Nuclear and kinetoplastic DNA replication cycles in normal and synchronously dividing Crithidia luciliae

Experimental Cell Research 6.5 (1971) 353-358

NUCLEAR IN NORMAL

AND KINETOPLASTIC

DNA REPLICATION

AND SYNCHRONOUSLY

DIVIDING

CYCLES

CRITHZDIA

LUCILIAE

S. VAN ASSEL and M. STEINERT Laboratoire de Morphologic UniversitP libre de Bruxelles,

animale, Facultt des Sciences, 1640 Rhode-St-Get&e, Belgium

SUMMARY Quantitative radioautography has been used to measure the incorporation of 3H-thymidine into nuclear and kinetoplastic DNA of Crithidia luciliae. Our results, obtained on randomly dividing cells as well as on cultures synchronized with hydroxyurea, show that the nuclear and kinetoplastic DNAs are labelled simultaneously during a part of the interphase. Thymidine incorporation is inhibited by hydroxyurea. although the initiation of new S phases still occurs in the presence of the drug.

The existence of genetic material in mitochondria and of a distinct and rather complete apparatus for its conservation and use is now firmly established. Since this organelle is only semi-autonomous, regulatory processes must harmonize mitochondrial and “host” cell syntheses and enzymatic activities; the balance between nuclear and mitochondrial DNA syntheses is one of the many facets of this partially integrated system. Most eukaryotic cells contain quite a large number of mitochondria, which form a population of rather constant size, although it appears to turn over independently from other cell constituents [l]. The fact that, in rat liver for instance, mitochondrial DNA is more rapidly labelled as compared with nuclear DNA is interpreted as an aspect of this independent and continuous wear and replacement of the organelle in differentiated cells [2-71. This independent mitochondrial DNA replication is achieved by a specific enzyme: a distinct mitochondrial DNA poly-

merase has been isolated and partially purified from rat liver [S-11] and from brewer’s yeast [12]. In most dividing cells, the duplication cycle of mitochondrial DNA is apparently unrelated to the S period of chromosomal DNA synthesis: it has been found to be a statistically continuous process in the slime mold Physurum [13-171 and in Tetrahymena [18-221, two species with numerous mitochondria in each cell. In yeast cells, which have only a few mitochondria, mitochondrial DNA replication occurs during a restricted part of the cell cycle, which differs, however, from the period of nuclear DNA synthesis [23, 241. A quite opposite situation seems to prevail in trypanosomidae: a single mitochondrion is present in each cell, and mitochondrial DNA, which is remarkable for its abundance and peculiar molecular characters [25, 261, forms by itself a kind of nucleoid of this mitochondrion, the kinetoplast. We found, some years ago, that kinetoplast DNA replication in Trypanosoma mega is Exptl Cell Res 65

354

S. Van Assel h M. Steinert no1 and air-dried. Radioautography and DNAase digested controls are prepared as described before [32]. A typical autoradiogram is shown in fig. 1.

RESULTS The cell cycle

Fig. I. Autoradiogram of two 3H-thymidine pulselabelled cells. This preparation, which has been exposed for a rather long time, shows that it is ouite easy to distinguish between nuclear and kinetoplastic labelling and also that the background is very low. k, kinetoplast; n, nucleus. x 3 200.

a discontinuous process which is almost simultaneous with nuclear DNA synthesis [27]. The present work extends these observations to another species, Crithidia luciliae, and also to synchronized cultures of this latter organism. It has been partially published in preliminary notes [28, 291. MATERIALS

AND METHODS

The strain and its cultivation have been described previously [30]. Synchronized cultures are obtained after release from hvdroxvurea (0.2 mglml) inhibition of early stationary phase cells, as reported elsewhere 1311.Total cell counts are made in a Thoma hemacytbmeter. DNA synthesis is determined as incorporation of SH-thymidine in DNAase-sensitive material, measured by quantitative autoradiography of dried smears. Before being pulse labelled, the samples of Crithidia luciliae are rapidly concentrated in their own medium, usually tenfold, by a low speed centrifugation. In order to keep the environment constant and to avoid the shift-up conditions which could arise from resuspending cells in fresh medium whenever the pulse is to be followed by a chase, the supernatant medium in excess is reserved for washing and resuspending the labelled cells. The wash is made with added carrier thymidine (1 mg/ml) and the cells are resuspended at annroximately the same cell density as- before the p&e. The smears are fixed in methanol, washed in carrier thymidine in water (1 mg/ml) for 1 h at 4”C, in deionized running water for 30 min, then rinsed in ethaExptl CeN Res 65

