THE INITIATION, MAINTENANCE AND TERMINATION OF DNA SYNTHESIS: A STUDY OF NUCLEAR DNA REPLICATION USING AMOEBA PROTEUS AS A CELL MODEL

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1 J. Cell Sci. 9, 1-21 (1971) Printed in Great Britain THE INITIATION, MAINTENANCE AND TERMINATION OF DNA SYNTHESIS: A STUDY OF NUCLEAR DNA REPLICATION USING AMOEBA PROTEUS AS A CELL MODEL M. J. ORD* Zoology Department, The University, Southampton, England SUMMARY By means of the nuclear transfer technique for amoeba, combinations of nuclei and cytoplasms from all parts of the cell cycle were available for examining the individual roles of the nucleus and cytoplasm in nuclear DNA replication. Neither 5-phase nor division sphere cytoplasm proved capable of initiating a new round of nuclear DNA synthesis in the Gj nucleus. There was some indication that G 2 nuclei which were transferred into early prophase cells, i.e. before the formation of a regular division sphere, did incorporate more [ 3 H]thymidine than control G, nuclei. Positive proof of the induction of DNA synthesis in 'immature' nuclei was observed in only two cases. When young G t nuclei were transplanted into late G. amoebae, the addition of the donor nucleus generally resulted in the older nucleus being held in a late G, phase until the younger nucleus passed through its Gj. Division of 90 % of heterophasic homokaryons was synchronous, with a subsequent synchrony of DNA synthesis. A study of variance in [ 3 H]thymidine incorporation by S nuclei sharing the same cytoplasm using binucleate, trinucleate and multinucleate homokaryons showed that nuclei through the peak-5 period synthesized DNA at approximately similar rates. The large differences in PHjthymidine incorporation by nuclei of amoebae of equal age appear due to differences in endogenous precursor pools. These would vary both with differences in food intake and with the draining of remote precursor pools for simultaneous cellular activities, particularly RNA synthesis. When sharing the same cytoplasm nuclei in peak 5 incorporated similar amounts of [ 3 H]thymidine. Though cytoplasm did not influence the progress of DNA replication by a nucleus, it did influence the use of exogenous [ 3 H]thymidine by the cell, and in so doing caused much of the variation observed in the labelling of nuclei during S. Nuclei sharing the same cytoplasm, and so subject to the same precursor pool changes, incorporated similar amounts of exogenous thymidine. Once DNA synthesis had been initiated it continued to completion regardless of the cytoplasm which surrounded it. Thus neither the maintenance nor termination of DNA synthesis required a special cytoplasmic state. INTRODUCTION As a cell model in the study of nuclear replication Amoeba proteus offers a unique opportunity for investigating more fully the individual roles of the nucleus and cytoplasm in the initiation and regulation of DNA synthesis. Such a study supplements the information already available from other cell models-homo- and heterokaryons obtained by cell fusion (Harris, 1968, 1970), nuclear transplants into amphibian egg Member of the M.R.C. Toxicology Unit, Carshalton, Surrey, England. 1 CEL 9

2 2 M. J. Ord cytoplasm (Gurdon & Woodland, 1968), grafting experiments with the ciliate, Stentor (De Terra 1969) and coalescence experiments using the multinucleate slime mould, Physarum (Guttes & Guttes, 1968). Though the lack of a G x phase in A.proteus contrasts with the situation in other cell model systems the availability of transfers containing combinations of nuclei and cytoplasms from all parts of the cell cycle has yielded conclusive evidence on the independence of the nucleus in the maintenance and termination of nuclear DNA replication. In the investigation to be described in this paper all phases of DNA synthesisinitiation, maintenance and termination - have been studied. The nuclear transfer technique of Comandon & DeFonbrune (1939) has been used to obtain the required nuclear/cytoplasmic combinations before exposing the resultant amoebae to tritiated thymidine. Errors which arise during the preparation of the amoebae for radioautography have also been investigated. Finally, the uptake and incorporation of exogenous thymidine and differences which may arise in interpretation of grain counts have been assessed in relation to the size of the endogenous DNA precursor pools. MATERIAL AND METHODS Material Amoebae were cultured at C using the Tetrahymena feeding technique of Prescott & James (1955). Four culture lines were used, all derived from a wheat culture strain of Amoeba proteus, PD<I, obtained in 1950 from J. A. Dawson of New York, U.S.A., and grown subsequently at King's College, London, and Southampton University. Though the lines differed slightly in the length of the cell cycle and the S period, no significant differences were noted in the comparative labelling of S and G % nuclei. Under the culture conditions used nuclear DNA synthesis followed the separation of the division sphere into 2 daughter cells; G t was negligible. Replication of the nuclear DNA occupied the first quarter of the cell cycle with the greater part of this synthesis occurring during a 1-5 h period. G,, which occupies a period of 30 or more hours, may be divided into early G 2 (12-24 h), mid G, (24-36 h) and late Gj (36 h to mitosis). A small amount of thymidine is incorporated in the nuclei during these periods. Cytoplasmic DNA synthesis (see Discussion) occurs throughout the entire cell cycle. Transfer method Nuclei were transferred to cytoplasm of the desired age using a DeFonbrune micromanipulator. Homotransfers were obtained by first removing the host nucleus. Homokaryons were produced by allowing the host nucleus to remain in the cytoplasm with the donor nucleus. When the donor and host nuclei were in different phases of the cell cycle the transfers are referred to as heterophasic homokaryons (Rao & Johnson, 1970). Four transfer types were used (Ord, 1969) to examine respectively S and G t nuclei in either S or G a cytoplasms: (1) transfer of the nucleus to anucleate cytoplasm; (2) transfer of the nucleus to a whole amoeba; (3) transfer of the nucleus to a binucleate amoeba; and (4) transfer of the nucleus to a binucleate amoeba from which one nucleus was first removed. Transfer types 1 and 4 were used to establish that the change in nuclear/cytoplasmic ratio did not affect the rate of DNA synthesis. In all other experiments, transfer types 2 and 3 were used. Transfer type 3 was particularly useful where little size difference existed between nuclei, the identical labelling of the binucleate's 2 nuclei serving to identify host from donor nuclei. The 4 types of transfer showed no significant differences in labelling. Control binucleate amoebae were obtained by exposing division spheres to a low ph Chalkley's medium (3-7~4'o) for approximately 40 min.

