Nucleic Acids Research
Volume 2 number 8 August 1 975
DNA replication in Physarum polycephalum: characterization of replication products in vivo
Steinar Funderud and Finn Haugli Institute of Medical Biology, University of Tromso,
Tromso.,
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 DN14molecules in the [ CJ-labelled pulse experiments. Co-sedimentation of 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 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 3 As with procaryotes, these two "models" of both strands3. 14 replication need not be mutually exclusive . Thus, much evidence has accumulated regarding the mechanism of DNA replication in eucaryotes as well as in procaryotes.
"replicating
1381
Nucleic Acids Research We have chosen to study tihe 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 and Physarum polycephalum has been studied by Brewer Brewer, Evans and Evans (1974i). 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 4 x 107 daltons. These molecules did not mature into DNA of any higher molecular weight, since even G2 DNA was of the same size.
(1972)6
MATERIALS AND METHODS
Strains and culture methods: Throughout this study the "WISCONSIN" diploid strain TU 291 was used (Haugli, 1972)8 (Mohberg, Babcock, Haugli and Rusch, 1973)9. Culture methods have been described (Daniel and Baldwin, 1964)10. In the present work Millipore membranes (code HAWPOOOO) 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 corc-bore (area 3 cm 2) and placed on a 100 pl droplet of medium, kept at 280 C, which was also the temperature for culturing the surface plasmodia. Such plasmodia contain approximately 2 x 107 nuclei in early S-phase, corresponding to roughly 100 ig of nuclear DNA (Mohberg et al. 1973)9. The incorporation medium contained regular growth medium and I3H]-thymidine (Amersham, TRK 120) at specific activity 15-19 Ci/mmole and at concentration 500 pCi/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
1382
Nucleic Acids Research placed on a 100 vl droplet or chase medium containing regular growth medium and unlabelled thymidine at 5 ug/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 pl droplet of chase medium every 10 minutes. This latter procedure is necessary, as the 100 yl droplets will support linear incorporation into DNA for only 10-15 minutes.
Isolation of nuclei and preparation for alkaline sucrose gradient analysis:
(1969)12
was The nuclear isolation method of Mohberg and Rusch used, with some modifications: 15 mM MgCl was used instead of CaCl2. The plasmodium was homogenized at 00 C - 40 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 centrifugation 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 107 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 precipitated with 3 ml of 0.6 M perchloric acid, 0.05 M 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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Nucleic Acids Research 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: 14 0)C]-labelled DNA was prepared from phage P2 by the method of Lindqvist (1971) 13 3. Phage X DNA was isolated by induction of the lysogen CR34 (l CI857 S7), concentration of phage particles on CsCl gradients and isolation of DNA by the phenolmethod. (Phage P2 was kindly provided by Dr. Bj0rn Lindqvist, the Institute of Medical Biology, University of Troms0, and the phage A lysogen by Dr. William F. Dove, the McArdle Laboratory, University of Wisconsin). The molecular weights of the DNA of these phages are 3.1 x 107 daltons for phage A (Burgi and Hershey, 1963)14 and 2.2 x 107 daltons for phage P2 (Mandel, 1967)15. With the use of Studiers equation (Studier, 1965) 16 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 [3H]-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 Oi{J-thymidine to plasmodial discs were analysed on sucrose gradients (Fig. 1).
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Nucleic Acids Research 14
12 10 8 6 4 2 24 10 E
8
-
6
m
I
l
L,1
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sec
30
sec
V
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D611 s Iw"'
I
I
b
VIV III II
4
4J2J2 0 0
12 ° 10 8 6 4 2
60 sec
+,
38 10 8 6 4 2
I
26; .
II
. V,V.6 min
V
v~~~~~~
90 sec
20k
9
min
Vb
2824201612 8 4 0 2824201612 8 4 0 Fraction number (from top to bottom).
