Timing of nuclear and kinetoplast DNA replication and early morphological

events in the cell cycle of Trypanosoma brucei

R. WOODWARD

Biological laboratory, The University, Canterbury, Kent CT2 7NJ, UK

and K. GULL*

Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road,Manchester M13 9PT, UK

* Author for correspondence

Summary

We have used immunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order todetermine the timing of initiation and the durationof nuclear and kinetoplast S-phases within theprocyclic stage of the Trypanosoma brucei cellcycle. Both nuclear and kinetoplast S-phases wereshown to be periodic, occupying 0.18 and 0.12 of theunit cell cycle, respectively. In addition, initiationof both of these S-phases were in approximatesynchrony, differing by only 0.03 of the unit cellcycle. We have also used a monoclonal antibodythat recognises the basal bodies of T. brucei inorder to visualise cells possessing a new pro-basalbody and hence determine the time of pro-basal

body formation within the cell cycle. Pro-basalbody formation occurred within a few minutes ofthe initiation of nuclear S-phase, at 0.41 of the unitcell cycle. This provides detection of the earliestknown cell cycle event in T. brucei at the level of thelight microscope. Cell cycle events including in-itiation of nuclear and kinetoplast DNA replicationand pro-basal body formation may be strictly co-ordinated in T. brucei in order to maintain theprecise single-mitochondrion (kinetoplast), single-flagellum status of the interphase cell.

Key words: T. brucei, bromodeoxyuridine, S-phase.

Introduction

An individual Trypanosoma brucei cell possesses a num-ber of organelles present in single copies, each of whichmust be duplicated once during the cell cycle. Thepresence of single-copy organelles and the precise mor-phology of the cell necessitates a highly ordered processof organelle duplication and segregation at cell division.As is the case in many eukaryotic cells, T. brucei containsa single nucleus. However, the cell also possesses othersingle-copy organelles such as the flagellum, the mito-chondrion and the network of mitochondrial DNAknown as the kinetoplast. The kinetoplast is situatedadjacent to the basal body of the flagellum and contains acomplex catenated network of DNA in the form of mini-and maxicircles. We have recently described a series ofevent markers that define particular stages of the T. bruceicell cycle (Sherwin and Gull, 1989). Electron microscopyshows that the first cytological indication of progressthrough the cell cycle is the elongation of the small pro-basal body lying adjacent to the mature basal bodysubtending the single flagellum. Cells progress through

Journal of Cell Science 95, 49-57 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

identifiable events that can describe unique stages in theinitiation and elongation of the new flagellum, divisionand separation of the kinetoplast, mitotic division of thenucleus and finally cytokinesis.

The classic description of events in the eukaryotic cellcycle involves the definition of four periods of nuclearactivity. S-phase, the period of nuclear DNA synthesisseparates two gap periods, Gi and G2, which are followedby mitosis (Howard and Pelc, 1953). We have describedthe events of cell division in T. brucei (Sherwin and Gull,1989; Sasse and Gull, 1989), so a description of theperiod of DNA synthesis (S-phase), and hence Gi andG2, in the T. brucei cell cycle is desirable. However, sucha description must include the kinetoplast DNA, since inthe trypanosomatid protozoa kinetoplast DNA repli-cation occurs in a single period of the cell cycle. Thus, anS-phase exists for both the nucleus (Sn) and kinetoplast

(SO-Studies of kinetoplast DNA synthesis in four other

members of the Trypanasomatidae (Crithidiafasciculata(Cosgrove and Skeen, 1970), Crithidia luciliae (VanAssel and Steinert, 1971), Trypanosoma mega (Steinert

49

and Steinert, 1962) and Leishmania tarentolae (Simpsonand Braly, 1970)), using asynchronous cultures, indicatethat there are variations in the precise sequence andrelative duration of the Sn and S^ phases. However, in allcases there appears to be a close relationship between theinitiation of the nuclear and kinetoplast S-phases.

All of the above studies were performed using auto-radiographic detection of [3H]thymidine incorporatedinto the nucleus and kinetoplast. Since Gratzner (1982)reported the production of a monoclonal antibody raisedagainst the thymidine analogues 5-iododeoxyuridine and5-bromodeoxyuridine it has been possible to detectimmunologically analogue-substituted DNA in culturedcells. Recently, both Nakamura et al. (1986) and Bravoand Macdonald-Bravo (1987) have used bromodeoxyuri-dine (BrdUrd) in a qualitative method to visualise thesites of DNA replication in mammalian cells.

