J. Cell Sci. 3, 483-491 (1968)
483
Printed in Great Britain
THE SYNTHESIS OF DNA THROUGH THE CELL
CYCLE OF AMOEBA PROTEUS
M. J. ORD*
Department of Zoology, The University, Southampton, England
SUMMARY
DNA synthesis has been investigated in Amoeba proteus by pulse-labelling cells of known
age with [3H]thymidine. Ninety per cent of the DNA so labelled was synthesized during the
first quarter of the cell cycle. Synthesis was in two peaks: the first occurring between 0-5 and 4 h
after division and the second at about 10 h. All cells labelled at both peaks. The authenticity
of the second peak was proved statistically.
Considerable variation was observed among amoebae of similar age. In experiments in which
daughter amoebae underwent different treatments, differences in the rates of incorporation of
[3H]thymidine due to differences in the nutrition of the cells were found to be a predominant
cause of variation. Heavy feeding reduced labelling; starvation increased labelling while at the
same time reducing variability.
INTRODUCTION
The synthesis of DNA during the cell cycle of Amoeba proteus has been studied
using pulse labelling with tritiated thymidine. Amoebae are particularly suitable for
studies of this kind because accurately aged cells can be obtained at any time throughout the cell cycle. In the following experiments the limits of the 5 period have been
determined and the validity of a second peak appearing about 10 h after division
established. Labelling of the nuclei of amoebae of similar age varied considerably and
an attempt has been made to ascertain some of the factors responsible for this variation.
MATERIAL AND METHODS
Culture technique
Amoeba proteus, strain Pj^Xd^ (Ord, 1968), cultured at 18-200 C using the tetrahymena-feeding technique of Prescott & James (1955), were used throughout the
experiments. At this temperature this strain has a cell cycle of 48-54 h, mitosis taking
30-35 min. The moment of separation into two daughter cells has been taken as o h.
In an experiment covering the whole of the cell cycle and requiring large numbers of
amoebae, accuracy of ageing was ±10 min; when more detailed investigations were
made of the early part of the cell cycle, particularly when establishing whether or not
a G1 period existed, accuracy of ageing was ± 2 min; that is, o-1 % of the cell cycle.
Binucleate amoebae were obtained by placing division spheres in Chalkley's medium
adjusted to pH 4 with 1 N HC1. Daughter amoebae of unequal size were obtained
• Member of the Toxicology Research Unit, Medical Research Council Laboratories, Carshalton, Surrey.
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M. J. Ord
either by sucking division spheres up and down a narrow pipette or by chopping off
half the cytoplasm from one daughter immediately after division. Exposure of amoebae
to unlabelled thymidine, in concentrations of up to 5 x io~3 M, for several days did
not change the length of the cell cycle and it was concluded that the exposures of 1 h
or less used in these experiments would not significantly affect the synthesis of DNA.
Thymidine labelling
For each experiment 200-300 division spheres were removed from mass cultures
of amoebae and kept after division in small watch glasses. Groups of 20-30 amoebae
were removed for labelling at intervals of 1, 2 or 4 h. Cells were exposed to pH]thymidine (0-2-1 me/ml; sp.act. 5 c/raM) for 15, 30 or 60 min. After exposure the amoebae
were washed free of the pHJthymidine in Chalkley's medium before chasing for 2 h in
unlabelled thymidine, 5 x io~3 M. Trial runs showed that once the pHJthymidine had
been incorporated into the DNA it was stable and could not be washed out; if sufficiently heavy, label could be observed in the nuclei of the progeny of an amoeba
exposed to [3H]thymidine eight cell cycles or more after exposure; that is, when the
original labelled cell had divided to form a clone of 250 or more cells.
Radioautographs
Amoebae were placed on slides and gently squashed beneath a coverslip with a
drop of 20 % acetic acid. The coverslip was removed by freezing over solid CO2 and the
amoebae further fixed in acetic acid/ethanol (1:3). Slides were air-dried after rinsing
in absolute and 95 % alcohol. A series of amoebae, usually from 6 to 8 different agegroups, were arranged in rows on each slide so that in most cases it was possible to
compare labelling for a number of age-groups using amoebae fixed on the same slide
and so subjected to identical treatment. Slides were coated with Gevaert 7:15 emulsion, exposed for 3 weeks, and developed with Kodak D 19 B. Before mounting they
were dipped in a dilute solution of light green to give the amoebae a faint stain. Slides
were made in triplicate.
Grain counts were made under oil immersion at a magnification of x 900 using an
eyepiece graticule. Because a thin layer of cytoplasm intervenes between the nucleus
and the emulsion, cytoplasmic labelling and background counts were taken into
account when evaluating nuclear labelling by subtracting the number of grains over
an equivalent area of cytoplasm from the number counted over the nucleus.