Figure 1: Alkaline sucrose gradient sed'mentation profiles of DMA pulse-labelled with [ HI-thymidine for time periods indicated in the graphs. Values are given as percent of total radioactivity. Fractions Arrow are numbered from top t4bottom of gradient. indicates position of [ CJ-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 position of 14C-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).
1385
Nucleic Acids Research The percentage of total activity found in the various regions of the gradients are given in table 1 class I class II dlass III IV
90" 9
2' 5
3'
6'
25
60"t 24
5
8
9
7
9
8
2 5
20
41
52
45
68
69
63
72
14
26
15
39
18
18
30
25
15"
30"
46 20
9' 1 2
V bottom Table 1.
The frequency distribution of single stranded DNA species after pulses of L3HJ-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 contains about 5 x 109 daltons of double stranded DNA,9 it appears unlikely that a single stranded DNA molecule of S-value 33-35 S (approximate molecular weight 107 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 daltons (34 S) after 9 minutes, 1.0 x 10- 1.9 x 107 daltons (34- 43 S) after 30 minutes, 3.8 x l0J - 5.5 x 101 daltons (57 - 66 S) after 60 minutes, daltons (65 - 75 S) after 90 minutes, 5.3 x 107 - 7.6 x 1.5 x 108 daltons (99 S) after 150 minutes and 2.0 x 108 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 108 daltons).
107
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Nucleic Acids Research 90 min
9 min 14 12 10 8 6 4 2
I I
CLE
28 14 o 12 + 10 0 8 ' 6 4 L a.2
13
1
150 min
30 mi n
-
16 14 12 10 8 6 4 2
I I 60 min
40
-
360 min
I I
S~ . . .E '. . .
28 24 20 16 12 8 4 0 28 24 20 16 12 8 4 0 Fraction number (from top to bottom)
Figure 2:
Alkaline sucrose gradient sedimentation3
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 bottom of gradient. Arrow indicates position top of [ Cl-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 In short-pulse experiments we have shown that the major product is a single stranded DNA molecule of 5-7 S, corresponding to approximately 1.4 x 105 daltons molecular weight (class I of fig. 1). Thus, our results suggest that DNA-synthesis i; discontinous on both strands in Physarum. can be summarized as follows:
1387
Nucleic Acids Research 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 molecule of 33-35 S, corresponding to approx*imately 1.0 x 107 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 106 daltons (class III fig. 1) and 30 S, or 8.0 x 106 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 molecules - 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 105 daltons and the final product of the first, rapid ligation period which has a molecular weight of about 107 daltons. In the chase experiments (Fig. 2) we have clear evidence for a second and slow maturation process, which eventually ligates the 107 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 109 daltons, this approaches chromosome-size single stranded DNA. In all gradients analyzed in this investigation Still, some care was taken not to overload gradients with DNA. in bulk DNA seemed to of always products replication trapping 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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Nucleic Acids Research 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 conclusions vary at some important points from those of Brewer (1972)6 and Brewer et al. (1974)7. 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 molec-ular weight 4 x 107 daltons. The shortest pulses employed by these workers was 4 minutes - and, as they point out, (Brewer et al. 1974)7 they might not have been in a position to discover short lived intermediates of low molecular weight. We propose that their "primary" replication product of 1.5 x 107 daltons corresponds to our 34 S molecule which in our calculations has a molecular weight of about 1.0 x 107 daltons. 6 In the chase experiments, Brewer (1972) and Brewer et al. (1974)7 found that the mature ligation product was a single stranded DNA molecule of molecular weight 4 x 107 daltons. This is likely to be a degradation product in our opinion, since extremely carefull lysis in our hands allows detection of 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) 17 in Ehrlich ascites tumor cells after short pulses found all label in single stranded DNA molecules of molecular weight 4 x 106 daltons and 2 x 107 daltons accumulated. 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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Nucleic Acids Research ACKNOWLEDGEMENTS We thank Miss Kerstin Wennberg for technical assistance during part of this work, and Dr. Unni Spaeren and Dr. Bjorn 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.
