Eur. J. Biochem. 80, 557-566 (1977)
The Replication of Ribosomal DNA in Physarum polycephalum Volker M. VOGT and Richard BRAUN Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York and Institute of General Microbiology, University of Bern (Received May 27, 1977)
The DNA coding for ribosomal RNA in Physarum polycephalum exists as a collection of extrachromosomal molecules of molecular weight 37 x lo6. We have investigated the replication of rDNA, with the following results. (a) Replication of rDNA is unscheduled. This means that molecules that are replicated at any particular time in one mitotic cycle have an equal probability of replicating again in each time interval in the subsequent cycle. Similarly, in a single cycle, some molecules replicate more than once, and some not at all. (b) Replication forks appear to move bidirectionally from points 45 % or 33 from one end of the DNA. Replicating molecules observed by electron microscopy are all linear.
In the acellular slime mold Physarumpolycephalum the DNA coding for ribosomal RNA, which comprises 1 - 2 % of total DNA, forms a heavy satellite band when sedimented to equilibrium in CsCl gradients. In contrast to the main-band DNA, whose time of replication defines the S period of the mitotic cycle (0-2.5 h after mitosis), ribosomal DNA (rDNA) replication proceeds throughout the G-2 period as well as through the latter half of the S period [1,2]. There is no G-1 period in Physarum plasmodia, DNA synthesis following immediately after the synchronous mitosis of the nuclei. The facts that rDNA can be purified by equilibrium sedimentation and can be labeled specifically with radioactive precursors in the G-2 period have facilitated studies of its structure. We have shown recently [3] that Physarum rDNA exists as a collection of linear DNA molecules of a unit size, 37 x lo6 M,. These molecules have a rotational axis of symmetry in the center, and thus are palindromes. One ribosomal transcription unit is located at each end of the molecule [4], and the large intervening spacer region contains blocks of short inverted repetitive sequences [3]. The curious timing of synthesis of rDNA led us to investigate its replication more closely. We show here that: (a) replication is unscheduled, and (b) replication proceeds by means of bidirectional fork displacement, apparently from fixed points on the linear molecules.
MATERIALS AND METHODS Growth and Labeling of Plasmodia Microplasmodia of Physarum polycephalum, strain MIIIC, were propagated in shaking cultures at 26 "C in semidefined medium [5]. Macroplasmodia were formed on paper filters by fusion of 0.15 ml of packed plasmodia diluted with an equal volume of water. They were grown over wire grids in the same medium. The time of mitosis was determined by microscopic examination under phase contrast. The time of mitosis I1 (MII) was generally 14 h after fusion. For density labeling, bromodeoxyuridine (BrdUrd, 100 pg/ ml), fluorodeoxyuridine (5 pg/ml), and uridine (100 pg/ ml) were added to the growth medium [8]. In all experiments involving further growth after density labeling, thymidine was added to 100 pg/ml after removal of BrdUrd, fluorodeoxyuridine, and uridine. In the presence of the BrdUrd medium, the mitotic cycle appeared to be lengthened by 0 - 1 h. Thymidine had no effect on the length of the mitotic cycle. The precursors used for radioactive labeling were either [3H]thymidine (usually at 50 pCi/ml) or [3H]deoxyadenosine (usually at 10 pCi/ml). About one half of the [3H]deoxyadenosine incorporated into nuclei by G-2 plasmodia was found in DNA, the rest being in RNA. Control experiments showed that after removal of radioactive label from plasmodia, incorporation ceased within 15 min. Preparation of DNA
Abbreviations. rDNA, ribosomal DNA; BrdUrd, bromodeoxyuridine.
Nuclei were prepared at 0 "C by homogenization followed by centrifugation [3]. They were resuspended
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in 3 mlO.05 M EDTA, pH 7.8, or in early experiments in 0.015 M sodium citrate, 0.15 M NaCl, 0.005 M EDTA. Selective extraction of DNA was done as described previously [6]. Total DNA was prepared by lysing resuspended nuclei by incubation with pronase at 1 mg/ml and sodium dodecyl sulfate at 2%. After 15 min at 37 "C, 2 vol. of ethanol were added and DNA was collected by spooling. DNA prepared either by this procedure or by selective extraction was redissolved in 3.5 ml of 0.05 M EDTA, adjusted to a density of 1.71 or 1.74 g/ml with CsCl, and centrifuged for 60 h at 33000 rev./min, 18 "C in a Ti50 rotor. Fractions were collected from the bottom of the gradient and aliquots counted. In some experiments, the fractions were adjusted to 0.3 M NaOH, heated for 20 min at 80 "C, and then precipitated with trichloroacetic acid before counting. Other Methods
DNA was observed by electron microscopy as described [3]. Lambda phage were purified by differential centrifugation from lysates of lysogenic E. coli that had been heat-induced for lambda cI857S7. RESULTS Timing of Replication
The main-band DNA of Physarum [7,8] as well as the DNA of other eucaryotes [9] replicates on a fixed schedule. This means that base sequences replicated at a particular time in one S phase are replicated again at the same relative time in subsequent mitotic cycles, as determined by density shift experiments. In outline, these experiments consist of radioactive labeling of DNA with a brief pulse of [3H]thymidine at a particular time in one S period, followed by density-labeling with bromodeoxyuridine (BrdUrd) at different times in the S phase of the subsequent mitotic cycle. If thymidine-labeled DNA replicates during the time when BrdUrd is present, the radioactive strand will become paired with a new strand of higher density. The resulting duplex of 'hybrid' density is then identified by equilibrium sedimentation in CsC1. We have used similar techniques to discover whether replication of ribosomal DNA is scheduled. The experiments rely on the fact that after selective extraction of nuclei prepared from plasmodia labeled in G-2, most of the radioactivity is found in rDNA. Selective extraction is a technique that separates low-molecular-weight DNA from the bulk of nuclear DNA and proteins [lo]. Applied to Physarum nuclei, it yields about two-thirds of the rDNA, with an enrichment of about 10-fold over total DNA [6] (and Gubler, Vogt and Braun, unpublished results). Fig. 1A
rDNA Replication in Phy.~urum
shows the pattern of selectively extracted, G-2-labeled DNA in CsCl equilibrium sedimentation. Most of the radioactivity forms a band to the dense side of the 32P-labeledlambda phage DNA included as an internal marker. This is the position of ribosomal DNA, 1.712 g/ml [l]. As seen in the same figure panel, purified main-band DNA forms a broader band centered at a density of 1.700 g/ml or four fractions to the light side of the phage marker. The profile in Fig. 1A is somewhat atypical; in most preparations of G-Zlabeled, selectively extracted DNA, a variable proportion of the radioactivity also is found in the position of the main band. In a typical experiment to investigate the scheduling of rDNA replication, a plasmodium containing about 10' nuclei was labeled 6.5 h after mitosis I1 (MII, the second mitosis after formation of the plasmodium by fusion) for 60 min with [3H]thymidine.The labeling was carried out in the presence of ethidium bromide to suppress replication of mitochondria1 DNA [I 11. Growth was allowed to continue in normal medium lacking radioactive thymidine until the subsequent mitosis, MIII, or about 9 h after MII. The plasmodium was then cut into six sectors, each of which was incubated separately in normal medium. At different times, the growing sectors were then transferred to medium containing BrdUrd and incubated for 2 or 6 h. As a control, one sector was incubated with unlabeled thymidine instead of with BrdUrd. After the density pulse, nuclei were isolated from each sector and low-molecular-weight DNA prepared from sodium dodecyl sulfate lysates by selective extraction. The DNA was then centrifuged to equilibrium in CsCl along with a 32P-labeled lambda phage DNA marker. Fig.1 shows the CsCl profiles of the radioactivity obtained. Fig. 1 A is from the sector receiving no BrdUrd. In Fig. 1B, representing the density pulse from 0-2 h after MIII, most of the radioactivity is still at the position of normal rDNA, with only a small peak of label at the density of hybrid DNA. This result is consistent with the observation that rDNA does not replicate in the first 1 - 1.5 h of S phase [l]. In Fig. 1 C, representing the density pulse from 2 - 4 h, a part of the label has shifted to the position of hybrid DNA, which in different experiments is centered at a density of 1.73- 1.74 g/ml. A similar fraction of radioactivity is found at hybrid density for pulses from 4-6 h (Fig. 1D) as well as from 6-8 h (Fig. 1E). The last profile, Fig. 1F, represents continuous density labeling from 2 - 8 h after MIII. Fig. 2 quantifies the radioactively labeled DNA that is found at the hybrid position after each density pulse. The sum of all radioactivity banding denser than the phage marker is defined as 1.00. (The justification for neglecting radioactivity in main-band DNA, i.e. in DNA on the light side of the phage marker, is given below.) With the exception of the first two hours,