For enzymic digestion of DNA, slides were immersed for 3 h at 350 C in a 0-5 mg/ml
solution of deoxyribonuclease made up in \ strength Mcllvaine's buffer (pH 7) with
a trace of MgSO4 added.
RESULTS
The DNA synthesis period
Labelling with pHJthymidine showed DNA synthesis to be taking place from
30 min after division in all amoebae. Nuclear grain counts of amoebae of different ages
gave two peaks: one at 0-5-4 n ^ieT division and a second small peak between Q and 13 h.
1500
48s
1500 A
B
1000
500
1000
ki{
500
1
, VI-.-.-.
2
4
6
8 10 12 14 16 18 20
0
2
4
6
8 10 12 14 16 18 20
2
4
6
8 10 12 14 16 18 20
0
2
4
6
10 12 14 16 18 20
1500 -
1000 -
500
Age of amoebae (h)
Fig. 1. Curves obtained from four experiments labelling synchronized amoebae during
the first part of the cell cycle when 90 % of the DNA is synthesized. Due to.the
variation in labelling between individual amoebae each point on the curves represents
the geometric mean obtained from counts over a number of nuclei. Fiducial limits have
been included to indicate the expected range. Each nuclear count was corrected for
cytoplasmic background as indicated (see Methods). Amoebae of ages from o to 20 h
were exposed to [ 3 H]thymidine: A, 0-2 me/ml for 1 h; each point derived from counts
on 10-15 amoebae. B, 1 me/ml for 30 min; each point derived from counts on
10 amoebae. C, 025 me/ml for 1 h; each point derived from counts on 8-22 amoebae.
D, 0-25 me/ml for 1 h; each point derived from counts on 15 amoebae. The effect of
access to food prior to division is shown in the timing of the first peak in labelling:
while in experiment A division spheres were removed from a heavily fed culture, and
in B and D from cultures with adequate food, in experiment C division spheres were
removed from a culture where almost all available food had been consumed. The timing
of the second smaller peak is also influenced by food intake, particularly intake
following division, as seen in D, where only a limited amount of food was given to the
young divided amoebae.
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M. J. Ord
A marked dip occurred between these peaks, the grain counts at this period frequently
being little above that of background. The first, or major peak, represented approximately 75 % of the total labelling occurring during the cell cycle, while the second,
or minor, peak represented a further 15 %; the period from 14 to 48 h accounted for
the residual 10 % of counts.
The authenticity of the second peak was tested in all four experiments shown in
Fig. 1. In the region of the second peak the grain counts are distributed statistically
in a way consistent with a log-normal distribution. The mean logarithms of the counts
at the peaks and at the time immediately before were shown to be different in every
instance (Fig. 1, A and D, P < o-oi; B, P < 0-002; C, P < o-ooi). Over this period
the grain counts showed greater variability than at other times, probably because not
all amoebae are exactly in phase and the time intervals are short.
The second peak could not be accounted for by heavy [3H] incorporated by a few
slow amoebae. All nuclei are labelled at the peaks (Fig. 2).
Variability of grain counts
The grain counts for each age-group were very variable (Fig. 2). They were not
normally distributed, but the counts were always consistent with a log-normal distribution, and the log-normal distribution has been assumed to be correct for statistical
analysis (Heath, 1967).
Three possible causes of this variation were investigated.
(1) Non-reproducibility of the technique, for example, in the degree of squashing. This
was not the major source of variation, as shown when single amoebae were heavily
labelled, and radioautographs made of their progeny 5, 6, 7 and 8 cell cycles after
labelling. Amoebae being highly polyploid, distribution of label at division is almost
equal and all the progeny should contain approximately the same amount of label.
In fact, the standard deviations on the progeny of the 5th and 6th cell cycles were
less than 20 % of the mean. This compares with variations 2-5 times greater within
groups of amoebae of the same age when pulse-labelled directly with [3H]thymidine.
(2) Confinement of DNA synthesis to specific periods. When amoebae of the same age
were exposed to [3H]thymidine for 0-25, 0-5, 0-75, i, 2, 3, 4 and 5 h, there was a
continual increase of labelling with time; but variations between amoebae were similar
at all times. Due to the heaviness of labelling with long exposures these observations
were made by eye, not by grain counting.
(3) Differences in rates of incorporation. Individual variations in the rates of incorporation of [3H]thymidine were the main source of variance; these differences were
chiefly dependent on the nutrition of the cell. In what follows variances were calculated for log-normal distributions, so differences between mean log values represent
ratios of values (Heath, 1967).
The nuclei of binucleate amoebae were very similarly labelled; the s.D. range of the
ratio between pairs was only 0-96-1-04. Nuclei of daughter pairs treated identically
after fission differed significantly more (s.D. range = 0-90-1-11, P < o-oi (F test,
Snedecor, 1946)) but the differences were still small. However, when daughters were
paired, and one of each pair was given ample access to food and the other was starved,
DNA synthesis in Amoeba proteus
487
two effects appeared. The nuclei of the starved members contained 1-7 times more
label than those of their fed sisters (t test, P < o-ooi), and this ratio was significantly
more variable than in sisters treated the same way (S.D. range = 1-46-1-93, F test,
P < o-oi).
