INTRODUCTION

The nucleolus is a widely studied nuclear structure in whichribosomal RNA synthesis, as well as assembly of preribo-somes, takes place (Hadjiolov, 1985). Under the electronmicroscope, three main domains are visible in this struc-ture: the dense fibrillar component (DFC), the granularcomponent (GC) and the fibrillar centres (FCs). Differenttypes of vacuoles and nucleolar interstices have also beenfound in both plant and animal cells, whereas peri- andintranucleolar chromatin are characteristic of animal cells(Goessens, 1984; Puvion and Moyne, 1980; Raska andDundr, 1993; Raska et al., 1992; Thiry et al., 1991); con-densed chromatin is not found inside the plant nucleolusexcept in the heterogeneous fibrillar centres (Risueño, 1993;Risueño and Medina, 1986; Risueño et al. 1982).

Various cytochemical techniques have been used tolocalize ribosomal genes, their products, and the proteinsinvolved in ribosomal transcription and RNA processing

within these components (see reviews by Deltour andMosen, 1987; Deltour and Motte, 1990; Derenzini et al.,1987, 1990; Derenzini and Ploton, 1991; Goessens, 1984;Hadjiolov, 1985; Hernandez-Verdun, 1991; Jordan, 1991;Raska et al., 1990; Risueño and Medina, 1986; Scheer andBenavente, 1990; Schwarzacher and Wachtler, 1991; Stahlet al., 1991). High-resolution autoradiography after shortpulses with tritiated uridine, together with several cyto-chemical methods for detecting DNA, histones and nucle-olar proteins, indicated that the FCs contained inactive ribo-somal genes and that the surrounding DFC was the site ofrRNA synthesis (reviewed by Goessens, 1984; Medina etal., 1983; Risueño and Medina, 1986; Risueño et al., 1982).

Nevertheless, in the last few years, there has been noagreement on the site of transcription in the fibrillar com-ponents. Immunocytochemical studies have demonstratedthe presence of proteins that play a key role in ribosomaltranscription, such as RNA polymerase I, DNA topoiso-merase I and upstream binding factors (UBF), on FCs

1333Journal of Cell Science 106, 1333-1346 (1993)Printed in Great Britain © The Company of Biologists Limited 1993

The distribution of ribosomal transcripts in the plantnucleolus has been studied by non-isotopic in situhybridization in ultrathin Lowicryl K4M sections andby high-resolution autoradiography after labelling withtritiated uridine. In parallel, cytochemical techniqueswere applied to localize RNA on different plant nucle-olar components of Allium cepa L. root meristematiccells and Capsicum annuum L. pollen grains.

For RNA/RNA in situ hybridization, several biotiny-lated single-stranded ribosomal RNA probes were usedfor mapping different fragments of the 18 S and the 25S rRNA gene transcribed regions. Ribosomal RNAs(from pre-rRNAs to mature 18 and 25 S RNAs) werefound in the nucleolus, in the dense fibrillar (DFC) andgranular components (GC). Hybridization signal wasfound at the periphery of some fibrillar centres (FCs)with probes recognizing both 18 and 25 S rRNA

sequences. A quantitative study was performed toanalyze the significance of this labelling.

Incorporation of tritiated uridine into roots was car-ried out and, later, after a long time-exposure, autora-diography revealed the presence of newly synthesizedRNA mainly in the DFC and at the periphery of theFCs. The presence of RNA in these areas was also con-firmed by the cytochemical techniques used in thisstudy.

Taken together, these data favour the hypothesis thattranscription can begin at the periphery of the FCs,although we cannot exclude the possibility that the DFCplays a role in this process.

Key words: high-resolution autoradiography, in situ hybridization,plant nucleoli, rRNA transcription, ultrastructural cytochemistry

SUMMARY

Ultrastructural rRNA localization in plant cell nucleoli

RNA/RNA in situ hybridization, autoradiography and cytochemistry

A. Olmedilla1,*, P. S. Testillano1,2, O. Vicente3, M. Delseny4 and M. C. Risueño1,†

1Centro de Investigaciones Biológicas, CSIC, Velázquez 144, E-28006 Madrid, Spain2Departamento CC, Morfológicas y Cirugía, Faculty of Medicine, University of Alcalá de Henares, Spain3Institut für Mikrobiologie und Genetik, Biozentrum, Wien, Dr Bohr-Gasse 9, A-1030 Wien, Austria4Laboratoire de Physiologie et Biologie Moléculaire Végétales, URA 565 CNRS, Université de Perpignan, F-66860Perpignan-Cédex, France

*Present adress: Estación Experimental del Zaidín. CSIC. Profesor Albareda 1, E-18008 Granada, Spain†Author for correspondence

1334

and/or DFC (Derenzini et al., 1990; Raska et al., 1989,1992; Rodrigo et al., 1992; Rose et al., 1988; Roussel etal., 1993; Scheer and Rose, 1984; Scheer et al., 1987).

Selective staining methods and immunogold techniquesallowed the detection of DNA in FCs and in the surround-ing DFC in both animal and plant cells (Derenzini et al.,1990, 1993; Martin and Medina, 1991; Motte et al., 1991;Olmedilla et al., 1992; Raska et al., 1992; Testillano et al.,1991, 1992a; Thiry et al., 1991). From these data, differ-ent interpretations arise: transcription occurs on FCs(Scheer and Benavente, 1990; Thiry and Goessens, 1992),at the border of the FCs (Derenzini et al., 1990; Derenziniand Ploton, 1991), or in the DFC (Hartung et al., 1990;Schwarzacher and Wachtler, 1991); for other hypothesessee Jordan (1991).

Ultrastructural non-isotopic in situ hybridization is apowerful method for visualizing nucleic acids and consti-tutes a very convenient way of studying the molecular com-position of different nucleolar components. However, con-tradictory results have been obtained when this techniqueis applied to different materials (Dundr and Raska, 1993;Ghosh and Paweletz, 1990; Hozák et al., 1993; Motte etal., 1991; Raska and Dundr, 1993; Thiry and Thiry-Blaise,1989, 1991; Thiry et al., 1991; Stahl et al., 1991; Watch-ler et al., 1990, 1992). The results obtained relating to thelocalization of rDNA using probes encompassing differentregions of the transcription unit and the results obtainedusing different materials are not in agreement: sometimesthey are located on DFC and at other times on FCs (Géraudet al., 1991; Puvion-Dutilleul et al., 1991a, 1992; Thiry andThiry-Blaise, 1989, 1991; Watchler et al., 1990, 1992).

