J. Cell Sci. aa, 521-530 (1976) 521

Printed in Great Britain

TIMING OF NUCLEOLAR DNA REPLICATION

IN AMOEBA PROTEUS

I. MINASSIAN AND L. G. E. BELLDepartment of Biology, Medical and Biological Sciences Building,University of Southampton, Southampton SOg 3 TU, England

SUMMARY

Light- and electron-microscope autoradiography have been used to follow the incorporationof [3H]thymidine at different stages during the interphase of synchronously growing populationsof Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e.DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesserdegree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA wasfound to be late replicating, i.e. it replicated during the Ga phase.

INTRODUCTION

Although there is general agreement between different authors concerning theexistence of DNA associated with the nucleoli (Busch & Smetana, 1970; Smetana &Busch, 1974), the timing of nucleolar DNA synthesis during interphase has been amatter of controversy for a number of years. Approximately half the reports shownucleolar DNA synthesis occurring during all or some part of S-phase, the remaindershow it occurring during Gly G2 or the whole of the interphase. For example, Nash &Plout (1965) and Balazs & Schildkrout (1971), looking at S-phase as a whole, reportedthat nucleolar DNA synthesis occurred with the rest of the nuclear DNA. But whenthe 5-phase was subdivided by other workers into 2 or more intervals, no commonpattern was found for its timing: in Chinese hamster cells the nucleolar DNA replicatedduring an early portion of 5-phase (Stambrook, 1974), whereas in a rat kangaroo cellline (Giacomoni & Finkel, 1972) the ribosomal cistrons replicated at the end of the5-phase.

Some workers suggest an independence between the biosynthesis of nucleolar andchromosomal DNA, i.e. they found no correlation between the timing of nucleolarDNA synthesis and the major chromosomal DNA synthesis. For example, in ratfibroblasts (Harris, 1959) nucleolar DNA synthesis preceded the main nuclear DNA-synthetic period, while in adult rat liver cells (Wintzerith et al. 1975) it was shown toreplicate in the absence of replication by the rest of the nuclear DNA. Using themultinucleate slime mould Physarumpolycephalutn where many thousands of accuratelysynchronized nuclei could be examined, Ryser, Fakan & Braun (1973) found that 'thegenes for the ribosomal RNA replicated to a large extent in the G2-phase'. In a studyof [3H]thymidine incorporation of Amoeba proteus, Ord (1968) showed 90% of thenuclear incorporation taking place during the first fifth of the cell cycle, referred to as

522 / . Minassian and L. G. E. Bell

5-phase. However, she also found a persistent, though low, level of nuclear incor-poration during the G2-phase. Since this work used a 'squashed whole cell technique'for autoradiography no prediction could be made as to whether the G2 nuclear labellingwas chromosomal or nucleolar.

In the present study using sections of labelled amoebae, the position of the late-replicating DNA has been visualized, with a clear distinction between labelling overthe peripherally located nucleoli and/or over the central chromatin region of thenucleus. By labelling cells, synchronized by selecting the detached mitotic cells, with[3H]thymidine at different intervals during interphase, differences in the degree of[3H]thymidine incorporation for the 2 regions have been studied.

MATERIALS AND METHODS

Culture

Cultures of Amoeba proteus, strain PDaX10, were maintained at 18-20 °C using the Tetra-hymena feeding technique of Prescott & James (1955).

A. proteus, strain PDaXti, was shown in earlier studies (Ord, 1968) to have a cell cycle of48-54 h: DNA synthesis occupied the first fifth of the cell cycle (with no significant G^, thelong G,-phase lasted some 38-44 h, mitosis took approximately 30-35 min. The strain PDaXlaused during this investigation had a slightly longer G,-phase, making the length of the cellcycle approximately 58-60 h. In this work the long G2-phase has been subdivided into earlyGt (13-28 h), mid G, (28-43 h) and late G% (43 h until mitosis).

Cells were synchronized by selecting the mitotic cells from mass cultures. After division cellsof the same age were grouped together and cultured normally until exposure to the [3H]thymidine.

