volume2 number8 August 1975
Nucleic Acids Research
DNA replication in Physarum polycephalum: characterization of replication
products in vivo
Steinar Funderud and Finn Haugli
Institute of Medical Biology, University of TromsfS, Troms0, Norway
Received 30 June 1975
ABSTRACT
Synchronous plasmodia of Physarum polycephalum in DNA
synthesis were pulse-labelled with I H ] - thymidine for time
periods of 15 seconds up to 9 minutes, or given a 30 seconds
pulse followed by chase periods of 9 minutes up to 6 hours.
Sedimentation analysis in alkaline sucrose gradients revealed
at least five species of single stranded DNAhinolecules in the
pulse experiments. Co-sedimentation of
[ C]-labelled
phage-DNA gave relative S-values of 5-7, 13-15, 23-25, 30 and
33-35 for these DNA molecules, all of which can be chased into
DNA of higher molecular weight.
INTRODUCTION
Studies on the mechanism of DNA replication in
eucaryotes have revealed that the DNA in chromosomes is
replicated bidirectionally in tandemly arranged subunits called
"replicating units" by Huberman and Riggs .
Within each
replicating unit DNA synthesis may proceed in continous fashion
on the template strand which is copied in the 5 ' — > 3' direction,
but in discontinous fashion on the strand which is copied with
opposite polarity .
Alternatively, DNA synthesis may proceed
via discontinous synthesis and formation of "Okazaki-pieces" on
both strands .
As with procaryotes, these two "models" of
replication need not be mutually exclusive .
Thus, much
evidence has accumulated regarding the mechanism of DNA replication in eucaryotes as well as in procaryotes.
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We have chosen to study the replication process in the
simple eucaryote Physarum polycephalum because this system
offers the unique advantage of natural, perfect synchrony in
DNA synthesis'5.
Aspects of the mechanism of in vivo DNA replication in
Physarum polycephalum has been studied by Brewer (1972) and
Brewer, Evans and Evans (1971*) • These researchers used longer
pulses than those applied in the present work, and concluded
that DNA synthesis in Physarum occurred by continous synthesis
of a DNA molecule of molecular weight H x 10 daltons. These
molecules did not mature into DNA of any higher molecular
weight, since even G2 DNA was of the same size.
MATERIALS AND METHODS
Strains and culture methods: Throughout this study the
"WISCONSIN" diploid strain TU 291 was used (Haugli, 1972) 8
Q
(Mohberg, Babcock, Haugli and Rusch, 1973) . Culture methods
have been described (Daniel and Baldwin, 1964) . In the
present work Millipore membranes (code HAWPOOO1O) were used
for support of synchronous surface cultures.
Labelling procedures: All experiments reported here were
started 30 minutes past synchrounous mitosis II or III, when
the rate of DNA synthesis is maximal . The mitotic stage was
determined by inspection of ethanol-fixed smears. Discs of
plasmodia supported on the Millipore membrane were cut out with
a core-bore (area 3 cm ) and placed on a 100 yl droplet of
medium, kept at 28° C, which was also the temperature for
culturing the surface plasmodia. Such plasmodia contain
•7
approximately 2 x 10 nuclei in early S-phase, corresponding
to roughly 100 yg of nuclear DNA (Mohberg et al. 1973 ) 9 . The
incorporation medium contained regular growth medium and
[ H]-thymidine (Amersham, TRK 120) at specific activity
15-19 Ci/mmole and at concentration 500 yCi/ml. The pulse was
terminated by quickly immersing the plasmodium in liquid nitrogen.
When a chase was to be included, the pulse was terminated,
and the chase started in the following way: excess radioactive medium was quickly removed on a pad of filter paper. The
disc was washed once on chase medium (5 seconds needed) and then
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placed on a 100 pi droplet or chase medium containing regular
growth medium and unlabelled thymidine at 5 pg/ml.
For chase
periods of 10 minutes or less, no change of medium was necessary.
For longer chase periods, plasmodia were either put on a large
(15 ml) reservoir of chase medium in a petrie-dish, or removed
to a new 100 pi droplet of chase medium every 10 minutes.
This
latter procedure is necessary, as the 100 pi droplets will
support linear incorporation into DNA for only 10-15 minutes.
Isolation of nuclei and preparation for alkaline sucrose
gradient analysis:
12
The nuclear isolation method of Mohberg and Rusch (1969)
was
used, with some modifications: 15 mM MgCl was used instead of
CaClp.