In this study we have used immunofluorescence detec-tion of BrdUrd incorporated into replicating DNA todetermine, for the first time, the timing of initiation andthe duration of nuclear and kinetoplast S-phases in the T.brucei cell cycle. We how that this technique is extremelysensitive when applied to these small cells. It has enabledus to place the periods of nuclear and kinetoplast DNAsynthesis onto the linear map of cell cycle events.Furthermore, we have used a monoclonal antibody thatrecognises the T. brucei basal bodies to show that pro-basal body formation occurs almost coincidently with theinitiation of nuclear DNA synthesis. This monoclonalantibody demonstrates the earliest cytological eventdetected to date in the T. brucei cell cycle at the level ofthe light microscope.

Materials and methods

Trypanosoma brucei brucei (strain 427) was cultured in tissueculture flasks in SDM-79 medium (Brun and Schonenberger,1979); 20 ml of mid-log phase culture (about 4x 106 cells ml"1)was split into two 10-ml samples. To one flask was added SO ftM-5-bromo-2-deoxyuridine (BrdUrd) (Sigma) and an equimolaramount of 2-deoxycytidine (Sigma); both flasks were incubatedat 27°C and 0.5-ml samples were taken from each culture athalf-hourly intervals for a period of 4h. The cells wereharvested by centrifugation in a microcentrifuge, washed oncein cold phosphate-buffered saline (PBS), pH7.4, and allowedto settle onto poly-L-lysine-coated slides. Cells were then fixedin 75 % ethanol at 4°C for a minimum of 1 h.

Immunofluorescence using an anti-BrdUrd monoclonalantibodyImmunofluorescence was performed essentially as described inthe Partec protocol for the use of an antibody to BrdUrd-substituted DNA (BioCell Consulting, Grellingen. Switzer-land). Briefly, the DNA of fixed cells, was denatured in 1.5 M-HC1 at room temperature for 30 min to allow the antibodyaccess to BrdUrd-substituted groups. Slides were washed inPBS, pH 7.4, to remove the acid and then incubated for 90 minat 25 °C in a moist atmosphere with an anti-BrdUrd antibody(Partec) diluted 1 in 100 with PBS, pH7.4, 0.5% Tween 20(Sigma) and 0.5 % bovine serum albumin (BSA, Sigma). Afterwashing in PBS, pH7.4, the slides were incubated with asecond antibody labelled with fluorescein isothiocyanate

(FITC, Sigma F-0257) diluted 1 in 75 with PBS, pH 7.4, 0.5 %Tween 20 and 1.5% neutral goat serum and incubated for 1 h at25 °C in a moist atmosphere. After further washing, cells werestained with the DNA intercalating dye, 4,6-diamino-2-phenylindole (DAPI), ljUgml"1, rinsed in PBS and then theslides were mounted in Moviol 4-88 (Harlow Chemical Com-pany Ltd) containing lmgml" 1 p-phenylenediamine as anantifade agent.

Two controls were performed: one with second antibody onlyand the other using cells that had not been cultured withBrdUrd.

Immunofluorescence using an anti-basal bodymonoclonal antibody

Samples were taken from a mid-log phase culture(4X106 cells ml" ), harvested as before, and after settling ontopoly-L-lysine-coated slides were fixed in methanol at —20°C for15 min, followed by rehydration in PBS, pH7.4. Samples werethen processed as before using the BBA4 monoclonal antibody,which recognises T. brucei basal bodies (Woods et al. 1989).

Growth characteristics of the culturesThe generation times of the cultures were determined bycounting the cell number ml~ at hourly intervals for 9 h, usinga haemocytometer.

Cell cycle analysisUsing asynchronous cultures the duration of a particular eventwithin the cell cycle can be determined from the frequencies ofcells showing that event, providing certain criteria are met: (1)the position of a particular stage in the cell cycle is known; (2)the culture is truly asynchronous; (3) the generation time isconstant. Using the analysis of Williams (1971), the durationsM and C for the nucleus and D and A for the kinetoplast can becalculated using the frequencies of cells within the populationexhibiting the relevant DAPI images (see description in Re-sults):

_ ln( 1-3-/2)

where x is the cumulative time within the cycle taken to reachthe end of the morphological stage in question, v is thecumulative % of cells up to and including the stage in question,expressed as a fraction of one unit, and j.l is the specific growthrate. A sample of 1000 cells was counted at 30-min intervals.

