An experimental approach to nucleolar organization in plant cells: a

morphological, cytochemical and quantitative study

FRANCISCO JAVIER MORENO*, ROSA MARIA RODRIGO and GREGORIO GARCIA-HERDUGO

Departamento de Biologfa Celular, Facultad de Biologfa, Universidad de Sevilla, 41012-Sevilla, Spain

•Author for correspondence

Summary

We have used morphological, cytochemical andimmunocytochemical techniques, along with tran-scription inhibitors, to try to understand the nu-cleolar organization in Allium cepa root meristemcells.

Our results reveal that the fibrillar componentshows intense staining after using the NOR-silvertechnique, and gold particle labelling after incu-bation with DNase-gold and RNase-gold com-plexes. These different techniques reveal a reticularstructure of the fibrillar component, supported by acentral axis that is circular in transverse section.These structures are usually considered to be fibril-lar centres, but we have called them clear areas,since we find in their interior neither Ag-NOR

proteins nor labelling after incubation with nu-clease-gold or anti-DNA.

Taking into account the specific localization ofthe Ag-NOR proteins, our cytochemical and quanti-tative results, after applying NOR-silver staining innucleoli of control cells and cells treated withtranscription inhibitors, help us to a better under-standing of the mechanisms of action of thesedrugs. Our results support a structural and func-tional role for the Ag-NOR proteins throughout thecourse of transcription.

Key words: Allium cepa, actinomycin D, anti-DNA,cordycepin, NOR-silver staining, nuclease-gold complex,nucleolus.

Introduction

The structure of the nucleolus is closely associated withthe assembly, processing and maturation of eukaryoticribosomal particles (Busch & Smetana, 1970; De la Torre& Gimenez-Martin, 1982; Fakan, 1986; Fakan & Her-nandez-Verdun, 1986; Goessens, 1984; Risuefio &Medina, 1986). At the ultrastructural level, five differentnucleolar components can be recognized. These are thegranular component, the dense fibrillar component, thefibrillar centres, the vacuoles and the nucleolus-associ-ated chromatin (Goessens, 1984; Jordan, 1984; Risuefio& Medina, 1986).

The nucleolar organizer regions (NORs) are the siteswhere the ribosomal genes are localized, and are associ-ated with a series of specific acidic proteins known asAg-NOR proteins (Fakan & Hernandez-Verdun, 1986;Hernandez-Verdun, 1986).

Biochemical (Herrera & Olson, 1986), cytological(Ochs et al. 1983; Pfeifle et al. 1986) and immunocyto-chemical studies (Christensen et al. 1986; Escande et al.1985; Gas et al. 1985; Spector et al. 1984) suggest thatthe Ag-NOR proteins correspond to highly phosphoryl-ated non-histone proteins and/or to the large RNApolymerase I subunit. Recently, different nucleolar pro-Journal of Cell Science 94, 51-59 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

teins have been assigned to the various nucleolar struc-tures (Ochs et al. 1983; Pfeifle et al. 1986; Reimer et al.1987; Spector et al. 1984; Thiry, 1988; Thiry et al.1988a).

In the interphase nuclei, the NOR-silver stainingdifferentiates the fibrillar centres from the dense fibrillarcomponent, due to their different reaction intensities(Moreno et al. 1985; Pebusque & Seite, 1981; Ploton etal. 1987). These fibrillar nucleolar components containrDNA chromatin in a de-condensed state that allows it tobe kept potentially active from a transcriptional point ofview (Hernandez-Verdun & Derenzini, 1983; Hernan-dez-Verdun et al. 1984; Medina et al. 1986).

By using monoclonal antibodies it has been demon-strated that both RNA polymerase I and DNA areconfined to the fibrillar centre exclusively (Reimer et al.1987; Scheer & Raska, 1987; Thiry etal. 1988a), whereasafter incubation with enzyme-gold complexes RNA hasbeen found over the dense fibrillar component (Thiry,1988). These results suggest that rDNA transcriptiontakes place predominantly in the periphery of the fibrillarcentres, close to the region of the surrounding densefibrillar component.

To gain more insight into this nucleolar structuralorganization by using cytochemical techniques we have

51

studied the drug-induced nucleolar disintegration anddissociation of the nucleolar components by using chemi-cals known to impair RNA synthesis and to inducecharacteristic alterations in the nucleolar architecture(Scheer et al. 1984; Wachtler et al. 1987).

