409Journal of Cell Science 109, 409-418 (1996)Printed in Great Britain © The Company of Biologists Limited 1996JCS4069

Nuclear architecture and ultrastructural distribution of poly(ADP-

ribosyl)transferase, a multifunctional enzyme

Wilhelm Mosgoeller1, Marianne Steiner1, Pavel Hozák2, Edward Penner3 and Józefa Wesierska-Gadek4,*1Institute of Histology and Embryology, University of Vienna, Schwarzspanierstrasse 17, A-1090 Wien, Austria2Academy of Sciences, Institute of Experimental Medicine, Prague, Czech Republic3University Clinic (AKH), Department of Gastroenterology and Hepatology, Vienna, Austria4Institute of Tumorbiology-Cancer Research, Borschkegasse 8a, A-1090 Vienna, Austria

*Author for correspondence

A monospecific autoimmune serum for poly(ADP-ribosyl)transferase (pADPRT) was used to localise theenzyme in ultrastructural cellular compartments. Wedetected enzyme in mitochondria of HeLa and Sertoli cells.Within the nucleoplasm the enzyme concentration was pos-itively correlated with the degree of chromatin conden-sation, with interchromatin spaces being virtually free ofpADPRT. During spermatogenesis we observed a gradualincrease of the chromatin associated pADPRT that paral-lelled chromatin condensation. The highest concentrationwas seen in the late stages of sperm differentiation, indi-cating the existence of a storage form in transcriptionallyinactive nuclei.

In nucleoli pADPRT is accumulated in foci within thedense fibrillar component. Such foci are seen in closespatial relationship to sites of nucleolar transcription asrevealed by high resolution immunodetection of bromouri-

dine uptake sites. It is suggested that nucleolar pADPRTplays a role in preribosome processing via the modificationof nucleolus specific proteins that bind to nascent tran-scripts and hence indirectly regulates polymerase I activity.The persisting binding of pADPRT to ribonucleoproteinsmay explain the observed disperse enzyme distribution atlower concentrations in the granular component. Thefibrillar centres seem to contain no pADPRT. We concludethat known compounds of fibrillar centres like polymeraseI are unlikely candidates for modification via directcovalent ADP-ribosylation.

Key words: Poly(ADP-ribosyl)transferase, ADP-ribose,Autoimmunity, Immunelectron microscopy, Immunfluorescencemicroscopy, Nucleolus, Ribosomal transcription, Humanspermatogenesis

SUMMARY

INTRODUCTION

Poly(ADP-ribosyl)transferase (pADPRT) catalyses the transferof ADP-ribose moieties to covalent linkages with variousacceptor proteins utilizing NAD+ as a substrate (for review see:Berger, 1985; Ueda and Hayaishi, 1985). pADPRT activity hasbeen found almost ubiquitously among animal tissues, plantsand lower eukaryotic organisms. Enzyme activity is mostlylocalized in the nucleus (Boulikas, 1991, 1993; Malanga andAlthaus, 1994; Nishizuka et al., 1967; Shall, 1994). The majorpart of nuclear pADPRT is located in chromatin in the internu-cleosomal space (Mullins et al., 1977; Niedergang et al., 1985).Enzyme activity has also been found associated with the nuclearmatrix (Wesierska-Gadek and Sauermann, 1985). Someextranuclear activity was detected in the ribosomal (Roberts etal., 1975) and in the mitochondrial fraction (Burzio et al., 1981;Kun et al., 1975; Richter et al., 1983).

This enzyme, strongly inducible by DNA strand breaks hasbeen suggested to play an important role in cellular events suchas DNA replication or DNA repair (Malanga and Althaus,1994; Muller et al., 1994; Yoshida and Simbulan, 1994) in

which breaking and rejoining of DNA strands may occur.There is evidence from different experimental systems thatpADPRT plays an important role in cell differentiation(Althaus et al., 1982; Farzaneh et al., 1982; Ueda et al., 1982).Recently pADPRT was found to modify specific nucleolarproteins in the nucleolus (Leitinger and Wesierska-Gadek,1993). The specific activity was comparable to that in wholenuclei thereby indicating that the nucleolar activity was not dueto chromatin contamination.

Since in the nucleolus at least three morphologically distinctcomponents can be defined and attributed to steps of ribosomalbiogenesis, we were interested in the distribution of pADPRTwithin the ultrastructural components of nucleoli in different celltypes. Using human testes for high resolution immunohisto-chemical studies, we were also able to trace the physicalrearrangements of pADPRT during sperm differentiation in situ.

MATERIALS AND METHODS

Anti-pADPRT antibodiesSerum was obtained from a patient who, following an allogenic bone

410 W. Mosgoeller and others

marrow transplantation (BMT), developed clinical complicationsdefined as graft versus host disease. He had been conditioned by totalbody irradiation (single dose of 3 Gy). While sera obtained pre-BMTand one and two months after transplantation were negative for anti-nuclear antibodies, specimens obtained at day 100 and thereafterrevealed a strong nuclear and nucleolar staining pattern. In this serum,which displayed homogenous nuclear and strong nucleolar staining inindirect immunofluorescence, anti-pADPRT and anti-histone H1autoantibodies were detected (data not shown). To purify specific anti-pADPRT we applied immunoaffinity chromatography.

