Journal of Cell Science 102, 71-78 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

71

PCNA and total nuclear protein content as markers of cell proliferation in

pea tissue

SANDRA CITTERIO, SERGIO SGORBATI, MARISA LEVI, BRUNO MARIA COLOMBO

and ELIO SPARVOLI

Dipartimcnto di Biologia, Sczione di Botanica Generate, Centro del C.N.R. per lo studio delta Biologia Cellulare e Molecolare delle Piante,via Celoria 26, 20100 Milano, Italy

Summary

The identification of cell proliferation markers has beenshown to be a useful tool with which to study basicmechanisms of cell cycle progression. The use ofimmunofluorescence techniques revealed the presence ofthe proliferating cell nuclear antigen (PCNA) in peatissue, where we observed a high PCNA expression inproliferating cells of the root meristem compared to non-cycling cells of the differentiated leaf. The presence ofPCNA was monitored also during the time-course ofseed germination, before, during and after the cell cycleresumption of the embryo cells. PCNA is present inembryo cells not only during and after resumption of the

cell cycle but also before, when cells have not yet begunreplicating their genome. A bivariate flow cytometricanalysis of DNA and nuclear protein content was used tolocalize precisely the cells of the examined pea tissues indifferent cell cycle phase subcompartments. A highcorrelation was found between the degree of cellproliferation and the protein content of Gj nuclei, on theone hand, and the percentage of PCNA positive cells onthe other.

Key words: PCNA, DNA/nuclear proteins, flow cytometry.

Introduction

There is increasing interest in studies on the regulatorymechanisms of the cell cycle. The ability of cells tobegin a new cell cycle is a prerequisite for cellreproduction (Pardee, 1989).

To study the transition of cells from the quiescent(Go) to the proliferating (Gi) condition and vice versa,it is necessary to know the kinetics and the molecularcharacteristics of the cell population in question and tohave appropriate markers for distinguishing betweencycling and non-cycling cells (Hall and Woods, 1990).The combination of flow cytometry and immunohisto-chemical techniques provides a powerful tool in thisregard. A bivariate cytofluorimetric analysis of cellkinetics, using methods permitting the simultaneousquantification of DNA and cellular or nuclear RNA orproteins has led to the identification in animal and inplant cells of well-defined cell cycle subcompartments:GIA, GIB, early S, late S, G2A and G2B (Bergounioux etal. 1988; Sgorbati et al. 1988; Darzynkiewicz et al.1990b). In a few instances, 2C cells showing a low RNAor protein content can be considered Go cells; that is,cells out of cycle (Darzynkiewicz et al. 1980a). Inparticular, the nuclear protein content, determined bybivariate flow cytometry, can be used as a marker fordistinguishing cycling from non-cycling cells (Pollack etal. 1984; Sgorbati et al. 1989).

Another approach serving to distinguish betweencycling and non-cycling cells is to reveal, by immuno-cytochemical techniques, the presence of specificproteins related to the proliferating or quiescent cellcondition. Changes in a wide range of nuclear com-ponents in association with the cell division cycle havebeen described in animal cells (Hall and Woods, 1990).Immunofluorescence and biochemical studies of cul-tured animal cells have suggested that an antigenassociated with cell proliferation is a 36 kDa proteinnamed Proliferating Cell Nuclear Antigen (PCNA;Bravo and Celis, 1980; Bravo et al. 1982; Takasaki et al.1984). It has been identified also as a DNA polymerase6 auxiliary protein (Fairman, 1990) and plays anessential role in eukaryote DNA replication (Bravo,1986; Zuber et al. 1989). PCNA is well conservedduring evolution with high homology between mam-malian PCNA and yeast. Recent data from Suzuka etal. (1989) have demonstrated the presence of the PCNAgene also in several plants such as rice, soybean andtobacco. Furthermore, PCNA may be involved in theDNA repair process, since redistribution of PCNA tosites of DNA damage after UV irradiation has beenreported in non-S phase cells (Celis and Madsen, 1986;Toschi and Bravo, 1988).

In this study we first set up a method for stainingpurified nuclei for immunofluorescence in order toverify the presence of PCNA or a homolog protein in

72 S. Citterio and others

pea tissue. We then investigated the possibility of usingPCNA as a specific marker of cell proliferation,verifying the presence and distribution of the antigen:(1) in active root meristem and differentiated leaf, inwhich cells are cycling and not cycling, respectively;(2) during seed germination, when cells progressivelyresume the cell cycle, passing from quiescence to theproliferating condition. In the same tissue, PCNAexpression was correlated with total nuclear proteincontent, a parameter that defines the cell cycle positionwhen determined by bivariate flow cytometry.