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Fig. 1. Equilibrium sedimentation of D N A lubeled with " H und BrdUrd in di/fbrent mitotic cycles. A plasmodium was incubated in the presence of 50 pCi/ml [3H]thymidine from 6.5-7.5 h after MII. Ethidium bromide at 10 pglml was present 30 min before and during this interval. At the time of MI11 (zero time), the plasmodium was cut into six sectors and each sector was incubated with fresh medium. At the times indicated, sectors were transferred to medium containing BrdUrd and incubated for 2 h or 6 h. One sector was incubated in normal medium as a control. Immediately after this labeling period, nuclei were prepared from the sectors and low-molecular-weight DNA isolated from them by selective extraction. After treatment with 20 pg/ml RNase A for 30 min at 37 "C in 1 mM Tris pH 7.8, 0.1 mM EDTA, the samples were sheared to an M , of about 15 x lo6 in a syringe, and then sedimented to equilibrium in CsCl together with phage lambda [3ZP]DNA.Fractions were collected and counted on filters after precipitation with 10 trichloroacetic acid. ( O d ) [3H]DNA; (. . . . .) phage [32P]DNA;only the position of the center of the 32Ppeak is marked in (B - F). (----) in (A) shows a typical profile of Physarum main-band DNA for comparison. The arrows mark the position of DNA of hybrid density, 1.735 gjml, in this experiment. (A) No BrdUrd, plasmodium harvested at 2 h. (B) BrdUrd from 0-2 h. (C) BrdUrd from 2-4 h. (D) BrdUrd from 4-6 h. (E) BrdUrd 6-8 h. (F) BrdUrd from2-8 h
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Fig.2. Quantification o f r D N A of hybrid density. In each panel of Fig. 1 the radioactivity of all fractions with density greater than that of the lambda DNA was summed and defined to be 1.00. The fraction of this radioactivity that appeared at hybrid density is given by the vertical bars. The open rectangle indicates the time of the radioactive labeling, and M indicates the time of mitosis 111. The data in (A) are taken from the experiment in Fig. 1, and those in (B) from a separate experiment
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an approximately equal fraction of rDNA, 0.1 1- 0.15, appears to be replicated in each interval of the mitotic cycle. These fractions are additive; the sum for the three pulses from 2 - 8 h is 0.41 while the fraction of hybrid DNA after continuous density labeling for that time (Fig. 1F) is 0.44. This suggests that BrdUrd is taken up without a significant lag time. Fig.2B shows that the proportion of labeled rDNA replicated in a given density pulse is not dependent on the time in the previous cycle when radioactivity was incorporated into DNA. When a plasmodium is labeled with r3H]thymidine early in G-2, from 3.5-4.5 h, again roughly equal fractions replicate in the intervals from 2- 8 h after the subsequent mitosis. The profiles in Fig. 1B (BrdUrd) from 0-2 h after MIII) and Fig. 1F (BrdUrd from 2- 8 h after MIII) are important controls for interpreting the other density shift experiments. They suggest that the variable amount of G-2 radioactivity found at the density of main band can be disregarded in the quantification in Fig. 2. Because at least 80 % of normal main-band DNA replicates in the first 2 h after mitosis [8,23] the near absence of tritium at the hybrid position in Fig. 1B implies that most of the labeled main-band DNA visible in the panels of Fig. 1 is not replicated normally. Moreover, Fig. 1F shows that in the time period 2 - 8 h, covering most of the rest of the mitotic cycle, again at least a major part of main-band radioactivity does not become hybrid, as seen by the lack of diminution of the labeled peak to the light side of the phage marker. These results suggest that the radioactive main band DNA observed in variable amounts after G-2 labeling is aberrant and probably does not replicate again in the subsequent mitotic cycle. Guttes and Guttes [12] have made a related observation, that the non-ribosomal DNA labeled in G-2 is in cytologically aberrant nuclei. We assume therefore that at least the majority of the radioactivity shifted to hybrid density in Fig. 1B - F represents rDNA. The results of these density shift experiments can be explained by the model of unscheduled DNA replication. According to this model, rDNA has no ‘memory’. Thus at any time in the latter half of S phase and throughout G-2, all rDNA molecules have the same probability of being chosen by the replication machinery, irrespective of whether or not they have been previously replicated. If N is the original number of rDNA molecules per nucleus after mitosis, then the probability that a given molecule will be chosen at the first rDNA replication is N-l, at the second replication is ( N + l)-’, and at the last rDNA replication before mitosis is (2 N - l)-‘, since there must be N replications in the cycle to double the quantity of rDNA from one mitosis to the next. The probabilities that a given molecule will not be replicated in the first round is then 1 - N - ’ = ( N - 1)/ N and not in the first two rounds (N - l)/N(N 1).
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Fig. 3. Equilibrium sedimentation of D N A labeled with BrdUrd and 3H in a single cycle. A plasmodium was incubated in medium containing BrdUrd from 1.5- 4.0 h, thymidine from 4- 6 h, and [3H]deoxyadenosine at 10 pCi/ml from 6-8 h. Ethidium bromide at 10 pg/ml was also present 1 h before and during the radioactive labeling. The times were measured from MIL At 8 h nuclei were prepared, and the DNA was isolated by selective extraction and analyzed in CsCl gradients similar to those in Fig.1. The arrow points to the peak of hybrid density
Thus the probability that a given molecule is not replicated throughout an entire mitotic cycle is the product N + l
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This fraction is nearly equal to l/2 for large N. The prediction from this calculation is that density labeling for an entire cycle should shift about onehalf of the rDNA radioactively labeled in a previous cycle to the hybrid position in CsCl. This prediction is born out. In three experiments in which BrdUrd was present for the entire periods from MI11 to MIV, 40 - 60 % of the selectively extracted G-2 radioactivity denser than phage DNA banded as hybrid DNA. The results obtained were very similar to the one shown in Fig. 1F. Under the same conditions more than 95 % of total nuclear DNA labeled with a pulse of 3H in the S phase was shifted to hybrid density (not shown). A further prediction from the model is that some rDNA molecules replicate more than once in a single mitotic cycle. Fig. 3 demonstrates that such double replication occurs. A plasmodium was incubated from 1.5-4 h after mitosis with BrdUrd so that all newly synthesized strands become dense. Incubation was continued for 2 h in the presence of thymidine to rapidly dilute unused BrdUrd, and then the same plasmodium was pulse-labeled for the following 2 h with [3H]deoxyadenosine. Low-molecular-weight DNA selectively extracted from nuclei was then centrifuged to equilibrium in CsCl. Of the label banding to the dense side
V. M. Vogt and R. Braun
of the phage marker, 22% was in the hybrid peak, centered at a density 20 mg/ml heavier than rDNA, as expected for duplex DNA with one strand fully substituted with BrdUrd. This result indicates that 22 % of the molecules that replicated during the radioactive pulse had also replicated previously during the density pulse. Alternative explanations for the appearance of radioactive DNA of hybrid density were excluded by two further experiments (not shown). In the first, the time when cultures were grown in the presence of thymidine was varied between 1 and 2 h. No significant alteration of the proportion of radioactive hybrid DNA was observed. In the second, the peak of hybrid density was rebanded in an alkaline CsCl gradient. All the radioactivity was found to be in light strands. Taken together, these data rule out the possibility of contemporaneous incorporation of BrdUrd and tritium, and the possibility of extensive recombination among rDNA molecules. According to the model, since any single DNA strand has the same chance of being a template in a replication event, the percentage of labeled hybrid DNA in the experiment in Fig.3 should be equal to the percentage of total single strands that are already dense at the time of the radioactive pulse. Assuming a linear increase of rDNA from 2N to 4N single strands, starting at 1.5 h after mitosis and ending at 9.5 h after mitosis, the 2.5-h density pulse in Fig.3 should label (2.5/8) (2N) = 0.62N strands. At 7 h after mitosis, at the middle of the radioactive pulse, there should be a total of 2N + (5.5/8) (2N) = 3.4N strands. The fraction of dense strands then is 0.62N/3.4N or 180/0, in approximate agreement with the experimental result. In other experiments of this kind, with varying times of density labeling and of radioactive labeling, all gave hybrid peaks, with 7 out of 8 matching the predicted value with 25 %. An objection that may be raised to the experiment described in Fig. 3 is that the hybrid DNA seen in the CsCl gradient was not shown directly to be rDNA. Main-band and mitochondria1 DNA incorporating BrdUrd would forms bands in CsCl of about the same density as hybrid rDNA. To provide direct evidence bearing on this point, an experiment like that described in Fig. 3 was performed on a preparative scale. The peaks of normal rDNA and the putative hybrid rDNA, and of the main-band peak as a control, were pooled separately, concentrated, and then analyzed by sedimentation as well as by restriction nuclease digestion. Both hybrid and normal rDNA sedimented in a sharp peak at about 40S, just ahead of lambda phage [‘“C] DNA, as expected for rDNA. The main band radioactivity sedimented heterogeneously from 25 S to 50 S (data not shown). Digestion with nuclease Hind111 is known to cut rDNA into pieces with M , 3.2, 5.2 and 21.5 x lo6 [3,4].The gel electrophoretic profiles of hybrid DNA and normal