In studies relating to the localization of the products oftranscription by non-radioactive in situ hybridization at theelectron microscope level, some discrepancies are alsofound. The rRNAs are reported to be localized by DNAprobes mainly on DFC and GC in Ehrlich, HeLa and mouse3T3 cells (Puvion-Dutilleul, 1991a, 1992; Thiry and Thiry-Blaise, 1989) although in the Vero cell line rRNA wasdetected on DFC but not on GC (Escaig-Haye et al., 1989).No labelling was found inside FCs of untreated HeLa and3T3 mouse cells whereas some infrequent hybridization sig-nals were found on FCs of cells treated with actinomycinD (Puvion-Dutilleul, 1991a,b, 1992). In Ehrlich tumourcells, the RNA is reported to be localized in the peripheralregions of the FCs (Thiry and Thiry-Blaise, 1989).

There are data on plant material showing the localizationof rDNA (Motte et al., 1991; Highett et al., 1993) but theuse of high-resolution in situ hybridization to detect RNAin plant material is not very extensive (Harris and Croy,1986; Mc Fadden, 1989; Mc Fadden et al., 1988; Olmedillaet al., 1993; Sato et al., 1991). Furthermore, these investi-gations have not addressed the question of the localizationof rRNAs in the nucleolus. The application of in situ hybrid-ization using 18 and 25 S rRNA probes for the first timeallowed the localization at the electron microscope level ofribosomal transcripts in different components of the plantnucleoli. On the other hand, it has been proven that highersensitivity is obtained by the use of RNA probes for RNAdetection (Mc Fadden, 1989). For this reason in the presentstudy we have developed a method of RNA/RNA in situhybridization with our plant material in order to study the

localization of the ribosomal RNAs. We have taken advan-tage of the fact that the 18 and 25 S coding regions arewell conserved throughout evolution and have used clonesfrom Arabidopsis or Raphanus to hybridize with the cellu-lar rRNA of Allium and Capsicum. The localization at theultrastructural level of these RNAs was also achieved byhigh-resolution autoradiography and using different cyto-chemical methods. The combination of in situ hybridizationwith different cytochemical methods provides additionaldata on the presence of these transcripts in the variousnucleolar components (dense fibrillar and granular compo-nents, and periphery of fibrillar centres).

MATERIALS AND METHODS

MaterialsThe materials used were root-tip meristematic cells from Alliumcepa L. bulbs germinated at 15°C under standard conditions, andanthers of Capsicum annuum L. flowers from plants grown in agreenhouse under controlled conditions.

Fixing and embedding For EDTA staining and autoradiography root tips were fixed in3% glutaraldehyde (Taab Lab. Equip. Ltd, Reading, UK) in 0.025M cacodylate buffer, pH 7, for 2 hours at room temperature. Post-fixation in 1% osmic acid for 1 hour in the same buffer was per-formed for autoradiography samples, and then, they were dehy-drated in ethanol, and embedded in Epon. Formvar-copper gridswere used to collect the sections.

For the methylation and acetylation (MA) method, and in situhybridization, meristematic onion root cells and pepper antherswere fixed in 4% formaldehyde (Merck, Germany) in PBS buffer,pH 7.3, at 4°C, for 4 hours or overnight, respectively, dehydratedin methanol and embedded in Lowicryl K4M at −20°C (Bendayanet al., 1987; Carlemalm et al., 1980). Ultrathin sections were col-lected on Formvar-coated gold grids.

EDTA technique Grids were floated in 5% aqueous uranyl acetate solution, 0.2 MEDTA and lead citrate according to Bernhard’s protocol (Bern-hard, 1969).

High-resolution autoradiography Roots attached to the bulbs were incubated for 30 minutes in watercontaining 100 µCi/ml [3H]uridine (sp. act. 25 Ci/mmol) (Radio-chemical Centre, Amersham, England). Grids with ultrathin sec-tions were coated with Ilford L4 emulsion by the loop technique(Bouteille et al., 1976). After exposure in the dark for 11-12months, at 4°C, developing was performed with Phenidon. Thesections were stained with uranyl acetate and lead citrate.

MA method on Lowicryl sections Sections on grids were dehydrated in a methanol series (70% for15 minutes followed by three changes of pure methanol for 15minutes each) and then treated with a freshly preparedmethanol:acetic anhydride (5:1,v/v) mixture at room temperatureovernight. After washing in water, sections were stained with 5%uranyl acetate for 60 minutes at 60°C (Tandler and Solari, 1982;Testillano et al., 1991).

Probe synthesis The 18 S rRNA Raphanus probe was synthesized from a rDNAfragment of 1.2 kb covering almost all the 18 S rDNA region

A. Olmedilla and others

1335rRNA detection in plant nucleoli by TEM

(Delseny et al., 1983) subcloned in the pGEM-7Zf(+) plasmid.Biotinylated RNA probes for nuclear encoded 18 S and 25 S rRNAwere synthesized from Arabidopsis rDNA fragments of 1.5 and2.3 kb, respectively (Gruendler et al., 1991), subcloned in Blue-script KS+ plasmid.

RNA probes were prepared from the linearized plasmids usingbiotinylated UTP according to the Promega protocol. Lineariza-tion of the plasmids for synthesizing the sense or antisense probeswas performed using restriction enzymes that cut at restrictionsites as far as possible from the corresponding RNA Pol tran-scription promotor that was used in each case (see Figs 4 and 5).For each preparation linearized plasmid DNA (1 µg) was incu-bated for 90 minutes at 40°C with an RNA polymerase solutioncontaining 2.5 mM ATP, CTP and GTP (Boehringer-Mannheim),and 0.5 mM biotinylated UTP (biotin-11-UTP) (Sigma), in thepresence of RNasin (Promega). Non-isotopic labelled RNA wasrecovered using a Sephadex G 25 column, followed by ethanolprecipitation. The probe was resuspended in hybridization buffer(50% formamide, 75 mM NaCl, 50 mM PIPES, pH 7.2, 5 mMEDTA, 0.02% (v/v) Ficoll, 0.02% (w/v) polyvinylpirrolidone,0.02% (w/v) bovine serum albumin and 100 µg/ml herring spermDNA.

In situ hybridization Prior to hybridization, ultrathin sections were treated with 1 µg/mlproteinase K (Boehringer-Mannheim) in 100 mM Tris-HCl, pH8.0, and 50 mM EDTA for 1 hour. RNA/RNA hybridization wascarried out in a moist chamber at 55°C overnight on 50% for-mamide hybridization buffer containing 2 ng/µl RNA biotinylatedprobe. Non-specific RNA hybridization was removed by washingin: 4× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate) (4×2 minutes), 2× SSC (4× 2 minutes), 1× SSC (1× 2 hours at 55°C)and in SC buffer (50 mM PIPES, 0.5 M NaCl, 0.5% Tween-20)(1× 15 minutes).