Purification of human pADPRTThe full-length human pADPRT was overproduced in insect cellsusing a baculovirus expression vector system (Giner et al., 1992).Infected insect Sf9 cells were lysed twice in a hypotonic buffer andcombined lysates were chromatographically fractionated in two con-secutive steps: on DNA-agarose (Sigma) and on Blue-Sepharose.Proteins eluted from DNA-agarose with buffer containing 1 M NaClwere diluted to a final concentration of 200 mM NaCl and loaded ona Blue-Trisacryl column (Pharmacia). pADPRT was eluted at a con-centration of 400 mM NaCl. To check the purity of isolated pADPRT,eluates were analysed on 10% SDS-polyacrylamide gels. Proteinswere visualised by Coomassie Blue and silver staining.

Covalent immobilization of pADPRTPurified to homogeneity human pADPRT was immobilized on MiniLeak Medium beads (Kem-En-Tec, Copenhagen, Denmark)according to the manufacturer’s instructions. Mini Leak is a divinylsulfone activated matrix of spherical agarose especially designed forgentle immobilization of biomolecules. pADPRT diluted withphosphate buffered saline (PBS) to a concentration of 0.5 mg/ml wasadded to the activated matrix and diluted with coupling buffer con-taining 30% polyethylene glycol 20,000 (PEG) to a final concentra-tion of 10% PEG and was incubated overnight. The supernatant wasdiscarded, the matrix was then washed and incubated with 0.2 Methanolamine-HCl, pH 9.0, for 4 hours to block excess active groups.Coupling yield was estimated by measurement of optical density at280 nm before and after coupling reaction.

Immunoaffinity chromatographyA 500 µl sample of patient’s serum was 5-fold diluted with PBS andloaded on a preequilibrated pADPRT affinity column. The columnwas washed with PBS until the basic level of optical density wasreached. Then autoantibodies were eluted with 150 mM NaCl inphosphate buffer (pH 3.0) and immediately neutralized with 2 M Tris-HCl, pH 9.0.

Purified antibodies were examined by immunoblotting using totalnuclear proteins from mycoplasma free HeLa cells and isolatedhuman pADPRT as antigen source. For control, affinity-purified anti-pADPRT antibodies were incubated for 1 hour at room temperaturewith purified pADPRT. The samples were centrifuged and clearsupernatant was used for immunoblotting.

ImmunostainingFor light microscopic (LM) investigations cells were grown oncoverslips, rinsed with PBS, fixed in 100% acetone for 10 minutes at−20°C, air dried and extracted in 0.2% Triton X-100 in PBS, pH 7.4,for 2.5 minutes at RT (room temperature) washed twice and kept inPBST (PBS, 0.025% Tween-20, pH 7.4) with 1% (w/v) BSA (bovineserum albumin Fraction V, Boehringer) added. Subsequently themonolayer was incubated in the purified anti-pADPRT serum for 60minutes at 37°C diluted 1:10 to a final antibody concentration of 0.03µg/µl in 1% BSA-PBST. After 3 washes in PBST and incubation withgoat anti-human FITC-labelled antibody (Sigma) diluted 1:100 in 1%BSA-PBST for one hour at room temperature, the coverslips werewashed again and mounted with anti-fade medium (Citiflour).Immunfluorescence signal was detected either by conventional flu-

orescence-microscopy (Leitz-Dialux) or by optical sectioning in aconfocal laser scanning microscope followed by digital imageenhancement (LSM-Zeiss).

For electron microscopic (EM) preparations HeLa cells grown oncoverslips were fixed with 2% freshly depolymerized paraformalde-hyde, with 0.5% glutaraldehyde added in 0.1 M sodium phosphatebuffer, pH 7.4, washed in buffer, postfixed in 1% OsO4, washed,dehydrated and flat embedded according to the method of Steiner atal. (1994). Testis material was obtained, fixed in aldehydes only andprocessed as earlier (Mosgoeller et al., 1993).

Thin sections on gold grids of the osmified material were deosmi-fied in 2% INaO4 in distilled water for 40 minutes at RT and washedextensively in distilled water. Following incubation with 5% BSA(w/v) in PBST the grids were kept in the anti-pADPRT antibodiesovernight in a moist chamber. Secondary antibodies were sheep anti-human Fab fragments labelled with TRITC (Boehringer) diluted 1:40in 1% BSA-PBST. The third antibody incubation was in rabbit anti-sheep IgG coupled with 5 nm gold (Chemicon) diluted 1:40 in 3%BSA-PBS (pH 8.2). The grids were washed 4 times in PBS (pH 8.2)followed by 6 washes in distilled water, which was pH adjusted to 8.0by the addition of a few grains of Tris (Merck). Following air dryingthe sections were contrasted in 1% (w/v) aqueous uranylacetate for 3minutes at room temperature, air dried again and investigated in atransmission electron microscope (Jeoul, EM 1200).

Controls were performed by omitting the first or second antibodyand absorption of the primary antibody by addition of excess purifiedenzyme. In all cases no significant label was observed.

During the evaluation of the ultrastructural images it becameevident that the grain distribution over some microscopic structureswas not random but tended to cluster. For the sake of objective dataevaluation we defined a signal cluster as an accumulation of a certainnumber of grains within a field of 50 nm in diameter. The number ofgrains was chosen for each experiment so that in correspondingcontrols using non immunogenic incubation less than 2.5% of theinvestigated cellular substructures would be scored as labelled byclusters. This procedure inherently controls also the quality ofsecondary gold coupled antibodies that tend to cluster when aged.