3 Thymidine labelling DNA replication in A.proteus 3 Cells were exposed to [ 3 H]thymidine, 02-1 mci/ml (sp. act. 5 Ct/mM) for periods of 30 min or 1 h. After exposure they were washed free of label and chased for at least 30 min in 2 x IO~'M unlabelled thymidine. Trial runs showed that though the thymidine incorporated into the DNA was stable and could not be washed out, a pool of labelled material remained during chasing, possibly exogenous thymidine already converted to thymidylic acid, and this continued to be incorporated into the DNA. Errors due to such added incorporation were checked by varying the chase periods and by preparing radioautographs from whole amoebae and from isolated nuclei. Where isolated nuclei were required for radioautographs each amoeba was burst by drawing it into a fine pipette, and the nucleus picked up as it floated free. Isolated nuclei were washed in several changes of either Chalkley's medium, 35 mm KC1 (Ord & Bell, 1970), or a 2 x io~ 3 M unlabelled thymidine solution in Chalkley's medium. A description of the method for preparing radioautographs and for grain counting has been given in a previous paper (Ord, 1968) RESULTS The initiation of DNA synthesis The inability of S cytoplasm to initiate replication in G 2 nuclei. If the ' switch on' of nuclear DNA synthesis depended on the presence of an initiator substance in the cytoplasm, or on a certain cytoplasmic state, one would expect such conditions to be met either during the S phase of the cell cycle, or just prior to it. Amoeba cytoplasm was examined for such a 'DNA-synthesis-initiating-condition' by transferring G 2 nuclei into (a) cells in peak S (Fig. 7), (b) cells in early S, and (c) division spheres (Fig. 2). After the transfer operation the amoebae were exposed to [ 3 H]thymidine to establish whether DNA synthesis was taking place in both S and G 2 nuclei. Examination of radioautographs of transfers using nuclei from all parts of the G 2 period showed that these nuclei - though surrounded by S cytoplasm and in company of an S nucleus which was replicating its DNA - did not incorporate exogenous thymidine beyond the small amount expected during G 2 itself (Tables 1, 2). Only when the G 2 nucleus was present from the early prophase stage of division, possibly undergoing some physical changes in the division sphere cytoplasm, was there a significant, though still small, increase in [ 3 H]thymidine incorporation. Control transfers of S nuclei into S cytoplasm showed that the operation had little effect on the DNA synthesis taking place in S nuclei. In most cases homokaryons were exposed to thymidine within an hour of the transfer operation. Since it is possible that a significant time is required for cytoplasm to effect a change in the G 2 nucleus, in a small proportion of experiments homokaryons were kept in dishes for periods of up to 5 h before exposure to fhjthymidine. The results of these radioautographs show that increasing the time of contact between the G 2 nucleus and S cytoplasm did not bring the G 2 nucleus into the condition required for DNA replication (Table 3). Since these findings, showing that G 2 nuclei are not 'switched on' by S cytoplasm, differ from observations of Prescott & Goldstein (1967) also working with A. proteus, further experiments were carried out examining the G 2 and S nuclei after different methods of radioautograph preparation. Whole mounts of amoebae,

4 4 M. J. Ord even when squashed, have a small amount of cytoplasm intervening between nucleus and emulsion. Since amoeba cytoplasm contains mitochondrial DNA which also incorporates the exogenous thymidine, grain counts over nuclei are always corrected for a cytoplasmic error by subtracting from the nuclear count the count over a similar area of adjacent cytoplasm. However, as the thickness of adjacent cytoplasm is greater than the layer of cytoplasm between the nucleus and emulsion this correction overcompensates for cytoplasmic label; and further, does not take into account absorption of nuclear activity by the cytoplasm. Both errors would tend to give too low a nuclear count and could hide small increases in label over the G 2 nuclei. Isolation of the Table i The effect of transferring early, mid- and late-g a nuclei to S amoebae followed by exposure to [ 3 H]thymidine and radioautograph preparation shows that G a nuclei, even from very late G t, are not induced to synthesize DNA by the 5 cytoplasm. Grain counts over G a nuclei seldom represented more than 5-10% of counts over S nuclei and averaged only 3-5 % of 5 nuclear grain counts. Type of transfer Early-Go nuclei in S amoebae Mid-G s nuclei in S amoebae No. of homokaryons Av. count over G 2 nucleus Av. count over 5 nucleus Ratio as % of GJS grain counts Late-G. nuclei in S amoebae Table 2 The effects of transferring G. nuclei into cells in peak S (groups 1, 2), cells in early S (3) and division spheres (4, 5), coupled with exposure to [*H]thymidine after first allowing 30 min 1 h for a possible 'switch on' of DNA synthesis in the G 5 nuclei. Differences seen in grain counts over 5 nuclei are due to differences in the age of the S nucleus at the time of exposure to [ a H]thymidine. Counts are particularly high in groups 3 and 4 where exposure to ['HJthymidine was during the first peak hour of S when labelling was maximal. Ratio of labelling in S/G, nuclei gives a comparison which avoids the complication of such variations. Type of transfer Gj nuclei transplanted into S amoebae Nuclei Nuclei examined in isolated for whole amoebae radioautograph G, nuclei transplanted into amoebae 5-20 min after division G. nuclei transplanted into division spheres (mid prophase to anaphase) G, nuclei transplanted into early prophase just before formation of sphere No. of homokaryons Av. count over G 2 nucleus Av. count over 5 nucleus Ratio as % of G.jS grain counts i '