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Fig.4. Restriction nuclease analysis of’ rDNA of hybrid density. A plasmodium was labeled and DNA prepared as described in Fig. 3, except that the concentration of [3H]deoxyadenosine was 20 pCi/ml. The peak fractions at the densities of hybrid DNA and normal rDNA were concentrated by ethanol precipitation, and then to aliquots of each was added 1 pg lambda phage DNA. The samples were digested with Hind111 nuclease and then analyzed by electrophoresis on a 1 % agarose gel. The gels were stained with ethidium bromide to locate the phage bands before being sliced and counted for radioactivity. Electrophoresis is from left to right. (A) Hybrid DNA; (B) rDNA of normal density
rDNA after Hind111 digestion are pictured in Fig.4. The restriction patterns for the two samples are identical, confirming that it is chiefly rDNA that constitutes the hybrid peak. From these experiments we conclude that at least the majority of rDNA sequences replicate in an un-
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Table 1. Electron microscopy of’replicuting rDNA Pulse-labeled ribosomal DNA was purified from total nuclear DNA and then observed by electron microscopy either before or after enrichment of replicating forms by sucrose gradient sedimentation, as described in the legend to Fig. 5. The replicating fraction corresponded to fractions 4-6 in Fig. 5A. These were concentrated by ethanol precipitation and spread in 1 M ammonium acetate tor microscopy. Only molecules that appeared on rapid scanning to be approximately the length of unit rDNA (70% of all molecules) were analyzed in the statistics
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scheduled manner. However, the data do not exclude that a small fraction of rDNA, for example a few genes integrated into chromosomal DNA, is replicated in a different way.
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To investigate by what mechanism rDNA replicates, we sought to visualize replicating molecules by electron microscopy. One would expect to find only a low percentage of such molecules in purified rDNA since (a) it takes about 8 h (an entire mitotic cycle minus the first hour) to double the population of about 300 ribosomal genes [2,13,23], or 150 rDNA molecules, and (b) a single molecule probably replicates in less than 30 min. The latter assumption is based on the known rate of fork displacement in higher eucaryotes, about 2 x lo6 g mol-l min-’ [14] and on our preliminary experiments with density labeling, which showed that intact rDNA of hybrid density appeared in less than 30 min after placing a G-2 plasmodium in medium with BrdUrd. The unit size of Physarum rDNA allows an enrichment of replicating molecules, since these should sediment at a different rate than intact linear DNA. The experiment in Fig. 5 confirms this expectation. A large fraction of rDNA that has been pulse-labeled for 15 min (Fig.5A) sediments more rapidly than rDNA that has been pulse-labeled and then allowed to continue replication (Fig. 5 B). In both cases the DNA was purified by three consecutive CsCl equilibrium sedimentations. Selective extraction was not used in these experiments because preliminary results
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Fig. 5. Velocity sedimmtation of pulse-labeled D N A . A plasmodium was labeled for 15 min at 4.5 h after MI1 with 20 pCi/ml [3H]deoxyadenosine. Nuclei were prepared from one half (‘pulse’) and the total DNA was isolated. For the other half incubation was continued for 2 h (‘chase’) and then total nuclear DNA was isolated. Each sample of DNA was sedimented to equilibrium in CsCl and aliquots were counted to determine the positions of rDNA. The rDNA peaks were sedimented to equilibrium twice more in order to remove any contaminating labeled main-band DNA. After dialysis; aliquots of the purified rDNA were mixed with lambda phage [14C]DNA and sedimented in gradients containing 5 - 20 % sucrose, 0.01 M Tris pH 7.8, 0.001 M EDTA. Sedimentation was at 18 “C for 110 min at 45000 rev./min in an SW 50.1 rotor. Fractions were collected from the bottom and counted for radioactivity. (0-0) 3H; ( A . . . . .A) I4C. (A) Pulse label. (B) Pulse label followed by chase
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Fig. 6 . Electron micrographs of’ replicuting rDNA. Pulse-labeled rDNA was purified in three consecutive CsCl gradients and then sedimented in a sucrose gradient, as described in Fig. 5 except that no phage DNA was added. The fractions corresponding to fractions 4- 6 in Fig.5A were pooled, concentrated by ethanol precipitation, and spread for electron microscopy in 1 M ammonium acetate. The bar represents 1 pm
indicated that pulse-labeled rDNA is preferentially lost by this procedure. From preparations similar to that shown in Fig. 5 A, the fractions constituting the heavy ‘replicating’ shoulder of the main rDNA peak (equivalent to fractions 4 - 6 in the figure) were pooled and prepared for electron microscopy. Table 1 lists the classes of
molecules observed in two independent preparations. Of all the molecules, 70% were judged by eye to be approximately unit length. The smaller molecules, which contained less than 1 % forked structures, were presumed to be fragments of rDNA or main band contaminants, since they do not fall into discrete size classes [ 3 ] . Of the large molecules counted,
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Fig I Diagramatic representation of replicating forms Molecules like those pictured in Fig 6 were selected randomly and measured Those with the length expected of rDNA (82 %) are pictured here In each molecule the double line represents the replicated region, or ‘eye’, and the single lines the linear ends over which the replication fork has not yet passed The molecules have been centered approximately, so that their 0 50 points fall on a vertical line Two classes of molecules are apparent, with eyes centered over points 0 45 or 0 33 from the left end of the DNA
14 % were forked. Most of these, or 12 % of the total, were molecules with two forks connected by an ‘eye’ of variable size. Examples of these eyes are pictured in Fig. 6. Since 82 % of the measured DNA molecules with eyes were the size of linear rDNA (1/2x the contour length of the circular eye plus the lengths of the two linear portions), we conclude that these forms are rDNA and not some contaminant. In all cases, the two sections of DNA making up the eye were of equal length within experimental error, suggesting strongly that they are replication intermediates. Moreover, as seen in the table, the pooled sucrose gradient fractions containing pulse-labeled rDNA are enriched for eye forms by a factor of 10 over total purified rDNA, as expected for replicating molecules. Fig. 7 pictures schematically a collection of randomly selected, unit-sized molecules with eyes. The lengths of the linear and semi-circular portions of the DNA are represented by the horizontal lines. The
molecules have been arranged so that their midpoints coincide, and so that the eye is on the left side. By inspection, it is apparent that about two thirds of the rDNA eyes are centered in one region, 0.45 times the unit length from one end, while the remaining one-third are centered at 0.33 unit from one end. The few molecules with very small eyes delimit these two regions most clearly. The simplest model that explains the pattern of eyes in rDNA appears to us to be the following. Replication is initiated in one of four possible points on the DNA, at 0.33, 0.45, 0.55, or 0.67 unit from one end. The points at 0.33 and 0.67, and at 0.45 and 0.55 are identical, respectively, since the DNA sequences are symmetrical about the middle, 0.50 [3]. Fork displacements then proceeds bidirectionally at approximately equal rates. Further discussion of this model is presented below. A small fraction of apparently forked molecules that were observed are not eyes. These fall into two classes, linears with one or two forks (273, and lariats (0.3 %). The linears we presume to be broken eyes or eyes in which one fork had passed over the end of the DNA. The lariat-shaped molecules, which might be construed to indicate a rolling circle mechanism of replication, appear to be artifacts. As seen in Table 1, sucrose gradient sedimentation of pulse-labeled DNA did not enrich these forms. Moreover, the entire contour length (circular plus linear portions) of 8 of 12 measured lariats was equal to the unit rDNA, M , = 3 7 x lo6, with the circular portion varying without obvious pattern between M , = 4.6 x lo6 and 25 x lo6. These measurements are inconsistent both with a rolling circle mechanism, and with unidirectional fork displacement from one end [17]. Instead, they suggest that in our hands one end of up to 1 % of the linear rDNA molecules lies very close to some interior stretch of the same DNA. Further analysis will be necessary to determine more carefully whether the lariat-shaped as well as the circular molecules in Physarum rDNA are indeed artificial or have some biological function.
