J. Cell Sci. aa, 521-530 (1976)
Printed in Great Britain
521
TIMING OF NUCLEOLAR DNA REPLICATION
IN AMOEBA PROTEUS
I. MINASSIAN AND L. G. E. BELL
Department of Biology, Medical and Biological Sciences Building,
University of Southampton, Southampton SOg 3 TU, England
SUMMARY
Light- and electron-microscope autoradiography have been used to follow the incorporation
of [3H]thymidine at different stages during the interphase of synchronously growing populations
of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e.
DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser
degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was
found to be late replicating, i.e. it replicated during the Ga phase.
INTRODUCTION
Although there is general agreement between different authors concerning the
existence of DNA associated with the nucleoli (Busch & Smetana, 1970; Smetana &
Busch, 1974), the timing of nucleolar DNA synthesis during interphase has been a
matter of controversy for a number of years. Approximately half the reports show
nucleolar DNA synthesis occurring during all or some part of S-phase, the remainder
show it occurring during Gly G2 or the whole of the interphase. For example, Nash &
Plout (1965) and Balazs & Schildkrout (1971), looking at S-phase as a whole, reported
that nucleolar DNA synthesis occurred with the rest of the nuclear DNA. But when
the 5-phase was subdivided by other workers into 2 or more intervals, no common
pattern was found for its timing: in Chinese hamster cells the nucleolar DNA replicated
during an early portion of 5-phase (Stambrook, 1974), whereas in a rat kangaroo cell
line (Giacomoni & Finkel, 1972) the ribosomal cistrons replicated at the end of the
5-phase.
Some workers suggest an independence between the biosynthesis of nucleolar and
chromosomal DNA, i.e. they found no correlation between the timing of nucleolar
DNA synthesis and the major chromosomal DNA synthesis. For example, in rat
fibroblasts (Harris, 1959) nucleolar DNA synthesis preceded the main nuclear DNAsynthetic period, while in adult rat liver cells (Wintzerith et al. 1975) it was shown to
replicate in the absence of replication by the rest of the nuclear DNA. Using the
multinucleate slime mould Physarumpolycephalutn where many thousands of accurately
synchronized nuclei could be examined, Ryser, Fakan & Braun (1973) found that 'the
genes for the ribosomal RNA replicated to a large extent in the G2-phase'. In a study
of [3H]thymidine incorporation of Amoeba proteus, Ord (1968) showed 90% of the
nuclear incorporation taking place during the first fifth of the cell cycle, referred to as
522
/ . Minassian and L. G. E. Bell
5-phase. However, she also found a persistent, though low, level of nuclear incorporation during the G2-phase. Since this work used a 'squashed whole cell technique'
for autoradiography no prediction could be made as to whether the G2 nuclear labelling
was chromosomal or nucleolar.
In the present study using sections of labelled amoebae, the position of the latereplicating DNA has been visualized, with a clear distinction between labelling over
the peripherally located nucleoli and/or over the central chromatin region of the
nucleus. By labelling cells, synchronized by selecting the detached mitotic cells, with
[3H]thymidine at different intervals during interphase, differences in the degree of
[3H]thymidine incorporation for the 2 regions have been studied.
MATERIALS AND METHODS
Culture
Cultures of Amoeba proteus, strain PDaX10, were maintained at 18-20 °C using the Tetrahymena feeding technique of Prescott & James (1955).
A. proteus, strain PDaXti, was shown in earlier studies (Ord, 1968) to have a cell cycle of
48-54 h: DNA synthesis occupied the first fifth of the cell cycle (with no significant G^, the
long G,-phase lasted some 38-44 h, mitosis took approximately 30-35 min. The strain PDaXla
used during this investigation had a slightly longer G,-phase, making the length of the cell
cycle approximately 58-60 h. In this work the long G2-phase has been subdivided into early
G t (13-28 h), mid G, (28-43 h) and late G% (43 h until mitosis).
Cells were synchronized by selecting the mitotic cells from mass cultures. After division cells
of the same age were grouped together and cultured normally until exposure to the [3H]thymidine.