The mathematical treatment of Stanners and Till (1960) wasused to calculate the times of G2 and S for both the kinetoplast(k) and the nucleus (n). For all phases of the cell cycle data werecollected separately and independently for the k and n. Thecells were classified as judged by the definitions in Results.

G2 was taken as the length of time for BrdUrd label to appearin an organelle classified as dividing. As there is variation in thelength of G2 the average G2 period was determined in two ways:by determining the time for 50% labelling of dividing organ-elles and also by taking the differential of the curves for thepercentage of labelled dividing organelle. A sample of 50organelles was counted at each time point.

The duration of the S-phase was calculated by counting over1000 cells at individual times and classifying them as eitherBrdUrd-labelled or unlabelled with respect to their nucleusand, separately, for their kinetoplast. These frequencies werethen analysed using the treatment derived by Stanners and Till(1960).

Pro-basal body formationA total of 2000 cells were scored as possessing either one or two

50 R. Woodward and K. Gull

basal body/pro-basal body pairs and, using the analysis ofWilliams (1971) as above, the timing of pro-basal body forma-tion was determined.

Results

We have approached the determination of the DNAsynthetic periods in the T. brucei cell cycle by allowingcells to incorporate BrdUrd into their DNA and thendetecting this incorporation via immunofluorescencewith a monoclonal antibody specific for BrdU-substitutedDNA. In applying the labelled cells methodology ofStanners and Till (1960) for cell cycle analyses one mustdetermine both the generation time and the position ofnuclear S-phase (Sn) and kinetoplast S-phase (Sk) in thecell cycle. Ordering the position of the periods of nuclearand kinetoplast S-phase in the cell cycle involves twoprocesses: first, defining the end of the S-phase (begin-ning of G2); and second, determining the duration andhence, indirectly, the start of S-phase.

First, therefore, we determined the end of S-phase forthe nucleus and kinetoplast. The period of the cell cyclebetween the end of S-phase and cell division in T. bruceiis more complicated than in many cells, in that we mustconsider a number of additional cytological events. In thenuclear cycle there will be a period of G2 after S-phaseand then mitosis. However, the T. brucei cell is bi-nucleate for some time (Sherwin and Gull, 1989).Therefore, we need to define three periods after the endof nuclear S-phase: G2, defined as the period between theend of S and the start of mitosis; M, mitosis, definedcytologically (Sasse and Gull, 1989); and C, a cytokinesisperiod that includes the binucleate (post-mitotic) periodplus the actual period of cell cleavage. Likewise, for thekinetoplast there is a cytologically definable divisionperiod, which we term D. Thus, for the kinetoplast weneed to consider three post-S-phase periods: first, G2;second, D, defined as the period in which kinetoplastdivision is occurring (dumbell or bilobed kinetoplast);and A, defined as the period of time when the predivisioncell contains two kinetoplasts.

Thus, from the DAPI-stained images and analyses ofcell populations it is possible to determine the duration ofboth M and C for the nucleus and D and A for thekinetoplast, since these are all cytologically definedperiods. Subsequently, analyses of the population of cellsthat have incorporated BrdUrd into their nucleus/

ox

S 6

6c55

ox 7

i 66

3

4 6Time (h)

10 2 4 6Time (h)

kinetoplast can provide an estimate of the position andduration of S-phase.

We performed the determinations of M, C, D and Aboth on normal cultures and on cultures grown withBrdUrd, since such cultures would be used for sub-sequent incorporation studies. Control cultures and cul-tures grown with 50 ̂ iM-BrdUrd and 50 //M-deoxycytidinewere sampled every 30min over a 4-h period. Cells wereprocessed for immunofluorescence of BrdUrd incorpor-ation and nuclei and kinetoplasts were counterstainedwith DAPI. Cell numbers in the cultures were monitoredand growth curves constructed. The results of thisexperiment are seen in Fig. 1A and B. The cell doublingtime estimated using linear regression analysis was 8.65 h(r=0.987) for the BrdUrd-treated cultures and 8.67 h(r=0.988) for the untreated controls. Thus, the con-ditions of treatment with BrdUrd had no effect on cellgrowth rate over the period studied. The DAPI images ofcells in the BrdUrd culture are shown in Fig. 2. Themicrographs in Fig. 2 show the same cells visualised byphase-contrast, immunofluorescence of BrdUrd incor-poration and finally the DAPI fluorescence detection ofnucleus and kinetoplast.