REFERENCES 1. 2.
3. 4.
5. 6. 7.
8.
9. 10. 11. 12. 13. 14. 15. 16. 17.
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Huberman, J.A. and Riggs, A.D. (1968) J. Mol. Biol. 32, 327-341. Hershey, H.V. and Taylor, J.H. (1974) Exptl. Cell Res. 85, 79-88. autschi, J.R. and Clarkson, J.M. (1975) Eur. J. Biochem. 50, 403-412. 6Tivera, B.M. and Bonhoeffer, F. (1972) Nature New Biol. 240, 233-235. Braun, R. and Wili, H. (1969) Biochim. Biophys. Acta 1714, 246-252. Brewer, E.N. (1972) J. Mol. Biol. 68, 401-412. Brewer, E.N., Evans, T.E. and Evans, H.H. (1974) J. Mol. Biol. 90, 335-342. Haugli, F.B. (1971) Ph.D. Thesis, University of Wisconsin 1971. Mohberg, J., Babcock, K.L., Haugli, F.B. and Rusch, H.P. (1973) Develop. Biol. 34, 228-245. Daniel, J.W. and Baldwin, H.H. (1964) In Methods in Cell Physiology Vol. I, 9-41. Academic Press, New York. Rusch, H.P. (1970) In Advances in Cell Biology Vol. I, 297-328. Appleton-Century-Crofts New York. J. Bact. 97, 1411-1418. Mohberg, J. and Rusch, H.P. (19695 Lindqvist, B.H. (1971) Molec. gen. Genet. 117 178-196. Burgi, E. and Hershey, A.D. (1963) Biophys. J. 3, 309-321. Mandel, M. (1967) Molec. gen. Genet. 99, 88-96 Studier, F.W. (1965) J. Mol. Biol.1.1, 373-390. Goldstein, N.O. and Rutman, R.J. (l1975) Nature New Biol. 244, 267-269.
Volume 2 number 8 August 1 975
DNA replication in Physarum polycephalum: characterization of replication products in vivo
Steinar Funderud and Finn Haugli Institute of Medical Biology, University of Tromso,
Tromso.,
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 DN14molecules in the [ CJ-labelled pulse experiments. Co-sedimentation of 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 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 3 As with procaryotes, these two "models" of both strands3. 14 replication need not be mutually exclusive . Thus, much evidence has accumulated regarding the mechanism of DNA replication in eucaryotes as well as in procaryotes.
"replicating
1381
Nucleic Acids Research We have chosen to study tihe 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 and Physarum polycephalum has been studied by Brewer Brewer, Evans and Evans (1974i). 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 4 x 107 daltons. These molecules did not mature into DNA of any higher molecular weight, since even G2 DNA was of the same size.
(1972)6
MATERIALS AND METHODS
Strains and culture methods: Throughout this study the "WISCONSIN" diploid strain TU 291 was used (Haugli, 1972)8 (Mohberg, Babcock, Haugli and Rusch, 1973)9. Culture methods have been described (Daniel and Baldwin, 1964)10. In the present work Millipore membranes (code HAWPOOOO) 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 corc-bore (area 3 cm 2) and placed on a 100 pl droplet of medium, kept at 280 C, which was also the temperature for culturing the surface plasmodia. Such plasmodia contain approximately 2 x 107 nuclei in early S-phase, corresponding to roughly 100 ig of nuclear DNA (Mohberg et al. 1973)9. The incorporation medium contained regular growth medium and I3H]-thymidine (Amersham, TRK 120) at specific activity 15-19 Ci/mmole and at concentration 500 pCi/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
1382
Nucleic Acids Research placed on a 100 vl droplet or chase medium containing regular growth medium and unlabelled thymidine at 5 ug/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 pl droplet of chase medium every 10 minutes. This latter procedure is necessary, as the 100 yl droplets will support linear incorporation into DNA for only 10-15 minutes.