Double labelling with nascent RNAHeLa cells were incubated with BrUTP fixed with aldehydes andembedded as described by Hozak et al. (1994). Thin sections on goldgrids were incubated in a mixture of mouse anti-BrdUTP IgG(Boehringer) diluted 1:5 in PBST with 5% (w/v) BSA and the anti-pADPRT serum, overnight in a moist chamber at room temperature.After three washes in PBS and preincubation for unspecific proteinbinding the grids were incubated with sheep IgG-Fab fragmentsspecific for the human immunglobulins (diluted 1:30 in 1% BSA-PBST, pH 7.4) for two hours. A third incubation was in polyclonalimmunoabsorption purified donkey anti-sheep IgG diluted 1:20 in 3%BSA-PBST, pH 8.2, coupled with 5 nm gold. The detection for theprimary anti-BrdUTP antibody was performed consecutively.Following 3 washes in PBS (pH 8.2) and blocking of unspecificbinding sites with 5% (v/v) normal rabbit serum in 0.2 M sodiumphosphate buffer (pH 7.5) the next incubation was with rabbit anti-mouse IgG coupled to 10 nm gold (Dako) diluted 1:60 in the blockingbuffer. All incubation times with secondary antibodies varied fromone to three hours and were performed at room temperature. The gridswere finally washed 6 times in 0.2 M phosphate buffer (pH 7.5) and6 times in double distilled boiled water (pH 8.2) and processed asabove.

For the double labelling additional controls were performed: (1) toevaluate the interaction and colocalization of the secondary antibodiesone or both of the primary antibodies were exchanged by non-immuneserum. In this case we could not observe interaction of the secondaryantibodies. (2) To rule out unspecific signal due to interaction of thefirst antibodies, both staining protocols were performed separately onconsecutive sections with one of them being turned upside down to

411Immunological pADPRT detection

enable a one sided ‘face en face’ staining. After superimposing thetwo corresponding face en face images of the same cells, the labeldistribution of both protocols gave the same result as the doublestained sections.

StatisticsAt the EM level it was difficult to judge the intensity of the colloidalgold stain in mitochondria due to very low signal. Similar to previ-ously described statistical techniques (Mosgoeller et al., 1993) grain

Fig. 1. Purification of human pADPRT and examination of affinity-purified anti-pADPRT antibodies. Proteins were separated on 10%SDS-polyacrylamide gels. M, marker proteins: 200 kDa myosin; 116kDa β-galactosidase; 94 kDa phosphorylase b; 68 kDa bovine serumalbumin; 55 kDa glutamic dehydrogenase; 36 kDa lactatedehydrogenase; 31 kDa carbonic anhydrase. Lane 1, lysate of controlinsect cells; lane 2, first lysate of transfected insects cells; lane 3,second lysate of transfected cells; lane 4, 1 M NaCl eluate fromDNA agarose column; lane 5, 400 mM NaCl eluate from Blue-Trisacryl column; lanes 6 and 7, proteins transferred onto membraneand Ponceau red stained; lanes 8 and 9, immunoblotting usingaffinity-purified anti-pADPRT antibodies in a dilution of 1:500;lanes 6 and 8, nuclear proteins of HeLa cells; lanes 7 and 9,chromatographically purified human pADPRT.

kDa

Fig. 2. HeLa cells after fluorescent staining of pADPRT (A) and DNA (laser microscope and digital contrast enhancement (C). In the standard flstained. The nucleoplasm, in particular the peripheral nuclear region andnucleoli are recognized as dark regions surrounded by bright fluorescenthe cytoplasm, arrowheads mark the signal along the nuclear membrane

densities were computed over a representative number of cytoplasmicsections. The Student’s t-test (Sokal and Rohlf, 1981) was used toestimate the significance of label differences between mitochondriaand the surrounding cytoplasm.

RESULTS

Anti-pADPRT serum purificationFig. 1, lane 5 shows the human pADPRT after expression inthe baculovirus system and chromatographical purification tohomogeneity, immobilized on activated agarose beads. Thisactivated matrix is especially designed for gentle coupling ofbiomolecules under mild conditions. The affinity-purified anti-pADPRT antibodies were then tested in immunoblotting usingtotal nuclear proteins of HeLa cells and purified enzyme (Fig.1, lanes 8 and 9).

Cytoplasmic immuno-pADPRT-staining in HeLacells and Sertoli cellsFig. 2 shows a HeLa cell after immunofluorescent pADPRTstaining. The cytoplasm reveals regional differences in labeldensity, particularly well seen in the confocal scanning opticalsection (Fig. 2C). Bright fluorescence appears around thenuclear envelope. Within the nuclear centre there are confinedregions with signal of a rather homogeneous fluorescence.Most of these homogeneously stained regions have anucleolus-like shape. Occasionally in some nuclei some flu-orescent spots can be recognized in addition to nucleolar signal(Fig. 2C).

After EM immunostaining of LR-White thin sections andcolloidal gold detection at the ultrastructural level, the signal dis-tribution was similar in both HeLa cells and Sertoli cells. In thecytoplasm a few dispersed grains were observed only occasion-ally. Although many mitochondrial sections were free of label,the signal density was, on average, significantly highercompared to the cytoplasmic staining (Fig. 3). We measured theaverage grain density over more than one hundred mitochon-

B), and optical sectioning of the same preparation using a confocaluorescent image (A) the nucleoli appear almost homogeneously the adjacent cytoplasm reveal fluorescence. In the DAPI image (B) the

ce. In the optical section (C) arrows point out bright fluorescent spots in. Bar, 10 µm.