DISCUSSION The density shift experiments described above imply that at least one half (and probably all) of the rDNA molecules participate in replication. This finding rules out replication schemes involving only a few ‘master’ DNA copies that replicate many times in a single mitotic cycle. In the proposed model, all rDNA molecules replicate with equal probability. This appears to us to be the simplest was to imagine a large collection of identical genes regularly being duplicated. The model would presumably require that some feedback mechanism regulates the rate of replication, in
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order to keep the haploid level of ribosomal genes constant. To date we have found no experimental evidence for such a mechanism. The eye-shaped rDNA molecules seen in electron micrographs of rDNA enriched for replicating intermediates strongly suggest that replication proceeds bidirectionally on linear DNA, as in chromosomal DNA of other eucaryotes. The low percentage of such molecules in purified rDNA is approximately as expected, given that it requires about 8 h to double the population, and that at a fork speed of 1 pm/min and bidirectional replication a single molecule would be doubled in about 10 min. In rDNA preparations the forked molecules other than eyes most likely are eyes that have been broken or partially completed, or are artifacts. We have found no evidence for replication of Physarum rDNA by a rolling circle mechanism, as reported by Bohnert et al. [15]. The sizes of circles and circular portions of lariat-shaped molecules seen in preparations of rDNA by these authors were multiples of M , about 8 x lo6, as found in amplified rDNA from Xenopus [16]. These molecular weights appear not to be related to the unit-sized Physarum rDNA described by us and by others [3,4]. This discrepancy has not yet been clarified. Since in vitro all known DNA polymerases synthesize in the 5 ' - + 3 'direction and require a primer, the findings that replicating rDNA molecules are linear raises the question by what mechanism the ends of the molecules are replicated [18]. One possibility is that rDNA exists in the form of circles or concatamers in vivo. Such structures might be held together by proteins, as in adenovirus DNA [19], or perhaps by base-pairing that is disrupted in vitro. Another possibility is that an inverted repetitious sequence at the ends of the molecule functions in replication of the ends [20- 221. It remains to be seen if replication of Physarum rDNA involves any of these features. The size and position of the measured replication eyes suggest that replication is initiated at points either 0.33 or 0.45 unit from one end of the DNA. Since each molecule is symmetrical about the middle, and since the molecules are diagrammed arbitrarily with the eye closest to the left end, presumably there would be identical origins at 0.55 and 0.67 unit. For several reasons these conclusions must be regarded as tentative, however. First, the nucleotide sequences of different rDNA molecules, and of the symmetrical halves of a given molecule, may not be identical. Thus, without direct evidence for more than one eye in a single molecule, it is not possible to conclude that any given rDNA molecule can be replicated from one of four sites. Second, without the untested assumption that both forks move equally fast, it is not possible to assign the molecules with medium or large eyes to one or the other of the two groups (initiation at 0.45 or 0.33) in Fig.7 or to exclude that there are
other points where replication is initiated. The eyes of only two molecules in the 0.33 group clearly fall outside of the 0.45 point, statistically a very small sample. Finally, the arbitrary placing of the eye closest to the left end means that a small eye actually at the center of a molecule will appear to the left of center in a digram because of chance errors in measurement. Thus it is possible to argue that, with the exception of the two molecules mentioned, replication in all the molecules in Fig.7 actually began at 0.50 instead of 0.45 or 0.33. This argument could be bolstered by the findings that replication of both mitochondria1 DNA [22] and extrachromosomal rDNA (Truett and Gall, personal communication) from Tetrahymena also appear to be initiated at a point close to the middle of linear DNA molecules. Does the measurement error support this last argument? Since they should be equal, the measured lengths of the opposite two sections making up the larger replication eyes give the best estimate of length errors in a given molecule. In the 23 molecules with eyes larger than 4 pm on a side, the mean difference between the sides is 2.5 % (standard deviation 1.75 %). By comparison, for the four smallest eyes that apparently are centered at 0.45, the differences between the arms bordering the eyes are 15 %, 21 %, 22 %, and 25 %. If we make the worst-case assumption that fork displacement has been unidirectional for these molecules, then the differences at the time of initiation might be reduced to 5 % , 12%, 15%, and 16%, still considerably larger than the estimated measurement error. Therefore, we favor the model in which replication actually begins at points either near 0.45 or near 0.33 on the DNA, and not in the middle. It is interesting that both of these points fall outside of the coding region (from about 0.075 - 0.25 [4]),but near the limits of the region of repetitive palindromes (from about 0.36-0.47 [3]). More detailed study will be necessary to confirm the existence of four separate initiation sites on a single molecule, however. We wish to thank H. Gautschi, L. Swofford, and J. Calvo for criticism and V. Kocher for technical assistance. This work was supported by grants (3.750.72 and 3.501.75) from the Swiss National Foundation, and from a grant (PCM 76-80440) from The American National Science Foundation.
REFERENCES 1. Zellweger, A., Ryser, U. & Braun, R. (1972) J . M d . B i d . 64, 681 -691. 2. Newlon, C. S., Sonenshein, G. E. & Holt, C. E. (1973) Biochemistry, 12,2338-2345. 3. Vogt, V. M. & Braun, R. (1976) J . Mol. Biol. 106, 567-587. 4. Molgaard, H. V., Matthews, H. R. & Bradbury, E. M. (1976) Eur. J . Biochem. 68, 541 - 549. 5. Daniel, J. W. & Baldwin, H. M. (1964) Methods in Cell Physiology (Prescott, D. C., ed.) pp, 9-41, Academic Press, New York.
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566 6. Braun, R. & Evans, T. E. (1969) Biochim. Biophys. Acta, 182,
7. 8. 9. 10. 11. 12. 13. 14.
511 - 522. Braun, K., Mittermayer, C. & Rusch, H. P. (1965) Proc. Nut/ Acad. Sci. U . S . A . 53, 924-931. Braun, R. & Wili, H. (1969) Biochim. Biophys. Acta, 174, 246- 252. Muller, G. C. & Kajiwara, K. (1966) Biochim. Biophys. Acfa, 114, 108-115. Hirt, B. (1967) J . Mol. Biol. 26, 365-369. Horowitz, H. B. & Holt; C. E. (1971) J . Cell. Riol. 49, 549553. Guttes, E. & Guttes, S. (1969) J . Cell. Biol. 43, 229-236. Hall, L., Turnock, G. & Cox, B. J. (1975) Eur. J . Biochem. 51, 459 - 465. Huberman, J. A. & Riggs, A. D. (1968) J . Mol. Biol. 32, 327341.
IS. Bohnert, H.-J., Schiller, B., Bohme, R. & Sauer, H. W. (1975) Eur. J . Biochem. 57, 361 369. 16. Hourcade, D., Dressier, D. & Wolfson, J. (1973) Proc. Nut/ Acad. Sci. U . S . A . 70, 2926-2930. 17. Goddard, J. M. & Cummings, D. J. (1975) J . Mol. Biol. 97, 593 609. 18. Watson, J. D. (1972) Nut. New Bid. 239, 197-201. 19. Robinson, A. J. & Bellet, A. J. D. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 523 - 531. 20. Cavalier-Smith, T. (1974) Nafure (Lond.) 250, 467-470. 21. Strauss, S. E., Sebring, E. D. & Rose, J. A. (1976) Proc. Nut1 Acad. Sci. U . S . A . 73, 742-746. 22. Arnberg, A. C., Van Bruggen, E. F. J., Clegg, R. A,, Upholt, W. B. & Borst, P. (1974) Biochirn. Biophys. Actu, 361, 266276. 23. Hall, L. & Turnock, G. (1976) Eur. J . Biochem. 62, 471 -477. -
-
V. M. Vogt, Department of Biochemistry, Molecular and Cell Biology, Wing Hall, Cornell University, Ithaca, New York, U.S.A. 14853
R. Braun, Institut fur Allgemeine Mikrobiologie der Universitat Bern, Altenbergrain 21, CH-3013 Bern, Switzerland
The Replication of Ribosomal DNA in Physarum polycephalum Volker M. VOGT and Richard BRAUN Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York and Institute of General Microbiology, University of Bern (Received May 27, 1977)
The DNA coding for ribosomal RNA in Physarum polycephalum exists as a collection of extrachromosomal molecules of molecular weight 37 x lo6. We have investigated the replication of rDNA, with the following results. (a) Replication of rDNA is unscheduled. This means that molecules that are replicated at any particular time in one mitotic cycle have an equal probability of replicating again in each time interval in the subsequent cycle. Similarly, in a single cycle, some molecules replicate more than once, and some not at all. (b) Replication forks appear to move bidirectionally from points 45 % or 33 from one end of the DNA. Replicating molecules observed by electron microscopy are all linear.