5 DNA replication in A. proteus 5 labelled nuclei before mounting for radioautographs should eliminate both errors, but a study of isolated nuclei showed that this method introduced its own problems. Examination of freeze-dried isolated nuclei with the Stereoscan microscope revealed that before washing nuclei are enveloped in a coat of cytoplasm. If isolated nuclei were treated with monocrotaline pyrrole, a pyrrole derivative which held this enveloping cytoplasm in place, the layer was sufficiently thick to peel off with a microneedle (M. J. Ord & R. Mattock, unpublished observations). Where nuclei with low label are isolated from highly labelled cytoplasm, even a very thin envelope of cytoplasm could seriously affect grain counting (Table 4). The chief danger when a nucleus is isolated is that no record exists of the level of cytoplasmic labelling of the cell from which it Table 3 Transfer of G a nuclei to S amoebae varying the time between the transfer operation and exposure to [ 3 H]thymidine. A gradual decrease occurs in grain counts over the S nuclei as these pass out of peak S. Since in these experiments nuclei were put into amoebae of 1-2 h age, when the period between transfer and exposure to ['HJthymidine exceeded 3 h the 5 nucleus would show a lower incorporation of [ 3 H]thymidine. Increasing the time of contact between G, nuclei and S cytoplasm did not produce any increase in incorporation of [ 3 H]thymidine by the G 2 nuclei. Time between transplant operation and exposure to [ 3 H]thymidine, h No. of homokaryons Av. count over G 2 nucleus Av. count over S nucleus 1 S I7S Table 4 Amoebae in groups 1 and 2 received similar treatment except that nuclei isolated for radioautographs were washed in 2 x IO~ 3 M unlubelled thymidine in Chalkley's solution in group 2, while nuclei of group i were mounted unwashed leaving the accompanying envelope of cytoplasm to dry down over the nucleus, greatly increasing the grain counts particularly over the much larger G, nucleus. In group 3 the G, nuclei were unlabelled but were transplanted into labelled and chased Samoebae; the small number of grains over the G. nucleus in this group would indicate either incorporation of radioactive material not' chased out' of the S amoeba, or insufficient washing of the isolated nuclei. In group 4, pairs of unlabelled C, and labelled S amoebae were burst together in drops of Chalkley's solution and their nuclei mounted without washing; the presence of labelled cytoplasm during isolation did not result in label over the G 3 nuclei. Type of amoeba [ 3 H]thymidine- [ 3 H]thymidine- G, nucleus Unlabelled G. labelled G a labelled G t transplanted into nuclei isolated in nucleus in S nucleus in 5 a pre-labelled the presence of amoeba amoeba and chased ['H]thymidine-labelled homokaryons homokaryons S amoeba cytoplasm of S amoebae No. of specimens Nuclei isolated without washing after washing after washing without washing then mounted Av. grains per 942/ / /1260 1/143 nucleus GJS Ratio (as %)

6 6 M. J. Ord came. Thoroughly washed nuclei seldom showed cytoplasmic contamination. The most reliable method of examining nuclei which contained only small amounts of [ 3 H]thymidine was to transfer such nuclei after labelling to fresh unlabelled cytoplasm, before isolating the nuclei for radioautographs (Figs. 3, 4). The ratio of grain counts over 5 and G 2 nuclei in 5 cytoplasm prepared for radioautographs after exposure to [ 3 H]thymidine using both whole amoebae and isolated nuclei preparations are compared in Table 2 (columns 1 and 2). Table 5 The lines of A. proteus used in these experiments proved very sensitive when handled or operated on during the pre-division period. Delay to division of a late-g a amoeba given a young G, nucleus was only partly due to the presence of an out-of-phase nucleus. In the majority of amoebae the division delay outspanned the time required for the young nucleus to pass through its G t. Division inhibition after nuclear transfers to give heterophasic homokarvons No. of specimens Av. delay to division, h Division inhibition after control micrurgy Young G t 1 * > nucleus Sucking in Stirring cyto- Exchange of transplanted and out of plasm with a equal-age into late-g a pipette microneedle nuclei amoeba Young G. nucleus transplanted into late-g. binucleate amoeba Young G. nucleus transplanted into late-g., binucleate amoeba with 1 nucleus removed The effect of division on nuclei which have not undergone a complete G 2 - To investigate whether a' switch on' signal for DNA synthesis may be present in amoebae during the period immediately preceding division, or more probably, whether the physical changes which occur at mitosis are required to initiate a new round of DNA replication, it was necessary to induce amoebae containing nuclei which had just completed replication to undergo division. In these experiments late-g 2 amoebae approaching the expected time for division were used as hosts for the young nuclei. Unfortunately, at this time the amoebae proved very sensitive when disturbed. Changes due to handling, or micrurgy, delayed their division (Table 5). Only cells which had already taken on the irregular pseudopodal form which indicates that the cell has entered the first recognizable early prophase stage (Ord, 1970) were able to pass into division. In all others division was inhibited, and in 75 % of cells the period of inhibition outspanned the time required for the younger nucleus to pass through its Go. In control homophasic binucleates, that is, binucleate amoebae containing identical nuclei, nuclear division results in 4 nuclei of equal size, which may be distributed at cytokinesis into 2, 3 or 4 offspring of equal or unequal size (Fig. 1 A). In heterophasic binucleates, that is, binucleate amoebae containing 2 nuclei of different ages, nuclear division in 90 % of cases resulted in 4 nuclei, one pair frequently being smaller than the other, with cytokinesis segregating the nuclei as in the homophasic binucleates.