Thymidinc labelling
Cells of known age were labelled with [Af«-!H]thymidine, specific activity 20 Ci/mM (purchased from the Radio Chemical Centre, Amersham) at a concentration of 0-5 mCi/ml for
exposure periods of 6 h. Four labelling intervals were used: 0-5-6-5 h, 15-21 h, 32-38 h, and
43-49 h. These periods were taken as being representative of S, early G2, mid G2 and late Ga
phases respectively. After exposure the cells were washed free of radioactive material, chased
with cold thymidine (concentration 12 x io~4 mg/ml) for 1 h and fixed.
Preparation for light- and electron-microscope autoradiography
Cells were fixed in a 1 % solution of osmium tetroxide in. 01 M cacodylate buffer pH 6-2 from.
60 to 90 min. Following fixation cells were washed with distilled water then dehydrated in a
graded series of ethanols, immersed in propylene oxide and embedded in Araldite. Sections of
1 /im thickness were cut from the blocks of labelled cells, put on clean slides and processed for
light-microscope autoradiography according to Prescott's (1964) dipping technique. The slides
were left under Ilford K5 nuclear emulsion (Ilford Ltd., England) for 3-4 weeks, developed,
then stained with 0-25 % toluidine blue made up with 0-25% borax in distilled water.
Sections of 60-80 nm were cut from blocks of labelled cells for the use of EM-ARG. These
were placed on copper grids and mounted on top of glass rods for application of emulsion by
the loop technique (Stevens, 1966). Ilford L4 nuclear emulsion diluted 2:3 with distilled water
was applied with a wire loop in the dark. Grids were left under emulsion at room temperature
for 8-10 weeks, then developed. After developing they were stained with uranyl acetate and
lead citrate and examined with a Philips 300 electron microscope. At the end of preparation
for EM-ARG, examination was made of serial sections to eliminate any error in interpretation
of nucleolar-localized grains which could arise due to the irregular shape of the amoeba nucleus.
In total, autoradiographs of more than 100 S and G, cells were examined by light microscopy
and a further 100 S and G, by EM-ARG.
Timing of nucleolar DNA replication
523
Enzymic digestion
Labelled amoebae were burst by drawing into, and blowing out of, a narrow-aperture pipette.
The nuclei were collected as they floated free and were placed on slides and fixed for 3 min in
acetic ethanol (113). After fixation the slides were rinsed and covered with 05 mg/ml of pancreatic deoxyribonuclease (BDH Biochemicals) made up with one-quarter strength Mcllvain's
buffer (pH 7). The slides were incubated foi 3 h at 35 CC, then rinsed, dehydrated and subsequently processed for light-microscope autoradiography as described above.
RESULTS
Differences in the localization of \?H]thymidine incorporation at S and at G 2
Autoradiographic study of cells treated with [3H]thymidine during the S-phase. Light-
microscope autoradiographic observations of synchronized cells exposed to [3H]thymidine for 6 h starting 30 min after mitosis revealed that the maximum incorporation occurred in the central region of the nucleus. The nucleoli — small and very
numerous bodies located at the marginal zones of the nucleus - showed very little
incorporation of [3H]thymidine. Silver grain counts performed on these cells showed
that most of the grains were localized over the central region; only a few were found
in the nucleolar region. The EM-ARG study confirmed the light-microscope results
and demonstrated very clearly the high concentration of grains in the nuclear central
region (Fig. 1 A, B). The few grains found on the nucleoli were over the perinucleolar
region.
There was no incorporation of [3H]thymidine by the helical structures of the amoeba
nucleus: structures believed to represent RNA and protein packaged together for
transportation to the cytoplasm (Stevens, 1967; Minassian & Bell, 1976). This is in
agreement with results of previous investigations suggesting that the helices are not
DNA-containing structures (Stevens, 1967; Wise, Stevens & Prescott, 1972).