Materials and methods

Plant material: growth conditions and samplingSeeds of Pisum sativum L. cv. Lincoln (Ingegnoli, Milan,Italy) were surface-sterilized with sodium hypoclorite (0.6%active chlorine) for 10 min, washed with running tap water for15 min and left to imbibe in water for 24 h; seeds were thengrown in Agriperlite (VIC, Italy) at 25(±1)°C in the dark.Root tips were collected after different times from thebeginning of imbibition. Leaves were cut from 15-day-oldplantlets. Fifty 0-2 mm root tips, and 100 mg of leaves wereused as samples.

Nuclear isolation and purificationSamples were fixed in 4% (w/v) paraformaldehyde (Poly-sciences, 10% solution, methanol-free) in Tris buffer (10 mMTris(hydroxy-methyl)aminomethane, 10 mM NaEDTA and100 mM NaCl, pH 7.4) for 30 min on ice and then washedthoroughly in the cold buffer for 10 min. Nuclei were isolatedfrom fixed pea roots or leaves in 2 ml of buffer according to aprevious protocol (Sgorbati et al. 1989). Briefly, samples werecrushed with a glass rod in Petri dishes on ice; nuclearsuspensions were filtered twice through 2 nylon meshes of 60and 15 fim pore size to remove gross particles. In order toremove small debris 2 ml of nuclear suspensions werecentrifugated on a 2 ml cushion of 2 M sucrose at 1000 g for 10min. The pellet of purified nuclei was resuspended in 1 ml ofcold buffer. Controls by microscopy and by flow cytometryshowed that nuclei were well preserved and that theirdistribution in the cell cycle phases was not affected bycentrifugation (data not shown).

Immunofluorescence analysisThe purified suspensions of nuclei obtained from the differentsamples were spread on slides (previously soaked in a 1%(w/v) condensed milk solution to reduce background fluor-escence) and air dried.

Slides were rehydrated in Tris buffer and incubatedovernight with diluted (1:100) anti-PCNA monoclonal anti-

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Fig. 1. Bivariate DNA/total protein flow cytogram of nuclei released from pea root meristem after 72 h from the beginningof seed imbibition (A). The distribution of nuclei released from wheat embryos (biological internal standard, W) is shownon the right of the cytogram. DNA and nuclear proteins were stained with DAPI and SR101, respectively. Cycling cells aredistributed in Gi, S and G2 cell cycle phases. The percentages of cells in the different cell cycle phases and the ratiobetween nuclear protein fluorescence of Gi nuclei and that of the internal standard are reported in Table 1. The samenuclei were stained for PCNA detection (C) and counterstained for DNA with DAPI (B). About 95% of the cells arePCNA positive (Table 1). The antigen is distributed all over the nucleus; in some cases it also appears to be associatedwith the nucleolus (arrowhead) or forms a ring round it.

Cell proliferation markers in pea 73

body (clone PC10, Novocastra, UK; Waseem and Lane, 1990)in a humidified chamber at 4°C. After 3 washes (5 min each) inTris buffer, slides were incubated for 30 min at roomtemperature with diluted (1:200) sheep anti-mouse Ig-biotin,F(ab')2 fragment antibody (Boehringer), washed 3 times inTris buffer, incubated for an additional 15 min with diluted(1:400) Texas Red/streptavidin (Amersham International)

and washed again 3 times in Tris. Slides were counterstainedwith 0.05 ^g/ml (0.14 iM in Tris) of the DNA-specificfluorochrome DAPI (4',6-diamidino-2-phenylindole, Sigma)and mounted in 90% glycerol in Tris, examined andphotographed with a Zeiss Axioplan microscope, usingKodak Ektachrome 100 film. A negative sample (withoutmAb) was included in each experiment. Slides were observed

DAPI (DNA)

Fig. 2. Bivariate DNA/total proteins cytogram of nuclei released from differentiated leaves (A). Non-cycling cells aredistributed in Gi (Go) and in G2 cell cycle phases; the mean nuclear protein fluorescence of Go nuclei with respect to thatof the standard (W) is markedly lower than that of root meristem proliferating cells (Table 1). No cells appear to have theminimum threshold level of antigen that we adopted to distinguish between PCNA negative and positive cells (C). Cellswere counterstained for DNA with DAPI (B).