Materials and methods

Material preparation

The material used for this study was the root meristem ofAllium cepa L. Onion bulbs were grown under standardconditions at 25°C (Cortes et al. 1982). Some of them weretreated with 50 \>% ml" cordycepin or 125^gml~ actinomycinD (AMD) during the last 6h prior to fixation (Fan & Penman,1971; Sanchez-Pina et al. 1984).

For the morphological study, roots were fixed in 4 % glutaral-dehyde in 0 1 M-cacodylate buffer, pH7-2, for 3 h at 4CC andpost-fixed in 1 % osmium tetroxide (OSO4) for 2h at 4CC.Samples were dehydrated in acetone at progressively higherconcentrations and embedded in resin following the method ofSpurr (1969).

For the cytochemical study roots were fixed in 1 -6 % glutaral-dehyde in O'l M-cacodylate buffer, p H 7 3 , for lOmin at roomtemperature, followed by a post-fixation in Carnoy's solution(1:3 (v/v), acetic acid-ethanol) for lOmin at 4°C.

For electron microscopic immunolocahzation roots werefixed in a mixture of 4% paraformaldehyde and 0-1 % glutaral-dehyde in 0-1 M-cacodylate buffer, pH7-3, for 60min at 4°C.

After the electron microscopic fixations, samples werewashed in 0 1 M-cacodylate buffer, pH7-3, and incubated in0 5 M-NH4CI for 4 h at room temperature to block free aldehydegroups. They were then washed in the same buffer again.

Lowicryl K4M-embedded sectionsLow temperature embedding with the hydrophilic resin Lowi-cryl K4M (Chemische Werke Lowi) was performed, with slightmodifications, according to the method described by Roth(1983). Samples were dehydrated in a series of graded meth-anols at progressively lower temperatures. During infiltrationwith Lowicryl K4M at -20°C, 90% methanol was used as thedehydration agent. Lowicryl was polymerized by indirect long-wave (360 nm) ultraviolet irradiation from a 15 W Philipsfluorescent lamp.

Ultrathin sections were cut on a Reichert-Jung Ultracut Eultramicrotome, then mounted on 300 mesh gold grids andphotographed in a Philips 300 EM transmission electronmicroscope (Servicio de Microscopfa Electr6nica, University ofSeville).

One-step silver stainingThis technique was applied, following Howell & Black (1980)and Ploton et al. (1982) with the modifications of Moreno et al.(1985). Grids were washed in distilled water for 5min, andtreated for 5 min at room temperature with a mixture containing1 volume of 2 % gelatin in 1 % formic acid and 2 volumes of50% silver nitrate solution (Merck). They were rinsed indistilled water, floated in 5 % thiosulphate solution for 10 minand thoroughly rinsed in distilled water again. In order todemonstrate the specificity of the staining, sections wereincubated for 60 min at 37 °C in one of the following enzymicsolutions: 0-1 % DNase in PBS containing 1 mM-MgCl2; 0 1 %RNase in PBS or 0 1 % Pronase in PBS (phosphate-bufferedsaline).

lmmunocytochemical labellingGrids were incubated by floating them, cell sections down, on a

drop of O - I M - ( P B S ) , pH7-4, containing 2% gelatin and 0-1%BSA (PBG). The next step of the treatment was incubationwith anti-DNA (Boehringer Mannheim) (15 jig ml"1) in PBSfor 2h at room temperature. After a rapid wash in PBG, thesecond incubation was performed with goat anti-mouse IgMcoupled to 10 nm diameter colloidal gold (Janssen Life Sci-ences, Belgium) diluted 1/25 in PBG, for 60 min at roomtemperature. After washing with PBG and PBS, the sectionswere postfixed in 2% glutaraldehyde in PBS. The grids werewashed with distilled water and contrasted with uranyl acetate.To demonstrate the specificity of the labelling, the followingcontrols were performed: (1) incubation with goat anti-mouseIgM-gold; (2) digestion with 0-1 % DNase for 60 min at 37°Cprior to the incubation with anti-DNA.