7 DNA replication in A. proteus 7 In i o % of cases, however, division of heterophasic binucleates produced offspring with a total of only 2 or 3 nuclei (Fig. 1 B). Radioautograph studies of the offspring of all heterophasic divisions led to the following conclusions. A. Division of homophaslc binucleate amoebae into 2, 3 or 4 daughter cells with a total of 4 nuclei B. Division of 10% of heterophasic binucleate amoebae producing offspring with a total of only 2 or 3 nuclei ' Oo Fig. 1 A. The division of homophasic binucleate amoebae resulting in 4 nuclei of equal size distributed with cytokinesis into 2, daughter cells. Division of 90% of heterophasic binucleate amoebae is similar to that of homophasic binucleate amoebae. B. Division of 10% of heterophasic binucleate amoebae resulting in 2 daughter cells containing a total of only 2 or 3 nuclei. Division in 90% of cases is in synchrony for nuclei sharing the same cytoplasm. After division these nuclei begin replication of their DNA in synchrony. Frequently 1 pair of nuclei, presumably that from the younger of the 2 G 2 nuclei, is smaller and the S phase of the smaller nuclei may be longer than that of the larger nuclei, even when sharing the same cytoplasm.

8 G 6-h nucleus in 2-h amoeba O-S < OOI 171 <^ OOI H 7-h nucleus in 2-h amoeba < OOI 23-2 < OOI I 8-h nucleus in 2-h amoeba '5 <^ OOI 203 <$ OOI J 9-h nucleus in 2-h amoeba <% OOI 167 <^ OOI K 10-h nucleus in 2-h amoeba <^ o-oi 49 <3 OOI Table 6 A comparison of the variation in labelling over S nuclei sharing the same cytoplasm using homokaryons with nuclei of equal (groups A-E) and unequal (groups F-K) age. In binucleate controls, where nuclei are identical, variation between the nuclear grain counts is negligible. Experimental binucleate and trinucleate homokaryons also have little difference in labelling among nuclei. In multinucleate homokaryons even though nuclei share the same cytoplasm incorporation of label shows some variation, but this is small when compared with the differences found between nuclei of early + late-s homokaryons (F-K). The 1 ate-s nuclei incorporate significantly less [ 3 H]thymidine even when sharing cytoplasm with an early-s nucleus. Group Type of homokaryon A Control binucleate amoeba B S nucleus in S amoeba C S nucleus in S binucleate amoeba D Multi S nuclei in Gj, binucleate amoeba E Multi S nuclei in binucleate amoeba F 5-h nucleus in 2-h amoeba Variance Degrees of freedom v x \v A P vx \v E p not significant not significant <OOI <So-oi O <S o-oi 7'S <3 OOI

9 DNA replication in A. proteus 9 In the 7% where the offspring of division contained only 2 nuclei, both nuclei incorporated [ 3 H]thymidine following division. It is thought that these represent amoebae where either the donor nucleus was extruded before division or where one of the homokaryon nuclei failed to reconstruct after division. Where division gave rise to offspring containing a total of 3 nuclei, that is, where 1 nucleus was carried through division without dividing (3 %) no reliable conclusions could be drawn concerning the initiation of DNA synthesis. In 2 of these homokaryon divisions both divided nuclei and the undivided nucleus incorporated [ 3 H]thymidine following division (Fig. 5); in 2 others, while the divided nuclei incorporated thymidine indicating DNA synthesis, the undivided nucleus incorporated only small amounts of [ 3 H]thymidine similar to G 2 nuclei transferred to early prophase cytoplasm (Table 2, column 5). In 27 % of heterophasic homokaryons division occurred before the younger nucleus had passed through the average time span of G 2. This suggests that the older cytoplasm and/or the accompanying older nucleus are sometimes able to shorten the G 2 period for the younger nucleus. The use of binucleates as hosts, giving trinucleate amoebae, though decreasing the division delay period in some cases, proved unsatisfactory for these studies. The synchrony of nuclear division, even in control homophasic trinucleates, can no longer be relied upon. While homophasic binucleates gave synchrony of nuclear division in 98% of cases, only in 60% of homophasic trinucleates, and 40% of homophasic tetranucleates, was nuclear division in synchrony. The maintenance of DNA synthesis In S cytoplasm. Peak DNA synthesis occupied a period of approximately 5 h near the beginning of the cell cycle in the lines of A. proteus used in these experiments. Comparison of the rates of synthesis for individual amoebae during this period is difficult, as small differences in precursor pools (e.g. due to variation in feeding) cause wide variations in the incorporation of exogenous thymidine. However, when nuclei share the same cytoplasm they would have the same supply of initial precursors for DNA synthesis and differences should be minimized. This supposition is upheld by an examination of the pairs of nuclei in binucleate amoebae. Variation in labelling between the twin nuclei of control binucleates is insignificant. Similarly, in experimental binucleates, that is, binucleates made by transplanting a second peak-*!? nucleus into a peak-5 amoeba (Table 6, Groups A and B), differences in labelling between sister nuclei sharing the same cytoplasm are insignificant. In experimental multinucleates 2 factors must be taken into account: the nuclei are no longer identical as in the control binucleates; and transplanted nuclei must undergo stresses caused both by the transfer operation itself and by adjustment to' foreign' cytoplasm. A comparison of control and experimental binucleate amoebae with experimental trinucleate and multinucleate amoebae (all containing peak-5 nuclei of roughly similar age) shows that the variation in incorporation of [ 3 H]thymidine among nuclei in the same cytoplasm, though still small, is greater in the multinucleate amoebae (Fig. 6). In group E, 4-6 nuclei were pushed into each S binucleate amoeba, that is, a total of 6-8 nuclei