Thymidinc labelling

Cells of known age were labelled with [Af«-!H]thymidine, specific activity 20 Ci/mM (pur-chased from the Radio Chemical Centre, Amersham) at a concentration of 0-5 mCi/ml forexposure periods of 6 h. Four labelling intervals were used: 0-5-6-5 h, 15-21 h, 32-38 h, and43-49 h. These periods were taken as being representative of S, early G2, mid G2 and late Gaphases respectively. After exposure the cells were washed free of radioactive material, chasedwith cold thymidine (concentration 12 x io~4 mg/ml) for 1 h and fixed.

Preparation for light- and electron-microscope autoradiography

Cells were fixed in a 1 % solution of osmium tetroxide in. 01 M cacodylate buffer pH 6-2 from.60 to 90 min. Following fixation cells were washed with distilled water then dehydrated in agraded series of ethanols, immersed in propylene oxide and embedded in Araldite. Sections of1 /im thickness were cut from the blocks of labelled cells, put on clean slides and processed forlight-microscope autoradiography according to Prescott's (1964) dipping technique. The slideswere left under Ilford K5 nuclear emulsion (Ilford Ltd., England) for 3-4 weeks, developed,then stained with 0-25 % toluidine blue made up with 0-25% borax in distilled water.

Sections of 60-80 nm were cut from blocks of labelled cells for the use of EM-ARG. Thesewere placed on copper grids and mounted on top of glass rods for application of emulsion bythe loop technique (Stevens, 1966). Ilford L4 nuclear emulsion diluted 2:3 with distilled waterwas applied with a wire loop in the dark. Grids were left under emulsion at room temperaturefor 8-10 weeks, then developed. After developing they were stained with uranyl acetate andlead citrate and examined with a Philips 300 electron microscope. At the end of preparationfor EM-ARG, examination was made of serial sections to eliminate any error in interpretationof nucleolar-localized grains which could arise due to the irregular shape of the amoeba nucleus.In total, autoradiographs of more than 100 S and G, cells were examined by light microscopyand a further 100 S and G, by EM-ARG.

Timing of nucleolar DNA replication 523

Enzymic digestion

Labelled amoebae were burst by drawing into, and blowing out of, a narrow-aperture pipette.The nuclei were collected as they floated free and were placed on slides and fixed for 3 min inacetic ethanol (113). After fixation the slides were rinsed and covered with 05 mg/ml of pan-creatic deoxyribonuclease (BDH Biochemicals) made up with one-quarter strength Mcllvain'sbuffer (pH 7). The slides were incubated foi 3 h at 35 CC, then rinsed, dehydrated and sub-sequently processed for light-microscope autoradiography as described above.

RESULTS

Differences in the localization of \?H]thymidine incorporation at S and at G2

Autoradiographic study of cells treated with [3H]thymidine during the S-phase. Light-microscope autoradiographic observations of synchronized cells exposed to [3H]-thymidine for 6 h starting 30 min after mitosis revealed that the maximum incorpora-tion occurred in the central region of the nucleus. The nucleoli — small and verynumerous bodies located at the marginal zones of the nucleus - showed very littleincorporation of [3H]thymidine. Silver grain counts performed on these cells showedthat most of the grains were localized over the central region; only a few were foundin the nucleolar region. The EM-ARG study confirmed the light-microscope resultsand demonstrated very clearly the high concentration of grains in the nuclear centralregion (Fig. 1 A, B). The few grains found on the nucleoli were over the perinucleolarregion.

There was no incorporation of [3H]thymidine by the helical structures of the amoebanucleus: structures believed to represent RNA and protein packaged together fortransportation to the cytoplasm (Stevens, 1967; Minassian & Bell, 1976). This is inagreement with results of previous investigations suggesting that the helices are notDNA-containing structures (Stevens, 1967; Wise, Stevens & Prescott, 1972).