DNase-goId and RNase-gold complexesPrepared and applied according to Bendayan (1981). Weobtained a gold particle size of 8 nm by following the method ofSlot & Geuze (1985). The specificity of the labelling wasassessed through three types of controls: (1) An enzyme-goldcomplex to which the corresponding substrate has been addedat a concentration of 0-1 % for 10 min prior to the incubation.(2) Incubation with protein A-gold complex. (3) Removing ofthe substrate from the tissue after treatment with the corre-sponding enzyme used at a concentration of 0-1 %, for 60 min at37°C, prior to incubation with the enzyme-gold complex.

Morphometric and stereological analysisThis study was carried out on 40 photographic plates per groupof nucleoli chosen at random, taken from ultrathin silver-stained sections at final magnifications of X12 300. The absoluteparameters measured from microscope sections were the nu-cleolar area, the nucleolar area contrasted with the NOR-silverstaining and the granular component area. The relative para-meters were measured by finding the relationship betweenvacuolar and nucleolar area (Vacuolar Vv) and the silver-labelled area in relation to the nucleolar area (Silver-stained Vv).

Measurements were made with the Automatic Image Ana-lyzer IBAS II (Kontron, FRG) (Department of Cell Biology,Faculty of Sciences, University of Cordoba). The area analysis(Weibel, 1987) was used to evaluate the volume density of thesilver precipitate in the nucleolus in relation to the nucleolarvolume (Silver-stained Vv).

Data were analysed statistically using the following pro-grammes written for the IBAS or IBM computer consoles:nested analysis of variance, Student's f-test or, for non-homogeneous data, the Kolmogorov-Smirnov test and the U-test. A statistical difference was considered when P < 0 - 0 1 .

Results

Nucleolar ultrastructure under control conditionsAt the ultrastructural level, nucleoli of Allium cepa inactively growing conditions are formed by a thread-likeorganized fibrillar component embedded in the granularcomponent. Occasionally, some clear areas can be ob-served inside these structures, consisting of a homo-geneous material and/or dense inclusions. Between thethreads of the fibrillar component the granular com-ponent is distributed, usually showing clear spaces corre-sponding to the nucleolar vacuoles (Fig. 1).

After NOR-silver staining, the silver precipitate waslocalized exclusively over the fibrillar component threads,whereas the clear areas, the granular component andnucleolar vacuoles were devoid of silver staining (Fig. 2).

52 F. J. Moreno et al.

Figs 1—2. Allium cepa root meristem cell nucleoli under control conditions.Fig. 1. Nucleolus formed by a thread-like fibrillar component if) embedded in a granular component (g). Note the clear areas(arrows) and vacuoles (arrowheads). Roots fixed in 4% glutaraldehyde, postfixed in 1 % osmium tetroxide and embedded inSpurr resin. Uranyl acetate and lead citrate staining. X30000.Fig. 2. NOR-silver staining showing a reticular distribution of the silver precipitate localized over the fibrillar component.Clear areas are unlabelled (arrows). Roots fixed in glutaraldehyde-Carnoy's and embedded in Lowicryl K4M. X30000.

Nucleolar organization in plant cells 53

8

After the application of the postembedding immuno-gold labelling method involving the monoclonal anti-DNA, which specifically reacts with DNA in bothdouble- and single-stranded forms, both the fibrillar andgranular components were devoid of gold markers. How-

ever, gold particles were present over the condensedchromatin and inside the clear areas with dense inclusions(Figs 3 and 4).

By using the DNase—gold complex, gold particles weredetected over the nuclear heterochromatic clumps, nu-

§4 F. J. Moreno et al.

Figs 3-9. Nucleoli of Allium cepa root meristem cells grownunder control conditions.Figs 3-4. Roots fixed in 4% paraformaldehyde and 0 1 %glutaraldehyde and embedded in Lowicryl K4M. Uranylacetate counterstaining. Fig. 3. Anti-DNA labelling over theheterochromatic clumps and inside the clear areas withinclusions (arrows). X20000. Fig. 4. Unlabelled clear area(arrow). X66000.Figs 5-9. Roots fixed in glutaraldehyde-Carnoy's andembedded in Lowicryl K4M. Uranyl acetate counterstaining.Figs 5-7. DNase-gold complex labelling. Fig. 5. Goldparticles are preferentially located over the fibrillarcomponent. Nuclear heterochromatic clumps are highlylabelled. X20000. Fig. 6. Unlabelled clear areas (arrow).X57 000. Fig. 7. Intense labelling over the inclusions incertain clear areas (arrow). X92000. Figs 8-9. RNase-goldcomplex labelling. Fig. 8. Gold particles are distributedthroughout the fibrillar and granular components. X20000.Fig. 9. Unlabelled clear areas (arrows). X46000.

cleolus-associated chromatin, nucleolar fibrillar threads,vacuoles, over the edges of the clear nucleolar areas andover the inclusions observed inside some of them (Figs5-7).