Table 1. Flow cytometric and immunocytochemical analysis of nuclei released from pea root during seedgermination (6-72 h) or from differentiated tissue (leaves)

Time afterimbibition (h)

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PCNApositive cells (%)

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7575757569393597

112338

252525252038273

0.470,480.470.450.480.510.570.39

60352038547595

The percentage of cells in the different cell cycle phases and the ratio between protein mean fluorescence of Gj nuclei of differentsamples and that of the internal standard were calculated from cytograms of Figs 1, 2 and 3. The percentage of PCNA positive nuclei persample is also reported. For each sample 4 slides were scored and the percentage was determined for 1000 nuclei per slide.

74 S. Citterio and others

DAPI (DNA)

by a Plan Neo-FLUAR xlOO immersion objective equippedwith an iris diaphragm. Nuclei were considered PCNApositive when visible to the eye with the diaphragm adjustedat the minimal aperture. The percentages of PCNA positivecells were determined on 1000 nuclei from 4 different slides.

Bivariate DNA/nuclear proteins flow cytometricanalysisAn internal biological standard is necessary to controlinstrumental and staining variation when comparing measure-ments referring to different samples. 2C nuclei released from

Cell proliferation markers in pea 75

Fig. 3. Bivariate cytograms of nuclei released fromquiescent embryo root after 6 (A) and 15 h (D) from thebeginning of seed imbibition. Cells are arrested in Gi andG2. Gi arrested cells (Go) have a lower mean proteincontent than the 2C proliferating cells of 72 h rootmeristem (Table 1). Nuclei of the same two samples werestained for PCNA detection (C and F, respectively) andcounterstained for DNA with DAPI (B and E). In the 6 hsample 60% of the nuclei show PCNA labelling above theminimum threshold level that we adopted to distinguishbetween PCNA positive and negative cells. The antigen isdistributed all over the nucleus and in some cases thenucleoli are not labelled (C). Passing from 6 h to 15 h thepercentage of PCNA positive nuclei drops (35%). Theantigen distribution is always all over the nucleus (F). Thearrowhead shows a PCNA negative nucleus.

quiescent embryos of Triticum aestivum L. were added toeach pea root or leaf sample in 1:1 (v/v) proportions beforestaining (Sgorbati et al. 1989). Nuclei were stained with twofluorochromes: DAPI to quantify the DNA content andSR101 (Sulforhodamine 101, Sigma) to measure nuclearprotein content, at final concentrations of 5.6 /iM and 50 M,respectively. The fluorescence intensity of nuclei wasmeasured with a Partec Pas II flow cytometer (Partec AB,Miinster, FRG), equipped with a HBO 100 mercury arc lampand a x40, 0.8 NA objective. An optical configuration for UVexcitation and simultaneous measurement of blue (DAPI)and red (SR101) fluorescence was used. KGi, BG38 (heatprotection), UG1 filters and dichroic mirror TK 420 were usedfor UV excitation; dichroic mirror TK 560 and barrier filtersRG 630 (red) and GG 435 (blue) were used to measurefluorescence emission. At least 20,000 nuclei were measuredfor each cytogram. Experiments were repeated at least 3 timeswith similar results. The Figures and Table report results ofone representative experiment.

Results

Meristematic and differentiated cellsThe bivariate cytograms of nuclei released from activelyproliferating cells of the root meristem and fromdifferentiated (Go) cells of pea leaves are reported inFigs 1A and 2A, respectively. Isolated nuclei werestained with DAPI to quantify DNA content and withSR101 to quantify protein content. The right side ofeach cytogram shows the DAPI/SR101 fluorescencedistribution of 2C nuclei of wheat embryos, which weused as an internal standard for the comparison ofprotein values from sample to sample. The percentagesof pea cells present in the different cell cycle compart-ments are presented in Table 1. The ratio between themean SR101 fluorescence of 2C pea nuclei releasedfrom root meristem and the mean fluorescence of theinternal standard (0.57) is greater than the same ratio(0.39) in the case of nuclei of differentiated cells(Table 1).

In Fig. 1C and 2C the nuclei released from the samesamples show the staining of a PCNA-like protein,detected by monoclonal antibodies (clone PC10).Nearly all the nuclei of meristematic cells are stained(Fig. 1C): the antigen is mostly distributed throughout

the nucleus and in some nuclei it also forms a ring roundthe nucleolus or is preferentially associated with it. Onthe contrary, in nuclei of non-cycling cells released fromdifferentiated leaves the PCNA-like protein, thoughdetectable, did not reach the threshold level that weadopted to distinguish between PCNA positive andnegative cells (Fig. 2C). These results regarding thegreater presence of PCNA-like protein in proliferatingcells are similar to those reported for cultured animalcells (Connolly et al. 1988).