These results indicated that feeding reduced labelling, and that amongst amoebae
with access to food a considerable part of the variation in labelling could be attributed
to differences in food intake. (Microscopic inspection of food vacuoles in amoebae
from the same culture dishes confirmed that food intake was variable.) Statistically
8
10
12
14
16
18
20
22
24
26
28
46
48
Age of amoebae (h)
Fig. 2. A diagrammatic presentation of the proportion of amoebae having counts
per nucleus of 100—200, 200—500, 500—1000, 1000 or more grains after 1 h exposure
to [3H]thymidine, 0-25 me/ml at intervals through the cell cycle. The diagram was
derived using over 500 amoebae. Counts represent the nuclear labelling after subtraction of background due to cytoplasmic labelling. Grain counts of 100 or more after
subtraction of cytoplasmic background were considered significant. Counts over a
similar area of cytoplasm reached this level in approximately 10 % of the amoebae.
this interpretation was confirmed by taking all the data for fasted amoebae and estimating their variance (s.D. range = 0-77-1-29, D.F. = 14) and comparing them with those
of fed amoebae over the same time-range (s.D. range = 0-58-1-71, D.F. = 40). The
variability of the fasted amoebae was significantly less (F test, P <^ o-oi) than of those
with access to food. It is obvious, however, from the quite big variation in fasted
amoebae that access to food is not the only factor.
Uptake was also influenced by the size of the nucleus, which is variable in amoebae
of similar age. When large size differences between nuclei were induced in daughter
pairs (see Methods) and the two daughters of each pair treated the same way, labelling
in the larger nucleus was always much greater. The labelling proved roughly proportional to the area of the nucleus, so that within pairs, with areas differing by about
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M. J. Ord
twofold, the counts/area in the larger nuclei were 1-15 times (S.E.M. range 1-12-1-19)
those in the smaller. However, the variations in nuclear size of amoebae of similar age
were generally small compared with the differences induced in the daughter pairs of
this experiment. They were not great enough to account for the whole variation in the
nuclei of starved amoebae where the nutritional factor was eliminated.
Not only did differences in feeding cause differences in intensity of label but also
shifts in the two peaks of DNA synthesis. For example, in Expt. A (Fig. 1), in which
amoebae were removed as division spheres from a heavily fed mass culture, labelling
was already maximal at 30 min, while in Expt. C, in which amoebae were removed as
division spheres from a mass culture with little food, labelling was not maximal until
4 h. The second peak of DNA synthesis occurred at 10 h in Expts. A, B and C (Fig. 1),
in which young divided amoebae were kept prior to labelling in a concentrated suspension of tetrahymena, but at 12 h in Expt. D in which amoebae were kept after division
in a dilute suspension of tetrahymena.
Synthesis during mitosis
In a number of nuclei DNA synthesis had already begun before the completion of
division. Amoebae labelling with [3H]thymidine at mitosis did so chiefly during early
prophase; little labelling occurred towards the end of division or immediately following the separation of an amoeba into two daughter cells. In one experiment using
division spheres and newly separated daughter cells the nuclear counts averaged:
350 per nucleus when the exposure to pHJthymidine (1 me/ml) was during the first
15 min of division; 150 per nucleus when the exposure was during the last 10 min of
division and the 5 min following division; and 900 per nucleus when the exposure was
during the 10-25 m m following separation into two daughter cells.
Cytoplasmic labelling
Tritiated thymidine labels both the nucleus and the cytoplasm of Amoeba proteus
(Plaut & Sagan, 1958; Wolstenholme & Plaut, 1964). Although in these experiments
the number of grains over an equivalent area of cytoplasm during the first quarter of
the cell cycle generally represented less than 10% of the total count, cytoplasmic
labelling accounted for most of the grains over the nuclei during the last half of the
cell cycle, when there was little nuclear DNA synthesis. In many cases it was possible
to identify cytoplasmic labelling, for grains due to nuclear labelling were evenly distributed over the nucleus whether labelling was dense or light, whereas grains due to
cytoplasmic labelling were frequently clumped. Deoxyribonuclease removed both
nuclear and cytoplasmic labelling.
DISCUSSION
Grain counts over nuclei after exposure to [3H]thymidine varied considerably and
according to a log-normal distribution. Some variation was caused by the nonreproducibility of the technique, but the greatest variation was due to differences in
the incorporation of pETlthymidine among individual amoebae. Though differences
DNA synthesis in Amoeba proteus
489
in nuclear size probably accounted for small differences in incorporation, differences
in the nutritional state of individual amoebae proved the main source of variance.