Autoradiographic study of cells treated with \*H~\thymidine during the G2-phase. Light-
microscope ARGs of i-/tm sections of amoebae exposed to [3H]thymidine at the
periods 15-21, 32-38 and 43-49 h. showed some incorporation of tritiated thymidine
by the nucleus throughout G2. The rate of incorporation was much lower than that
of an S-phase nucleus. The grains were mainly localized on the peripherally located
nucleolar region. Only a few were observed in the central region of the nucleus.
Examination of labelled mid-G2 nuclei at the electron-microscope level confirmed that
the grain density was high in the region of the peripherally located nucleoli and low
in the central region of the nucleus. There was a preferential labelling of the marginal
zones of the nucleoli, the perinucleolar region (Fig. 2 A, B).
These experiments attempted to cover the whole of G2 by exposing cells at 3 different
intervals. Though the same grain distribution pattern, i.e. localization over the
nucleoli with few grains over the central region, was found in each case, a detectable
increase in thymidine incorporation was observed in cells treated during mid G2. Such
an increase could indicate greater nucleolar activity in the mid G2-phase. However,
fluctuations would be expected in the endogenous DNA precursor pools, in the
quantity and/or activity of DNA replicating enzymes as the cell passed from the
524
/. Minassian and L. G. E. Bell
•
«
1 A
Timing of nucleolar DNA replication
525
5-phase into the G2-phase, and again as it approached mitosis. Such fluctuations
would account for differences in the level of pHJthymidine incorporation noted for
early, mid and late G2-phases. Since it was impossible in this type of experiment to
estimate the sizes of the endogenous DNA precursor pools, or to establish the level
of activity of DNA replicating enzymes, no attempt has been made to quantify the
pHJthymidine incorporation during these 3 different G2-periods.
Quantitive differences in ^K\thymidine incorporation within the nucleus
Having established by light and EM autoradiography that there was a very different
localization of grains in the nucleus during 5-phase and G2, it was considered reasonable to attempt to compare the levels of [3H]thymidine incorporated over nucleoli and
central nuclear region during S and G2 by grain counting. For this purpose the second
G2-phase, i.e. mid G2, was chosen as representative of G2 since it was furthest removed
both from changes taking place as the cell moved from S into G2) or as the cell
approached division. Grain counts over 5 and G2 nuclei produced the following
results: (1) 5-phase nuclei had a total average grain count 4 times greater than G2phase nuclei. (2) In 5-phase nuclei 95 % of the grains were located in the central main
chromatin region of the nucleus, only 5 % being found in the nucleolar region. And
(3) in G2-phase nuclei 90 % of the grains were located peripherally, i.e. over or around
the nucleoli, the remaining 10% being scattered in the central region of the nuclei.
See Table 1.
Cytoplasmic labelling
Observation of the cytoplasm using light microscope ARGs revealed an increase in
cytoplasmic labelling coincident with the period when nucleolar DNA was replicating.
Thus grain counts showed that pH]thymidine incorporation was 3 times higher in
G2-phase cytoplasm than in 5-phase cytoplasm (Table 1). It was impossible to identify
the location within the cytoplasm of pHJthymidine incorporation using light-microscope ARG, since the resolution does not allow identification of structures in the size
range of mitochondria. However, previous EM studies on A.proteus (Minassian, 1974)
had already shown that much of the cytoplasmic label was located over the mitochondria, while the remainder was generally associated with endoplasmic vesicles. A
recent study performed on Tetrahymena pyriformis (Engberg, Nilsson, Pearlman &
Leick, 1974) had shown that nucleolar and mitochondrial DNA replication are under
a control independent of that for the replication of bulk DNA. If this is so for amoeba,
then an increase in the cytoplasmic grain count, coincident with the time of nucleolar
DNA replication might be expected.
Fig. 1. High-iesolution autoradiograph of a cell labelled with [3H]thymidine for 6 h
during S-phase. Silver grains are confined to the central regions of the nucleus, the
peripherally located nucleoli appear almost unlabelled. c, cytoplasm; he, honeycomb
layer of the nuclear envelope; n, nucleus; no, nucleolus. A and B, X 9600 and 18500,
respectively.