The five cells in Fig. 2 are arranged in order of positionthrough the cell cycle defined by their DAPI images. Thefirst cell shows a single nucleus and a single kinetoplast(Fig. 2C) and therefore represents a cell at an early pointin the cell cycle. The cell in Fig. 2F shows an elongated,slightly bilobed kinetoplast characteristic of a cell in theD period. The three remaining cells (Fig. 21, L and O)illustrate later points in the cell cycle, but in terms oftheir kinetoplast morphologies all three cells are definedas being in the A period. The five cells seen in Fig. 2 alsoillustrate the nuclear morphology in the cell cycle. Thecells in Fig. 2C, F and I show the mononucleatecondition whilst Fig. 2L shows a cell with a mitoticnucleus. The cell shown in Fig. 20 shows the post-mitotic arrangement of two nuclei characteristic of the Cperiod.

We then performed a quantification of the proportionof cells within an asynchronous exponentially growingculture that possessed each of the nuclear and kinetoplastconfigurations defined above. Sample sizes of 1000 cellswere scored each 30min during a 4-h period. The datashown in Table 1 are the average results obtained fromthis experiment. The consistency of these results showsthat the culture is truly asynchronous in growth and so

Fig. 1. Growth curves of BrdUrd-treated anduntreated cultures. A mid-log phase culture (about4x 106 cells ml"1) was subdivided into two, eachwas incubated at 27°C; one in SDM-79 medium(A) and the other in SDM-79 plus SO^M-BrdUrd(B). Cell numbers were determined each hour inorder to construct a growth curve from which thegeneration time was calculated to be 8.67 h(r=0.988) for the untreated control and 8.65 h(r=0.987) for the BrdUrd-treated culture. BrdUrdwas added at zero time.

DNA synthesis in Trypanosoma brucei 51

10

Phase FITC DAPI Fig. 2. Nuclei or kinetoplasts containing BrdUrd-substitutedDNA were detected immunologically. Samples were takenfrom the culture every 0.5 h and processed forimmunofluorescence using an antibody that recognisesBrdUrd-substituted DNA as primary antibody. In additioncells were stained with the DNA intercalating dye DAPI.The DAPI fluorescence indicates cells at progressive stages inthe T. brucei cell cycle. Initially the kinetoplast elongates andseparates; this precedes nuclear division and cell division. Anelongated kinetoplast representative of the D period isillustrated in F, whilst kinetoplasts that have already divided,representative of the A period, are shown in I, L and O. Anelongated nucleus is illustrated in L, the same cell possessinga mitotic spindle J; both are features of the M period. Twoseparated nuclei representative of the C period are shown inO. In addition B, E, K, H and N illustrateimmunofluorescent staining patterns using an antibody thatrecognises BrdUrd-substituted DNA. The proportion of cellscontaining BrdUrd-substituted DNA in their nucleus orkinetoplast can be used to determine the duration of Sn andSk, respectively. Bar, Sum.

Table 1. Percentage of cells within the populationexhibiting particular nuclear and kinetoplast

configurations

Organelletype

Singleorganelle

Dividingorganelle

Post-divisionorganelle

NucleusKinetoplast

85.3±0.371.210.8

6.0+0.17.9±0.4

8.7±0.320.910.5

Table 2. A. Durations of the cell cycle with respect tonuclear events

Stage

G,snGzMC

Proportion ofcell cycle

0.400.180,220.080.12

B. Durations of the cell cycle with

Stage

G,skG2DA

kinetoplast events

Proportion ofcell cycle

0.370.120J50.090.27

Hours

3.431.511.870.691.06

respect to

Hours

3.220.991.240.762.35

M

Durations of the cell cycle occupied by defined events werecalculated using the analysis of Williams (1971). Generationtime=8.65h.

justifies the use of the analysis after Williams (1971). Thedata shown in Table 1 were therefore used in an analysisbased on that of Williams (1971) and described inMaterials and methods. Essentially, the percentage of thepopulation with particular organelle configurations given

in Table 1 is converted to estimates of the duration oftime taken for particular events in the cell cycle to occur.This analysis produces estimates of the duration of the Mand C periods for the nucleus and D and A periods for thekinetoplast (Table 2).