Probe detection Blocking of non-specific antigens was performed by floating thegrids on 1% BSA in SC buffer for 15 minutes at room tempera-ture. The hybrids were detected with a rabbit anti-biotin antibody(Enzo Biochemicals Inc. NY, USA), 1:50 in SC for 1 hour at roomtemperature. The antibody was rinsed off with SC buffer (4× 2minutes). Then, grids were floated on a drop of goat anti-rabbitIgG conjugated to colloidal gold particles of 10 nm diameter(Jansen Biotech, NV; Olen/Belgium) diluted 1:20 in SC for 45minutes. Finally, sections were washed in SC (4× 2 minutes) anddistilled water, air dried and stained with uranyl/lead.

Statistical analysisThe hybridization signal that appears on FCs, DFC and GC wasevaluated in each micrograph by counting gold particles per areaunit in these components and in the cytoplasmic vacuoles (wheregold particles represent the background); 20-25 cell micrographswere used for each experiment. Therefore, labelling density incytoplasmic vacuoles was used as an internal control and its mainvalue was in estimation of the background.

FCs were counted in 20-25 nucleoli for each experiment, con-sidering FCs: (1) without gold particles; (2) with gold particlesforming clusters covering also the DFC; and (3) with gold parti-cles inside. Only FCs whose labelling density value was muchhigher than the mean value for the estimated background (densitylabelling on cytoplasmic vacuoles) were counted as labelled.

In situ hybridization combined with MA followedby EDTA After the hybridization some grids were postfixed in 3% glu-taraldehyde in PBS for 5 minutes, rinsed in the same buffer and

in distilled water. Finally, the MA procedure was applied asdescribed above, followed by EDTA/staining.

RESULTS

Plant nucleoli have a well established morphology (Figs 1-3). They are constituted of dense fibrillar component (DFC)and granular component (GC); the latter being almostabsent in inactive nucleoli. In active nucleoli the GC isintermingled with the DFC (Fig. 3) but in moderately orless-active nucleoli the GC is present at the peripheralregion of the nucleoli (Figs 1 and 2). Immersed in the densefibrillar component clear areas are visible that constitute thefibrillar centres (FCs). Depending on the degree of activitytwo types of FC can be found on the nucleolus. The homo-geneous FCs are small and numerous and contain decon-densed chromatin fibres. The heterogeneous FCs are largerand less numerous, containing cores of condensed chro-matin. An intermediate type of FC can also be observedwith small cores of condensed chromatin associated withdecondensed chromatin fibres. Vacuoles of different sizes,sometimes connected to FCs, can also be present in thenucleolus (Figs 1 and 2). We have used different techniquesto determine the composition of nucleolar components.

Cytochemistry for nucleic acidsThe application of the methylation and acetylation (MA)method followed by uranyl staining blocks the staining ofproteins and results in a clear distinction between the densefibrillar and the granular components of the nucleolus.Extranucleolar condensed chromatin masses show a highelectron density. With this method we observed the con-densed chromatin cores of the heterogeneous FCs with avery high contrast as well as some fibrils and granules witha lower contrast (Fig. 1).

The nucleolus after EDTA/staining, which is preferentialfor ribonucleoproteins, showed the dense fibrillar and gran-ular components well contrasted while extranucleolar chro-matin patches were not contrasted. Within the fibrillar cen-tres some bleached areas similar to those forming chromatinmasses were found as well as some stained fibrils and gran-ules (Fig. 2).

Autoradiography High-resolution autoradiography was performed after 30minutes incubation of tritiated uridine and a long period ofexposure (12 months). In Allium cepa 30 minutes is a shortincubation time, due to the problems of reagent penetrationinto plant cells and shorter periods result in non-appreci-able incorporation. In the nucleolus, the dense fibrillar com-ponent was labelled very frequently in regions close to thefibrillar centres and, in some cases, silver grains also cov-ered certain areas of the fibrillar centres. The granular com-ponent was also labelled (Fig. 3). Uridine incorporation wasalso evident in inter- and perichromatin regions.

In situ hybridizationRNA/RNA in situ hybridization at the electron microscopelevel was performed using three biotinylated probes. Matureregions of 18 S rRNA from Raphanus and 18 S and 25 S

1336 A. Olmedilla and others

Fig. 1. The methylation-acetylation (MA) method followed by uranyl staining. Nucleolus from Allium cepa L. meristematic root cells.Differential contrast between RNA- and DNA-containing structures was observed. F, dense fibrillar component; G, granular component;chr, chromatin masses. Fibrillar centres show structures with different degrees of contrast. Heterogeneous FCs (arrowheads) andhomogeneous FCs (arrows). v, vacuoles. Bar, 0.5 µm.

Fig. 2. EDTA ribonucleoprotein preferential stain on an Epon section. Meristematic onion root cell. Stained nucleolus showing densefibrillar component (F) intermingled with granular component (G). Fibrillar centres showing bleached areas (arrowheads) and somecontrasting material (arrows). Chromatin masses (chr) show absence of staining whereas fibrils and granules in the peri- andinterchromatin regions appear contrasted. Bar, 0.5 µm.

1337rRNA detection in plant nucleoli by TEM

rRNA from Arabidopsis were used for synthesizing theprobes (Figs 4 and 5). In situ hybridization with these threeprobes revealed a similar pattern of labelling on both Alliumcepa L. and Capsicum annuum L. pollen grains (Figs 6, 7and 8). Most of the hybridization signal was located overthe nucleolus and cytoplasm, and some labelling was alsofound on the nucleoplasm; in contrast, condensed chromatinmasses appeared to be free of gold particles (Figs 6A,D,7A and 8A,C). In the cytoplasm ribosome-rich areas werecompletely covered with particles while vacuoles and cyto-plasmic organelles such as mitochondria appeared not to belabelled (Figs 6A,D, 7A and 8A). In the nucleolus, goldparticles were concentrated on the dense fibrillar and gran-ular components (Figs 6A, 7A and 8A). In order to better

visualize the structures where probes are localized we havecombined the MA method and EDTA/staining with the insitu hybridization technique. By this means we can see thatthe density of labelling is higher over the granular compo-nent, and gold particles are also present on the dense fib-rillar component. In FCs some stained fibrils were visual-ized covered by gold particles (Fig. 9). The areascorresponding to fibrillar centres were, in some cases, notlabelled (Figs 6A,B,C, 7A,B,C and 8A,B) and in the caseswhere gold particles were present, the particles formed clus-ters that also covered the surrounding dense fibrillar com-ponent (Figs 6A,B,C, 7A,B,C and 8A,C). Nevertheless,when the particles appeared close to the central area of theFCs they were usually isolated (Figs 6B, 7C and 8B).

Fig. 3. Autoradiograph of nucleoli from meristematic onion root cells labelled with tritiated uridine for 30 minutes. After a long exposure(12 months) the preparations were developed with Phenidon. Dense fibrillar component (F) appears labelled, especially in regions close tothe FCs. In some cases FCs are also labelled (thin arrows). Heterogeneous FCs (arrowhead). In the granular component (G) and in someperichromatin regions some silver grains are visible (arrows). chr, chromatin masses. Bar, 1 µm.