412 W. Mosgoeller and others

Fig. 3. Cytoplasm with two mitochondria of a human Sertoli cellafter EM immunostaining and 5 nm colloidal gold detection ofpADPRT. One mitochondrium is labeled in the central compartment.Bar, 0.2 µm.

Fig. 4. HeLa cell after EM-pADPRT staining. Arrowheads indicatelabel over the entire nucleoplasm (np) which is more intense close tothe nuclear envelope (ne). Bar, 0.5 µm.

drial and extramitochondrial regions. By means of the Student’st-test the average mitochondrial labelling (30.1±23.0 s.d.grains/µm) was found to be significantly higher (P<0.001) ascompared with surrounding cytoplasm (5.2±8.0 s.d. grains/µm).

Chromatin stainingLM optical sections showed brightly fluorescing dots at theperipheral nucleus (Fig. 2C). At the EM level all of thechromatin was labelled with increasing signal density over thechromatin associated with the nuclear membrane (Fig. 4) andnucleolus associated chromatin (Figs 5-7). The interchromatinspace is more or less devoid of signal. Occasional chromatininclusions in nucleoli were also labelled.

In spermatogonia the labelling distribution was similar toHeLa cells. The gold grains decorated chromatin all over thenucleus (Fig. 7). In the course of meiosis and in later stages ofsperm differentiation, the grain density increases as the con-densation of chromatin advances to form chromatids and lateron the highly compacted nucleus of the spermatid (Fig. 8A-C).The synaptonemal complex remains free of label (Fig. 8A).The highest grain density in our material was observed overthe compacted chromatin of spermatids (Fig. 8C).

Nucleolar stainingThe standard light microscopic fluorescent images reveal ahomogeneous signal emitted from the nucleolus (Fig. 2A). Thenucleolar signal density after confocal scanning imagerecording and digital contrast enhancement shows regionalintensity differences (Fig. 2C). At the EM level we observedgrain cluster over the DF and occasionally some grains overthe GC. We did not see label clearly attributable to fibrillarcentres (FCs). However, the nucleolus of HeLa cells is a verydynamic structure which is reflected in a rather complexarrangement of components. In a major part of the sectionssignal allocation to a particular component can be difficult andin some cases not possible. For a better ultrastructural resol-ution of nucleolar components the human Sertoli cell providesa good model due to a very large FC and a distinct segregationof the other components (Cataldo et al., 1988). Fig. 6 shows asection of a Sertoli cell revealing all three nucleolar compo-nents. The FC and the major part of the strands of DF are free

of label. However, within the DF there are confined regionsdecorated by gold clusters. No such clusters were observedover the granular component, which typically revealed fewsingle grains dispersed over the section. A similar label distri-bution was also seen in nucleoli of spermatogonia (Fig. 7).

Colocalization of pADPRT and nucleolartranscription In HeLa cells that were incubated with a short pulse of BrUTPprior to fixation and embedding, we were able to performdouble labelling for nascent transcription sites and pADPRTsimultaneously using different gold grain sizes as markers.Within the nucleoli we found many more foci revealing incor-poration of BrUTP as compared with the labelling sites forpADPRT. To investigate the relation of transcription foci tofocal accumulations of pADPRT we scored more than ahundred nucleoli. Not in a single case were the two grain sizesfrom the different detection systems found intermingled aswould be expected in a mixed cluster. Table 1 gives a summaryof the evaluation. Typically, labelling clusters were observedover the DF but independent from each other. In 11% of allcluster events the two different grain sizes were arranged in aside by side manner as shown in Fig. 9.

DISCUSSION

pADPRT antibodiesInterestingly, the native serum did not react with B23, C23,

413Immunological pADPRT detection

Fig. 5. HeLa cell nucleolus after EM-pADPRT staining. Arrowheads indicategold grains in chromatin and densefibrillar component (df). Bar, 0.6 µm.

Fig. 6. Sertoli cell nucleolus after EM-pADPRT staining. Arrowheads indicatelabel in the chromatin, the densefibrillar component (df) and a fewsingle grains in the granular component(gc). The fibrillar centre (fc) is notlabelled. Bar, 0.5 µm.

414 W. Mosgoeller and others

Table 1. Colocalization of BrUTP uptake and pADPRT pADPRT + Negative control +

BrUTP BrUTPn=127 n=111

Independent pADPRT cluster 18.9% 1.8%Independent BrUTP cluster 70.1% 98.2%Side by side (2 clusters within 100 nm) 11.0% 0%

Fig. 7. pADPRT EM-immunostainingof a spermatogonium nucleolusattached to the nuclear envelope (ne).Arrowheads indicate signal in thechromatin and in the dense fibrillarcomponent (df). Bar, 0.5 µm.

RNA polymerase I and fibrillarin (data not shown), commonnucleolar antigens in human pathology (Wesierska-Gadek etal., 1992), but revealed strong reactivity with nucleolarpADPRT.

The immunoabsorbed serum reacted solely with the enzymethereby demonstrating its purity and specificity. Due to theorigin of the anti-pADPRT serum we consider them of highspecificity and reactivity with naturally occurring epitopes andhence for cytobiological studies superior to monoclonal anti-bodies.