10 io M.J.Ord occupied the equivalent of 2 volumes of S cytoplasm, while in Group D, 4 nuclei were pushed into each G 2 binucleate amoeba, that is, a total of 6 nuclei occupied 2 volumes of G 2 cytoplasm or the equivalent of 4 volumes of S cytoplasm (Fig. 10). Since the latter operations, where fewer nuclei were pushed into a large volume of cytoplasm, were technically easier, the damage sustained by each nucleus should be less. Table 6 shows the variation in phjthymidine incorporation is less for nuclei of homokaryons in Group D than E. Table 7 Grain counts over S nuclei in G t cytoplasms. Labelling over G a nuclei was negligible in all cases. Labelling over S nuclei shows that incorporation of ['H]thymidine continues even in the absence of S cytoplasm. Incorporation of [ 3 H]thymidine is lower, possibly due to the larger volume of cytoplasm producing an increased supply of precursor materials. When labelling was delayed for 1-4 h after transfer, incorporation of [ 3 H]thymidine shows DNA synthesis continuing; but when delayed for 15 or more hours lack of incorporation suggests that the 5 nucleus has completed replication of its DNA.) Time between transfer and exposure to [ 3 H]thymidine, h ^ No. of * * 17 s specimens [ 3 H]thymidine cone, in mci/ml Exposure time, hi Av. grain count o o over S nucleus Homokaryons with 4 5 nuclei per G s amoeba (Fig. 10). Though homokaryons containing a number of peak-s nuclei showed that during this period nuclei sharing the same cytoplasm incorporated approximately equal amounts of pitjthymidine, this did not apply to all nuclei throughout the 5 period. When nuclei from late 5 were put into peak-5 amoebae and exposed to [ 3 H]thymidine, radioautographs showed that the pairs of nuclei had incorporated quite different quantities of the exogenous thymidine (Fig. 8). In groups F to K (Table 6) the difference in labelling between pairs of early- and late-5 nuclei sharing the same cytoplasm are seen to be highly significant even when variance is related to group E, the most variable of the peak-s multinucleate amoebae. From this study of exogenous thymidine incorporation by nuclei sharing the same cytoplasm the following facts emerge. (1) Over much of peak S, nuclei synthesize DNA at approximately the same rate. (2) The addition of extra nuclei does not interfere with the synthesis of DNA by an S nucleus, though when a number of nuclei share the same cytoplasm the drain on the precursor pools for DNA synthesis can increase the amount of exogenous thymidine used by the cell. (3) Once DNA synthesis has been initiated it is not substantially affected by the transplant operation. (4) G 2

11 DNA replication in A. proteus 11 nuclei when added to the S amoebae neither incorporate [ 3 H]thymidine themselves, nor affect the replication taking place in the S nuclei. In G 2 cytoplasm. Transfers of S nuclei to G 2 cytoplasms were used to establish whether S cytoplasm itself was necessary for the continuation of replication, i.e., whether special conditions existed in the S cytoplasm which were necessary to allow replication to proceed. Radioautographs of 150 such transfers showed that S nuclei continued to incorporate phjthymidine even when surrounded by G 2 cytoplasm. Since S nuclei were generally transplanted to late-g 2 binucleate amoebae, the volume of cytoplasm available to supply precursors for DNA synthesis was substantially greater than the volume of their own cytoplasm. For this reason the amount of exogenous thymidine incorporated during DNA synthesis should be less (Table 7). With low concentrations of [ 3 H]thymidine (concentrations which gave adequate labelling to S nuclei in S cytoplasm) labelling of 5 nuclei in G 2 cytoplasm was poor (column 2). Good incorporation in binucleate hosts was obtained only when concentrations of 0-5 mci/ml [ 3 H]thymidine were used (Fig. 9), or when mononucleate G 2 amoebae were used as hosts giving a smaller volume of cytoplasm for precursor supplies, or when a number of nuclei were added to one G 2 binucleate so that precursors for synthesis were shared among them (Fig. 10). The continued synthesis of DNA by S nuclei in G 2 cytoplasm suggests that once initiated a special cytoplasmic state is not necessary to maintain DNA synthesis. Experiments where 5 nuclei were pulse labelled 2, 3 or 4 h after transplantation to G 2 cytoplasm excluded any possibility that labelling was only a short burst made possible by small pools of precursors carried over in the transplanted nuclei (Table 7, columns 4-7). G 2 cytoplasm would appear able to supply the necesssary precursors for replication of DNA, though the number of counts per S nucleus decreased as the nuclei (generally 2-h when transferred) passed out of their peak-5 period. The termination of DNA synthesis The rate of DNA synthesis in S nuclei decreased after 5-6 h and, providing that the amoebae had been well fed both before and during S and that the nuclei were of normal size, little synthesis occurred beyond 10 h. Observations of S nuclei in G 2 cytoplasm, and of late-5 nuclei in early S cytoplasm, were used to establish whether cytoplasm played a part in 'switching off' DNA synthesis. S nuclei in G 2 cytoplasm. It has already been observed that 5 nuclei transferred to G 2 cytoplasm can synthesize DNA as shown by the incorporation of phjthymidine, and that this synthesis continues for at least 4 h. When S nuclei were transferred to G 2 cytoplasm but not exposed to [ 3 H]thymidine for 15 or more hours following the transfer operation, that is, allowing time for the S nuclei to complete the replication of their DNA, no incorporation of phjthymidine occurred (Table 7, columns 8 and 9). This suggested that DNA synthesis was able to continue to completion in these nuclei, and that when the pulse of phjthymidine was given, 15 or 18 h later, these nuclei had already passed into G 2. Transfers of late S nuclei to early S cytoplasm. The difference in labelling of pairs of nuclei in S cytoplasm homokaryons carrying a late-5 with an early-5 nucleus (see p. 9)