Calculated from cell counts in log-phase cultures, the mean duration of the division cycle is found to vary from one experiment to the other, from 5 h 30 min to more than 7 h. The sequence of different steps of cell division can be determined from the observations in table 1. It appears clearly that, statistically, the nucleus divides before the kinetoplast and, using as before [27] an equation adapted from Stanners & Till [33], it is found that, in this particular culture which had a mean generation time of 5 h 39 min, the nucleus and the kinetoplast statistically divide 44 min and 30 min before cytokinesis, respectively. The replication cycle of randomly dividing C. luciliae is analysed by the labelled mitoses wave method, as developed by Quastler & Sherman [34] and later tested, on theoretical bases, by Takahashi [35, 361. Results of two experiments are shown in fig. 2. The curves plotted for labelled kinetoplasts are almost exactly superimposed on those for the labelled Table 1. Stained smears font a log-phase culture are observed and 1 600 cells classified according to the presence of resting nucleus (1 N), dividing or divided nucleus (2N), resting kinetoplast (IK) and dividing or divided kinetoplast (2K). The cells in the last column, without kinetoplast (IN.-), are abnormal and probably produced by asymmetric division Class

lN.lK

2N.lK

lN.2K

2N.2K

lN.-

Cells. %

90.1

3.4

0.2

6.1

0.2

DNA replication cycles in Crithidia luciliae

355

4

Fig. 2. Labelled mitoses waves in Crithidiu bciliae. Log-phase cells are pulse labelled for 15 min with 3H-thymidine (50 @X/ml), washed and chased as described in Methods. Cells are sampled after increasing periods of chase and prepared for radioautography. The percentage of dividing cells with labelled nucleus (-•-) and with labelled kinetoplast (-0-) is plotted against time beginning at mid-pulse. As typical mitosis cannot be observed in trypanosomidae, acell is considered here as being in division whenever it contains a dividing ordivided nucleus. Diagram a shows a short experiment in which only the second half of the S phase can be outlined, but with somewhat more accuracy than in experiment of fig. 26, which shows the entire labelled mitosis wave.

nuclei, disclosing simultaneity of nuclear and kinetoplastic labelling. If we assume that label uptake into DNA characterizes the whole S phase of DNA replication, the length of this S phase as well as that of the G2 + 4 M periods can be calculated from the 50 % intercepts, as recommended by Takahashi [35]. The M (mitosis) period has been defined here as the time elapsed between incipient separation of daughter nuclei and cytokinesis (see legend fig. 2), and we have seen above that in log-phase cells, it is approx. 44 min long. Taking this into account, experiments (a) and (b), fig. 2, give, as mean length of the G2 phase, 67 min and 57 min respectively. The S phase, measured in fig. 2b, takes about 3 h 40 min. The DNA synthesis cycle has also been investigated in synchronized cells, as illustrated in fig. 3. In fact, the ability to incorporate 3H-thymidine has been tested immediately before and during the incubation of the cells

with hydroxyurea, as well as for two complete cell cycles thereafter. Let us first consider diagram (a). Owing to the fact that early stationary phase cells are used, the proportion of dividing cells at time 0 is low, as compared to data in table 1. The duration of the cell cycle in synchronous growth after release from hydroxyurea inhibition, measured as the interpeak distance, is 3 h 20 min, which is much shorter than the mean division time in normally growing log-phase cultures, as described above. Considering the curves in fig. 3b, one observes that, after a quick and limited decrease, the proportion of pulse-labelled cells increases steadily during their incubation with hydroxyurea. As soon as the drug is washed off, a first short labelling wave takes place, soon followed by a second one. An approximate value for the G2 +&M period may be tentatively calculated from the 50 % intercept of the descending slope of the Exptl Cdl Res 65

356 S. Van Assel & M. Steinert 40

r

Fig. 3. 3H-thymidine incorporation in synchronized Crithidia luciliae. The arrows point to the beginning

and end of the hydroxyurea treatment. Small samples of the cells are taken at different times, given a 15 min pulse of 3H-thymidine (50 ,&i/ml) and then immediately spread and dried on slides for radioautography and cell counts. Three variables are plotted against an identical time scale: the proportion, per cent, of dividing cells (a); the proportion of cells with labelled nucleus (-•-) and labelled kinetoplast (-0-), whatever amount of label is present (b); the amount of thymidine incorporated, expressed as the mean number of silver grains per organelle, counted over the nucleus (-•-) and the kinetoplast (-0-) in a random sample of the whole population (c). Each experimental point is established from the examination of at least 1 000 cells.

