the transcription unit of ribosomal genes is attached to the nuclear skeleton

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EXPERIMENTAL CELL RESEARCH 227, 374–379 (1996) ARTICLE NO. 0287 The Transcription Unit of Ribosomal Genes Is Attached to the Nuclear Skeleton KLARA WEIPOLTSHAMMER,* CHRISTIAN SCHO ¨ FER,* FRANZ WACHTLER,* AND PAVEL HOZA ´ K² ,1 *Institute of Histology and Embryology, University of Vienna, Schwarzspanierstrasse 17, A-1090 Vienna, Austria; and ²Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vı B den d ska B 1083, 142 20 Prague 4-Krc ˇ, Czech Republic proposed that active polymerases form part of the The relationship between various loci of the ribo- nucleoskeleton while the genes pass through such fixed somal gene repeat and the nucleoskeleton was exam- polymerizing sites [for review, see 22]. Thus, there is ined in agarose-embedded HeLa cells. The accessibil- convincing evidence that the nucleoskeleton is more ity of intranucleolar structures to molecular probes than a passive network with only structural functions. was improved by dispersing the granular component Little is known about the nature of the nucleoskele- of nucleoli, and unattached DNA was removed from ton that is present in nucleoli, sometimes called the permeabilized nuclei under ‘‘physiological’’ conditions nucleolar matrix or skeleton. Nevertheless, a presence by enzymatic digestion and subsequent electroelution. of filamentous skeletal structures in nucleoli has been The cells were then hybridized in situ with various shown [23, 24]. As the nucleoli are places of simultane- human rDNA probes for the transcription unit or for ous transcription of many ribosomal genes, they repre- the intergenic spacer. A strong signal was detected sent a unique and convenient model for studying the with probes for the transcription unit but no signal relationship between DNA and the nucleoskeleton. Bio- was seen with probes for the intergenic spacer. These chemical studies agree on the existence of attachment results show that only the transcription unit is sites in rDNA; however, their character is controversial strongly attached to the nucle(ol)ar skeleton and im- [for review, see 25]. Some authors report that the at- ply that rDNA is probably attached to the skeleton pri- tachment sites are found exclusively in the intergenic marily via RNA polymerase complexes rather than via (nontranscribed) spacer [26 – 28]; others report that sequence-specific attachment sites. Nucleolar fibrillar they are found both in the intergenic spacer and in the centers, embedded into the nucle(ol)ar skeleton, pro- external transcribed spacer [29]. In contrast, Keppel vide structural support for these attachments. q 1996 [30] found attachments of rDNA, in actively transcrib- Academic Press, Inc. ing cells, within the entire gene repeat and, similarly, no ‘‘universal attachment sequence’’ was identified INTRODUCTION [31]. The discrepancy between results is probably due to the different, and frequently very unphysiological First reports on structures forming a network within and disruptive, procedures used for preparations of the the nucleus—the nuclear matrix or skeleton—date to nuclear matrix [for reviews, see 4, 32]. Using more the early sixties [1–3, for reviews, see 4–7]. Recently, physiological conditions, Dickinson et al. [33] showed some additional components of the nucleoskeleton have in a biochemical experiment that RNA polymerase I been identified [e.g., 8 – 12] and, more importantly, the activity remained within the nucleus when DNA was basic nuclear activities — replication and transcrip- digested and electroeluted out of the cells. This sug- tion—were shown to be connected with the nuclear gested a connection between RNA polymerase I and the matrix. For example, newly synthesized DNA is tightly nucleoskeleton. However, the nucleoli are very dense bound to the nuclear skeleton [2, 13–16]; the replica- structures and the transcribing genes are buried in tion apparatus forms specific nuclear structures — the dense fibrillar component covered by an additional known as ‘‘replication factories’’ — which are attached layer of the granular component. This result could be to the nucleoskeleton [17]. Similarly, transcription oc- explained by retaining the rDNA fragments together curs in discrete nuclear foci [18, 19] and RNA-synthe- with polymerase complexes under the ‘‘cover’’ of the sizing activities as well as splicing complexes are compact nucleolar components. attached to the nucleoskeleton [20, 21]. It has also been We address this question using an alternative in situ approach. HeLa cells were first encapsulated in aga- rose beads, the granular component of nucleoli was dis- 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (/42-2)-475 27 82; E-mail: [email protected]. persed by a brief hypotonic treatment, and unattached 374 0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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EXPERIMENTAL CELL RESEARCH 227, 374–379 (1996)ARTICLE NO. 0287