Variations were markedly less in starved amoebae than in those with access to food.
This suggests that when there is a continuous supply of food in a culture there is no
synchrony in the feeding habits of the amoebae; some may be well fed while others
are searching for food and in a metabolic state approaching starvation. This was confirmed by observation of the food vacuoles.
Though starvation could reduce variability it was not desirable to starve the amoebae,
as this was likely to slow the processes under investigation. Consequently most experiments were carried out with 'fed' amoebae, and large numbers were used to get
significant results.
The nuclei of starved amoebae took up more label than did the nuclei of fed
amoebae. As in starvation the label was presumably less diluted with endogenous
thymidine, a greater uptake of labelled thymidine could even have corresponded to a
smaller incorporation of thymidine into DNA—that is, to slower DNA synthesis.
In the strain of Amoeba proteus used in these experiments, 90 % of the DNA was
synthesized during the first quarter of the cell cycle. Goldstein & Prescott (1967) found
in their strain of Amoeba proteus that all labelling took place in the first 3-6 h following
division. The present results differ—only 75 % of the labelling took place in the first
6 h. A second, shorter period of DNA synthesis occurred 9-13 h after division. Thus
there was a very rapid DNA synthesis at the beginning of the cell cycle, during which
all amoebae incorporated [3H]thymidine, a pause during which little incorporation
occurred in any of the amoebae, followed by the synthesis of a further 15 % of the
DNA when all amoebae again incorporated pHJthymidine.
The grain count was especially variable just before and during the second peak. As
partial starvation can delay the second peak (Fig. 1D), in amoebae even from the
same culture the peaks may be asynchronous (see above), so big variations between
individual amoebae are to be expected.
The results show that DNA synthesis occurs in two waves. Howard & Dewey (1961)
suggested this to account for results on the labelling of nuclei in the root cells of Vicia
fabia seedlings; but, as they could not label their cells at known stages in the cell cycle,
they were unable to disprove the possibility that the cells were in two classes, one of
which labelled DNA ahead of the other. Two peaks of labelling have also been observed
in tissue culture cells (Newton & Wildy, 1959).
The existence of two peaks is consistent with asynchronous duplication of chromosomes. This may occur when different parts of chromosomes duplicate at different
stages of the S period—for example, the chromosome arms in the root-tip cells of
Tradescantia paludosa (Wilber, 1961)—or when different chromosomes duplicate at
different times—for example, the sex chromosomes as observed in mammalian cells
in tissue culture (Taylor, i960; Grumbach, Morishima & Taylor, 1963). Due to the
smallness of the chromosomes (about 1 fi) in Amoeba and the very large number, no
attempt was made to determine by observation whether asynchronous duplication of
chromosomes occurred.
No G± period was found in these amoebae. All cells were incorporating [3H]thymid-
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M. J. Ord
ine by 30 min after division; some cells even incorporated [3H]thymidine during
mitosis. Incorporation at division was chiefly during early prophase; during the later
stages of division and immediately after cytokinesis there was little incorporation.
The absence of a G± period has also been observed in fission yeast (Bostock, Donachie,
Masters & Mitchison, 1966), grasshopper neuroblasts (Gaulden, 1956), the slime
mould Physarum (Braun, Mittermayer & Rusch, 1965) and the micronucleus of
Tetrahymena (McDonald, 1962).
The importance of nutrition in studies of DNA synthesis is obvious from the
preceding discussion. As the DNA synthesis period for Amoeba proteus begins at or
just after division, the control of feeding before division is as important as that of the
young daughter amoebae between division and labelling. Difficulties may arise when
comparing normal cells with cells which have undergone treatment with chemicals or
irradiation, since these treatments may themselves interfere with the nutrition of the
cell. It would appear that while labelling with pHJthymidine can be used as an indication of DNA synthesis, the amount of label may not always be an accurate measure of
the rate of synthesis.
It has been reported that cells may be damaged by the /?-radiation from incorporation of pHJthymidine (Trowell, 1966). The doses of pHJthymidine used in these
experiments are much higher than those recommended for use on tissue culture cells.
Amoebae are highly resistant to irradiation, doses of no000 r being needed to cause
lethal damage (Ord & Danielli, 1956). In these experiments the [3H]thymidine, as long
as exposure was limited to 1 h, did not appear to affect the amoebae. However, when
amoebae were exposed to doses of 1 me/ml for 12 h or more immediately following
division, so that very large quantities of pHJthymidine were incorporated into their
DNA, the division following exposure was generally delayed; even so, no abnormalities
showed in the offspring of such amoebae.
The author is greatly indebted to Dr D. F. Heath for the statistical analyses made in this
paper. Her thanks are also due to Mr R. Legg for technical assistance with radioautographs.
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{Received 19 January 1968)