Fig. 4. Map corresponding to the ribosomal DNA of Raphanus sativus L.Localization of the 18 S rRNA probe within this rDNA is shown. The position ofrestriction sites used for the subcloning in the pGEM-7Zf+ plasmid, as well as therestriction sites for linearizing the plasmid before sense or antisense RNA probesynthesis are also indicated.

1338

As mentioned above, a similar pattern of labelling wasobserved with each of the three probes used and also ineach tissue tested: meristematic onion root and pepperpollen grains. Only slight differences were observed in thehybridization signal obtained with the different probes; goldgrains are more abundant after hybridization with the 18 SrRNA probes than with the 25 S rRNA probe (compareFigs 6 and 7 with Fig. 8). The hybridization signal obtainedfrom the 18 S probe of Raphanus was stronger than thatobtained with the 18 S probe of Arabidopsis (compare Fig.6 with 7). With respect to the differences between materi-als, the nucleoli of both cell types have some differencesin ultrastructure, the nucleolus of Capsicum pollen grainsbeing smaller and almost exclusively formed of DFC (com-pare Figs 6A, 7A and 8A with Figs 6D and 8C). The goldparticles detected were more abundant in onion root meris-tematic cells than in pepper pollen grains, irrespective ofthe probe used.

Controls were performed in order to verify the specificityof the in situ hybridization reaction. Sense biotinylatedrRNA probes from the three plasmids were synthesized andused for in situ hybridization in the same conditions. Witheach of the sense probes the labelling was not significant(Fig. 7D). Also, no signal was observed when anti-biotinantibodies were replaced by PBS buffer to detect thebiotinylated hybrids.

Quantitative studyFor the quantitative study we have taken into account thatthere is no clear border between DFC and FCs; thus todecide to which of these components gold particles shouldbe assigned we assumed that a gold particle is on a FCwhen it is not over a stained fibre connected with the adja-cent DFC. The results on the distribution of labelling den-sity in the different nucleolar components (DFC, GC andFCs), as well as in cytoplasmic vacuoles, are shown in Fig.10. The data show the highest labelling density on the DFCand GC (174.75 particles/µm2), and a much lower value onFCs (24.47 particles/µm2), while the corresponding densityfor cytoplasmic vacuoles is comparatively very low (2.16particles/µm2).

We have also performed a statistical analysis of thelabelling on FCs in order to differentiate the hybridizationsignal from the background in these structures (Fig. 11).We have taken as reference the density of label on cyto-plasmic vacuoles where gold particles correspond to back-

ground. We have counted the density of gold particles perunit area in FCs and in cytoplasmic vacuoles that appearin the same micrograph. The x-axis corresponds to the den-sity of gold labelling per unit area in FCs and the y-axis tothe density of gold particles per unit area in cytoplasmicvacuoles. The enormous dispersion of the cloud of pointsfor these pairs of values indicates that there is no definedcorrelation between the two densities of labelling. There-fore, these data indicate that the labelling on FCs does notcorrespond to background but it represents hybridizationsignal. Moreover, by applying to this group of points thestatistic computation of the regression line, a correlationcoefficient of −0.43 with 14 degrees of freedom has beenobtained for the best fit (after using different models ofregression analysis: linear, exponential, reciprocal, multi-plicative, and trying with either the original data, or thetransformed data: log, ln, square root, arcsen). This valueshows no correlation between the two densities of labelling(Fig. 11).

FCs showed three types of labelling: without gold parti-cles, with gold particles forming clusters covering bothDFC and FCs, and with gold particles inside (see the threeclasses shown schematically in Fig. 12). After quantitativeanalysis, we found that the most abundant pattern was thatof FCs without gold particles, between 49 and 63%; FCsshowing the second pattern of labelling comprised 10-21%,and FCs with gold particles inside between 23 and 36%.

DISCUSSION

In this paper the localization of transcriptional products hasbeen studied by high-resolution in situ hybridization usingdifferent RNA biotinylated probes allowing the detectionof rRNA precursors and mature 18 and 25 S rRNAs inplants. Moreover, ultrastructural cytochemistry and autora-diographical assays provide additional information on thepresence of those transcripts in the different nucleolar com-ponents. On the basis of this study we find that rRNA ispresent in GC, DFC and at the periphery of some FCs. Allthese data suggest that transcription is initiated at theperiphery of the FCs but a role for the DFC in this processshould be considered.

The cytochemical method of MA followed by uranylstaining is a procedure that avoids the uranyl staining ofproteins because it blocks their amino and carboxyl groups

A. Olmedilla and others

Fig. 5. Map corresponding to the ribosomal DNA ofArabidopsis thaliana L. Localization of the 18 S and25 S rRNA probes within this rDNA. The positions ofrestriction sites used for the subcloning in theBluescript plasmid as well as the restriction sites forlinearizing the plasmid prior to the synthesis of senseor antisense RNA probe synthesis are also indicated.

1339rRNA detection in plant nucleoli by TEM

Fig. 6. RNA/RNA hybridization on Allium (A, B and C) and Capsicum (D) Lowicryl sections. 18 S rRNA Raphanus antisense probe.Hybridization signal is found on the nucleolus where is possible to see arrays of gold particles (crossed arrow). Labelled probe is locatedmainly over the dense fibrillar (F) and granular (G) component; fibrillar centres show no labelling (double arrows) in many cases,although there are also FCs with gold particles inside (thin arrows) and with arrays of particles that also cover the dense fibrillarcomponent (arrows) (A,B and C). Ribosome-rich cytoplasmic areas (cyt) are covered by particles whereas vacuoles (v) and mitochondria(m) are not labelled (A). (D) Somatic cells of Capsicum anthers. The same pattern of labelling is observed: the dense fibrillar component(F), which exclusively forms this type of nucleoli, appears labelled, but the distribution of particles is not as homogeneous over thenucleolus as in meristematic cells, i.e. not all of the dense fibrillar component is covered by gold particles. The nucleoplasm contains onlya few gold particles (arrowheads). Ribosomal-rich regions of the cytoplasm are labelled (D). Bars, 0.5 µm.

1340

(Tandler and Solari, 1982). The adaptation of this methodfor plant material embedded in Lowicryl is a helpful cyto-chemical method to use with nucleic acid-containing struc-

tures (Risueño, 1993; Testillano et al., 1991). The MAmethod makes it possible to distinguish between fib r i l l a rand granular ribonucleoproteins; this difference is not

A. Olmedilla and others

Fig. 7. RNA/RNA hybridization on Allium Lowicryl sections. 18 S rRNA Arabidopsis probe. (A, B and C) Antisense probe: in the nucleigold particles are located predominantly over the nucleolus, being numerous on the dense fibrillar (F) and granular (G) components. Goldparticles appear in arrays (crossed arrow). Fibrillar centres are in many cases not labelled (double arrows) and in the cases where goldparticles are present, the particles form clusters (arrows) that also cover the dense fibrillar component (F); the particles closer to the centreusually appear isolated (thin arrows) (B and C). Some gold particles are visible on the nucleoplasm (arrowheads). Ribosomal cytoplasmicregions (cyt) are highly labelled whereas vacuoles (v) and other organelles do not show any labelling (A). (D) Sense probe: very few goldparticles are visible in comparison with the hybridization with the antisense RNA probe. nu, nucleolus; n, nucleoplasm; v, vacuoles. Bars,0.5 µm.