In the acellular slime mold Physarumpolycephalum the DNA coding for ribosomal RNA, which comprises 1 - 2 % of total DNA, forms a heavy satellite band when sedimented to equilibrium in CsCl gradients. In contrast to the main-band DNA, whose time of replication defines the S period of the mitotic cycle (0-2.5 h after mitosis), ribosomal DNA (rDNA) replication proceeds throughout the G-2 period as well as through the latter half of the S period [1,2]. There is no G-1 period in Physarum plasmodia, DNA synthesis following immediately after the synchronous mitosis of the nuclei. The facts that rDNA can be purified by equilibrium sedimentation and can be labeled specifically with radioactive precursors in the G-2 period have facilitated studies of its structure. We have shown recently [3] that Physarum rDNA exists as a collection of linear DNA molecules of a unit size, 37 x lo6 M,. These molecules have a rotational axis of symmetry in the center, and thus are palindromes. One ribosomal transcription unit is located at each end of the molecule [4], and the large intervening spacer region contains blocks of short inverted repetitive sequences [3]. The curious timing of synthesis of rDNA led us to investigate its replication more closely. We show here that: (a) replication is unscheduled, and (b) replication proceeds by means of bidirectional fork displacement, apparently from fixed points on the linear molecules.
MATERIALS AND METHODS Growth and Labeling of Plasmodia Microplasmodia of Physarum polycephalum, strain MIIIC, were propagated in shaking cultures at 26 "C in semidefined medium [5]. Macroplasmodia were formed on paper filters by fusion of 0.15 ml of packed plasmodia diluted with an equal volume of water. They were grown over wire grids in the same medium. The time of mitosis was determined by microscopic examination under phase contrast. The time of mitosis I1 (MII) was generally 14 h after fusion. For density labeling, bromodeoxyuridine (BrdUrd, 100 pg/ ml), fluorodeoxyuridine (5 pg/ml), and uridine (100 pg/ ml) were added to the growth medium [8]. In all experiments involving further growth after density labeling, thymidine was added to 100 pg/ml after removal of BrdUrd, fluorodeoxyuridine, and uridine. In the presence of the BrdUrd medium, the mitotic cycle appeared to be lengthened by 0 - 1 h. Thymidine had no effect on the length of the mitotic cycle. The precursors used for radioactive labeling were either [3H]thymidine (usually at 50 pCi/ml) or [3H]deoxyadenosine (usually at 10 pCi/ml). About one half of the [3H]deoxyadenosine incorporated into nuclei by G-2 plasmodia was found in DNA, the rest being in RNA. Control experiments showed that after removal of radioactive label from plasmodia, incorporation ceased within 15 min. Preparation of DNA
Abbreviations. rDNA, ribosomal DNA; BrdUrd, bromodeoxyuridine.
Nuclei were prepared at 0 "C by homogenization followed by centrifugation [3]. They were resuspended
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in 3 mlO.05 M EDTA, pH 7.8, or in early experiments in 0.015 M sodium citrate, 0.15 M NaCl, 0.005 M EDTA. Selective extraction of DNA was done as described previously [6]. Total DNA was prepared by lysing resuspended nuclei by incubation with pronase at 1 mg/ml and sodium dodecyl sulfate at 2%. After 15 min at 37 "C, 2 vol. of ethanol were added and DNA was collected by spooling. DNA prepared either by this procedure or by selective extraction was redissolved in 3.5 ml of 0.05 M EDTA, adjusted to a density of 1.71 or 1.74 g/ml with CsCl, and centrifuged for 60 h at 33000 rev./min, 18 "C in a Ti50 rotor. Fractions were collected from the bottom of the gradient and aliquots counted. In some experiments, the fractions were adjusted to 0.3 M NaOH, heated for 20 min at 80 "C, and then precipitated with trichloroacetic acid before counting. Other Methods
DNA was observed by electron microscopy as described [3]. Lambda phage were purified by differential centrifugation from lysates of lysogenic E. coli that had been heat-induced for lambda cI857S7. RESULTS Timing of Replication
The main-band DNA of Physarum [7,8] as well as the DNA of other eucaryotes [9] replicates on a fixed schedule. This means that base sequences replicated at a particular time in one S phase are replicated again at the same relative time in subsequent mitotic cycles, as determined by density shift experiments. In outline, these experiments consist of radioactive labeling of DNA with a brief pulse of [3H]thymidine at a particular time in one S period, followed by density-labeling with bromodeoxyuridine (BrdUrd) at different times in the S phase of the subsequent mitotic cycle. If thymidine-labeled DNA replicates during the time when BrdUrd is present, the radioactive strand will become paired with a new strand of higher density. The resulting duplex of 'hybrid' density is then identified by equilibrium sedimentation in CsC1. We have used similar techniques to discover whether replication of ribosomal DNA is scheduled. The experiments rely on the fact that after selective extraction of nuclei prepared from plasmodia labeled in G-2, most of the radioactivity is found in rDNA. Selective extraction is a technique that separates low-molecular-weight DNA from the bulk of nuclear DNA and proteins [lo]. Applied to Physarum nuclei, it yields about two-thirds of the rDNA, with an enrichment of about 10-fold over total DNA [6] (and Gubler, Vogt and Braun, unpublished results). Fig. 1A
rDNA Replication in Phy.~urum
shows the pattern of selectively extracted, G-2-labeled DNA in CsCl equilibrium sedimentation. Most of the radioactivity forms a band to the dense side of the 32P-labeledlambda phage DNA included as an internal marker. This is the position of ribosomal DNA, 1.712 g/ml [l]. As seen in the same figure panel, purified main-band DNA forms a broader band centered at a density of 1.700 g/ml or four fractions to the light side of the phage marker. The profile in Fig. 1A is somewhat atypical; in most preparations of G-Zlabeled, selectively extracted DNA, a variable proportion of the radioactivity also is found in the position of the main band. In a typical experiment to investigate the scheduling of rDNA replication, a plasmodium containing about 10' nuclei was labeled 6.5 h after mitosis I1 (MII, the second mitosis after formation of the plasmodium by fusion) for 60 min with [3H]thymidine.The labeling was carried out in the presence of ethidium bromide to suppress replication of mitochondria1 DNA [I 11. Growth was allowed to continue in normal medium lacking radioactive thymidine until the subsequent mitosis, MIII, or about 9 h after MII. The plasmodium was then cut into six sectors, each of which was incubated separately in normal medium. At different times, the growing sectors were then transferred to medium containing BrdUrd and incubated for 2 or 6 h. As a control, one sector was incubated with unlabeled thymidine instead of with BrdUrd. After the density pulse, nuclei were isolated from each sector and low-molecular-weight DNA prepared from sodium dodecyl sulfate lysates by selective extraction. The DNA was then centrifuged to equilibrium in CsCl along with a 32P-labeled lambda phage DNA marker. Fig.1 shows the CsCl profiles of the radioactivity obtained. Fig. 1 A is from the sector receiving no BrdUrd. In Fig. 1B, representing the density pulse from 0-2 h after MIII, most of the radioactivity is still at the position of normal rDNA, with only a small peak of label at the density of hybrid DNA. This result is consistent with the observation that rDNA does not replicate in the first 1 - 1.5 h of S phase [l]. In Fig. 1 C, representing the density pulse from 2 - 4 h, a part of the label has shifted to the position of hybrid DNA, which in different experiments is centered at a density of 1.73- 1.74 g/ml. A similar fraction of radioactivity is found at hybrid density for pulses from 4-6 h (Fig. 1D) as well as from 6-8 h (Fig. 1E). The last profile, Fig. 1F, represents continuous density labeling from 2 - 8 h after MIII. Fig. 2 quantifies the radioactively labeled DNA that is found at the hybrid position after each density pulse. The sum of all radioactivity banding denser than the phage marker is defined as 1.00. (The justification for neglecting radioactivity in main-band DNA, i.e. in DNA on the light side of the phage marker, is given below.) With the exception of the first two hours,
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Fig. 1. Equilibrium sedimentation of D N A lubeled with " H und BrdUrd in di/fbrent mitotic cycles. A plasmodium was incubated in the presence of 50 pCi/ml [3H]thymidine from 6.5-7.5 h after MII. Ethidium bromide at 10 pglml was present 30 min before and during this interval. At the time of MI11 (zero time), the plasmodium was cut into six sectors and each sector was incubated with fresh medium. At the times indicated, sectors were transferred to medium containing BrdUrd and incubated for 2 h or 6 h. One sector was incubated in normal medium as a control. Immediately after this labeling period, nuclei were prepared from the sectors and low-molecular-weight DNA isolated from them by selective extraction. After treatment with 20 pg/ml RNase A for 30 min at 37 "C in 1 mM Tris pH 7.8, 0.1 mM EDTA, the samples were sheared to an M , of about 15 x lo6 in a syringe, and then sedimented to equilibrium in CsCl together with phage lambda [3ZP]DNA.Fractions were collected and counted on filters after precipitation with 10 trichloroacetic acid. ( O d ) [3H]DNA; (. . . . .) phage [32P]DNA;only the position of the center of the 32Ppeak is marked in (B - F). (----) in (A) shows a typical profile of Physarum main-band DNA for comparison. The arrows mark the position of DNA of hybrid density, 1.735 gjml, in this experiment. (A) No BrdUrd, plasmodium harvested at 2 h. (B) BrdUrd from 0-2 h. (C) BrdUrd from 2-4 h. (D) BrdUrd from 4-6 h. (E) BrdUrd 6-8 h. (F) BrdUrd from2-8 h