Following incubation with the RNase-gold complex,labelling was seen preferentially located over the nuclearheterochromatin periphery. Over the nucleolus intenselabelling was found homogeneously distributed through-out the fibrillar and granular components, whereas label-ling was scarce over the intranucleolar chromatin. We didnot observe gold particles inside the clear areas withhomogeneous content (Figs 8 and 9). Cytochemical andimmunocytochemical controls did not show specificstaining or labelling over the different nucleolar com-ponents.

Nucleolar ultrastructure under the effect of inhibitorsNucleoli of cells treated with cordycepin and AMD for6h are constituted mainly of the fibrillar component.Under treatment with cordycepin large vacuoles arefrequently detected with perichromatinic granules intheir interior. These granules can also be detected at thenucleolar periphery (Figs 10 and 11).

After treatment with cordycepin, NOR-silver stainingwas found distributed all over the nucleolus, whereas inAMD-treated cells a peripheral zone devoid of silverstaining was detected. Vacuoles and perichromatinicgranules did not show a silver precipitate (Figs 12 and13).

After incubation with the DNase-gold complex in-tense labelling was found over the nuclear heterochroma-tin, whereas over the nucleolus labelling was moderate.Some particles were localized inside certain vacuoles(Fig. 14A and B).

Labelling with the RNase-gold complex was moreintense over the nucleolus than over the nuclear hetero-chromatin and inside the vacuoles (Fig. 15).

After the postembedding immunolabelling techniqueusing anti-DNA antibody on cells treated with bothcordycepin and AMD, nucleoli were free of gold par-ticles. In contrast, the nuclear heterochromatic clumpswere strongly labelled (Figs 16 and 17).

Table 1. Morphometric values obtained from electronmicroscopy

Nucleolar areaTreatment

Ag-stained area(Mm2)

Granular area

ControlCordycepinAMD

9-21 ±06-24 ± 06-73 ±0

•44*•22f•41*

120

•31 ±0-116•18±0-126f•81 ± 0-121t

7-77 ± 03-73 ± 05-86±0

•366•152t•327f±

•Mean ± S.E.M.•)•/>< 0-01 compared with control group.

compared with cordycepin group.

Table 2. Stereological values obtained from electronmicroscopy

Treatment Vacuolar Vv Silver-stained W

ControlCordycepinAMD

0-0125 ± 0-0014*0-0505 ±0-0126f0-0093 ± 0-0029-t

0-1411 ±0-0090-3492 ±0-013t01127 ±0-013±

• Mean ± S.E.M.fP<0-01 compared with control group.

compared with cordycepin group.

The quantitative analysis at the ultrastructural leveldemonstrated that the nucleolar area of cells treated withcordycepin and AMD decreased when compared withthat of the control (Table 1). After analysis by theAg-NOR technique it was found that the silver-stainedVvand area, and nucleolar vacuolar Vv, were statisticallyincreased in cells treated with cordycepin. After treat-ment with AMD only the silver-stained area was de-creased in relation to that found in control cells (Tables 1and 2).

Discussion

The direct relationship between transcriptional activityand nucleolar size has been demonstrated widely (De laTorre & Gimenez-Martin, 1982; Deltour, 1985; Deltour& De Barsy, 1985; Goessens, 1984; Hernandez-Verdun,1986; Mirre & Knibiehler, 1984; Moreno et al. 1989;Risuefio & Medina, 1986). Our data confirm that thisactivity was provoked in this case in the nucleoli oi Alliumcepa root meristem cells by the assimilation of certaindrugs known to inhibit rRNA synthesis.