Cell cycle resumption during germinationA correlated DNA/nuclear proteins and PCNA analysiswas carried out also during the time-course of pea seedgermination. Fig. 3, Fig. 4 and Fig. 5 show the resultsobtained by cytometric and immunofluorescence analy-sis of nuclei isolated from pea root apices at differenttimes during germination. It can be observed that, at 6,8, 15 h and until 30 h from the beginning of imbibition,cells have not yet begun replicating their genome (nocells are observed in S phase; Fig. 3A,D, Fig. 4A andTable 1). At the same times, in comparison with thestandard, the mean protein fluorescence of 2C cells islower than that found in the corresponding cellpopulations at 40, 48 and 72 h when cells areprogressively beginning DNA synthesis (Fig. 4D andFig. 5A) but greater than in differentiated (Go) cells(Fig. 2A, Table 1). With regard to the PCNA staining at6 h, 60% of the nuclei are stained and the PCNAfluorescence in these nuclei is often greater then inproliferating cells (Fig. 3C). PCNA is distributedthroughout the nucleus. The percentage of labellednuclei decreases up to 15 h (Table 1); after 30 h and upto 72 h the percentage of positive nuclei increases inparallel with the increase in the percentage of cells in Sphase and in the mean value of nuclear proteinfluorescence detected by flow cytometry (Table 1).There is also a change in the staining pattern of PCNAduring the time-course of germination. Up to 30 h theantigen is visible in all nuclei as points scatteredthroughout the nucleus (in some cases the nucleolus isnot labelled; Fig. 3C,F and Fig. 4C). At 40 h somenuclei show the antigen staining not only as points in thenucleoplasm, but also as a ring of fluorescence aroundthe nucleolus or as a strong fluorescence associated withthe nucleolus (Fig. 4F). The number of nuclei showingthese staining patterns increases with the percentage ofcells in S-phase, i.e. from 40 to 72 h from the beginningof seed imbibition (Fig. 4F, Fig. 5C and Table 1).

Discussion

The presence and the important role of PCNA in DNAsynthesis has been indicated in several animal cell types(Madsen and Celis, 1985; Bravo, 1986; Bauer andBurgers, 1988) and recently also in plants (Suzuka et al.1989). Our work shows that also in pea tissue a highlevel of PCNA is detected by immunofluorescence inproliferating cells of the root meristem, whereas nucleiof differentiated leaf cells appear to be very weakly

76 5. Citterio and others

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Fig. 4. Bivariate cytograjris of nuclei released from plantlet root meristem after 30 h (A) and 40 h (D) from the beginningof seed imbibition. At 30 h cells have not yet begun to replicate their DNA, whereas at 40 h cells start to enter the Sphase after the resumption of their first cell cycle. While the mean nuclear protein content does not increase significantlyfrom 30 h to 40 h, the percentage of PCNA positive nuclei increases considerably from 38% to 54% (Table 1). Nuclei ofthe same two samples were stained for PCNA detection (C and F, respectively) and counterstained for DNA with DAPI(B and E). At 30 h the antigen is distributed throughout the nucleus (with or without labelled nucleoli (C)), whereas at40 h (F) some nuclei also show a ring round the nucleolus (arrow).

Cell proliferation markers in pea 77

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DAPI (DNA)Fig. 5. Bivariate cytogram of nuclei released from root meristem after 48 h from the beginning of seed imbibition (A). Mostcells are proliferating and distributed during G1; S and G2 cell cycle phases. At this time after imbibition cells have a highermean nuclear protein content and most of them (75%) are PCNA positive (Table 1). C shows the antigen distribution throughoutthe nucleus and partially associated with the nucleolus. B shows the same nucleus counterstained for DNA with DAPI.

stained (Fig. 1 and Fig. 2). Furthermore, differentiatednuclei released from differentiating root segmentsappear weakly stained (data not shown). These immu-nohistochemical results have been confirmed in ourlaboratory by biochemical investigations demonstratingthat a 34-35 kDa protein is recognized in pea prolifer-ating tissue extract by the same commercial monoclonalantibody (clone PC10) as we used for immunofluor-escence (unpublished data).