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Fig.2. Quantification o f r D N A of hybrid density. In each panel of Fig. 1 the radioactivity of all fractions with density greater than that of the lambda DNA was summed and defined to be 1.00. The fraction of this radioactivity that appeared at hybrid density is given by the vertical bars. The open rectangle indicates the time of the radioactive labeling, and M indicates the time of mitosis 111. The data in (A) are taken from the experiment in Fig. 1, and those in (B) from a separate experiment
rDNA Replication in Physarum
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an approximately equal fraction of rDNA, 0.1 1- 0.15, appears to be replicated in each interval of the mitotic cycle. These fractions are additive; the sum for the three pulses from 2 - 8 h is 0.41 while the fraction of hybrid DNA after continuous density labeling for that time (Fig. 1F) is 0.44. This suggests that BrdUrd is taken up without a significant lag time. Fig.2B shows that the proportion of labeled rDNA replicated in a given density pulse is not dependent on the time in the previous cycle when radioactivity was incorporated into DNA. When a plasmodium is labeled with r3H]thymidine early in G-2, from 3.5-4.5 h, again roughly equal fractions replicate in the intervals from 2- 8 h after the subsequent mitosis. The profiles in Fig. 1B (BrdUrd) from 0-2 h after MIII) and Fig. 1F (BrdUrd from 2- 8 h after MIII) are important controls for interpreting the other density shift experiments. They suggest that the variable amount of G-2 radioactivity found at the density of main band can be disregarded in the quantification in Fig. 2. Because at least 80 % of normal main-band DNA replicates in the first 2 h after mitosis [8,23] the near absence of tritium at the hybrid position in Fig. 1B implies that most of the labeled main-band DNA visible in the panels of Fig. 1 is not replicated normally. Moreover, Fig. 1F shows that in the time period 2 - 8 h, covering most of the rest of the mitotic cycle, again at least a major part of main-band radioactivity does not become hybrid, as seen by the lack of diminution of the labeled peak to the light side of the phage marker. These results suggest that the radioactive main band DNA observed in variable amounts after G-2 labeling is aberrant and probably does not replicate again in the subsequent mitotic cycle. Guttes and Guttes [12] have made a related observation, that the non-ribosomal DNA labeled in G-2 is in cytologically aberrant nuclei. We assume therefore that at least the majority of the radioactivity shifted to hybrid density in Fig. 1B - F represents rDNA. The results of these density shift experiments can be explained by the model of unscheduled DNA replication. According to this model, rDNA has no ‘memory’. Thus at any time in the latter half of S phase and throughout G-2, all rDNA molecules have the same probability of being chosen by the replication machinery, irrespective of whether or not they have been previously replicated. If N is the original number of rDNA molecules per nucleus after mitosis, then the probability that a given molecule will be chosen at the first rDNA replication is N-l, at the second replication is ( N + l)-’, and at the last rDNA replication before mitosis is (2 N - l)-‘, since there must be N replications in the cycle to double the quantity of rDNA from one mitosis to the next. The probabilities that a given molecule will not be replicated in the first round is then 1 - N - ’ = ( N - 1)/ N and not in the first two rounds (N - l)/N(N 1).
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Fig. 3. Equilibrium sedimentation of D N A labeled with BrdUrd and 3H in a single cycle. A plasmodium was incubated in medium containing BrdUrd from 1.5- 4.0 h, thymidine from 4- 6 h, and [3H]deoxyadenosine at 10 pCi/ml from 6-8 h. Ethidium bromide at 10 pg/ml was also present 1 h before and during the radioactive labeling. The times were measured from MIL At 8 h nuclei were prepared, and the DNA was isolated by selective extraction and analyzed in CsCl gradients similar to those in Fig.1. The arrow points to the peak of hybrid density
Thus the probability that a given molecule is not replicated throughout an entire mitotic cycle is the product N + l
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This fraction is nearly equal to l/2 for large N. The prediction from this calculation is that density labeling for an entire cycle should shift about onehalf of the rDNA radioactively labeled in a previous cycle to the hybrid position in CsCl. This prediction is born out. In three experiments in which BrdUrd was present for the entire periods from MI11 to MIV, 40 - 60 % of the selectively extracted G-2 radioactivity denser than phage DNA banded as hybrid DNA. The results obtained were very similar to the one shown in Fig. 1F. Under the same conditions more than 95 % of total nuclear DNA labeled with a pulse of 3H in the S phase was shifted to hybrid density (not shown). A further prediction from the model is that some rDNA molecules replicate more than once in a single mitotic cycle. Fig. 3 demonstrates that such double replication occurs. A plasmodium was incubated from 1.5-4 h after mitosis with BrdUrd so that all newly synthesized strands become dense. Incubation was continued for 2 h in the presence of thymidine to rapidly dilute unused BrdUrd, and then the same plasmodium was pulse-labeled for the following 2 h with [3H]deoxyadenosine. Low-molecular-weight DNA selectively extracted from nuclei was then centrifuged to equilibrium in CsCl. Of the label banding to the dense side
V. M. Vogt and R. Braun
of the phage marker, 22% was in the hybrid peak, centered at a density 20 mg/ml heavier than rDNA, as expected for duplex DNA with one strand fully substituted with BrdUrd. This result indicates that 22 % of the molecules that replicated during the radioactive pulse had also replicated previously during the density pulse. Alternative explanations for the appearance of radioactive DNA of hybrid density were excluded by two further experiments (not shown). In the first, the time when cultures were grown in the presence of thymidine was varied between 1 and 2 h. No significant alteration of the proportion of radioactive hybrid DNA was observed. In the second, the peak of hybrid density was rebanded in an alkaline CsCl gradient. All the radioactivity was found to be in light strands. Taken together, these data rule out the possibility of contemporaneous incorporation of BrdUrd and tritium, and the possibility of extensive recombination among rDNA molecules. According to the model, since any single DNA strand has the same chance of being a template in a replication event, the percentage of labeled hybrid DNA in the experiment in Fig.3 should be equal to the percentage of total single strands that are already dense at the time of the radioactive pulse. Assuming a linear increase of rDNA from 2N to 4N single strands, starting at 1.5 h after mitosis and ending at 9.5 h after mitosis, the 2.5-h density pulse in Fig.3 should label (2.5/8) (2N) = 0.62N strands. At 7 h after mitosis, at the middle of the radioactive pulse, there should be a total of 2N + (5.5/8) (2N) = 3.4N strands. The fraction of dense strands then is 0.62N/3.4N or 180/0, in approximate agreement with the experimental result. In other experiments of this kind, with varying times of density labeling and of radioactive labeling, all gave hybrid peaks, with 7 out of 8 matching the predicted value with 25 %. An objection that may be raised to the experiment described in Fig. 3 is that the hybrid DNA seen in the CsCl gradient was not shown directly to be rDNA. Main-band and mitochondria1 DNA incorporating BrdUrd would forms bands in CsCl of about the same density as hybrid rDNA. To provide direct evidence bearing on this point, an experiment like that described in Fig. 3 was performed on a preparative scale. The peaks of normal rDNA and the putative hybrid rDNA, and of the main-band peak as a control, were pooled separately, concentrated, and then analyzed by sedimentation as well as by restriction nuclease digestion. Both hybrid and normal rDNA sedimented in a sharp peak at about 40S, just ahead of lambda phage [‘“C] DNA, as expected for rDNA. The main band radioactivity sedimented heterogeneously from 25 S to 50 S (data not shown). Digestion with nuclease Hind111 is known to cut rDNA into pieces with M , 3.2, 5.2 and 21.5 x lo6 [3,4].The gel electrophoretic profiles of hybrid DNA and normal