1341rRNA detection in plant nucleoli by TEM

always evident on Lowicryl sections. The fibrillar mater-ial stained on FCs using this method represents DNA andRNA-containing structures. On the other hand, due to the

increase in contrast of the condensed chromatin we havec o n firmed, using this method, that the unique condensedchromatin structures observed in the plant nucleolus are

Fig. 8. RNA/RNA hybridization on Allium cepa L. (A and B) and Capsicum annuum L. (C). Lowicryl sections. 25 S rRNA Arabidopsisantisense probe. Meristematic onion root cells (A and B). The nucleolus appears labelled. Gold particles are located over the densefibrillar component (F), including areas surrounding fibrillar centres (double arrows), and over its granular component (G). In somefibrillar centres isolated gold particles (thin arrows) are visible (B). Arrays of gold particles covering both FCs and the dense fibrillarcomponent are also present (arrows). Cytoplasmic regions enriched in ribosomes (cyt) appear labelled. A few particles are also present innucleoplasm (arrowheads) (A) corresponding to the fast transport of preribosomes to the cytoplasm throughout the nucleolar envelope(ne) (open arrows). (C) Pepper pollen grain. The labelling is less abundant than in onion root cells but is located on the same structures.Gold particles appear to decorate certain areas of the dense fibrillar component (F) and some peripheral regions of the fibrillar centres(arrows). cyt, cytoplasm; ne, nuclear envelope. Bars, 0.5 µm.

1342

the chromatin cores of heterogeneous FCs, since there areno other areas of densely stained material except the onethat appears in these FCs (Deltour and Motte, 1990;

Medina et al., 1993, Risueño et al., 1982; Testillano et al.,1991).

After autoradiography with four months of exposure,RNA was mainly detected at the dense fibrillar componentsurrounding fibrillar centres in Allium cepa L. but no sig-nificant labelling was found on FCs (Risueño et al., 1982).However, after a longer exposure (10-12 months) theappearance of silver grains very close to, or inside the fib-rillar centres increases, indicating the presence of newlysynthesized RNA. In these components ribonucleoproteinmaterial is also detected after EDTA/staining (Bernhard,1969). In a previous paper in which an autoradiographicaland cytochemical study was made on the Allium cepa L.nucleolus, the presence of some material stained by theEDTA technique inside FCs was also described, althoughthe presence of this material was not confirmed, by RNasedigestion or uridine incorporation (Risueño et al., 1982).The differences between those data and the results pre-sented here could be explained, because only after longexposure times has autoradiographic labelling beenobtained on FCs, as reported in Ehrlich tumor cells (Thiryand Goessens, 1991). The fact that the RNA detected onFCs could constitute another RNA type such as the snRNAsdoes not seem possible because different immunocyto-chemical studies have demonstrated the presence of these

A. Olmedilla and others

Fig. 9. RNA/RNA hybridization on AlliumLowicryl sections with a 25 S rRNAArabidopsis antisense probe combined withMA, followed by EDTA/staining. Nucleoluscovered by gold particles. Granularcomponent (G) showing more gold particlesthan the dense fibrillar one (F). Some fibrilsare stained in the interior of FCs and appearlabelled (arrows). Bar, 1 µm.

Fig. 10. Labelling density after the in situ hybridizationexperiments. The ordinate represents the number of gold particlesper µm2. Data show the mean number of gold particles per µm2

and the standard deviation; *P<0.02, **P<0.05.

1343rRNA detection in plant nucleoli by TEM

snRNAs on DFC and GC but not on FCs (Puvion-Dutilleulet al., 1992; Raska et al. 1992; Testillano et al., 1992a,b).

Most of the reported studies show the presence of RNAin DFC, but there are only a few reports showing RNA inFCs: by in situ/in vitro transcription applied to Lowicrylsections of Ehrlich tumor cells (Thiry and Goessens, 1991);by polyadenylate nucleotidyl transferase/immunogold inhuman Sertoli cells (Thiry, 1993a,b); or by in situ hybrid-ization in Ehrlich or actinomycin D-treated HeLa cells(Thiry and Thiry-Blaise 1989; Puvion-Dutilleul et al., 1992;Raska et al., 1992). In Ehrlich tumor cells, the presence of

some RNA-containing material on FCs was also reportedusing RNase/gold (Thiry, 1988) or RNase treatment(Yasuzumi and Sugihara, 1965).

The presence of rRNA in the DFC and GC of the plantnucleolus demonstrated by in situ hybridization experi-ments correlates with the data on human and animal cells.In our data, the hybridization signal found in the FCs rep-resents RNA as was proven by the lack of correlationbetween the labelling on the FCs and the labelling on cyto-plasmic vacuoles that constitutes background. The signal inFCs is very low but the labelling density is many timeshigher than that of the corresponding background. Thehybridization labelling found on nucleoplasm is also verylow but clearly represents the very fast passage of theseproducts from nucleolus to cytoplasm.

Based on the statistical studies, in which we have con-sidered the labelling density of the cytoplasmic vacuoles asan internal control of the background, the regression curveobtained with a correlation coefficient of −0.43 with 14degrees of freedom clearly indicates that the labelling den-sity in both structures (FCs and cytoplasmic vacuoles) arenot correlated. These data suggest that the hybridizationsignal in FCs can be considered significant and does notcorrespond to the background.

Thus, three different classes of labelling on FCs havebeen found and after counting the label almost 50% of fib-rillar centres contain rRNA in the centre and/or in theperiphery. Two different interpretations can be made for thepresence of in situ hybridization labelling at the peripheryof the FCs, which also covers the DFC: they could be eitherfibres of RNA synthesized in the DFC that enter into theFCs, or vice versa. The hybridization signal on the centreof FCs is not very significant because the gold particlesusually appear isolated in these cases.

With in situ hybridization using 18 and 28 S DNA probesno rRNA is reported to be detected in the interior of FCs

Fig. 11. Regression curve. The abscissa represents the Napierianlogarithm of the value representing gold particles per unit area inthe fibrillar centres and the ordinate Napierian logarithm of thevalue representing gold particles per unit area in cytoplasmicvacuoles. No correlation between these parameters is found. Thehighest correlation coeficient found is −0.43 with 14 degrees offreedom.