Autoradiographic study of cells treated with \*H~\thymidine during the G2-phase. Light-microscope ARGs of i-/tm sections of amoebae exposed to [3H]thymidine at theperiods 15-21, 32-38 and 43-49 h. showed some incorporation of tritiated thymidineby the nucleus throughout G2. The rate of incorporation was much lower than thatof an S-phase nucleus. The grains were mainly localized on the peripherally locatednucleolar region. Only a few were observed in the central region of the nucleus.Examination of labelled mid-G2 nuclei at the electron-microscope level confirmed thatthe grain density was high in the region of the peripherally located nucleoli and lowin the central region of the nucleus. There was a preferential labelling of the marginalzones of the nucleoli, the perinucleolar region (Fig. 2 A, B).

These experiments attempted to cover the whole of G2 by exposing cells at 3 differentintervals. Though the same grain distribution pattern, i.e. localization over thenucleoli with few grains over the central region, was found in each case, a detectableincrease in thymidine incorporation was observed in cells treated during mid G2. Suchan increase could indicate greater nucleolar activity in the mid G2-phase. However,fluctuations would be expected in the endogenous DNA precursor pools, in thequantity and/or activity of DNA replicating enzymes as the cell passed from the

524 /. Minassian and L. G. E. Bell

• «

1 A

Timing of nucleolar DNA replication 525

5-phase into the G2-phase, and again as it approached mitosis. Such fluctuationswould account for differences in the level of pHJthymidine incorporation noted forearly, mid and late G2-phases. Since it was impossible in this type of experiment toestimate the sizes of the endogenous DNA precursor pools, or to establish the levelof activity of DNA replicating enzymes, no attempt has been made to quantify thepHJthymidine incorporation during these 3 different G2-periods.

Quantitive differences in ^K\thymidine incorporation within the nucleus

Having established by light and EM autoradiography that there was a very differentlocalization of grains in the nucleus during 5-phase and G2, it was considered reason-able to attempt to compare the levels of [3H]thymidine incorporated over nucleoli andcentral nuclear region during S and G2 by grain counting. For this purpose the secondG2-phase, i.e. mid G2, was chosen as representative of G2 since it was furthest removedboth from changes taking place as the cell moved from S into G2) or as the cellapproached division. Grain counts over 5 and G2 nuclei produced the followingresults: (1) 5-phase nuclei had a total average grain count 4 times greater than G2-phase nuclei. (2) In 5-phase nuclei 95 % of the grains were located in the central mainchromatin region of the nucleus, only 5 % being found in the nucleolar region. And(3) in G2-phase nuclei 90 % of the grains were located peripherally, i.e. over or aroundthe nucleoli, the remaining 10% being scattered in the central region of the nuclei.See Table 1.

Cytoplasmic labelling

Observation of the cytoplasm using light microscope ARGs revealed an increase incytoplasmic labelling coincident with the period when nucleolar DNA was replicating.Thus grain counts showed that pH]thymidine incorporation was 3 times higher inG2-phase cytoplasm than in 5-phase cytoplasm (Table 1). It was impossible to identifythe location within the cytoplasm of pHJthymidine incorporation using light-micro-scope ARG, since the resolution does not allow identification of structures in the sizerange of mitochondria. However, previous EM studies on A.proteus (Minassian, 1974)had already shown that much of the cytoplasmic label was located over the mito-chondria, while the remainder was generally associated with endoplasmic vesicles. Arecent study performed on Tetrahymena pyriformis (Engberg, Nilsson, Pearlman &Leick, 1974) had shown that nucleolar and mitochondrial DNA replication are undera control independent of that for the replication of bulk DNA. If this is so for amoeba,then an increase in the cytoplasmic grain count, coincident with the time of nucleolarDNA replication might be expected.

Fig. 1. High-iesolution autoradiograph of a cell labelled with [3H]thymidine for 6 hduring S-phase. Silver grains are confined to the central regions of the nucleus, theperipherally located nucleoli appear almost unlabelled. c, cytoplasm; he, honeycomblayer of the nuclear envelope; n, nucleus; no, nucleolus. A and B, X 9600 and 18500,respectively.