Cytoplasmic stainOur LM data (Fig. 2A,C) show that a considerable amount ofpADPRT is contained in the cytoplasm. Using optical section-ing (Fig. 2C) it becomes apparent that the enzyme is concen-trated in some small structure. A statistical test at the EM levelsuggests that the major portion of cytoplasmic pADPRTresides in the mitochondria (Fig. 3). The label distribution oncross sections suggests that the enzyme resides in central com-partments and not at the outer membrane. The fixation andembedding of our material was designed to preserve anti-genicity which naturally compromises on structure preserva-tion. Although membranes are difficult to distinguish, the outermitochondrial membrane can be clearly located. It seems thatwe visualized pADPRT which has been found in the innermembrane by biochemical means (Burzio et al., 1981; Richterand Schlegel, 1993), where it binds to the mitochondrialprotein-DNA complex (Masmoudi et al., 1993).

Nucleolar localization of pADPRT The fluorescent pattern in the nucleoli and the size of labelledregions indicates that the enzyme resides in more than onenucleolar component (Fig. 2A). When superimposing bothimages the pADPRT-fluorescence covers the entire nucleolus(as recognized in the DAPI staining) and some adjacent DNArich chromatin (see Fig. 2A and B). The comparison of signalintensity of the pADPRT staining with the correspondingDAPI-DNA stain (Fig. 2A,B) suggests that within thenucleolus the enzyme exists in a form not associated with DNAand/or in a concentration highly in excess of DNA.

At the ultrastructural level we were able to observe twodifferent labelling patterns in DF and GC of the nucleolus. Thetypical cluster-like arrangement of gold grains suggests a focalaccumulation of pADPRT within confined regions of the DF(Figs 5-7, 9). The major portion of DF was devoid of label; weestimate that in Sertoli cells less than 10% of the entire DFcontains the enzyme. Wachtler et al. (1989) could show thatthe DF is inhomogeneous by means of nucleolus specific silverstaining. In agreement with this, a previous observation on thedistribution of DNA in nucleoli (Mosgoeller et al., 1993) andthe present study clearly show that the DF as recognized inroutine electron micrographs consists of domains withdifferent molecular compositions. This kind of heterogeneitywithin the nucleolus seems to occur in the DF only; in our datawe have no evidence for a similar molecular focality in theother components, i.e. FCs or GCs.

Fakan et al. (1988) succeeded in locating pADPRT onnascent transcripts as visualized on chromatin spread prepara-tions. With the technique available at that time it was notpossible to investigate intranucleolar transcription. Wecombined the recently developed non-autoradiographic highresolution detection of rDNA transcription (Hozak et al., 1994;Schöfer et al., 1993) with EM pADPRT immunostaining. Thisapproach enabled us to directly visualize nucleolar transcrip-tion and pADPRT simultaneously in situ, without spreadinginduced artifacts. We found a close relationship between

415Immunological pADPRT detection

Fig. 8. Stages of chromatincondensation during spermiogenesis.In spermatocytes I (A) when DNAcondenses around the synaptonemalcomplex (sc) the concentration ofimmunogold-detected pADPRT alsoincreases in the surrounding region. Inlater stages (B) when chromosomestake shape the condensed chromatin isheavily labelled by gold particles.Little or no label is seen in thenucleoplasm (np). In final stages ofsperm differentiation (C) the highestsignal density is seen over the mosttightly packed chromatin of thespermatid. Bar, 0.5 µm.

pADPRT foci and nascent intranucleolar RNA in the DF (Fig.9). Our data do not allow us to suggest a strict colocalizationof pADPRT sites and transcription foci in one point, since thegrain cluster of the different labelling systems did not overlap.Our interpretation of the label distribution in double labellingexperiments would rather be in favour of a side by sidearrangement of individual foci, which theoretically may reflectan involvement of pADPRT sites either before or directly aftertranscription.

The nascent rRNA has several binding sites for nucleolinand B23 (Dumbar et al., 1989; Ghisolfi et al., 1990). Sincesome binding sites of nucleolin depend on a native hair pinloop structure of ribosomal RNA and most binding sites arelocalized in the 18 S and 28 S sequence, an important role ofthe protein in the preribosome assembly was suggested(Ghisolfi et al., 1990). Interestingly enough, both proteins havebeen shown to be poly(ADP-ribose) acceptors (Leitinger andWesierska-Gadek, 1993) despite their acid nature. Since

416 W. Mosgoeller and others

Fig. 9. Section of a HeLa cell nucleolus afterBrUTP incorporation and EM double immuno-staining of BrUTP incorporation sites (largegrains) and pADPRT (small grains). Focalaccumulations of pADPRT-label (arrowheads)and BrUTP-label (arrows) may occur completelyindependent of each other within the densefibrillar component (df) or may be arranged sideby side. The fibrillar centre (fc) is not labelled.Bar, 0.5 µm.

intranucleolar transcription almost exclusively accounts forribosomal RNA production (Hozak et al., 1993) we suggestthat most if not all of the pADPRT, which we detected in theDF, might be associated with nascent ribosomal RNA. Theassociation may be mediated by proteins like B23 or nucleolin.In eukaryotic cells it was not possible to show a direct modi-fication of polymerase I by pADPRT (Momii and Koide, 1982)although some kind of mutual dependence between the twoenzymes has been established (Taniguchi et al., 1982).However, the time course given for the pADPRT induced RNApolymerase I downregulation by Taniguchi et al. (1982) ishighly suggestive of an indirect mechanism.