Fig. 12. Threeclasses of fibrillarcentres are defined:those without goldparticles (1); thosewith gold particlesforming ropes, whichalso cover the densefibrillar component(2); and those withgold particles inside(3). Percentageswere obtained foreach class. The mostabundant class is theone formed by FCswithout labelling (1).

1344

of HeLa cells (Puvion-Dutilleul et al., 1991a,b, 1992).When DNA probes containing the external transcribedspacer were used the peripheral region of FCs appearedlabelled in studies with Ehrlich and HeLa cells (Thiry andThiry-Blaise, 1989; Puvion-Dutilleul, 1991a,b, 1992). Thedifference from our results using 18 S and 25 S rRNAprobes may be associated not only with the difference inmaterials used, but also with using RNA probes, whichmake the signal more abundant, as is expected inRNA/RNA hybridization (Mc Fadden et al., 1988).

When we used probes encompassing mature 18 and 25S regions of the transcriptional unit we were able to detectnot only the mature rRNAs but also pre-rRNAs. In additionwe were able to detect hybridization labelling on FCs.Finally, when we combined in situ hybridization with MAand EDTA we were able to find stained fibrils showinghybridization signal, which provided additional data on thepresence of RNA on FCs.

On the other hand, recently, the presence of rDNA hasbeen detected by in situ hybridization and confocalmicroscopy of Pisum sativum, which seems to cover areasbigger than FCs (Highett et al., 1993), this suggests that theDNA detected on DFC could be rDNA. In plant nucleolithere are no chromatin inclusions except for those that appearin the interior of heterogeneous FCs. DNA has been local-ized on DFC and on FCs (Martin and Medina, 1991;Olmedilla et al., 1991, 1992; Risueño, 1993; Risueño et al.,1991; Testillano et al. 1991, 1992a). Therefore, in this mate-rial the argument based on finding DNA only on FCs to sup-port transcription taking place only in these structures wasnot confirmed by those results. Moreover, although RNApolymerase I has not been directly localized by immunocy-tochemistry there are some data that seem to indicate thatthis enzyme can be localized on both fibrillar componentsin plants (Martin and Medina, 1991). The RNA polymeraseI transcription factor UBF (upstream binding factor) has beenlocalized on different phylogenetically separate species(including Allium cepa) in the DFC (Rodrigo et al., 1992).Finally, nucleolar snRNAs and fibrillarin, known to play anactive role at early stages of maturation, to date have notbeen localized on FCs in plant material (Testillano et al.,1992b,c, 1993). These data are in favour of the hypothesisthat ribosomal transcription can start at the periphery of FCs,but the possibility cannot be excluded that the DFC close tothe FCs also plays a role in this process. To confirm thishypothesis, experiments using in situ hybridization withRNA probes for the transcribed external spacer in order tolocalize these earlier transcripts, and different rDNA probesto localize the corresponding genes, are in progress.

We thank Prof. Dr Schweizer, Institute of Botany, Universityof Vienna, for providing us with the E. coli strains containing theArabidopsis rRNA genes; Mr J. Blanco for his technical assis-tence with the photographic work, Dr A. Moreno (Inst. Acústica,CSIC, Madrid), Dr R. González (Est. Exp. Zaidín, CSIC, Granada)and Dr J. Renau-Piqueras (Hospital La Fe, Valencia) for thestatistical work, and Mrs B. Ligus-Walker for revision of the Eng-lish. This work was supported by projects of the DIGICYT PB87-0332-C02-01/PB92-0079-C03-01 and Spanish-Austrian interna-tional cooperation.

REFERENCES

Bendayan, M., Nancy, A. and Kan, F. W. K. (1987). Effect of tissueprocessing on colloidal gold cytochemistry. J. Histochem. Cytochem. 35,983-996.

Bernhard, W. (1969). A new staining procedure for electron microscopicalcytology. J. Ultrastruct. Res. 27, 250-265.

Bouteille, M., Laval, M. and Dupuy-Coin, A. M. (1976). Efficiency,resolution, grain size and grain shape in routine electron microscopeautoradiography. J. Microsc. Biol. Cell 27, 171-176.

Carlemalm, E. W., Villiger, W. and Acetarin, J. D. (1980). Advances inspecimen preparation for electron microscopy. I. Novel low-temperatureembeding resins and a reformulated Vestopal. Experientia 36, 740-744.

Delseny, M., Cooke, R. and Penon, P. (1983). Sequence heterogeneity inradish nuclear ribosomal RNA genes. Plant Sci. Lett. 30, 107-119.

Deltour, R. and Motte, P. (1990). The nucleolonema of plant and animalcells: a comparison. Biol. Cell 68, 5-11.

Deltour, R. and Mosen, H. (1987). Proposals for the macromolecularorganization of higher plant nucleolonema. Biol. Cell 60, 75-86.

Derenzini, M., Farabegoli, F. and Trere, D. (1993). Localization of DNAin the fibrillar components of the nucleoli: a cytochemical andmorphometric study. J. Histochem Cytochem. 41, 829-836.

Derenzini, M., Hernandez-Verdun, D., Farabegoli, F., Pession, A. andNovello, F. (1987). Structure of ribosomal genes of the mammalian cellsin situ. Chromosoma 95, 63-70.

Derenzini, M. and Ploton, D. (1991). Interphase nucleolar organizerregions in cancer cells. Int. Rev. Exp. Pathol. 32, 149-192.

Derenzini, M., Thiry, M. and Goessens, G. (1990). Ultrastructuralcytochemistry of the mammalian cell nucleolus. J. Histochem. Cytochem.38, 1237-1256.

Dundr, M. and Raska, I. (1993). Non-isotopic ultrastructural mapping oftranscription sites within the nucleolus. Exp. Cell Res. (in press).

Escaig-Haye, F., Grigoriev, V. and Fournier, J. G. (1989). Détectionultrastructurale d’ARN ribosomal par hybridation in situ à l’aide d’unesonde biotinylée sur coupes ultrafines de cellule animale en culture. C. R.Hebd. Séanc. Acad. Sci., Paris [III] 309, 429-437.

Géraud, M., Herzog, M. and Soyer-Gobillar, M. O. (1991). Nuclearlocalization of rRNA coding sequences in Prorocentrum micans Ehr.(dinomastigote, kingdom Protoctist) by in situ hybridization. BioSystems26, 61-74.

Goessens, G. (1984). Nucleolar structure. Int. Rev. Cytol. 87, 107-158. Gosh, S. and Paweletz, N. (1990). Localization of ribosomal cistrons at the

ultrastructural level by in situ hybridization technique. Cell Biol. Int. Rep.14, 521-525.

Gruendler, P., Unfried, I., Pascher, K. and Schweizer D. (1991). rDNAintergenic region from Arabidopsis thaliana structural analysis,intraspecific variation and functional implications. J. Mol. Biol. 221,1209-1222.