52 R. Woodward and K. Gull

We are now able to determine the length of the nuclearand kinetoplast G2 periods. These values can be derivedby adding BrdUrd to a culture and then determining thekinetics of appearance of BrdUrd-substituted DNA individing kinetoplasts or nuclei. The configuration of suchcells is shown in Fig. 2E and Fig. 2K for the kinetoplastand nucleus, respectively. These kinetics of appearanceare shown in Fig. 3A and B. Samples were taken at 0.5-hintervals and the time taken for 50 % of the dividingorganelles to become labelled was taken as the averageduration of G2 (from Fig. 3A and B). However, we alsocalculated the length of G2 from the peak of the differen-tial of these original curves, Fig. 3C and D (Stanners andTill, 1960). Both methods gave very similar results andthe average G2 durations for nucleus and kinetoplast areshown in Table 2.

It is now possible to use the Stanners and Till (1960)analysis to determine the duration of S-phase, in this casefor both nucleus and kinetoplast. This was achieved byincubating cells with BrdUrd and determining the per-centage of cells with labelled nuclei or kinetoplastsdetermined separately (examples of such cells are shownin Fig. 2). These percentages can then be used tocalculate the duration of S-phase knowing the generationtime and the duration of the total post-S period of the cellcycle that we have calculated above (Stanners and Till,1960). The formula used in this calculation is shown inequation (1):

S=\/a\n[L + e^]-(z + t), (1)

where <*=ln Z/T and z = G2+M + C for the nucleus andG2+D+A for the kinetoplast, T equals the generationtime, t is the duration of the BrdUrd labelling period andL is the proportion of cells exhibiting either labellednuclei or kinetoplasts. We have performed this analysisfor two different time periods of BrdUrd incorporation: 2

and 2.5 h for the nucleus and 1 and 1.5 h for thekinetoplast. Again, the data derived from each timeperiod were similar and the average values of the durationof Sn and Sk are given in Table 2.

Analysis of these population-derived data shows thatSn is 31 min longer than Sk, and since we know that G2

for the kinetoplast begins 44 min before G2 for thenucleus there must be an overlap of these two periodswithin the overall cell cycle. In fact these population datasuggest that there is both a non-coordinate start andfinish to the S-phases in the nucleus and kinetoplast(Fig. 4). This suggests that we should observe twodistinct sub-types of BrdUrd-labelled cells in the popu-lation: first, cells in which the kinetoplast has incorpor-ated BrdUrd but the nucleus has not (since Sk is initiatedslightly earlier than Sn); and second, a cell type in whichthe nucleus has incorporated BrdUrd but the kinetoplasthas not (since Sk terminates before Sn). Indeed, both ofthese individual cell types are observable in the BrdUrd-substituted populations (Fig. 2B and 2H, respectively).

The conclusion of this cell cycle analysis is the calcu-lation of the Gi period for the nucleus and kinetoplast,which is achieved by merely subtracting all of the abovecalculated periods from the total cell cycle time(Table 2). The overall order and duration of each of theT. brucei cell cycle periods: Gi, S, G2, M and C for thenucleus, and Gi, S, G2, D and A for the kinetoplast, aresummarized graphically in Fig. 4.

In addition to determining the timings and duration ofthe Sn and S^ phases of the T. brucei cycle we have soughtprobes that might allow us to detect early events occur-ring in the cell cycle at the light-microscope level. In thiscontext we have recently described a monoclonal anti-body, BBA4, which detects the T. brucei flagellum basalbodies (Woods et al. 1989). We know from our previousstudies (Sherwin and Gull, 1989) that flagellum growth is

100r B

1 2 3Time (h)

Fig. 3. The duration of the G2 period for thenucleus and kinetoplast was determined byculturing cells with BrdUrd and immunologicallydetecting the progressive appearance of BrdUrd-substituted DNA in dividing kinetoplasts (A-) andnuclei (B). The time taken for 50% of dividingorganelles to become labelled was used as theaverage G2 period, this was 1.23 h for thekinetoplast and 1.86 h for the nucleus. Forcomparison, the length of G2 was also determinedfrom the differential of these curves, the peak ofthe differential was taken as the average G2. C andD, for kinetoplast and nucleus, respectively.