The binding of pADPRT to ribonucleoproteins (RNPs)seems to persist throughout later stages of ribosome biogen-esis in the GC (Figs 6, 7) and even to the stage of cytoplasmicribosomes where pADPRT activity has been described(Roberts et al., 1975). We did find pADPRT in the GC, whereit appears to be distributed at random over the entirecomponent (Figs 6, 7). However, the concentrations asrevealed by immunochemisty are low.

We used cells with nucleoli that have clearly distinctfibrillar centres. However, after evaluating a large number ofsections in the different cell types used, we were not able todetect significant amounts of pADPRT in the FCs. Thefunction of FCs in nucleoli is still a matter of controversialdiscussion (for review see Schwarzacher and Wachtler, 1993).FCs are a highly dynamic structure that change shape, numberand size during nucleolar activation (Hozak et al., 1989).Although they contain many different proteins (including tran-scription enzymes) they do not necessarily participate in tran-scription or transcript processing (Hozak, 1995). Since thereis also no nucleosomal DNA present in this component(Derenzini et al., 1985), this might explain the low concen-tration or absence of pADPRT in FCs. Furthermore, our datasuggest that no other proteins localized in FCs (e.g. poly-merase I, topoisomerase I) are typical candidates for pADPRTdependent modification.

Chromatin associated pADPRT Our results concerning the distribution of pADPRT inchromatin are in agreement with those of Fakan et al. (1988).It is distributed at random in the condensed parts (Figs 4, 6).The enzyme may be located in the internucleosomal space(Mullins et al., 1977; Niedergang et al., 1985) where it may beinvolved in a histone shuttling mechanism (Althaus et al.,1994; Wesierska-Gadek and Sauermann, 1988) or in chromatinrelaxation (de Murcia et al., 1988). Furthermore, there are indi-cations that pADPRT can be associated with nuclear matrixproteins (Wesierska-Gadek and Sauermann, 1985). Duringmeiosis the pADPRT label density gives a good reflection ofthe grade of chromatin condensation (Fig. 8A-C). This char-acteristic may well account for the relatively high signal innucleolus associated condensed chromatin (Fig. 6) and inchromatin near the nuclear envelope (Fig. 4).

There is evidence from different cell systems that pADPRTplays an important role in cell differentiation (Althaus et al.,1982; Farzaneh et al., 1982) without being dependent on cell pro-liferation (Ueda et al., 1982). Interestingly pADPRT remainschromatin associated throughout human sperm differentiation.The highest enzyme concentrations we observed occurred in thedifferentiated sperm head (Fig. 8C). Transcription processes(Simbulan et al., 1993; Yoshida and Simbulan, 1994) or involve-ment in gene expression regulation (Qu et al., 1994; Yamagoe etal., 1991) are unlikely to occur at this late stage of spermato-genesis in inactive cells. During spermiogenesis the nucleolusdecreases in activity and size, while the chromatin condenses assperm maturation advances (Kierszenbaum and Tres, 1978; Stahlet al., 1991). The function of the enzyme at this stage remainsunclear. It may be a ‘dormant, stand-by form’ awaiting involve-ment in chromatin relaxation (Aubin et al., 1983; Niedergang etal., 1985). More likely it may bind to DNA breaks (Gradwohl etal., 1990; Ikejima et al., 1990) that decrease constraints on thesupercoiled DNA. In this case it would be crucially involved inDNA repair and strand ligation (Creissen and Shall, 1982) beforethe chromatin can be decondensed and the genes reactivated.

417Immunological pADPRT detection

This work was supported by: the Oesterreichische Nationalbank,grant no. 4478; the Welcome Trust; the Grant Agency of the Academyof Sciences of the Czech Republic (no. 539402); and the GrantAgency of the Czech Republic (no. 304/94/0148). We are grateful toDr G. de Murcia for the most generous gift of the baculovirusconstruct, expressing the full length human pADPRT. We thank DrP. Kier for the serum and Dr H. G. Schwarzacher and Dr F. Wachtlerfor helpful discussion and comments.

REFERENCES

Althaus, F. R., Lawrence, S. D., He, Y. Z., Sattler, G. L., Tsukada, Y. andPitot, H. C. (1982). Effects of altered [ADP-ribose]n metabolism onexpression of fetal functions by adult hepatocytes. Nature 300, 366-368.

Althaus, F. R., Hofferer, L., Kleczkowska, H. E., Malanga, M., Naegeli, H.,Panzeter, P. L. and Realini, C. A. (1994). Histone shuttling by poly ADP-ribosylation. Mol. Cell. Biochem. 138, 53-59.

Aubin, R. J., Frechette, A., de Murcia, G., Mandel, P., Lord, A., Grondin,G. and Poirier, G. G. (1983). Correlation between endogenous nucleosomalhyper(ADP-ribosyl)ation of histone H1 and the induction of chromatinrelaxation. EMBO J. 2, 1685-1693.

Berger, N. A. (1985). Symposium: cellular response to DNA damage: the roleof poly(ADP-ribose). Radiat. Res. 101, 4-15.

Boulikas, T. (1991). Relation between carcinogenesis, chromatin structure andpoly(ADP-ribosylation) (review). Anticancer Res. 11, 489-527.