Hadjiolov, A. (1985). In The Nucleolus and Ribosome Biogenesis. CellBiol. Monographs, vol. 12, pp. 1-263. Springer-Verlag, Vienna.

Harris, N. and Croy, R. R. D. (1986). Localization of mRNA for Pealegumin: In situ hybridization using a byotinilated cDNA. Protoplasma130, 57-67.

Hartung, M., Watchler, F., de Lanversin, A., Fouet, C., Schwarzacher,H. G. and Stahl, A. (1990). Sequential changes in the nucleoli of humanspermatogonia with special reference to rDNA location and transcription.Tissue & Cell22, 25-37

Hernandez-Verdun, D. (1991). The nucleolus today. J. Cell Sci. 99, 465-471.

Highett, M. I., Rawlins, D. J. and Shaw, P. J. (1993). Different patterns ofrDNA distribution in Pisum sativum nucleoli correlate with differentlevels of nucleolar activity. J. Cell Sci. 104, 843-852.

Hozák, P., Schöfer, C., Sylvester, J. and Wachtler, F. (1993). A study onnucleolar DNA: isolation on DNA from fibrillar components andultrastructural localization of different DNA probes. J. Cell Sci. 104,1199-1205.

Jordan, E. G. (1991). Interpreting nucleolar structure: where are thetranscribing genes? J. Cell Sci. 98, 437-442.

Martin, M. and Medina, F. J. (1991). A Drosophila anti-RNA polymeraseII antibody recognizes a plant nucleolar antigen, RNA polymerase I,which is mostly localized in fibrillar centres. J. Cell Sci. 100, 99-107.

Mc Fadden, G. I. (1989). In situ hybridization in plants: from macroscopicto ultrastructural resolution. Cell Biol. Int. Rep. 13, 3-22.

Mc Fadden, G. I., Boing, I., Cornish, E. C. and Clark, A. E. (1988). A

A. Olmedilla and others

1345rRNA detection in plant nucleoli by TEM

simple fixation and embedding medium method for use in hybridizationhistochemistry on plant tissues. Histochem. J. 20, 575-586.

Medina F. J., Risueño, M. C. and Moreno Díaz de la Espina, S. (1983). 3-D reconstruction and morphometry of the fibrillar centres in plant cells inrelation to the nucleolar activity. Biol. Cell 48, 31-38.

Motte, P. M., Loppes, R., Menager, M. and Deltour, R. (1991). Three-dimensional electron microscopy of ribosomal chromatin in two higherplants: a cytochemical, immunocytochemical and in situ hybridizationapproach. J. Histochem. Cytochem. 39, 1495-1506.

Olmedilla, A. and Risueño, M. C. (1993). Localización de ácidosnucléicos mediante hibridación in situ. In Progresos en Biología Celular(ed. J. Becerra et al.), pp. 147-150. Univ. Málaga, Spain.

Olmedilla, A., Testillano, P. S., Raska, I. and Risueño, M. C. (1992). Insitu nick translation and anti-BrdU techniques as convenient tools tostudy the functional regions of chromatin in plants. In ElectronMicroscopy. EUREM 92. vol. 3 (ed. L. Megías, M. I. Rodríguez-García,A. Ríos and J. M. Arias), pp. 193-194. University of Granada, Spain.

Olmedilla, A., Testillano, P. S., Sánchez-Pina, M. A., Müller, S. andRisueño, M. C. (1991). Simultaneous detection of histones and DNA inplant nuclei by immunogold labelling and NAMA-Ur. In Proc. 12thEuropean Workshop on the Cell Nucleus (ed. S. Fakan), p. 67. LesDiablerets, Switzerland.

Puvion, E. and Moyne, G. (1980). In situ localization of RNA structures. InThe Cell Nucleus, vol. VIII (ed. H. Busch), pp. 59-115. Acad. Press, Inc.,NY.

Puvion-Dutilleul, F., Mazan, S., Nicoloso, M., Pichard, E., Bachellerie,J. P. and Puvion, E. (1992). Alterations of nucleolar ultrastructure andribosome biogenesis by actinomycin D. Implications for U3 snRNPfunction. Eur. J. Cell Biol. 58, 149-162.

Puvion-Dutilleul, F., Bachellerie, J. P. and Puvion, E. (1991a). Nucleolarorganization on HeLa cells as studied by in situ hybridization.Chromosoma 100, 395-409.

Puvion-Dutilleul, F., Mazan, S., Nicoloso, M., Christensen, M. E. andBachellerie, J. P. (1991b). Localization of U3 RNA molecules innucleoli of HeLa and mouse 3T3 cells by high resolution in situhybridization. Eur. J. Cell Biol. 56, 178-186.

Raska, I. and Dundr, M. (1993). Compartmentalization of the cell nucleus:case of the nucleolus. In Chromosomes Today, vol. 11 (ed. A. T. Sumnerand A. C. Chandley), pp. 111-119. Chapman and Hall, London.

Raska, I., Reimer, G., Jarnik, M., Kostrouch, Z. and Raska, K. (1989).Does the synthesis of ribosomal RNA take place within nucleolar fibrillarcentres or dense fibrillar component? Biol. Cell 65, 79-82.

Raska, I., Ochs, R. L. and Salamin-Michel, L. (1990).Immunocytochemistry of the cell nucleus. Electron Microsc. Rev. 3, 301-353.

Raska, I., Dundr, M. and Koberna, K. (1992). Structure-functionsubcompartments of the mammalian cell nucleus as revealed by theelectron microscopic affinity cytochemistry. Cell Biol. Int. Rep. 16, 771-789.

Risueño, M. C. (1993). Aspectos estructurales y funcionales del nucleolo.In Progresos en Biología Celular (ed. J. Becerra et al.), pp. 129-134.Univ. Málaga, Spain.

Risueño, M. C. and Medina, F. J. (1986). The nucleolar structure in plantcells. Cell Biol Rev. 7, 1-140.

Risueño, M. C., Medina, F. J. and Moreno Díaz de la Espina, S. (1982).Nucleolar fibrillar centres in plant meristematic cells: ultrastructure,cytochemistry and autoradiography. J. Cell Sci. 58, 313-329.

Risueño, M. C., Testillano, P. S., Olmedilla, A., Sánchez-Pina, M. A.,Ollacarizqueta, M. A., Müller, S. and Tandler, C. J. (1991). Nucleolarchromatin: A cytochemical and immunocytochemical study. In Proc.12th European Workshop on the Cell Nucleus (ed. S. Fakan), p. 82. LesDiablerets. Switzerland.

Rodrigo, R., Rendón, M. C., Torreblanca, J., García-Herdugo, G. andMoreno, F. J. (1992). Characterization and immunolocalization of RNApolymerase I transcription factor UBF with anti-NOR serum in protozoa,higher plant and vertebrate cells. J. Cell Sci. 103, 1053-1063.