Isolation of nuclei and preparation for alkaline sucrose gradient analysis:
(1969)12
was The nuclear isolation method of Mohberg and Rusch used, with some modifications: 15 mM MgCl was used instead of CaCl2. The plasmodium was homogenized at 00 C - 40 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 centrifugation 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 107 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 precipitated with 3 ml of 0.6 M perchloric acid, 0.05 M 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
1383
Nucleic Acids Research 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: 14 0)C]-labelled DNA was prepared from phage P2 by the method of Lindqvist (1971) 13 3. Phage X DNA was isolated by induction of the lysogen CR34 (l CI857 S7), concentration of phage particles on CsCl gradients and isolation of DNA by the phenolmethod. (Phage P2 was kindly provided by Dr. Bj0rn Lindqvist, the Institute of Medical Biology, University of Troms0, and the phage A lysogen by Dr. William F. Dove, the McArdle Laboratory, University of Wisconsin). The molecular weights of the DNA of these phages are 3.1 x 107 daltons for phage A (Burgi and Hershey, 1963)14 and 2.2 x 107 daltons for phage P2 (Mandel, 1967)15. With the use of Studiers equation (Studier, 1965) 16 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 [3H]-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 Oi{J-thymidine to plasmodial discs were analysed on sucrose gradients (Fig. 1).
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Nucleic Acids Research 14
12 10 8 6 4 2 24 10 E
8
-
6
m
I
l
L,1
V
sec
30
sec
V
2mi
3 min
16
D611 s Iw"'
I
I
b
VIV III II
4
4J2J2 0 0
12 ° 10 8 6 4 2
60 sec
+,
38 10 8 6 4 2
I
26; .
II
. V,V.6 min
V
v~~~~~~
90 sec
20k
9
min
Vb
2824201612 8 4 0 2824201612 8 4 0 Fraction number (from top to bottom).
Figure 1: Alkaline sucrose gradient sed'mentation profiles of DMA pulse-labelled with [ HI-thymidine for time periods indicated in the graphs. Values are given as percent of total radioactivity. Fractions Arrow are numbered from top t4bottom of gradient. indicates position of [ CJ-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 position of 14C-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).
1385
Nucleic Acids Research The percentage of total activity found in the various regions of the gradients are given in table 1 class I class II dlass III IV
90" 9
2' 5
3'
6'
25
60"t 24
5
8
9
7
9
8
2 5
20
41
52
45
68
69
63
72
14
26
15
39
18
18
30
25
15"
30"
46 20
9' 1 2
V bottom Table 1.
The frequency distribution of single stranded DNA species after pulses of L3HJ-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 contains about 5 x 109 daltons of double stranded DNA,9 it appears unlikely that a single stranded DNA molecule of S-value 33-35 S (approximate molecular weight 107 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 daltons (34 S) after 9 minutes, 1.0 x 10- 1.9 x 107 daltons (34- 43 S) after 30 minutes, 3.8 x l0J - 5.5 x 101 daltons (57 - 66 S) after 60 minutes, daltons (65 - 75 S) after 90 minutes, 5.3 x 107 - 7.6 x 1.5 x 108 daltons (99 S) after 150 minutes and 2.0 x 108 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 108 daltons).
107
1386
Nucleic Acids Research 90 min
9 min 14 12 10 8 6 4 2
I I
CLE
28 14 o 12 + 10 0 8 ' 6 4 L a.2
13
1
150 min
30 mi n
-
16 14 12 10 8 6 4 2
I I 60 min
40
-
360 min
I I
S~ . . .E '. . .