Starting at 40 h from the beginning of seed imbi-bition, there is a parallel increase in the percentage ofPCNA positive cells and cells observed in S phase byflow cytometry (Table 1). When the first cells begin toreplicate their DNA (Fig. 4, Fig. 5) the PCNAdistributions appear to be similar to those described byCelis and Celis (1985) as nuclear staining patternscharacteristic of the S phase in animal cells. Thepercentage of these distributions increases with theincreasing number of cells in S phase. In animal cellsPCNA has been demonstrated biochemically to bepresent at a low level in quiescent cells and at higherlevels in all cell cycle phases of proliferating cells, with asynthesis peak in early S phase (Morris and Mathews,1989). However, the percentage of PCNA positive cellsdetectable by immunofluorescence, for a given PCNAantibody, is largely due to methods of cell fixation; theuse of methanol and detergents is responsible for therelease of a PCNA fraction not closely linked to DNA

replication sites (Bravo and Macdonald-Bravo, 1987;Landberg et al. 1990).

Our results demonstrate by immunofluorescence thatin actively proliferating cells of the root meristem (Fig.1) 95% of nuclei are PCNA positive (Table 1). Thesefindings indicate that our method of fixing, isolating andpurifying nuclei, and the use of the clone PC10 mAbseems to allow good preservation of the immunofluor-escence staining of the PCNA-like protein present inpea tissue. This means that, in our material, it would bepossible to use PCNA staining as a proliferation markerto identify the cells that, more or less competent toprogress through cell cycle, show a correspondingmajor or minor PCNA staining level. In fact, in the rootmeristem of 72 h plantlets, most but not all cells areproliferating; this Go cell fraction has been recentlydistinguished from Gi proliferating cells after aphidi-colin block and synchronization (Sgorbati et al. 1991,and unpublished data). Work is in progress to verify thepresence and the level of PCNA staining in this Gofraction after its physical separation from Gi prolifer-ating cells by sorting.

An alternative approach to distinguishing betweenGo and Gi cells in the root meristem could be thequantification of PCNA staining by means of a flowcytometric bivariate DNA/PCNA analysis of the cellcycle, similar to the recently method performed onhuman hematopoietic cell lines (Landberg et al. 1990).

78 5. Citterio and others

With regard to time-course of seed germination bothPCNA positive nuclei and the total nuclear proteinfluorescence increase between 15 and 72 h from thebeginning of seed imbibition (Table 1). However, in thisperiod of time the increase in the percentage of stainednuclei (from 20 to 95%) is much greater and precedesthe increase (from 0.47 to 0.57) in nuclear proteinfluorescence. Therefore, PCNA staining could beregarded as a more accurate marker of proliferative cellconditions in the root meristem than the total nuclearprotein content; besides, it allows the identification ofthose d cells that, before entering the S phase, becomecompetent to proliferate.

Our results also show that at 6 h from the beginningof seed imbibition, when all cells are non-cycling, thepercentage of stained nuclei (60%) is much higher thanexpected and decreases up to 15 h (Table 1). This highpercentage of positive nuclei (most of which arestrongly stained), during the first hours of germination,suggests that the protein could be present before seeddehydration and be conserved during quiescence;afterwards and up to 15 h, the PCNA-like protein couldbe, at least partially, destroyed or undetectable, untilthe cells approach the resumption of their first cellcycle. A tentative explanation of this high level ofPCNA staining at 6 h takes into account the possibilitythat PCNA could be involved not only in DNAreplication, but also in DNA repair (Toschi and Bravo,1988) and/or in DNA topological manipulation associ-ated with the DNA topoisomerase I enzyme. Thequiescence of pea embryo cells arrested in Gi and G2during seed dehydration is very different from that ofdifferentiated leaf cells or that of animal cell culture. Asa consequence of dehydration, DNA could havesuffered damage and/or assumed a specific confor-mation; PCNA, during the first hours of seed imbi-bition, could be involved in DNA repair and/or DNAre-arrangement activities. This hypothesis is supportedby our preliminary data demonstrating the presence ofDNA topoisomerase I in 6 h pea tissue.

The authors are grateful to Drs Elena Cappellini and PaolaBrusa for technical assistance and to Antonio Grippo forphotographic printing. This research was supported by ItalianMURST and the National Research Council of Italy, SpecialProject RAISA, Subproject no. 2. Paper no. 337.

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(Received 13 November 1991 • Accepted 28 January 1992)