The plasmodium was homogenized at 0° C - 4° C in 20 ml
of isolation medium in a 50 ml stainless steel chamber of
Sorvall Omnimixer set at speed 2 for 20 seconds.
After centri-
fugation at 2 000 rpm for 15 minutes in an International/Damon
refrigerated centrifuge (rotor no 269) nuclei were quickly resuspended in 1 ml of 0.15 M NaCl and recentrifuged.
The
sodiumchloride wash improves subsequently lysis, probably because
some polysaccharide is removed from the nuclear membrane.
About 2 -x 10
nuclei from each plasmodial disc, isolated
as described, were lysed in 0.5 ml of 0.3 M NaOH, 2 % N-Lauroyl
Sarcosine, 0.01 M EDTA.
Shaking of the lysate was avoided and
after 15 minutes on ice the lysate was gently layered on top of
a 34 ml gradient of 4-20 % sucrose in 0.1 M NaOH, 0.2 % NLauroyl Sarcosine.
The gradients were centrifuged for 18 hours
in the SW-27 rotor of Beckman preparative centrifuge.
The rotor
speed was 27 000 rpm in the pulse experiments and 15 000 rpm and
20 000 rpm in the chase experiments (see figure 2 for details).
Gradients were collected in 28-31 fractions from the top with
the help of a Buchler Auto-Densiflow connected to a peristaltic
pump.
Determination of radioactivity:
The fractions were preci-
pitated with 3 ml of 0.6 M perchloric acid, 0.05 H sodiumpyrophosphate in presence--of 125 vg of bovine serum albumin as
carrier, collected on Whatman glass filters GF/C and washed
with 30 ml of 0.3 M perchloric acid with 0.05 M sodiumpyrophosphate, and finally with 5 ml of ethanol.
Filters were
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dried, treated with Soluene 350 (Packard) and counted in 5 ml
scintillator liquid (6 g PPO
and 0.6 g bis-(O-methylstyryl)-
benzene per liter toluene) in a Packard liquid scintillation
counter.
Preparation of marker DNA and estimation of relative S-values
and molecular weights:
[
C]-labelled DNA was prepared from phage P2 by the method of
Lindqvist (1971)
.
Phage A DNA was isolated by induction of
the lysogen CR34 (\ CIo,-7 S _ ) , concentration of phage particles
on CsCl gradients and isolation of DNA by the phenolmethod.
(Phage P2 was kindly provided by Dr. BjszSrn Lindqvist, the
Institute of Medical Biology, University of Troms(zS, and the
phage \ lysogen by Dr. William P. Dove, the McArdle Laboratory,
University of Wisconsin) .
The molecular weights of the DNA of these phages are
7
14
3.1 x 10
daltons for phage ^ (Burgi and Hershey, 1963)
2.2 x 1 0 7 daltons for phage P2 (Mandel, 1 9 6 7 ) 1 5 .
use of Studiers equation (Studier, 1965)
and
With the
we calculate the
sedimentation coefficients for the corresponding single stranded
alkaline DNA to be 40 for phage A and 35 for phage P2.
Assuming a linear relationship between S-values and
position in gradients, the approximate S-values of all labelled
fractions of DNA can be calculated from the position of marker
DNA, and from these the approximate molecular weights in daltons
can be calculated.
RESULTS
Pulse labelling experiments:
Preliminary experiments designed
to study the products of DNA synthesis after short pulses of
[ H]-thymidine showed that the method used to stop the reaction
was critical to the results obtained.
Thus, stopping the
reactions by immersing the plasmodia in icecold nuclear isolation medium appeared to allow transport of the shorter pieces
of newly made DNA into DNA of higher molecular weight.
This
problem was avoided by quickly immersing the plasmodia in
liquid nitrogen to stop reactions.
The products obtained
after short (15 seconds to 90 seconds) and long (2 minutes to
9 minutes) pulses of [ H]-thymidine to plasmodial discs were
analysed on sucrose gradients (Pig. 1 ) .
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15 sec
i
2 min
3 min
60 sec
26
6 min
20
9 min
III
90 sec
I
28 24 20 16 12 8 4 0 28 24 20 16 12 8 4 0
Fraction number (-from top to bottom).