Boulikas, T. (1993). Poly(ADP-ribosyl)ation, DNA strand breaks, chromatinand cancer. Toxicol. Lett. 67, 129-150.

Burzio, L. O., Saez, L. and Cornejo, R. (1981). Poly(ADP-ribose) synthetaseactivity in rat testis mitochondria. Biochem. Biophys. Res. Commun. 103,369-375.

Cataldo, C., Souchier, C. and Stahl, A. (1988). Three dimensionalultrastructure and quantitative analysis of the human Sertoli cell nucleolus.Biol. Cell 63, 277-285.

Creissen, D. and Shall, S. (1982). Regulation of DNA ligase activity bypoly(ADP-ribose). Nature 296, 271-272.

de Murcia, G., Huletsky, A. and Poirier, G. G. (1988). Modulation ofchromatin structure by poly(ADP-ribosyl)ation. Biochem. Cell Biol. 66, 626-635.

Derenzini, M., Pession, A., Licastro, F. and Novello, F. (1985). Electronmicroscopic evidence that ribosomal chromatin of human circulatinglymphocytes is devoid of histones. Exp. Cell Res. 157, 50-62.

Dumbar, T. S., Gentry, G. A. and Olson, M. O. J. (1989). Interaction ofnucleolar phosphoprotein B23 with nucleic acids. Biochemistry 28, 9495-9501.

Fakan, S., Leduc, Y., Lamarre, D., Brunet, G. and Poirier, G. G. (1988).Immunoelectron microscopical distribution of poly(ADP-ribose)polymerasein the mammalian cell nucleus. Exp. Cell Res. 179, 517-526.

Farzaneh, F., Zalin, R., Brill, D. and Shall, S. (1982). DNA strand breaks andADP-ribosyl transferase activation during cell differentiation. Nature 300,362-366.

Ghisolfi, L., Joseph, G., Erard, M., Escoubas, J. M., Mathieu, C. andAmalric, F. (1990). Nucleolin - pre-rRNA interactions and preribosomeassembly. Mol. Biol. Rep. 14, 113-114.

Giner, H., Simion, F., de Murcia, G. and Menissier de Murcia, J. (1992).Overproduction and large-scale purification of the human poly(ADP-ribose)polymerase using a baculovirus expression system. Gene 114, 279-283.

Gradwohl, G., Menissier de Murcia, J. M., Molinete, M., Simonin, F.,Koken, M., Hoeijmakers, J. H. and de Murcia, G. (1990). The secondzinc-finger domain of poly(ADP-ribose) polymerase determines specificityfor single-stranded breaks in DNA. Proc. Nat. Acad. Sci. USA 87, 2990-2994.

Hozak, P., Novak, J. T. and Smetana, K. (1989). Three-dimensionalreconstruction of nucleolus-organizing regions in PHA-stimulated humanlymphocytes. Biol. Cell 66, 225-233.

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

Hozak, P., Cook, P. R., Schöfer, C., Mosgöller, W. and Wachtler, F. (1994).Site of transcription of ribosomal RNA and intranucleolar structure in HeLacells. J. Cell Sci. 107, 639-648.

Hozak, P. (1995). Catching RNA polymerase I in flagranti: Ribosomal genes

are transcribed in the dense fibrillar component of the nucleolus. Exp. CellRes. 216, 285-289.

Ikejima, M., Noguchi, S., Yamashita, R., Ogura, T., Sugimura, T., Gill, D.M. and Miwa, M. (1990). The zinc fingers of human poly(ADP-ribose)polymerase are differentially required for the recognition of DNA breaks andnicks and the consequent enzyme activation. Other structures recognizeintact DNA. J. Biol. Chem. 265, 21907-21913.

Kierszenbaum, A. L. and Tres, L. L. (1978). RNA transcription andchromatin structure during meiotic and postmeiotic stages ofspermatogenesis. Fed. Proc. 37, 2512-2516.

Kun, E., Zimber, P. H., Chang, A. C., Puschendorf, B. and Grunicke, H.(1975). Macromolecular enzymatic product of NAD+ in liver mitochondria.Proc. Nat. Acad. Sci. USA 72, 1436-1440.

Leitinger, N. and Wesierska-Gadek, J. (1993). ADP-ribosylation ofnucleolar proteins in HeLa tumor cells. J. Cell Biochem. 52, 153-158.

Malanga, M. and Althaus, F. R. (1994). Poly(ADP-ribose) molecules formedduring DNA repair in vivo. J. Biol. Chem. 269, 17691-17696.

Masmoudi, A., el Fetouaki, J., Weltin, D., Belhadj, O. and Mandel, P.(1993). Association of mitochondrial ADP-ribosyl transferase activity withthe DNA-protein complex. Biochem. Mol. Biol. Int. 29, 77-83.

Momii, A. and Koide, S. S. (1982). RNA synthesis and poly(adenosinediphosphoribosyl) synthetase activity in developing mouse testis. Arch.Androl. 8, 277-283.

Mosgoeller, W., Schöfer, C., Derenzini, M., Steiner, M., Maier, U. andWachtler, F. (1993). Distribution of DNA in human Sertoli cell nucleoli. J.Histochem. Cytochem. 41, 1487-1493.