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Fig.4. Restriction nuclease analysis of’ rDNA of hybrid density. A plasmodium was labeled and DNA prepared as described in Fig. 3, except that the concentration of [3H]deoxyadenosine was 20 pCi/ml. The peak fractions at the densities of hybrid DNA and normal rDNA were concentrated by ethanol precipitation, and then to aliquots of each was added 1 pg lambda phage DNA. The samples were digested with Hind111 nuclease and then analyzed by electrophoresis on a 1 % agarose gel. The gels were stained with ethidium bromide to locate the phage bands before being sliced and counted for radioactivity. Electrophoresis is from left to right. (A) Hybrid DNA; (B) rDNA of normal density
rDNA after Hind111 digestion are pictured in Fig.4. The restriction patterns for the two samples are identical, confirming that it is chiefly rDNA that constitutes the hybrid peak. From these experiments we conclude that at least the majority of rDNA sequences replicate in an un-
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Table 1. Electron microscopy of’replicuting rDNA Pulse-labeled ribosomal DNA was purified from total nuclear DNA and then observed by electron microscopy either before or after enrichment of replicating forms by sucrose gradient sedimentation, as described in the legend to Fig. 5. The replicating fraction corresponded to fractions 4-6 in Fig. 5A. These were concentrated by ethanol precipitation and spread in 1 M ammonium acetate tor microscopy. Only molecules that appeared on rapid scanning to be approximately the length of unit rDNA (70% of all molecules) were analyzed in the statistics
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3 Fraction number
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scheduled manner. However, the data do not exclude that a small fraction of rDNA, for example a few genes integrated into chromosomal DNA, is replicated in a different way.
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To investigate by what mechanism rDNA replicates, we sought to visualize replicating molecules by electron microscopy. One would expect to find only a low percentage of such molecules in purified rDNA since (a) it takes about 8 h (an entire mitotic cycle minus the first hour) to double the population of about 300 ribosomal genes [2,13,23], or 150 rDNA molecules, and (b) a single molecule probably replicates in less than 30 min. The latter assumption is based on the known rate of fork displacement in higher eucaryotes, about 2 x lo6 g mol-l min-’ [14] and on our preliminary experiments with density labeling, which showed that intact rDNA of hybrid density appeared in less than 30 min after placing a G-2 plasmodium in medium with BrdUrd. The unit size of Physarum rDNA allows an enrichment of replicating molecules, since these should sediment at a different rate than intact linear DNA. The experiment in Fig. 5 confirms this expectation. A large fraction of rDNA that has been pulse-labeled for 15 min (Fig.5A) sediments more rapidly than rDNA that has been pulse-labeled and then allowed to continue replication (Fig. 5 B). In both cases the DNA was purified by three consecutive CsCl equilibrium sedimentations. Selective extraction was not used in these experiments because preliminary results
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Fig. 5. Velocity sedimmtation of pulse-labeled D N A . A plasmodium was labeled for 15 min at 4.5 h after MI1 with 20 pCi/ml [3H]deoxyadenosine. Nuclei were prepared from one half (‘pulse’) and the total DNA was isolated. For the other half incubation was continued for 2 h (‘chase’) and then total nuclear DNA was isolated. Each sample of DNA was sedimented to equilibrium in CsCl and aliquots were counted to determine the positions of rDNA. The rDNA peaks were sedimented to equilibrium twice more in order to remove any contaminating labeled main-band DNA. After dialysis; aliquots of the purified rDNA were mixed with lambda phage [14C]DNA and sedimented in gradients containing 5 - 20 % sucrose, 0.01 M Tris pH 7.8, 0.001 M EDTA. Sedimentation was at 18 “C for 110 min at 45000 rev./min in an SW 50.1 rotor. Fractions were collected from the bottom and counted for radioactivity. (0-0) 3H; ( A . . . . .A) I4C. (A) Pulse label. (B) Pulse label followed by chase
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Fig. 6 . Electron micrographs of’ replicuting rDNA. Pulse-labeled rDNA was purified in three consecutive CsCl gradients and then sedimented in a sucrose gradient, as described in Fig. 5 except that no phage DNA was added. The fractions corresponding to fractions 4- 6 in Fig.5A were pooled, concentrated by ethanol precipitation, and spread for electron microscopy in 1 M ammonium acetate. The bar represents 1 pm
indicated that pulse-labeled rDNA is preferentially lost by this procedure. From preparations similar to that shown in Fig. 5 A, the fractions constituting the heavy ‘replicating’ shoulder of the main rDNA peak (equivalent to fractions 4 - 6 in the figure) were pooled and prepared for electron microscopy. Table 1 lists the classes of
molecules observed in two independent preparations. Of all the molecules, 70% were judged by eye to be approximately unit length. The smaller molecules, which contained less than 1 % forked structures, were presumed to be fragments of rDNA or main band contaminants, since they do not fall into discrete size classes [ 3 ] . Of the large molecules counted,
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~
c
A 33% 50%
5Km
Fig I Diagramatic representation of replicating forms Molecules like those pictured in Fig 6 were selected randomly and measured Those with the length expected of rDNA (82 %) are pictured here In each molecule the double line represents the replicated region, or ‘eye’, and the single lines the linear ends over which the replication fork has not yet passed The molecules have been centered approximately, so that their 0 50 points fall on a vertical line Two classes of molecules are apparent, with eyes centered over points 0 45 or 0 33 from the left end of the DNA
14 % were forked. Most of these, or 12 % of the total, were molecules with two forks connected by an ‘eye’ of variable size. Examples of these eyes are pictured in Fig. 6. Since 82 % of the measured DNA molecules with eyes were the size of linear rDNA (1/2x the contour length of the circular eye plus the lengths of the two linear portions), we conclude that these forms are rDNA and not some contaminant. In all cases, the two sections of DNA making up the eye were of equal length within experimental error, suggesting strongly that they are replication intermediates. Moreover, as seen in the table, the pooled sucrose gradient fractions containing pulse-labeled rDNA are enriched for eye forms by a factor of 10 over total purified rDNA, as expected for replicating molecules. Fig. 7 pictures schematically a collection of randomly selected, unit-sized molecules with eyes. The lengths of the linear and semi-circular portions of the DNA are represented by the horizontal lines. The
molecules have been arranged so that their midpoints coincide, and so that the eye is on the left side. By inspection, it is apparent that about two thirds of the rDNA eyes are centered in one region, 0.45 times the unit length from one end, while the remaining one-third are centered at 0.33 unit from one end. The few molecules with very small eyes delimit these two regions most clearly. The simplest model that explains the pattern of eyes in rDNA appears to us to be the following. Replication is initiated in one of four possible points on the DNA, at 0.33, 0.45, 0.55, or 0.67 unit from one end. The points at 0.33 and 0.67, and at 0.45 and 0.55 are identical, respectively, since the DNA sequences are symmetrical about the middle, 0.50 [3]. Fork displacements then proceeds bidirectionally at approximately equal rates. Further discussion of this model is presented below. A small fraction of apparently forked molecules that were observed are not eyes. These fall into two classes, linears with one or two forks (273, and lariats (0.3 %). The linears we presume to be broken eyes or eyes in which one fork had passed over the end of the DNA. The lariat-shaped molecules, which might be construed to indicate a rolling circle mechanism of replication, appear to be artifacts. As seen in Table 1, sucrose gradient sedimentation of pulse-labeled DNA did not enrich these forms. Moreover, the entire contour length (circular plus linear portions) of 8 of 12 measured lariats was equal to the unit rDNA, M , = 3 7 x lo6, with the circular portion varying without obvious pattern between M , = 4.6 x lo6 and 25 x lo6. These measurements are inconsistent both with a rolling circle mechanism, and with unidirectional fork displacement from one end [17]. Instead, they suggest that in our hands one end of up to 1 % of the linear rDNA molecules lies very close to some interior stretch of the same DNA. Further analysis will be necessary to determine more carefully whether the lariat-shaped as well as the circular molecules in Physarum rDNA are indeed artificial or have some biological function.