12 12 M.J.Ord suggested that a slow down in DNA synthesis occurred in the latter half of S. Decrease in [ 3 H]thymidine incorporation could be due to change in DNA or nuclear activity, or to cytoplasmic influence, e.g. the decrease in cytoplasm of an inducer substance or the presence in cytoplasm of a switch off signal. To investigate whether some cytoplasmic change occurred during S which was necessary to initiate a slow down of DNA synthesis, 4-h nuclei were transplanted to i-h amoebae and the resultant homokaryons left for 3-5 h before exposing to [ 3 H]thymidine. By this time the incorporation of thymidine by the older nucleus would be expected to have decreased if it had remained in its own cytoplasm. Radioautographs of such homokaryons show that the decrease has taken place even though the late-5 nucleus spent all of its DNAsynthesizing period in peak-5 cytoplasm (Table 8). Table 8 The ability of late-s nuclei to decrease their incorporation of [ 3 H]thymidine when in peak-s cytoplasm is seen in columns i and 2. In column i the late-s nuclei were exposed to peak-s cytoplasm throughout their entire 75 h of 5 by transferring them at 35 h from their own cytoplasm into l-h-old amoebae. Column 4, with nuclei of equal age, shows little difference in labelling of nuclei.) Late-5 nuclei surrounded throughout S by peak-s cytoplasm Late-S nuclei transferred to peak-s amobae just before exposure to [ 3 H]thymidine Late-5 nuclei in late-5 cytoplasm compared with peak-s nuclei in peak-s cytoplasm S nuclei transferred to S amoebae No. of specimens of each S Age of late-s nucleus 7-5 when exposed to ['HJthymidine, h Age of peak-s nucleus 4^5 when exposed to [ l H]thymidine, h Ratio as % of grains in low- 23 labelled nucleus to grains in high-labelled nucleus DISCUSSION From the experiments presented in this paper 2 facts emerge clearly. (1) Once nuclear DNA synthesis has been initiated, it is completed regardless of the surrounding cytoplasm or the activities of other nuclei which may accompany it. (2) A second round of DNA synthesis cannot be initiated in a G 2 nucleus by 5 cytoplasm, though G 2 nuclei which have been exposed to division sphere cytoplasm may incorporate phjthymidine to a greater extent than control G 2 nuclei (see Table 2, column 5). That G 2 nuclei are not induced to synthesize DNA has also been established using fusion of G 2 - and 5-phase tissue culture cells (Rao & Johnson, 1970), and coalescence of G 2 - and 5-phase pieces of the multinucleate slime mould, Physarum (Guttes & Guttes, 1968). The drawback of these 2 systems is that the G 2 cytoplasm is diluted, rather than replaced, by S cytoplasm.

13 DNA replication in A. proteus 13 The lack of a G x phase in the amoeba leaves the search for a 'switch on' signal for DNA synthesis incomplete. The presence in the cytoplasm, or action through the cytoplasm, of an inducer substance for DNA synthesis has been detected in other systems. Thus the fusion of G ± and S tissue culture cells rapidly induced DNA synthesis in the Gj-phase nuclei (Harris, 1970). Likewise when the G 1 macronucleus of Stentor was transferred to an S cell, DNA synthesis was initiated (DeTerra, 1969). Finally, liver, intestine, blastula or brain cell nuclei transplanted into Xenopus egg cytoplasm (Graham, Arms & Gurdon, 1966) were induced to synthesize DNA even though nuclei from some cells, e.g. brain cells, were not able to support development and would not normally undergo DNA synthesis (Gurdon, 1967). The probability that the initiation of DNA synthesis in G x nuclei is due to an inducer substance present in the S cytoplasm is supported by results on multinucleate tissue culture cells which show induction to be dose dependent, i.e. the greater the proportion of S cells to G 1 cells in the homokaryon the more rapid the initiation of DNA synthesis in the G 1 nucleus (Rao & Johnson, 1970). The inability of S cytoplasm to 'switch on' DNA synthesis in the G 2 nucleus of amoebae would suggest a difference in the chromatin of the G 2 cell rather than an absence of an inducer. Similar results were obtained using both G 2 -phase tissue culture cells, where experiments with G x - and 5-phase homokaryons had already indicated the presence of an inducer in the S-phase cytoplasm, and Xenopus egg cytoplasm, where the proportion of intestine or liver cell nuclei able to support development without first being 'switched on' by the egg cytoplasm was equivalent to the proportion of cells thought to be in the G 2 phase (Gurdon & Woodland, 1968). Whether or not an inducer, if present, is essential during the whole of the S phase remains in doubt. In amoebae, 5 nuclei transferred to G 2 cytoplasm completed the replication of their DNA. In tissue culture homokaryons, radioautographs of 5-phase with either G x - or G 2 -phase cells showed DNA synthesis continuing in the S nucleus. In Stentor, however, though fusion experiments where the homokaryon contained both S and G x cytoplasm showed the S nucleus capable of synthesizing DNA, nuclear transplant experiments where the S nucleus was surrounded by G x cytoplasm showed synthesis depressed in the S nucleus (DeTerra, 1969). This suggests the necessity of the inducer during S; but such results would also be expected if there was a lack of precursors for synthesis during the G x phase or an effect on the S nucleus by either a substance in, or the physical state of, the G x cytoplasm. In an attempt to investigate the 'switch on' of DNA synthesis by forcing nuclei from early G 2 to pass through division in homokaryons, it was found that nuclear division in binucleates was synchronous whether nuclei were of equal or unequal age. Division was inhibited in most homokaryons for a period which outspanned that required for the young nucleus to pass through its entire G 2. When division did occur, DNA synthesis began in synchrony for all nuclei. Johnson & Rao (1970) obtained similar results using tissue culture cells; that is, when tissue culture cells from different stages of the cell cycle were fused. Here division of all nuclei was synchronous, but in many cases the older nuclei were held in an early prophase rather than a late G a state.