/. Minassian and L. G. E. Bell
*
*
2A
T
I
1
n
:
Ml
'•'
V »
•
•
•->
B• •
Timing of nucleolar DNA replication
527
Enzymic digestion using DNase on isolated nuclei
There is always some possibility that exogenous thymidine is degraded and the 3H
subsequently incorporated into nuclear molecules other than DNA. This is partially
avoided by using thymidine with the label on the methyl group, where degradation
should avoid a preferential use in RNA. However, enzymic digestion experiments
using DNase were thought necessary to eliminate any uncertainty that the PH]thymidine could be finding its way into molecules other than DNA. Since section
digestion by DNase of nuclei or amoeba has given poor results in the past (Wise &
Goldstein, 1972) digestion experiments in this work were carried out on isolated nuclei
as described in the Methods section. The results showed that all label was removed
from those slides which had been treated with DNase.
Table 1. Numbers of grainsjunit area over the nucleus of cells exposed to \?H]thymidine
during 6 h of either the S-phase or the mid G2-phase
•S-phase
MidG.
Extranucleolar
grain count/
25 /tm1
Nucleolar
grain count/
6665 ±(11 0)
I-83±(OI)
3-92 ±(0-2)
16-00 ± (1)
25 fitn1
Cytoplasmic
grain count/
Extranucleolar
Nucleolar
grain count/ grain count/
total nuclear total nuclear
2-5 /im'
count, %
112 | ( o - 2 )
95
5
3-90 ± (0-2)
10
90
count, %
Grains were located either over the central region of the nucleus (extranucleolar) or over the
nucleoli. Though a few grains fell over the nuclear membrane, these were considered insignificant and have been excluded from the values above. Background count was negligible: o-i ±
001 grains/25 /im*. Each reading is the average grain count obtained fiom 350—400 sections.
This repiesents 50-60 S-phese and 50-60 G,-phase cells. Observations, without grain count,
were made on a further 80-100 cells. The values in parentheses are the standard errors.
DISCUSSION
Investigating the incorporation of pHJthymidine during interphase, using both
light and EM-ARG techniques, showed 2 main patterns in A. proteus. (1) A major
incorporation occurring during the first quarter of the cell cycle showed almost all
grains localized over the central region of the nucleus; few grains were found over the
peripherally located nucleoli. (2) A minor incorporation occurring during the rest of
interphase showed almost all grains over the nucleolar region; few were found on
the extranucleolar chromatin.
In an earlier cell cycle autoradiographic study, using the same strain of A. proteus,
Ord (1968) found heavy incorporation of piTJthymidine by the nucleus during the
Fig. 2. Electron-microscope autoradiograph of a cell labelled with [3H]thymidine for
6 h during mid Ga, illustrating the localization of silver grains predominantly over
the nucleoli. The label is confined to the perinucleolar region, c, cytoplasm; he, honeycomb layer of the nuclear envelope; n, nucleus; no, nucleolus. A and B, x 7800 and
13800, respectively.
528
/. Minassian and L. G. E. Bell
first quarter of the cell cycle: this she termed 5-phase. However, she also found that
during the rest of the cell cycle, termed G2, there was a consistent, though low, level
of [3H]thymidine incorporation by the nucleus. A similar high level of incorporation
of pHJthymidine by the nucleus during the early part of the cell cycle has been found
in other strains or species of Amoeba (Ron & Prescott, 1969; Narasimha Rao &
Chatterjee, 1974). In the present work we have adopted the S and G2 terminology of
these workers.
Autoradiographic studies on Amoeba have generally been made using a squash
technique of whole cells, i.e. the whole nucleus contributes to the grain count. A
change in the distribution of grains over different nuclear areas would go undetected.
It was this change in distribution of grains, first observed during cell cycle ARG
studies, which stimulated the present investigation. Since the G2 incorporation of
PHJthymidine was very low it was necessary to change from the i-h pulses used by
Ord (1968) to exposure periods of 6 h or more. Fortunately, as amoebae are able to
tolerate high doses of radiation (Ord, 1973) this did not lead to any loss of viability of
the cells. Furthermore, amoebae have a well developed salvage DNA pathway: they
are able to take up and utilize exogenous thymidine throughout the whole of the cell
cycle (e.g. 5-phase nuclei incorporate [3H]thymidine while in G2 cytoplasm (Ord,
1971)). This may not be so for all types of cells as the development of the salvage pathway varies from one cell type to another (Kornberg, 1974) and the activity of their
thymidine kinase is under strict regulation (Okazaki & Kornberg, 1964).