Figure 1: Alkaline sucrose gradient sedimentation
profiles of DMA pulse-labelled with [ H]-thymidine
for time periods indicated in the graphs. Values are
given as percent of total radioactivity. Fractions
are numbered from top to^bottom of gradient. Arrow
indicates position of [ C]-labelled P2-phage DNA.
Experiments with short pulses show that 5 distinct
species of single stranded DNA can be found in replicating
DNA in Physarum (I, II, III, IV and V in Fig. 1 ) . These can
all be chased into larger molecules (Fig. 2 ) . From the posi14
tion of
C-labelled P2 phage DNA relative S values were
calculated to be 5-7 for class I, 13-15 for class II, 23-25
for class III, 30 for class IV and 33-35 for class V. With
longer pulses there appeared to be a slight shift of some of
the class V DNA molecules to higher molecular weights
(class V b, Fig. 1 ) .
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The percentage of total activity found in the various
regions of the gradients are given in table 1
class I
class II
class III
IV
V
bottom
Table 1.
2'
9
90"
9
7
41
52
45
68
26
15
39
18
46
30"
25
60"
24
20
8
20
in
15"
6-
91
2
5
1
2
69
63
72
18
30
25
3'
5
8
5
9
The frequency distribution,of single stranded DNA
species after pulses of [ H]-thymidine ranging from
15 seconds to 9 minutes. Fractions are divided into
4 groups: class I, fraction 1-6 from top, class II
fraction 7-10 from topp, class III-IV-V pooled fractions
12-26. Fraction 26-31 is the bottom fraction. Numbers
give percent of total radioactivity found in the various
groups of DNA molecules with increasing pulse-lengths.
Pulse-chase experiments: Since an average Physarum chromosome
Q~
9
contains about 5 x 10 daltons of double stranded DNA, it
appears unlikely that a single stranded DNA molecule of S-value
y
33-35 S (approximate molecular weight 10 daltons) should be
the mature replication product. Experiments where a 30 second
pulse was followed by chase periods of 9 minutes up to 6 hours
showed that the S-values of the chase product increased
steadily with time from 34 S (after 9 minutes) to 110 S and
more after 6 hours (Fig. 2 ) .
Intermediates in this ligation process, which appears
to give an almost linear increase in molecular weight over the
first 2.5 hours, includes single stranded DNA molecules of
molecular weights 1.0 x 10 7 daltons (34 S) after 9 minutes,
1.0 x 1O 7 -1.9 x 10 7 daltons (34-43 S) after 30 minutes,
3.8 x 10 7 - 5.5 x 10 7 daltons (57 - 66 S) after 60 minutes,
5.3 x 10 7 - 7.6 x 10 7 daltons (65 - 75 S) after 90 minutes,
1.5 x 10 daltons (99 S) after 150 minutes and 2.0 x 10 daltons
(110 S) after 6 hours.
In the 6 hour chase one can also discern a shoulder at 137 S,
which is the heaviest DNA species for which molecular weight
could be estimated during these studies (3.4 x 10 daltons).
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9 min
14
12
10
8
6
4
2
28
14
12
10
8
6
4
2
16
14
12
10
8
6
4
2
90 min
.
K
A\
/
\ / \
•\J.. V
I
/
\
150 min
30 min
I
/ \
v.
\y
60 min
40
A'
360 min
I
/
/ \
\
LJ V
28 24 20 16 12 8 4 0 28 24 20 16 12 8 4 0
Fraction number (from top to bottom)
Figure
;ure 2: Alkaline sucrose gradient sedimentation,
profiles of DNA given a 30 seconds pulse with [ H ] thymidine and then chased for the time periods indicated in the graphs. Values are given as percent
of total radioactivity. Fractions are numbered from
top to bottom of gradient. Arrow indicates position
of [ C]-labelled X-phage DNA. Centrifugation speed
was 20 000 rpm in the 9 and 30 minutes chase experiments and 15 000 rpm in the 60, 90, 150 and 360 minutes
chase experiments.
DISCUSSION
Our results and the conclusions to be drawn from them
can be summarized as follows:
In short-pulse experiments we
have shown that the major product is a single stranded DNA
molecule of 5-7 S, corresponding to approximately l.U x 10
daltons molecular weight (class I of fig. 1 ) . Thus, our results
suggest that DNA-synthesis is. discontinous on both strands in
Physarum.
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This primary product of replication very rapidly becomes
ligated into single stranded DNA molecules of higher molecular
weight.