14 14 M. J. Ord Apart from the general errors common to radioautography (Rodgers, 1967), there are 2 particular and quite separate problems when using [ 3 H]thymidine with amoebae. The first is due to the DNA bodies of the cytoplasm (Rabinovitch & Plaut, 1962), which incorporate thymidine throughout the cell cycle and so lead to confusion with nuclear grain counts. Transfer of labelled nuclei to unlabelled cytoplasm before using either the whole cells or isolated nuclei for radioautographs avoids this complication and is advisable where nuclear incorporation is low in comparison with cytoplasmic incorporation. The second problem is due to changes in the endogenous pools of material which supply the precursors for DNA; other cells would be subject to pool variation, but in amoebae it is exaggerated due to the discontinuous method of feeding. Quastler (1963) introduced a 4-point model for thymidine incorporation into DNA involving: (1) the entrance of endogenous precursors into the immediate precursor pool, a pool assumed to contain the 3 thymidine precursors, thymidine monophosphate, thymidine diphosphate and thymidine triphosphate (TTP); (2) the entrance of exogenous [ 3 H]thymidine into the pool after its change to thymidylic acid; (3) the exit from the pool of TTP for incorporation into DNA; (4) the exit of precursors from the pool following a degradation pathway. Cleaver (1967) using L strain tissue culture cells found TTP to be the major component of the pool. But TTP, apart from affecting remote precursors by inhibition of a number of enzyme systems, has a direct effect on the use of external thymidine through its inhibitory action on thymidine kinase. Since both dilution with endogenous precursors and inhibition of thymidine kinase would affect the use of [ 3 H]thymidine by the cell, the larger the precursor pool, the smaller the incorporation of labelled material. The availability of the remote endogenous precursors which determine pool size would depend on both the food supply of the cell (Ord, 1968) and other simultaneous cellular activities, e.g. RNA synthesis. Controlled feeding, both before and after division, decreases variation in labelling among nuclei of similar age. Variation in labelling due to changes in cellular activities during S can only be controlled by nuclei sharing the same cytoplasm. A study of variance in phjthymidine incorporation for such nuclei - using binucleate, trinucleate and multinucleate homokaryons - showed that the rate of DNA synthesis changes little through the peak S period of 1-5 h. Where a number of nuclei shared the same volume of cytoplasm, so making greater demands on the precursor pool, or where food intake was limited giving a smaller precursor pool, incorporation of pitjthymidine was increased, not because of a change in the rate of DNA synthesis by these nuclei but by a decrease in the dilution of the exogenous thymidine and in the inhibition of thymidine kinase. Thus, though cytoplasm does not appear to play an active role in the maintenance, rate, or termination of DNA synthesis, it does have a considerable influence on the amount of external thymidine incorporated into DNA through its control over precursor pools. Nuclei of similar age and sharing the same cytoplasm incorporate the same amount of phjthymidine. Experiments are in progress to identify the site of thymidine incorporation into the G 2 nucleus, whether chromosomal or nucleolar.

15 DNA replication in A. proteus 15 The author is indebted to Dr D. F. Heath for the statistical analysis of labelling in the section on the maintenance of DNA synthesis in S cytoplasm (p. 9 and Table 6), and to Mr R. F. Legg for technical assistance with radioautography and photography. REFERENCES CLEAVER, J. E. (1967) Thymidine metabolism and cell kinetics. North Holland Research Monographs Frontiers of Biology, vol. 6 (ed. A. Neuberger & E. L. Tatum). Amsterdam: North Holland Pub. Co. COMANDON, J. & DEFONBRUNE, P. (1939). Greffe nucleaire totale, simple ou multiple chez une Amibe. C. r. Stone. Soc. Biol. 130, DE TERRA, N. (1969). Cytoplasmic control over the nuclear events of cell reproduction. Int. Rev. Cytol. 25, GRAHAM, C. F., ARMS, K. & GURDON, J. B. (1966). The induction of DNA synthesis by frog egg cytoplasm. Devi Biol. 14, GURDON, J. B. (1967). On the origin and persistence of a cytoplasmic state inducing nuclear DNA synthesis in frogs' eggs. Proc. natn. Acad. Set. U.S.A. 58, GURDON, J. B. & WOODLAND, H. R. (1968). The cytoplasmic control of nuclear activity in animal development. Biol. Rev. 43, GUTTES, S. & GUTTES, E. (1968). Regulation of DNA replication in the nuclei of the slime mold, Physarum polycephalum. J. Cell Biol. 37, HARRIS, H. (1968). Nucleus and Cytoplasm. Oxford: Clarendon Press. HARRIS, H. (1970). Cell Fusion. Oxford: Clarendon Press. JOHNSON, R. T. & RAO, P. N. (1970). Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature, Lond. 226, ORD, M. J. (1968). The synthesis of DNA through the cell cycleof Amoeba proteus. J. Cell Sci ORD, M. J. (1969). Control of DNA synthesis in Amoeba proteus. Nature, Lond. 221, ORD, M. J. (1970). Amoeba proteus as a cell model in toxicology. In Mechanism of Toxicity (ed. W. N. Aldridge), pp London: Macmillan. ORD, M. J. & BELL, L. G. E. (1970). Viability of isolated nuclei. Nature, Lond. 226, PRESCOTT, D. M. & GOLDSTEIN, L. (1967). Nuclear-cytoplasmic interaction in DNA synthesis. Science, N. Y. 155, PRESCOTT, D. M. & JAMES, T. W. (1955). Culturing of Amoeba proteus on Tetrahymena. Expl Cell Res. 8, QUASTLER, H. (1963). Effects of irradiation on synthesis and loss of DNA. In Actions chimiques et biologiques des Radiations (ed. M. Haissinsky), pp Paris: Masson et Cie. RABINOVITCH, M. & PLAUT, W. (1962). Cytoplasmic DNA synthesis in Amoeba proteus. I. On the particulate nature of the DNA-containing elements. J. Cell Biol. 15, RAO, P. N. & JOHNSON, R. T. (1970). Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature, Lond. 225, RODGER, A. W. (1967). Techniques of Autoradiography. Amsterdam, London and New York: Elsevier. {Received 10 November 1970)