DISCUSSION The density shift experiments described above imply that at least one half (and probably all) of the rDNA molecules participate in replication. This finding rules out replication schemes involving only a few ‘master’ DNA copies that replicate many times in a single mitotic cycle. In the proposed model, all rDNA molecules replicate with equal probability. This appears to us to be the simplest was to imagine a large collection of identical genes regularly being duplicated. The model would presumably require that some feedback mechanism regulates the rate of replication, in
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order to keep the haploid level of ribosomal genes constant. To date we have found no experimental evidence for such a mechanism. The eye-shaped rDNA molecules seen in electron micrographs of rDNA enriched for replicating intermediates strongly suggest that replication proceeds bidirectionally on linear DNA, as in chromosomal DNA of other eucaryotes. The low percentage of such molecules in purified rDNA is approximately as expected, given that it requires about 8 h to double the population, and that at a fork speed of 1 pm/min and bidirectional replication a single molecule would be doubled in about 10 min. In rDNA preparations the forked molecules other than eyes most likely are eyes that have been broken or partially completed, or are artifacts. We have found no evidence for replication of Physarum rDNA by a rolling circle mechanism, as reported by Bohnert et al. [15]. The sizes of circles and circular portions of lariat-shaped molecules seen in preparations of rDNA by these authors were multiples of M , about 8 x lo6, as found in amplified rDNA from Xenopus [16]. These molecular weights appear not to be related to the unit-sized Physarum rDNA described by us and by others [3,4]. This discrepancy has not yet been clarified. Since in vitro all known DNA polymerases synthesize in the 5 ' - + 3 'direction and require a primer, the findings that replicating rDNA molecules are linear raises the question by what mechanism the ends of the molecules are replicated [18]. One possibility is that rDNA exists in the form of circles or concatamers in vivo. Such structures might be held together by proteins, as in adenovirus DNA [19], or perhaps by base-pairing that is disrupted in vitro. Another possibility is that an inverted repetitious sequence at the ends of the molecule functions in replication of the ends [20- 221. It remains to be seen if replication of Physarum rDNA involves any of these features. The size and position of the measured replication eyes suggest that replication is initiated at points either 0.33 or 0.45 unit from one end of the DNA. Since each molecule is symmetrical about the middle, and since the molecules are diagrammed arbitrarily with the eye closest to the left end, presumably there would be identical origins at 0.55 and 0.67 unit. For several reasons these conclusions must be regarded as tentative, however. First, the nucleotide sequences of different rDNA molecules, and of the symmetrical halves of a given molecule, may not be identical. Thus, without direct evidence for more than one eye in a single molecule, it is not possible to conclude that any given rDNA molecule can be replicated from one of four sites. Second, without the untested assumption that both forks move equally fast, it is not possible to assign the molecules with medium or large eyes to one or the other of the two groups (initiation at 0.45 or 0.33) in Fig.7 or to exclude that there are
other points where replication is initiated. The eyes of only two molecules in the 0.33 group clearly fall outside of the 0.45 point, statistically a very small sample. Finally, the arbitrary placing of the eye closest to the left end means that a small eye actually at the center of a molecule will appear to the left of center in a digram because of chance errors in measurement. Thus it is possible to argue that, with the exception of the two molecules mentioned, replication in all the molecules in Fig.7 actually began at 0.50 instead of 0.45 or 0.33. This argument could be bolstered by the findings that replication of both mitochondria1 DNA [22] and extrachromosomal rDNA (Truett and Gall, personal communication) from Tetrahymena also appear to be initiated at a point close to the middle of linear DNA molecules. Does the measurement error support this last argument? Since they should be equal, the measured lengths of the opposite two sections making up the larger replication eyes give the best estimate of length errors in a given molecule. In the 23 molecules with eyes larger than 4 pm on a side, the mean difference between the sides is 2.5 % (standard deviation 1.75 %). By comparison, for the four smallest eyes that apparently are centered at 0.45, the differences between the arms bordering the eyes are 15 %, 21 %, 22 %, and 25 %. If we make the worst-case assumption that fork displacement has been unidirectional for these molecules, then the differences at the time of initiation might be reduced to 5 % , 12%, 15%, and 16%, still considerably larger than the estimated measurement error. Therefore, we favor the model in which replication actually begins at points either near 0.45 or near 0.33 on the DNA, and not in the middle. It is interesting that both of these points fall outside of the coding region (from about 0.075 - 0.25 [4]),but near the limits of the region of repetitive palindromes (from about 0.36-0.47 [3]). More detailed study will be necessary to confirm the existence of four separate initiation sites on a single molecule, however. We wish to thank H. Gautschi, L. Swofford, and J. Calvo for criticism and V. Kocher for technical assistance. This work was supported by grants (3.750.72 and 3.501.75) from the Swiss National Foundation, and from a grant (PCM 76-80440) from The American National Science Foundation.
REFERENCES 1. Zellweger, A., Ryser, U. & Braun, R. (1972) J . M d . B i d . 64, 681 -691. 2. Newlon, C. S., Sonenshein, G. E. & Holt, C. E. (1973) Biochemistry, 12,2338-2345. 3. Vogt, V. M. & Braun, R. (1976) J . Mol. Biol. 106, 567-587. 4. Molgaard, H. V., Matthews, H. R. & Bradbury, E. M. (1976) Eur. J . Biochem. 68, 541 - 549. 5. Daniel, J. W. & Baldwin, H. M. (1964) Methods in Cell Physiology (Prescott, D. C., ed.) pp, 9-41, Academic Press, New York.
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566 6. Braun, R. & Evans, T. E. (1969) Biochim. Biophys. Acta, 182,
7. 8. 9. 10. 11. 12. 13. 14.
511 - 522. Braun, K., Mittermayer, C. & Rusch, H. P. (1965) Proc. Nut/ Acad. Sci. U . S . A . 53, 924-931. Braun, R. & Wili, H. (1969) Biochim. Biophys. Acta, 174, 246- 252. Muller, G. C. & Kajiwara, K. (1966) Biochim. Biophys. Acfa, 114, 108-115. Hirt, B. (1967) J . Mol. Biol. 26, 365-369. Horowitz, H. B. & Holt; C. E. (1971) J . Cell. Riol. 49, 549553. Guttes, E. & Guttes, S. (1969) J . Cell. Biol. 43, 229-236. Hall, L., Turnock, G. & Cox, B. J. (1975) Eur. J . Biochem. 51, 459 - 465. Huberman, J. A. & Riggs, A. D. (1968) J . Mol. Biol. 32, 327341.
IS. Bohnert, H.-J., Schiller, B., Bohme, R. & Sauer, H. W. (1975) Eur. J . Biochem. 57, 361 369. 16. Hourcade, D., Dressier, D. & Wolfson, J. (1973) Proc. Nut/ Acad. Sci. U . S . A . 70, 2926-2930. 17. Goddard, J. M. & Cummings, D. J. (1975) J . Mol. Biol. 97, 593 609. 18. Watson, J. D. (1972) Nut. New Bid. 239, 197-201. 19. Robinson, A. J. & Bellet, A. J. D. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 523 - 531. 20. Cavalier-Smith, T. (1974) Nafure (Lond.) 250, 467-470. 21. Strauss, S. E., Sebring, E. D. & Rose, J. A. (1976) Proc. Nut1 Acad. Sci. U . S . A . 73, 742-746. 22. Arnberg, A. C., Van Bruggen, E. F. J., Clegg, R. A,, Upholt, W. B. & Borst, P. (1974) Biochirn. Biophys. Actu, 361, 266276. 23. Hall, L. & Turnock, G. (1976) Eur. J . Biochem. 62, 471 -477. -
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V. M. Vogt, Department of Biochemistry, Molecular and Cell Biology, Wing Hall, Cornell University, Ithaca, New York, U.S.A. 14853
R. Braun, Institut fur Allgemeine Mikrobiologie der Universitat Bern, Altenbergrain 21, CH-3013 Bern, Switzerland