Muller, S., Briand, J. P., Barakat, S., Lagueux, J., Poirier, G. G., deMurcia, G. and Isenberg, D. A. (1994). Autoantibodies reacting withpoly(ADP-ribose) and with a zinc-finger functional domain of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA. Clin.Immunol. Immunopathol. 73, 187-196.

Mullins, D. W. Jr, Giri, C. P. and Smulson, M. (1977). Poly(adenosinediphosphate-ribose) polymerase: the distribution of a chromosome-associated enzyme within the chromatin substructure. Biochemistry 16, 506-513.

Niedergang, C. P., de Murcia, G., Ittel, M. E., Pouyet, J. and Mandel, P.(1985). Time course of polynucleosome relaxation and ADP-ribosylation.Correlation between relaxation and histone H1 hyper-ADP-ribosylation.Eur. J. Biochem. 146, 185-191.

Nishizuka, Y., Ueda, K., Nakazawa, K. and Hayaishi, O. (1967). Studies onthe polymer of adenosine diphosphate ribose. I. Enzymic formation fromnicotinamide adenine dinuclotide in mammalian nuclei. J. Biol. Chem. 242,3164-3171.

Qu, Z., Fujimoto, S. and Taniguchi, T. (1994). Enhancement of interferon-gamma-induced major histocompatibility complex class II gene expressionby expressing an antisense RNA of poly(ADP-ribose) synthetase. J. Biol.Chem. 269, 5543-5547.

Richter, C., Winterhalter, K. H., Baumhuter, S., Lotscher, H. R. andMoser, B. (1983). ADP-ribosylation in inner membrane of rat livermitochondria. Proc. Nat. Acad. Sci. USA 80, 3188-3192.

Richter, C. and Schlegel, J. (1993). Mitochondrial calcium release induced byprooxidants. Toxicol. Lett. 67, 119-127.

Roberts, J. H., Stard, P., Giri, C. P. and Smulson, M. (1975). Cytoplasmicpoly(ADP-ribose) polymerase during the HeLa cell cycle. Arch. Biochem.Biophys. 171, 305-315.

Schöfer, C., Müller, M., Leitner, M.-D. and Wachtler, F. (1993). The uptakeof uridine in the nucleolus occurs in the dense fibrillar component. Immuno-gold localization of incorporated digoxigenin-UTP at the electronmicroscopic level. Cytogenet. Cell Genet. 64, 27-30.

Schwarzacher, H. G. and Wachtler, F. (1993). The nucleolus. Anat. Embryol.188, 515-536.

Shall, S. (1994). The function of poly (ADP-ribosylation) in DNA breakageand rejoining. Mol. Cell Biochem. 138, 71-75.

Simbulan, C. M., Suzuki, M., Izuta, S., Sakurai, T., Savoysky, E., Kojima,K., Miyahara, K., Shizuta, Y. and Yoshida, S. (1993). Poly(ADP-ribose)polymerase stimulates DNA polymerase alpha by physical association. J.Biol. Chem. 268, 93-99.

Sokal, R. R. and Rohlf, F. J. (1981). Biometry. 2nd edn (ed. J. Wilson and S.Cotter), pp. 145-147. W. H. Freeman and Company, New York.

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

Steiner, M., Schöfer, C. and Mosgoeller, W. (1994). In situ flat embedding of

418 W. Mosgoeller and others

monolayers and cell relocation in the acrylic resin LR White for comparativelight and electron microscopy studies. Histochem. J. 26, 934-938.

Taniguchi, T., Agemori, M., Kameshita, I., Nishikimi, M. and Shizuta, Y.(1982). Participation of poly(ADP-ribosyl)ation in the depression of RNAsynthesis caused by treatment of mouse lymphoma cells withmethylnitrosourea. J. Biol. Chem. 257, 4027-4030.

Ueda, K., Ogata, N., Kawaichi, M., Inada, S. and Hayaishi, O. (1982). ADP-ribosylation reactions. Curr. Top. Cell Regul. 21, 175-187.

Ueda, K. and Hayaishi, O. (1985). ADP-ribosylation. Annu. Rev. Biochem.54, 73-100.

Wachtler, F., Hartung, M., Devictor, M., Wiegant, J., Stahl, A. andSchwarzacher, H. (1989). Ribosomal DNA is located and transcribed in thedense fibrillar component of human Sertoli cell nucleoli. Exp. Cell Res. 184,61-71.

Wesierska-Gadek, J. and Sauermann, G. (1985). Modification of nuclear

matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix. Eur. J. Biochem. 153, 421-428.

Wesierska-Gadek, J. and Sauermann, G. (1988). The effect of poly(ADP-ribose) on interactions of DNA with histones H1, H3 and H4. Eur. J.Biochem. 173, 675-679.

Wesierska-Gadek, J., Penner, E., Hitchman, E., Kier, P. and Sauermann,G. (1992). Nucleolar proteins B23 and C23 as target antigens in chronicgraft-versus-host disease. Blood 79, 1081-1086.

Yamagoe, S., Kohda, T. and Oishi, M. (1991). Poly(ADP-ribose) polymeraseinhibitors suppress UV-induced human immunodeficiency virus type 1 geneexpression at the posttranscriptional level. Mol. Cell Biol. 11, 3522-3527.

Yoshida, S. and Simbulan, C. M. G. (1994). Interaction of poly(ADP-ribose)polymerase with DNA polymerase. Mol. Cell Biochem. 138, 39-44.

(Received 4 September 1995 - Accepted 13 November 1995)