/. Minassian and L. G. E. Bell

T I

n

: M l

* *

2 A

1

' • ' V » • • •-> B • •

Timing of nucleolar DNA replication 527

Enzymic digestion using DNase on isolated nuclei

There is always some possibility that exogenous thymidine is degraded and the 3Hsubsequently incorporated into nuclear molecules other than DNA. This is partiallyavoided by using thymidine with the label on the methyl group, where degradationshould avoid a preferential use in RNA. However, enzymic digestion experimentsusing DNase were thought necessary to eliminate any uncertainty that the PH]-thymidine could be finding its way into molecules other than DNA. Since sectiondigestion by DNase of nuclei or amoeba has given poor results in the past (Wise &Goldstein, 1972) digestion experiments in this work were carried out on isolated nucleias described in the Methods section. The results showed that all label was removedfrom those slides which had been treated with DNase.

Table 1. Numbers of grains junit area over the nucleus of cells exposed to \?H]thymidineduring 6 h of either the S-phase or the mid G2-phase

•S-phaseMidG.

Extra-nucleolar

grain count/25 /tm1

6665 ±(11 0)I - 8 3 ± ( O I )

Nucleolargrain count/

25 fitn1

3-92 ±(0-2)

16-00 ± (1)

Cytoplasmicgrain count/

2-5 /im'

112 |(o-2)

3-90 ± (0-2)

Extra-nucleolar

grain count/total nuclear

count, %

951 0

Nucleolargrain count/total nuclear

count, %

590

Grains were located either over the central region of the nucleus (extranucleolar) or over thenucleoli. Though a few grains fell over the nuclear membrane, these were considered insignifi-cant and have been excluded from the values above. Background count was negligible: o-i ±001 grains/25 /im*. Each reading is the average grain count obtained fiom 350—400 sections.This repiesents 50-60 S-phese and 50-60 G,-phase cells. Observations, without grain count,were made on a further 80-100 cells. The values in parentheses are the standard errors.

DISCUSSION

Investigating the incorporation of pHJthymidine during interphase, using bothlight and EM-ARG techniques, showed 2 main patterns in A. proteus. (1) A majorincorporation occurring during the first quarter of the cell cycle showed almost allgrains localized over the central region of the nucleus; few grains were found over theperipherally located nucleoli. (2) A minor incorporation occurring during the rest ofinterphase showed almost all grains over the nucleolar region; few were found onthe extranucleolar chromatin.

In an earlier cell cycle autoradiographic study, using the same strain of A. proteus,Ord (1968) found heavy incorporation of piTJthymidine by the nucleus during the

Fig. 2. Electron-microscope autoradiograph of a cell labelled with [3H]thymidine for6 h during mid Ga, illustrating the localization of silver grains predominantly overthe nucleoli. The label is confined to the perinucleolar region, c, cytoplasm; he, honey-comb layer of the nuclear envelope; n, nucleus; no, nucleolus. A and B, x 7800 and13800, respectively.

528 /. Minassian and L. G. E. Bell

first quarter of the cell cycle: this she termed 5-phase. However, she also found thatduring the rest of the cell cycle, termed G2, there was a consistent, though low, levelof [3H]thymidine incorporation by the nucleus. A similar high level of incorporationof pHJthymidine by the nucleus during the early part of the cell cycle has been foundin other strains or species of Amoeba (Ron & Prescott, 1969; Narasimha Rao &Chatterjee, 1974). In the present work we have adopted the S and G2 terminology ofthese workers.

Autoradiographic studies on Amoeba have generally been made using a squashtechnique of whole cells, i.e. the whole nucleus contributes to the grain count. Achange in the distribution of grains over different nuclear areas would go undetected.It was this change in distribution of grains, first observed during cell cycle ARGstudies, which stimulated the present investigation. Since the G2 incorporation ofPHJthymidine was very low it was necessary to change from the i-h pulses used byOrd (1968) to exposure periods of 6 h or more. Fortunately, as amoebae are able totolerate high doses of radiation (Ord, 1973) this did not lead to any loss of viability ofthe cells. Furthermore, amoebae have a well developed salvage DNA pathway: theyare able to take up and utilize exogenous thymidine throughout the whole of the cellcycle (e.g. 5-phase nuclei incorporate [3H]thymidine while in G2 cytoplasm (Ord,1971)). This may not be so for all types of cells as the development of the salvage path-way varies from one cell type to another (Kornberg, 1974) and the activity of theirthymidine kinase is under strict regulation (Okazaki & Kornberg, 1964).