28 24 20 16 12 8 4 0 28 24 20 16 12 8 4 0 Fraction number (from top to bottom)
Figure 2:
Alkaline sucrose gradient sedimentation3
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 bottom of gradient. Arrow indicates position top of [ Cl-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 In short-pulse experiments we have shown that the major product is a single stranded DNA molecule of 5-7 S, corresponding to approximately 1.4 x 105 daltons molecular weight (class I of fig. 1). Thus, our results suggest that DNA-synthesis i; discontinous on both strands in Physarum. can be summarized as follows:
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Nucleic Acids Research 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 molecule of 33-35 S, corresponding to approx*imately 1.0 x 107 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 106 daltons (class III fig. 1) and 30 S, or 8.0 x 106 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 molecules - 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 105 daltons and the final product of the first, rapid ligation period which has a molecular weight of about 107 daltons. In the chase experiments (Fig. 2) we have clear evidence for a second and slow maturation process, which eventually ligates the 107 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 109 daltons, this approaches chromosome-size single stranded DNA. In all gradients analyzed in this investigation Still, some care was taken not to overload gradients with DNA. in bulk DNA seemed to of always products replication trapping 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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Nucleic Acids Research 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 conclusions vary at some important points from those of Brewer (1972)6 and Brewer et al. (1974)7. 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 molec-ular weight 4 x 107 daltons. The shortest pulses employed by these workers was 4 minutes - and, as they point out, (Brewer et al. 1974)7 they might not have been in a position to discover short lived intermediates of low molecular weight. We propose that their "primary" replication product of 1.5 x 107 daltons corresponds to our 34 S molecule which in our calculations has a molecular weight of about 1.0 x 107 daltons. 6 In the chase experiments, Brewer (1972) and Brewer et al. (1974)7 found that the mature ligation product was a single stranded DNA molecule of molecular weight 4 x 107 daltons. This is likely to be a degradation product in our opinion, since extremely carefull lysis in our hands allows detection of 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) 17 in Ehrlich ascites tumor cells after short pulses found all label in single stranded DNA molecules of molecular weight 4 x 106 daltons and 2 x 107 daltons accumulated. 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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Nucleic Acids Research ACKNOWLEDGEMENTS We thank Miss Kerstin Wennberg for technical assistance during part of this work, and Dr. Unni Spaeren and Dr. Bjorn 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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Huberman, J.A. and Riggs, A.D. (1968) J. Mol. Biol. 32, 327-341. Hershey, H.V. and Taylor, J.H. (1974) Exptl. Cell Res. 85, 79-88. autschi, J.R. and Clarkson, J.M. (1975) Eur. J. Biochem. 50, 403-412. 6Tivera, B.M. and Bonhoeffer, F. (1972) Nature New Biol. 240, 233-235. Braun, R. and Wili, H. (1969) Biochim. Biophys. Acta 1714, 246-252. Brewer, E.N. (1972) J. Mol. Biol. 68, 401-412. Brewer, E.N., Evans, T.E. and Evans, H.H. (1974) J. Mol. Biol. 90, 335-342. Haugli, F.B. (1971) Ph.D. Thesis, University of Wisconsin 1971. Mohberg, J., Babcock, K.L., Haugli, F.B. and Rusch, H.P. (1973) Develop. Biol. 34, 228-245. Daniel, J.W. and Baldwin, H.H. (1964) In Methods in Cell Physiology Vol. I, 9-41. Academic Press, New York. Rusch, H.P. (1970) In Advances in Cell Biology Vol. I, 297-328. Appleton-Century-Crofts New York. J. Bact. 97, 1411-1418. Mohberg, J. and Rusch, H.P. (19695 Lindqvist, B.H. (1971) Molec. gen. Genet. 117 178-196. Burgi, E. and Hershey, A.D. (1963) Biophys. J. 3, 309-321. Mandel, M. (1967) Molec. gen. Genet. 99, 88-96 Studier, F.W. (1965) J. Mol. Biol.1.1, 373-390. Goldstein, N.O. and Rutman, R.J. (l1975) Nature New Biol. 244, 267-269.