Rose, K. M., Szopa, J., Han, F., Cheng, Y., Richter, A. and Scheer, U.(1988). Association of DNA topoisomerase I and RNA polymerase I: apossible role for topoisomerase I in ribosomal gene transcription.Chromosoma 96, 411-416.

Roussel, P., André, C., Masson, C., Géraud, G. and Hernandez-Verdun,D. (1993). Localization of the RNA polimerase I transcription factorhUBF during the cell cycle. J. Cell Sci. 104, 327-333.

Sato, S., Jones, K., Alché, J. D. and Dickinson, H. G. (1991). Cytoplasmic

nucleoloids of Lillium male reproductive cells contain rDNA transcriptsand share features of development with nucleoli. J. Cell Sci. 100, 109-118.

Scheer, U. and Rose, K. (1984). Localization of RNA polymerase I ininterphase cells and mitotic chromosomes by light and electronmicroscopic immuno-cytochemistry. Proc. Nat. Acad. Sci. USA 81, 1431-1435.

Scheer, U. and Benavente, R. (1990). Functional and dinamic aspects ofthe mammalian nucleolus. BioEssays 12, 14-21.

Scheer, U., Messner, K., Hazan, R., Raska, I., Hansmann, P., Falk, H.,Spiess, E. and Franke, W. W. (1987). High sensitivityimmunolocalization of double and single-stranded DNA by a monoclonalantibody. Eur. J. Cell Biol. 43, 358-371.

Schwarzacher, H. G. and Wachtler, F. (1991). The functional significanceof nucleolar structures. Ann. Génét. 34, 151-160.

Stahl, A., Wachtler, F., Hartung, M., Devictor, M., Schöfer, C.,Mosgöller, W., de Lanversin, A., Fouet, C. and Schwarzacher, H. G.(1991). Nucleoli, nucleolar chromosomes and ribosomal genes in thehuman spermatocyte. Chromosoma 101, 231-244.

Tandler, C. J. and Solari, A. J. (1982). Methanol-acetic anhydride: anefficient blocking agent for electron microscope cytochemistry. Itsapplication to mouse testis and other tissues. Histochemistry 76, 351-361.

Testillano, P. S., Sánchez-Pina, M. A., Olmedilla, A., Ollacarizqueta, M.A., Tandler, C. J. and Risueño, M. C. (1991). A specific ultrastructuralmethod to reveal DNA: The NAMA-Ur. J. Histochem. Cytochem. 39,1427-1438.

Testillano, P. S., Olmedilla, A., Sánchez-Pina, M. A., Raska, I. andRisueño, M. C. (1992a). The use of ultrathin cryosections forimmunoelectron microscopy in plant cell nucleus. Electron MicroscopyEUREM 92, vol. 3(ed. L. Megías, M. I. Rodríguez-García, A. Ríos and J.M. Arias), pp. 87-88. University of Granada, Spain.

Testillano, P. S., Olmedilla, A., Sánchez-Pina, M. A. and Risueño, M. C.(1992b). Detection of small nuclear RNAs in plant nuclei. In NewApproaches for in situ Detection of DNA-RNA Molecules (ed. J. M.Bricall), pp. 38-39. CFIME. University of Barcelona, Spain.

Testillano, P. S., Sánchez-Pina, M. A., López-Iglesias, C., Olmedilla, A.,Christensen, M. E. and Risueño, M. C. (1992c). Distribution of B36nucleolar protein in relation to transcriptional activity in plant cells.Chromosoma 102, 41-49.

Testillano, P. S., Sánchez-Pina, M. A., Olmedilla, A., Fuchs, J. P. andRisueño, M. C. (1993). Characterization of the interchromatin region asthe nuclear domain containing snRNPs in plant cells. A cytochemical andimmunoelectron microscopy study. Eur. J. Cell Biol. 61, 349-361.

Thiry, M. (1988). Study of RNA distribution in the nucleolar componentsof Ehrlich cell using RNase-gold method. Histochemistry 89, 231-236.

Thiry, M. (1993a). Immunodetection of RNA on ultra-thin sectionsincubated with polyadenylate nucleotidyl transferase. J. Histochem.Cytochem. 41, 657-665.

Thiry, M. (1993b). Ultrastructural distribution of DNA and RNA within thenucleolus of human Sertoli cells as seen as by molecularimmunocytochemistry. J. Cell Sci. 105, 33-39.

Thiry, M. and Goessens, G. (1991). Distinguishing the sites of pre-rRNAsynthesis and accumulation in Ehrlich tumor cell nucleoli. J. Cell Sci. 99,759-767.

Thiry, M. and Goessens, G. (1992). Where, within the nucleolus, are therRNA genes located? Exp. Cell Res. 200, 1-4.

Thiry, M. and Thiry-Blaise, L. (1989). In situ hybridization at the electronmicroscope level: an improved method for precise localization ofribosomal DNA and RNA. Eur. J. Cell Biol. 50, 235-243.

Thiry, M. and Thiry-Blaise, L. (1991). Locating transcribed and non-transcribed rDNA spacer sequences within the nucleolus by in situhybridization and immunoelectron microscopy. Nucl. Acids Res. 1 9, 11-15.

Thiry, M., Scheer, U. and Goessens, G. (1991). Localization of nucleolarchromatin by immunocytochemistry and in situ hybridization at theelectron microscopic level. Electron Microsc. Rev. 4, 85-110.

Wachtler, F., Schöfer, C., Mosgöller, W., Weipoltshammer, K.,Schwarzacher, H. G., Guichaoua, M., Hartung, M., Stahl, A., Bergé-Lefranc, J. L., González, I. and Sylvester, J. (1992). Human ribosomalRNA gene repeats are localized in the dense fibrillar component ofnucleoli: Light and electron microscopic in situ hybridization in humanSertoli cells. Exp. Cell Res. 198, 135-143.

Wachtler, F., Mosgöller, W. and Schwarzacher, H. G. (1990). Electronmicroscopic in situ hybridization and autoradiography: localization and

1346

transcription of rDNA in human lymphocyte nucleoli. Exp. Cell Res. 187,346-348.

Yasuzumi, G. and Sugihara R. (1965). The fine structure of nuclei asrevealed by electron microscopy. Exp. Cell Res. 37, 207-229.

(Received 23 July 1993 - Accepted 8 September 1993)

Note added in proofResults recently obtained in plant cells by different cyto-

chemical and immunocytochemical methods demonstratethe presence of DNA and DNA/RNA hybrids in regions ofthe DFC near the FCs (Risueño and Testillano, 1993,invited review, Micron; Testillano, Gorab and Risueño,1993, J. Histochem. Cytochem., in press). These data sup-port the hypothesis presented in this paper that the periph-ery of the FCs and/or regions of the DFC near the FCs areinvolved in transcription.

A. Olmedilla and others