first labelling peak and the mid-peak abscissa of the next division wave (fig. 3a). One finds 32 min, which is much shorter than the equivalent period in randomly dividing cells. As the length of the M phase of synchronized cells is not known, the particular value of G2 cannot be ascertained. Neither is it possible, in this experiment, to determine precisely the duration of the S phase, but an approximate value of 2 h may be deduced for the second wave in fig. 3 b, from 50 % intercepts. The same value is found for each one of the two organelles, although labelling appears to Exptl Cell Res 65

occur a very short time later in the kinetoplast. Silver grain counts, as shown in fig. 3 c, show the mean relative thymidine incorporation rates into nuclear and kinetoplastic DNAs at different times of the experiment. A first piece of information found in this diagram is the strong drop in the labelling which very rapidly follows the addition of hydroxyurea. Some relaxation of this inhibition appears to take place at the end of the treatment. Coordination between kinetoplastic and nuclear labelling is clearly apparent during the syn-

DNA

chronized growth which ensues after the drug treatment. A curious observation has been made here: during the first synchronized replication phase, the ratio of kinetoplastic to nuclear incorporation of 3H-thymidine is abnormally low. This ratio is best obtained from the surface ratio of the corresponding peaks in diagram (c). Although a rough approximation only may be expected from this diagram, the anomaly is quite obvious, the value measured being 0.22 as compared to a ratio of 0.43 observed in this experiment before the addition of the hydroxyurea. The ratio found in the same way for the second replication wave is much closer to normal with the value of 0.35. DISCUSSION There is good evidence, from our experiments on both log-phase and synchronized cultures, that the labelling of nuclear and kinetoplastic DNAs by 3H-thymidine are closely coordinated in time, as observed before on T. mega [27]. Before this can be taken as proof that the DNAs are really replicating simultaneously, further information is required concerning, for instance, the dynamics and size of the precursors pool(s) or probable variations in the level of enzymes like thymidine- and thymidylate kinases. Assuming that hydroxyurea interferes with DNA synthesis at the level of nucleotide reduction [37, 381, our observation that both nuclear and kinetoplastic thymidine uptake are very rapidly blocked by the drug is consistent with a small pool size, or would at least indicate a rapid exhaustion of some of its essential constituents. Coordinated synthesis of kinetoplastic and nuclear DNAs in synchronized Leishmania tarentolae, another trypanosomidae, has been found by Simpson [39]. Somewhat discrepant conclusions have been published [40] with respect to Crithidia fasci-

replication

cycles in Crithidia

luciiiae

357

however in the absence of clear quantitative data, it is difficult to ascribe a different behavior to this species. We found that, in normal log-phase conditions, the S replication phase takes 3 h 40 min and the G2 phase approx. 1 h. These values are close to, although not identical with those which were published previously on the ground of preliminary experiments [28]. That the length of the whole cycle, and of its different phases, is liable to extensive variations is clearly illustrated in our experiments with synchronized cells, in which the cell cycle, as well as the S phase, are reduced to nearly half their usual value. The fact that, whatever the duration of this S phase, it remains of equal Iength and almost simultaneous in the two DNA synthesizing bodies brings strong evidence for the existence of some common regulating process. No such close coordination seems to exist with respect to the rate of DNA synthesis: we found indeed that the ratio of kinetoplastic to nuclear DNA labelling is lower during the first synchronized replication cycle than in normal conditions. As yet, we have no good explanation for this. The chromosomal system might be able to increase the number of simultaneously active replication forks more readily than the kinetoplast; if this were the case, the cells of the first synchronized generation would contain a somewhat reduced amount of kinetoplast DNA. Once more, it is difficult to exclude any effect of variable pool size and composition. Our results on thymidine incorporation during the hydroxyurea treatment call for some comments. The fact that, under hydroxyurea inhibition, the labelling rate remains low although most of the cell population enters the S phase (fig. 3b, c), is consistent with the idea that the initiation of DNA synthesis is not affected, but that the rate of DNA chain elongation is limited by an impaired culata,

Exprl Cell Res 68

358 S. Van Assel h M. Steinert supply of deoxyribonucleotides. The partial relaxation of hydroxyurea inhibition observed in fig. 3c, at the end of the 6 h 30 min incubation time, could be attributed to derepressed synthesis of ribonucleotide reductases [41]. The accumulation of high amounts of these enzymes during the incubation with hydroxyurea might also have an activating effect on the rate of the deoxynucleotide pool formation after removal of the drug, allowing DNA replication to proceed at a higher rate. The authors are grateful to Dr Pamela Malpoix for kind assistance in revising the English text. This work has been supported by financial aid from the World Health Organization (Geneva) and from Fonds de la Recherche Fondamentale Collective.

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