It is clear from our investigation, where observations have been made on serialsections through 100 or more 5-phase and G2-phase cells, that the incorporation ofpHJthymidine during G2 is localized on or in the immediate vicinity of the nucleoli.This suggests that the replication of nucleolar DNA is out of synchrony with thereplication of the main bulk of the nuclear DNA (referred to as extranucleolar DNA).

Asynchrony of nucleolar and extranucleolar DNA was not unexpected in that thishas been reported for a number of cell types (Charret, 1969; Giacomoni & Finkel,1972; Ryser et al. 1973; Stambrook, 1974; Andersen & Engberg, 1975; Wintzerithet al. 1975). The chief controversy concerning nucleolar DNA synthesis has been inits timing during the cell cycle, rather than its correlation with the rest of the nuclearDNA. This controversy may be due to species differences, for example tissue culturecell and protozoa may have genuine differences in the sequence of events in the cellcycle. However, this does not account for the differences in results obtained in thefollowing 2 cases. (1) In T.pyriformis, where cell age was determined by morphologicalcharacteristics, Charret (1969) has shown that nucleolar DNA replicated during theG2-phase. In contrast to this finding Andersen & Engberg (1975), using the heat-shocktechnique for synchronizing their cells, demonstrated that nucleolar DNA replicatedat the onset of the macronuclear «S-phase. (2) In Chinese hamster cells results ofexperiments performed by Amaldi, Giacomoni & Zito-Bignami (1969) using thethymidine-block technique for synchronization showed that nucleolar DNA replicatedduring mid 5-phase. In contrast, Stambrook (1974) working on cells synchronized byselecting mitotic cells, showed that nucleolar DNA replicated at an early stage in5-phase. The differences found in each of these cases could result from the use of

Timing of nucleolar DNA replication 529

different synchronization techniques and such a possibility has been suggested byStambrook (1974). Further information about the effect of different synchronizationtechniques on cells is provided by the work of Pica-Mattoccia & Attardi (1972).Investigating the pattern of mitochondrial DNA synthesis in HeLa cells, they foundthat in cells synchronized by selecting the detached mitotic cells, mitochondrial DNAsynthesis started in S-phase and reached a maximum during G2; while in cells syn-chronized by applying the double thymidine block, mitochondrial DNA synthesisoccurred at a constant rate throughout the cell cycle. Mitchison (1971a) in his workon the cell cycle, has emphasized the possibility that induced synchrony may causecell cycle distortions; thus, results obtained using the selective technique should bemore reliable. In our studies cells were synchronized by selecting detached mitoticcells from mass cultures of A. proteus, and should therefore be free of any distortions,which might occur with induced synchronization. Our results are in good agreementwith work done on Physarumpolycephalum (Guttes & Guttes, 1969; Guttes & Telatnyk,1971; Ryser et al. 1973) and with Charret's work on T. pyriformis (1969): two studieswhere cell age was determined without resort to any induced-synchronization methods.

The finding in this work of a fraction of nuclear DNA which replicates outside the5-phase is not consistent with the classic subdivisions of the cell cycle into Glt S, G2,and mitosis introduced by Howard & Pelc (1953). They defined 5-phase as a restrictedperiod in interphase during which the DNA is replicated. We believe that there areno sharp boundaries between 5- and G2-phases in A. proteus and that the syntheticactivities during these periods are continuous. As information accumulates about themetabolism of the cell during interphase, it is clear that the initial very useful sub-divisions proposed by Howard & Pelc (1953) cannot be taken to have rigid boundaries.The dissociation that can occur between various aspects of cell cycle metabolism,particularly as a result of synchronization techniques, is reviewed by Mitchison (1971 b).

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{Received 25 March 1976)