16 16 M.J. Ord Fig. 2. A late-gj nucleus (46-h-old) transplanted into a dividing amoeba at prophase. (Division of the host nucleus is unaffected by the operation; the donor nucleus does not take part in division.) Cytokinesis was inhibited and the resultant trinucleate amoeba exposed to [ 3 H]thymidine (1 mci/ml for 1 h) 30 min after transfer, followed by a S-h chase in 2 x io~ 3 M unlabelled thymidine before mounting and coating with emulsion. The radioautograph shows the 2 S nuclei well labelled; the larger G 2 nucleus has labelling only a little above cytoplasmic level and no more than found in control G, nuclei. Nuclei stained with light green, x 800. Fig. 3. A mid-g,, nucleus (25-h) transplanted into a 2-h S-phase amoeba. The homokaryon was exposed to [ 3 H]thymidine, 0-2 mci/ml for 2 h. After chasing the nuclei were isolated and washed in unlabelled thymidine before mounting side by side on a slide. The small amount of label in the G s nucleus could be [ 3 H] thymidine incorporated by the G 2 nucleus or labelling due to cytoplasmic contamination. Nuclei stained with light green, x 800.

17 DNA replication in A. proteus V.* '' CEL 9

18 18 M.J.Ord Fig. 4. A mid-g t nucleus (25 -h) transplanted into a 2-h S-phase amoeba. After i-h exposure to 0-5 mci/ml [ 3 H]thymidine and a short chase in unlabelled thymidine the 2 nuclei were transferred to unlabelled enucleate cytoplasm before isolating for radioautography. While labelling over the 5 nucleus indicates DNA synthesis, only a small number of grains are found over the G, nucleus. Nuclei stained with light green, x 800. Fig. 5. An early G, nucleus (18-h) transplanted into a late-gj binucleate amoeba (42-h) with one of the host nuclei removed immediately prior to the addition of the donor nucleus. This homokaryon divided 24 h later (i.e. when the younger nucleus was 42 h old). Division resulted in 2 offspring, a binucleate with 2 small nuclei and a mononucleate with 1 large nucleus. The offspring were exposed to [ 3 H]thymidine, 1 mci/ml from h age. The radioautograph shows all 3 nuclei heavily labelled indicating that the undivided homokaryon nucleus incorporated [ 3 Ff]thymidine. Amoebae stained with light green, x 50. Fig. 6. Three S nuclei transplanted into an S binucleate amoeba (with all nuclei of equal age). The homokaryon was exposed to [ 3 H]thymidine (1 mci/ml) for 1 h and chased for 4 h in unlabelled thymidine. The radioautograph shows all nuclei have incorporated approximately similar amounts of exogenous thymidine. Nuclei stained with light green, x 800.

19 DNA replication in A. proteus 2-2

20 20 M. J. Ord Fig. 7. A late-gj nucleus (48-h) in an 5 amoeba (3-h) with the homokaryon exposed for 1 h to 0-25 mci/ml ['HJthymidine. Amoeba stained with light green, x 250. Fig. 8. A late-5 nucleus (7-h) transplanted into a peak-5 binucleate anioeba (z-h) with the homokaryon exposed to ['H]thymidine, 0-25 mci/ml for 1 h. Since the 2 host nuclei label similarly, the nucleus with less label is the donor nucleus, i.e. the nucleus from the late-5 amoeba. (2, nuclei from 2-h host; 7, nucleus from 7-h transplant.) Amoeba stained with light green, x 250. Fig. 9. A peak-5 nucleus transplanted into a late-g 2 binucleate amoeba. The resultant homokaryon was exposed for 1 h to 0-5 mci/ml ['HJthymidine and chased with 2 x IO~ 3 M unlabelled thymidine. Photographed with phase microscopy, since the unstained and unlabelled G s nuclei are otherwise invisible, x 400. Fig. 10. Four peak-5 nuclei (2-h) transplanted into an early-g, binucleate amoeba (16-h); 3 h after the transfer operation, the homokaryon was exposed to [ 3 H]thymidine, 0-25 mci/ml for 1 h. The radioautograph shows the 4 5 nuclei (centre) have incorporated thymidine even though labelling was delayed for 3 h after transfer. The G 2 nuclei are unlabelled. Amoeba stained with light green, x 250.

21 DNA replication in A. proteus 21 7 I S 10

22