It is clear from our investigation, where observations have been made on serial
sections through 100 or more 5-phase and G2-phase cells, that the incorporation of
pHJthymidine during G2 is localized on or in the immediate vicinity of the nucleoli.
This suggests that the replication of nucleolar DNA is out of synchrony with the
replication of the main bulk of the nuclear DNA (referred to as extranucleolar DNA).
Asynchrony of nucleolar and extranucleolar DNA was not unexpected in that this
has been reported for a number of cell types (Charret, 1969; Giacomoni & Finkel,
1972; Ryser et al. 1973; Stambrook, 1974; Andersen & Engberg, 1975; Wintzerith
et al. 1975). The chief controversy concerning nucleolar DNA synthesis has been in
its timing during the cell cycle, rather than its correlation with the rest of the nuclear
DNA. This controversy may be due to species differences, for example tissue culture
cell and protozoa may have genuine differences in the sequence of events in the cell
cycle. However, this does not account for the differences in results obtained in the
following 2 cases. (1) In T.pyriformis, where cell age was determined by morphological
characteristics, Charret (1969) has shown that nucleolar DNA replicated during the
G2-phase. In contrast to this finding Andersen & Engberg (1975), using the heat-shock
technique for synchronizing their cells, demonstrated that nucleolar DNA replicated
at the onset of the macronuclear «S-phase. (2) In Chinese hamster cells results of
experiments performed by Amaldi, Giacomoni & Zito-Bignami (1969) using the
thymidine-block technique for synchronization showed that nucleolar DNA replicated
during mid 5-phase. In contrast, Stambrook (1974) working on cells synchronized by
selecting mitotic cells, showed that nucleolar DNA replicated at an early stage in
5-phase. The differences found in each of these cases could result from the use of
Timing of nucleolar DNA replication
529
different synchronization techniques and such a possibility has been suggested by
Stambrook (1974). Further information about the effect of different synchronization
techniques on cells is provided by the work of Pica-Mattoccia & Attardi (1972).
Investigating the pattern of mitochondrial DNA synthesis in HeLa cells, they found
that in cells synchronized by selecting the detached mitotic cells, mitochondrial DNA
synthesis started in S-phase and reached a maximum during G2; while in cells synchronized by applying the double thymidine block, mitochondrial DNA synthesis
occurred at a constant rate throughout the cell cycle. Mitchison (1971a) in his work
on the cell cycle, has emphasized the possibility that induced synchrony may cause
cell cycle distortions; thus, results obtained using the selective technique should be
more reliable. In our studies cells were synchronized by selecting detached mitotic
cells from mass cultures of A. proteus, and should therefore be free of any distortions,
which might occur with induced synchronization. Our results are in good agreement
with work done on Physarumpolycephalum (Guttes & Guttes, 1969; Guttes & Telatnyk,
1971; Ryser et al. 1973) and with Charret's work on T. pyriformis (1969): two studies
where cell age was determined without resort to any induced-synchronization methods.
The finding in this work of a fraction of nuclear DNA which replicates outside the
5-phase is not consistent with the classic subdivisions of the cell cycle into Glt S, G2,
and mitosis introduced by Howard & Pelc (1953). They defined 5-phase as a restricted
period in interphase during which the DNA is replicated. We believe that there are
no sharp boundaries between 5- and G2-phases in A. proteus and that the synthetic
activities during these periods are continuous. As information accumulates about the
metabolism of the cell during interphase, it is clear that the initial very useful subdivisions proposed by Howard & Pelc (1953) cannot be taken to have rigid boundaries.
The dissociation that can occur between various aspects of cell cycle metabolism,
particularly as a result of synchronization techniques, is reviewed by Mitchison (1971 b).
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{Received 25 March 1976)