DNA synthesis in Trypanosoma brucei 53

0.2bb

0.4pfr

0.6 0.8 1.0 unit Phase/FITC FITC DAPI

0 1

G,G,

2 3

S

S

14

G2

G2

5

D

6

M C

A

7 8 8

Fig. 4. Summary of the calculated durations and sequencesof the periods Gi, S, G2, D or M and C or A represented ona linear map of the T. brucei cell cycle. In addition the timesof initiation of basal body duplication (bb) and initiation ofparaflagellar rod synthesis (pfr) are also indicated.Fig. S. The formation of new pro-basal bodies can bedetected immunologically. Cells from an asynchronouslydividing population were prepared for immunofluorescencestaining using a monoclonal antibody that recognises basalbodies as the primary antibody. The cells were alsocounterstained with DAPI to indicate the position that a celloccupies within the unit cell cycle (C, F, I, M and O). Fromthe immunofluorescence images cells were classified as eitherpossessing one pro-basal body/basal body pair (B), or twopro-basal body/basal body pairs (E, H, K and N). Theseproportions were then used to determine the point of pro-basal body formation within the cell cycle. The first visiblesigns of formation of two pro-basal body/basal body pairs areillustrated in E. The two pairs then separate as the cell cycleprogresses (H, K and N). Bar, 5jUm.

initiated around 0.4 of the unit cell cycle and so dupli-cation of the basal bodies appears likely to occur early inthe cell cycle and so might provide such a marker. Thus,we have repeated the above analysis of cell cycle timingsafter quantification of the different cell types defined inthe population by immunofluorescence staining with theBBA4 monoclonal antibody. We can recognise five dis-tinct cell types with this antibody and these are shown inFig. 5. First, cells early in the cell cycle (defined by theirDAPI image) show a basal body/pro-basal body pair(Fig. 5B). Later in the cell cycle we can recognise cellsthat express two basal body/pro-basal body pairs(Fig. 5E). Subsequently, these two basal body/pro-basalbody pairs segregate to well-defined positions in the cellin readiness for cytokinesis (Fig. SH, K and L).

Using the previously described analysis technique ofWilliams (1971) and immunofluorescence staining of thebasal bodies in an exponentially growing asynchronousculture, we determined the position of pro-basal bodyformation within the cell cycle. First, we used a quantifi-cation of the DAPI images of the cells in the culture toshow that this culture was indeed directly comparablewith that used earlier for the S-phase analysis. We thencounted 2000 cells and classified them into two groups,containing either one or two basal body/pro-basal bodypairs. A total of 993 cells exhibited one pair and 1007 cellspossessed two pairs. Analysis of these data shows thatpro-basal body formation occurs at 0.41 of the unit cellcycle. This now provides a marker for the earliestcytological event within the T. brucei cell cycle. More-over, it is an event that is almost coincident with ourcalculation of the initiation of S-phase in the nucleus(Fig. 4).

M

Discussion

We have previously described the duration of a series ofmorphological events within the T. brucei cell cycle(Sherwin and Gull, 1989). This present study has now

54 R. Woodward and K. Gull

established these earlier cytological events within a cellcycle description that includes the periodic DNA-syn-thetic periods in the nucleus and mitochondrion (kin-etoplast). Moreover, we have been able to establish thetiming of an early event marker in the cell cycle, i.e. pro-basal body formation. In establishing this description ofevents in the T. brucei cell cycle we have provided a newdefinition of phases relevant to the nucleus and kineto-plast. The nuclear cycle in T. brucei encompasses theclassical Gj, Sn, G2 and M, together with a cytokinesis,C, phase. It is important to define this C phase, since itrepresents a reasonably lengthy period in which the twopost-mitotic nuclei acquire particular positions in readi-ness for the actual longitudinal cytokinesis event. SinceDNA synthesis is periodic in the kinetoplast we candefine Gi, Sk and G2 phases in the cell cycle relative tothat organelle. However, we can also define a divisionperiod, D, in which the kinetoplast is characterised by itsbilobed image in DAPI fluorescence, and an apportion-ing phase, A, during which the cell contains two kineto-plasts. This A phase of the kinetoplast is extensive,occupying 0.27 of the unit cell cycle. Presumably, thislong period is important for establishing the correctsegregation of the two daughter mitochondria and kin-etoplasts at cell division. In this context it is important tonote that the A period of kinetoplast segregation isinitiated some time, 0.07 of the unit cycle, before thenucleus divides in mitosis.