We have observed a specific silver precipitate over thefibrillar component after using NOR-silver staining. Asthese Ag-NOR proteins are localized over the rDNA andRNP chains in transcription (Hernandez-Verdun, 1986;Medina et al. 1986; Risuefio & Medina, 1986), thisreticular appearance could correspond to a section of thearborescent nucleolonema described by Deltour & Mosen(1987). This nucleolonema consists of a central axisformed by fibrillar centres surrounded by dense fibrillarcomponent. The absence of silver staining over the clearareas in our experiments supports the hypothesis pro-posed by Motte et al. (1988) concerning the differencesbetween the animal and plant cell molecular compositions

Nucleolar organization in plant cells 55

of these structures, which are considered as fibrillarcentres by several authors. The absence of gold particles,after incubation with anti-DNA and DNase-gold com-plex, over the clear areas with an homogeneous contentconstitutes another argument to support the hypothesis

of the different molecular organization and/or compo-sition of these structures in plant cells.

Labelling after incubation with anti-DNA was foundover the heterochromatic clumps. The nucleolus usuallyappeared as a zone of exclusion of gold particles, except

56 F. J. Moreno et al.

Figs 10-15. Nucleoli of Allium cepa root meristem cellstreated with rRNA synthesis inhibitors.Figs 10-11. Roots fixed in 4% glutaraldehyde, postfixed in1 % osmium tetroxide and embedded in Spurr resin. Uranylacetate and lead citrate counterstaining. Fig. 10. Nucleolus ofa cell treated with cordycepin mainly comprising fibrillarcomponent. Some vacuoles can be observed showingperichromatinic granules in their interior (arrow). X29000.Fig. 11. Nucleolus of a cell treated with AMD principallyformed of fibrillar component. X32 000.Figs 12-15. Roots fixed in glutaraldehyde-Carnoy andembedded in Lowicryl K4M. Figs 12-13. NOR-silverstaining. Fig. 12. After treatment with cordycepin, silverprecipitate is seen distributed throughout the nucleolus.Vacuoles are free of staining. X17 000. Fig. 13. Aftertreatment with AMD the periphery of the nucleolus shows nosilver staining. Vacuoles are free of staining. X17 000.Figs 14-15. Treatment with cordycepin. Nuclease-goldcomplex labelling. Uranyl acetate counterstaining.Fig. 14. A. DNase-gold labelling distributed over thenucleolus. Nuclear heterochromatin shows an intenselabelling. X19000. B. Higher-magnification detail of certainvacuoles showing some gold particles. X31 000. Fig. 15. Anintense RNase-gold labelling detected over the nucleolus.Inside the vacuoles and over the nuclear heterochromatinlabelling is moderate. X17 500.

for small 'islands' of gold grain clusters within thenucleolar body that can represent invaginations of thenucleolus-associated chromatin and that correspond tothe clear areas with inclusions. These results are inagreement with these obtained by Scheer et al. (1987)after labelling with the DNA antibody AK 30-10.

These results, obtained after the application of thepostembedding immunogold labelling method, led us tobelieve that this monoclonal antibody is preferentiallybound to zones of the chromatin with a certain degree ofcondensation, being, therefore, nucleolar chromatinwithout labelling, as it is kept in a de-condensed state toallow the transcriptional processes to occur (Fakan, 1986;Hernandez-Verdun, 1986; Medina, 1988). Moreover,concentrations of rDNA existing at the exposed surfaceof the thin sections can be below the limit of detection ofthis method. In the same way, Scheer et al. (1987)established that these are the limiting factors in theidentification of DNA by means of this type of antibody.

Using treatment with transcription inhibitors, ourresults with the immunocytochemical method are inaccord with those obtained by Thiry et al. (19886), asthey did not find any labelling over the nucleolus. Theseauthors explain the absence of nucleolar labelling aftertreatment with AMD as caused by inactivation of rRNAgene transcriptional activity. However, we believe thatthis problem is still unresolved, because in that study theauthors found labelling, during interphase, over othernuclear areas where the condensation and inactivation ofthe chromatin is obvious.

Our results obtained after labelling with the DNase-gold and RNase-gold complexes show that DNA andRNA chains in transcription are preferentially locatedover the nucleolonema threads and inside the vacuoles

Figs 16-17. Roots fixed in 4% paraformaldehyde and 0-1 %glutaraldehyde and embedded in Lowicryl K4M. Anti-DNAlabelling. Uranyl acetate counterstaining. Unlabellednucleolus under treatment with cordycepin (Fig. 16;X22500) and AMD (Fig. 17; X22500).

containing dense inclusions; the gold labelling corre-sponding to condensed nuclear chromatin in this ca9e.