While the end product of this rapid ligation is a DNA
7
molecule of 33-35 S, corresponding to approximately 1.0 x 10
daltons molecular weight (class V of fig. 1 ) , there is a
transient accumulation of DNA in three intermediate size
classes corresponding to 13-15 S, or ca. 1.0 x 10 daltons
(class II fig.l) 23-25 S, or ca. 4.0 x 10 6 daltons (class III
fig. 1) and 30 S, or 8.0 x 10 6 daltons (class IV fig. 1 ) .
We can not be definite about the relationship between
these intermediates. However, the calculated molecular weights
suggest that molecules in class II, III, IV and V are made up
of 8, 31, 55 and 71 class I molecules respectively. Furthermore, molecules in class III, IV and V could be made up of 4,
7 and 9 class II molecules. Since the size difference between
class IV and class V molecules is only about 30 % it is unlikely
that these have a direct relationship. It appears possible
from the sizes found that 2 class III molecule's - of somewhat
varying size make up both the class IV and the class V molecules.
Although the details of the ligation process must remain somewhat speculative at this stage, our results definitely suggests
that the ligation process occurs in a discontinous manner,
since otherwise one should not expect distinct size classes
between the primary replication product of 1.4 x 10 daltons
and the final product of the first, rapid ligation period which
has a molecular weight of about 10 daltons.
In the chase experiments (Fig. 2) we have clear evidence
for a second and slow maturation process, which eventually
ligates the 10 daltons molecular weight product into a single
stranded DNA molecule of molecular weight at least 2-4 x 10
daltons. Since the average molecular weight of single stranded
DNA in a Physarum chromosome can not be more than approximately
2.5 x 10 daltons, this approaches chromosome-size single
stranded DNA. In all gradients analyzed in this investigation
care was taken not to overload gradients with DNA. Still, some
trapping of replication products in bulk DNA always seemed to
take place. Because of the kinetics in our results we do not
believe that this has interfered with the quality of the observed
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replication products, although it certainly has influenced the
quantity observed in the various fractions. There is, however,
nothing to suggest that trapping has been selective (table 1)
and thus we feel confident tnat our observations reflect true
replication behaviour.
Total recovery of activity put on
gradients were 80 % or better.
Our observations and conclu-
sions vary at some important points from those of Brewer (1972)
and Brewer et al. (1974) .
In similar experiments based on
longer pulses these authors suggested that DNA replication in
Physarum proceeded via the continous synthesis of pieces of
DNA with molecular weight 4 x 10
daltons.
The shortest pulses
employed by these workers was 4 minutes - and, as they
y
out, (Brewer et al. 1974) they might not have been in
to discover short lived intermediates of low molecular
We propose that their "primary" replication product of
point
a position
weight.
y
1.5 x 10
daltons corresponds to our 34 S molecule which in our calcuy
lations has a molecular weight of about 1.0 x 10 daltons.
In the chase experiments, Brewer (1972)
y
al. (1974)
and Brewer et
found that the mature ligation product was a single
stranded DNA molecule of molecular weight 4 x 10
daltons.
This is likely to be a degradation product in our opinion,
since extremely carefull lysis in our hands allows detection of
p
molecules with molecular weights of 2-4 x 10
daltons, at least.
While our results are somewhat different from these in vivo
investigations in Physarum polycephalum, they appear to agree
well with what other workers have found in other eucaryotic
cells in vivo.
Goldstein and Rutman (1973)
in Ehrlich ascites
tumor cells after short pulses found all label in single
stranded DNA molecules of molecular weight 4 x 10
y
2 x 10
daltons accumulated.
daltons and
They also mentioned evidence for
DNA molecules of much higher molecular weight.
Gautschi and Clarkson (1975) , similarly, found DNA
synthesis in mouse P-815 cells to occur in discontinous fashion
on both strands, with primary synthesis of very small fragments, which in about 2-8 minutes became ligated into
molecules of 20-60 S.
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ACKNOWLEDGEMENTS
We thank Miss Kerstin Wennberg for technical assistance
during part of this work, and Dr. Unni Spaeren and
Dr. Bj0rn Lindqvist for helpfull and critical discussions.
We would also like to thank Dr. Justin McCormick of the
Michigan Cancer Foundation for helpfull suggestions on
alkaline sucrose gradient analysis.
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