We have carefully chosen this separate terminology fornuclear and kinetoplast events, since it allows particularpoints in the overall cell cycle to be defined in respect ofevents that are particular to the individual organelle.Previous work on other trypanosomatids has highlightedthe existence of a periodic S phase for both the nuclearand mitochondrial DNA (Cosgrove and Skeen (1970) forCrithidia fasciculata; Steinert and Steinert (1962) forTrypanosoma mega; Van Assel and Steinert (1971) forCrithidia luciliae; Simpson and Braly (1970) for Leish-mania tarentolae). The general positions of Sn and Sk inthe cell cycle of other trypanosomatids described in theseearly studies appear reasonably comparable with thepositions that we have now defined for T. brucei. How-ever, the relative timings of kinetoplast division, D, andnuclear division, M, appear to vary depending uponwhich particular trypanosomatid is being considered.Thus we can contrast our results with T. brucei, wheredivision of the kinetoplast is completed, some time beforemitosis, with those for Crithidia luciliae, in which thenucleus divides before the kinetoplast (Van Assel andSteinert, 1971). It is likely that this phenomenon may bea reflection of the morphology of the cell in question.Crithidia cells are chonomastigotes, which means that thepositions of nucleus, kinetoplast and basal body in theserather rounded cells produce a natural bilateral symmetrysoon after organelle duplication. In contrast, the elongatecells of the trypomastigote T. brucei would produce arather tandem arrangement of the duplicated organelles.Thus extensive rearrangement of the organelles is necess-ary to provide the bilateral symmetry required forcytokinesis. This presumably provides the explanationfor the long A and C periods that we have described

above for T. brucei. It takes a substantial period for the T.brucei cell to locate the daughter flagella, nuclei andmitochondria in the correct predivision configuration(Sherwin and Gull, 1989).

Our use of immunological detection of BrdUrd-substi-tuted DNA to reveal DNA-synthetic periods in trypano-somes has many advantages over the previously usedautoradiographic techniques. The method appears ex-tremely sensitive, gives low backgrounds and is veryrapid, producing results in hours rather than days orweeks. Also, the protocols we have used give excellentcytological preservation of the trypanosome cells and,moreover, allow a precise co-localisation of nucleus andkinetoplast by DAPI fluorescence. We have analysed thedata without consideration of the time required forequilibration with the endogenous thymidine pools. Wehave little information on the size of such pools. How-ever, it appears that BrdUrd is rapidly taken up and madeavailable for incorporation into DNA, since we havedetected BrdUrd substitution in newly formed DNAafter less than IS min of culture. The BrdUrd exper-iments have allowed us to place the timings and durationsof the nuclear and kinetoplast S phases within the overallcell cycle. We have been able to add further event markersto the cell cycle by using the DAPI fluorescence detectionof the nucleus and kinetoplast division cycles and theimmunological detection of other organelle-biogenesisevents. This results in the overall description of the cellcycle seen in Fig. 4.

Cell cycle kinetic studies of populations such as thesesuffer from variation in individual cell cycle times.Within any population of growing cells there is variationin the individual cell cycle times and consequently theduration of cell cycle phases. Our analysis is based ondata taken from the population as a whole. Fig. 3illustrates the variation in the duration of the G2 period.If G2 was constant the accumulation of BrdUrd-labelleddividing organelles would occur in a single step from 0 to100% at the end of G2. We have observed BrdUrd-labelled kinetoplasts leaving G2 from 0.76 h to 1.76 hafter initiation of labelling; the G2 duration of 1.25 h thatwe quote was taken when 50 % of the dividing kineto-plasts were labelled. Similar variation is evident in thenuclear G2 period: Fig. 3B shows this to be from 1.44 to2.5 h; our average was 1.87 h. Variation in G2 affects thecalculated durations of Sk and Sn, since the G2 duration isinvolved in equation (1). If one determines the Sk valuesusing the two extremes of the G2 duration range Sk

becomes 0.9 h for the shorter G2 and 1.09 h for the longerG2; our average value was 0.99 h. This variation occursfor Sn, where the duration is from 1.07 h to 1.7 h. Sincewe are unable to study individual motile cells at presentwe do not know how variation in G2 times relates to thebehaviour of the other cell cycle phases or whether asimilar increase or decrease is relevant to Gi, S, M/D andC/A cell cycle phases.

