similar experimental approach. They inhibited protein synthesis by
low doses of cycloheximide, while RNA synthesis continued. In general, all experiments using inhibitors are
open to criticism because side effects cannot be excluded.
Particularly, the use of protein synthesis inhibitors seems
to be alarming. Disturbing of enzyme systems or the
syntheses of other proteins involved in the initiation of
DNA replication or other reproductive processes can
occur even at the low doses of inhibitors applied.
More physiological treatments (deprivation of light
energy or withdrawal of exogenous supplies of phosphorus and nitrogen) were used in the present study to
uncouple RNA and protein synthesis. Surprisingly, such
non-specific treatments as phosphorus or nitrogen starvation were found to cause the specific inhibition of RNA
synthesis (Parenti et al. 1971; Lien & Knutsen, 1973;
Zachleder et al. 1988; Ballin et al. 1988). However,
protein synthesis continued undisturbed for the whole
cell cycle in the case of phosphorus starvation or for
several hours after nitrogen withdrawal. It was also
proved that the course of the reproductive processes
themselves was not affected by both phosphorus and
nitrogen starvation and dark treatment (Setlfk et al. 1972;
Zachleder et al. 1975, 1988).
(3) Neither RNA nor protein amount increase is
required for DNA replication and mitosis. Several papers
have been recently published, which tend to cast doubt
on the role of accumulation of both ribosomal RNA and
protein (R0nning & Seglen, 1982; R0nning & Lindmo,
1983; R0nning & Petersen, 1984; Grummt et al. 1979;
Mercer et al. 1984; Zetterberg & Engstrom, 1983). All
these papers dealt with mammalian cells and the authors
found that G\ cells were able to proceed under certain
conditions through DNA replication and cell proliferation even if RNA or protein synthesis were stopped.
These findings are in discrepancy with those of Darzenkiewicz et al. (1980) who proved that only those mammalian cells that had an RNA amount above a threshold
level were able to enter S phase.
It is well known that the reproductive processes of cells
in a wide range of organisms can be triggered and
terminated in the absence of concomitant growth processes. A cleavage of amphibian oocytes can serve as an
extreme example. Similarly, in algal cells, the multiple
reproductive processes can be triggered and terminated
during dark periods without any concomitant increase in
net RNA or protein content (for review see Setlfk &
Zachleder, 1984).
The simple explanation for this phenomenon is that
the multiple growth without any concomitant reproductive processes is realized before both the onset of
cleavage of these amphibian oocytes and the initiation of
reproductive processes in algae. The same rules were
proved to be valid in bacterial cells (Helmstetter, 1974) as
well as in mammalian cells lacking a Gi phase (Liskay et
al. 1979, 1980).
However, even in organisms with a Gi phase the
growth required for a given cell cycle is performed
partially or completely during the preceding cell cycle
(during S, G 2 and M phases) (Cooper, 1979, 1984; Setlfk
& Zachleder, 1984). This growth can be sometimes
sufficient for attaining commitment to enter S phase or
even for the onset of cellular division. We, therefore,
assume that the mammalian cells that were able to
perform DNA replication or cellular division without
RNA or protein synthesis in a given cell cycle attained the
threshold levels of RNA and protein during growth in the
preceding cell cycle.
We would like to stress that the points where the
threshold levels of RNA or protein have been attained can
be relatively well separated in time from the onset of
DNA replication or mitosis itself. Therefore, all papers
trying to find a regulatory role of growth processes at the
very beginning of DNA replication of mitosis (e.g.
Craigie & Cavalier-Smith, 1982; Fantes & Nurse, 1978)
argue from a misinterpretation, because the amount of
RNA or protein was determined by growth that followed
the attainment of critical points, and it has nothing to do
with the regulation of those reproductive events that take
place at the same time.
Contmls of DNA replications and mitosis
537
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(Received 2 August I9SS - Accepted 26 August 19SS)
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