According to the model proposed by Deltour & Mosen

Nucleolar organization in plant cells 57

(1987), after treatment with cordycepin and AMD,shrinkage or loss of the proteinaceous central axis wouldoccur, the remaining rDNA helix around a central axis istoo thin to be observed under the electron microscope(Deltour & Mosen, 1987). Without this proteinaceousstructure, the rDNA would form fibrillar filamentsembedded in a mass composed mainly of Ag-NORproteins and RNA, and therefore the control nucleolarstructural organization would be lost.

After treatment with cordycepin, inside the nucleolarvacuoles we have found some particles that could,because of their size and morphology, correspond toribosomal precursors (Fakan, 1986; Greimers & Deltour,1984). These vacuoles would have as their main functionsthe storage of nucleolar products and/or the facilitationof their transport to the outside, by providing an increasein exchange surface (Chouinard, 1982; Deltour, 1985;Deltour & De Barsy, 1985; Deltour et al. 1986; Goes-sens, 1984; Hernandez-Verdun, 1986; Mirre & Kni-biehler, 1984; Risueno & Medina, 1986). The effects ofcordycepin, together with the impairment of 45 S rRNAand the increased degradation of pre-rRNA (Fan &Penman, 1971; Lill et al. 1969) would lead to a progress-ive loss of the granular component.

Taking into account that, after using the adenosineanalogue DRB, other workers have noted labelling overrDNA after applying anti-RNA polymerase I antibodies(Scheer et al. 1984), we suggest that cordycepin wouldact in a similar way to permit the synthesis of rRNAchains smaller than 45 S. These chains would then bereleased and integrated into the nucleolar structure.

Our results lead us to suggest that the Ag-NORproteins remain joined to the rDNA and unfinishedrRNA chains, whether released or not, forming a mor-phologically homogeneous fibrillar mass. This suggestionis supported by the increased silver-stained area aftertreatment with cordycepin as compared to control nu-cleoli. However, in AMD-treated nucleoli, we havefound decreased staining of the Ag-NOR proteins. Thisresult could be explained by the studies of several authors(Busch et al. 1987; Reimer et al. 1987; Thiry et al.1988a), who, after using immunocytochemical tech-niques, have demonstrated that rDNA transcription isinhibited after treatment with AMD, which causes theblocking of RNA polymerase I (Miller, 1987). Theseresults again corroborate the functional role performedby the Ag-NOR proteins in the rDNA transcriptionprocesses, apart from their role in maintaining thestructure of rDNA and its transcriptional products.

Our results, obtained after applying different morpho-logical, cytochemical and immunocytochemical tech-niques, support the model for nucleolar organization inplant cells proposed by Deltour & Mosen (1987). Thismodel proposes that there exists a large loop of activerDNA belonging to the NOR-bearing chromosome. ThisrDNA, which can be labelled with the DNase-goldcomplex, forms a double helix around a central axiscontaining scaffolding proteins that are not NOR-silver-stained. Each transcriptional unit of active rDNA,together with the RNA chains undergoing transcription,labelled with the DNase-gold and RNase-gold com-

plexes, would be located around the central protein axis.The clear areas with dense inclusions labelled with bothanti-DNA and the DNase-gold complex could corre-spond to the described heterogeneous fibrillar centresthat contain inclusions of dense non-transcribing nu-cleolar chromatin inserted from place to place within thenucleolonemal axis (Deltour & Mosen, 1987; Risuefio &Medina, 1986). This macromolecular structure is consist-ent with an arborescent or reticulate nucleolonema struc-ture, formed by fibrillar component, where it is notpossible to distinguish fibrillar centres constituted ofrDNA, pre-rRNA and Ag-NOR proteins, embedded ina thick layer of the granular component.

We are most grateful to Dr R. Deltour for critical reading ofthe manuscript, Dr F. Gracia-Navarro for his help in themorphometric and stereological analysis, and R. Garcfa-Navarro and J. M. Sanabria for their technical assistance. Thiswork was partially supported by the DGICYT (project no.666/86) and the Junta de Andalucfa, Consejerfa de Educaci6n yCiencia (project no. 5404/87).

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(Received 14 February I9S9 -Accepted, in revised form, 31 Mav1989)

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