We are now able to position an individual cell withinparticular areas of the cell cycle with respect to nuclearand kinetoplast DNA synthesis and organelle division byuse of the DAPI fluorescence and monoclonal antibodiesto specific organelles. For example, a cell that exhibits the

DNA synthesis in Trypanosoma brucei 55

elongated, bilobed kinetoplast by DAPI staining (the Dkinetoplast period) is in the G2 period with respect to thenucleus, i.e. the Sn period must have been traversed andterminated. Likewise, pro-basal body formation marksthe time of entry to the Sn phase. Thus, if cells are stainedwith the BBA4 antibody to detect basal bodies, and theR0D1 antibody to detect the new paraflagellar rod, thenone can classify cells into a number of the classic cell cyclestages. Cells that exhibit one basal body/pro-basal bodypair are in the nuclear Gi phase; cells with two probasalbody/basal body pairs with no new paraflagellar rod arein the Sn period of the cell cycle. Subsequently, consider-ation of the length of the new flagellum (defined by theparaflagellar rod detection) can give a reasonable idea ofthe cell's position in the remainder of the cell cycle(Sherwin and Gull, 1989). It is interesting that ourresults show that pro-basal body formation occurs almostcoincidently with the start of the nuclear S phase. Thisjuxtaposition of the morphological event and S phase isvery similar to the situation of centriole morphogenesiswithin some mammalian cell cycles (Robbinsei al. 1968;Vorobjev and Chentsov, 1982).

Eukaryotic cells usually possess multiple mitochondriaand in most cells studied synthesis of mitochondrialDNA is not restricted to any one period of the cell cycle.Rather, studies with both eukaryotic microbes such asPhysarum (Guttes et al. 1967; Braun and Evans, 1969;Brewer et al. 1967) and mammalian cells (Meyer andSimpson, 1968; Schultz and Nass, 1969; Kalf and Ch'il,1968) show continuous synthesis of mitochondrial DNAthroughout the cycle. Thus, the periodic S phase ofkinetoplast DNA is an intriguing exception against thisbackground.

Our studies show that Sn and Sk start very closetogether. However, DNA synthesis in the kinetoplastfinishes before DNA synthesis in the nucleus. This is animportant distinction and means that there appears to beintra-organelle control over DNA synthesis termination.Kinetoplast DNA synthesis can be terminated in acellular environment where nuclear DNA synthesis con-tinues. The shorter Su may merely be a reflection of thesmaller amount of DNA to be replicated in the mitochon-drial kinetoplast. Nevertheless, it is clear that somecontrol must be exerted to stop the continuous re-replication of kinetoplast DNA during the remainingperiod of nuclear S-phase. This argues for some localintra-organelle control over DNA synthesis. Harland andLaskey (1980), and Harland (1981) showed that DNAintroduced into unfertilized Xenopus eggs was able toreplicate once per cell cycle with no re-replication. Blowand Watson (1987) found the same to be true when usingcell-free systems devised by Lohka and Masui (1983). Amodel for the block to re-replication of DNA has beenproposed whereby a 'licencing factor' becomes associatedwith DNA when the nuclear envelope breaks down atmitosis; only DNA that is associated with this factor isavailable for replication during the next cell cycle (Blow,1989; Blow and Laskey, 1988). It is suggested that thelicencing factor associated with DNA becomes dis-sociated when that piece of DNA has been replicated,rendering this DNA unavailable to further replication.

The same model is applicable in systems where thenuclear envelope does not break down. In these cases apermeabilization of the envelope may occur, allowinglicencing factor access to the DNA sites. This model mayalso be applicable to the kinetoplast DNA and nuclearDNA in trypanosomatids, allowing one round of repli-cation in each organelle after the coordinate initiation ofS-phase. This would, of course, necessitate a biochemicalpermeabilisation phenomenon that is applicable to boththe nucleus and the mitochondrion. The signal for thenear-coincident initiation of kinetoplast DNA and nu-clear DNA synthesis remains to be identified.

This work was funded by the Science and EngineeringResearch Council. This investigation received financial supportfrom the UNDP/World Bank/WHO special programme forResearch and Training in Tropical Diseases. We also thank DrIan Salmon for his valued advice on the data analysis.

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{Received 25 July 1989 - Accepted 17 October 1989)

DNA synthesis in Trypanosoma brucei 57