The Transcription Unit of Ribosomal GenesIs Attached to the Nuclear Skeleton

KLARA WEIPOLTSHAMMER,* CHRISTIAN SCHOFER,* FRANZ WACHTLER,* AND PAVEL HOZAK†,1

*Institute of Histology and Embryology, University of Vienna, Schwarzspanierstrasse 17, A-1090 Vienna, Austria; and †Institute ofExperimental Medicine, Academy of Sciences of the Czech Republic, VıB dend skaB 1083, 142 20 Prague 4-Krc, Czech Republic

proposed that active polymerases form part of theThe relationship between various loci of the ribo- nucleoskeleton while the genes pass through such fixed

somal gene repeat and the nucleoskeleton was exam- polymerizing sites [for review, see 22]. Thus, there isined in agarose-embedded HeLa cells. The accessibil- convincing evidence that the nucleoskeleton is moreity of intranucleolar structures to molecular probes than a passive network with only structural functions.was improved by dispersing the granular component Little is known about the nature of the nucleoskele-of nucleoli, and unattached DNA was removed from ton that is present in nucleoli, sometimes called thepermeabilized nuclei under ‘‘physiological’’ conditions nucleolar matrix or skeleton. Nevertheless, a presenceby enzymatic digestion and subsequent electroelution. of filamentous skeletal structures in nucleoli has beenThe cells were then hybridized in situ with various shown [23, 24]. As the nucleoli are places of simultane-human rDNA probes for the transcription unit or for ous transcription of many ribosomal genes, they repre-the intergenic spacer. A strong signal was detected sent a unique and convenient model for studying thewith probes for the transcription unit but no signal

relationship between DNA and the nucleoskeleton. Bio-was seen with probes for the intergenic spacer. Thesechemical studies agree on the existence of attachmentresults show that only the transcription unit issites in rDNA; however, their character is controversialstrongly attached to the nucle(ol)ar skeleton and im-[for review, see 25]. Some authors report that the at-ply that rDNA is probably attached to the skeleton pri-tachment sites are found exclusively in the intergenicmarily via RNA polymerase complexes rather than via(nontranscribed) spacer [26–28]; others report thatsequence-specific attachment sites. Nucleolar fibrillarthey are found both in the intergenic spacer and in thecenters, embedded into the nucle(ol)ar skeleton, pro-external transcribed spacer [29]. In contrast, Keppelvide structural support for these attachments. q 1996

[30] found attachments of rDNA, in actively transcrib-Academic Press, Inc.

ing cells, within the entire gene repeat and, similarly,no ‘‘universal attachment sequence’’ was identified

INTRODUCTION [31]. The discrepancy between results is probably dueto the different, and frequently very unphysiological

First reports on structures forming a network within and disruptive, procedures used for preparations of thethe nucleus—the nuclear matrix or skeleton—date to nuclear matrix [for reviews, see 4, 32]. Using morethe early sixties [1–3, for reviews, see 4–7]. Recently, physiological conditions, Dickinson et al. [33] showedsome additional components of the nucleoskeleton have in a biochemical experiment that RNA polymerase Ibeen identified [e.g., 8–12] and, more importantly, the activity remained within the nucleus when DNA wasbasic nuclear activities—replication and transcrip- digested and electroeluted out of the cells. This sug-tion—were shown to be connected with the nuclear gested a connection between RNA polymerase I and thematrix. For example, newly synthesized DNA is tightly nucleoskeleton. However, the nucleoli are very densebound to the nuclear skeleton [2, 13–16]; the replica- structures and the transcribing genes are buried intion apparatus forms specific nuclear structures— the dense fibrillar component covered by an additionalknown as ‘‘replication factories’’—which are attached layer of the granular component. This result could beto the nucleoskeleton [17]. Similarly, transcription oc- explained by retaining the rDNA fragments togethercurs in discrete nuclear foci [18, 19] and RNA-synthe- with polymerase complexes under the ‘‘cover’’ of thesizing activities as well as splicing complexes are compact nucleolar components.attached to the nucleoskeleton [20, 21]. It has also been We address this question using an alternative in situ

approach. HeLa cells were first encapsulated in aga-rose beads, the granular component of nucleoli was dis-1 To whom correspondence and reprint requests should be ad-

dressed. Fax: (/42-2)-475 27 82; E-mail: [email protected]. persed by a brief hypotonic treatment, and unattached

3740014-4827/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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375ATTACHMENT SITES IN RIBOSOMAL GENES

chromatin fragments were then removed by electroelu-tion. Remnants of the nucleolar chromatin, stillattached to the nuclear matrix, were then in situ hy-bridized with various DNA probes for the transcribedpart of the ribosomal gene and for the intergenicspacer. Our results show an attachment of the tran-

FIG. 1. Schematic representation of the human ribosomal genescribed part of the ribosomal gene repeat to the nucle(o- repeat showing the positions of the three probes used for in situl)ar skeleton and suggest that the transcriptional com- hybridization. The arrow marks the 45S rRNA precursor that yieldsplexes of RNA polymerase I are responsible for these 18S, 5.8S, and 28S rRNA (solid rectangles). The transcription unit

was detected using EcoRI fragments A and B. The A-fragment con-attachments.tains the 3 * end of 18S rDNA, the 5.8S rDNA, two internal tran-scribed spacers, and most of the 28S rDNA. The B-fragment containsthe 5* external transcribed spacer and most of the 18S rDNA. TheMATERIAL AND METHODSintergenic spacer was detected using a probe cocktail containing theEcoRI fragment C and three smaller probes within the EcoRI frag-Agarose embedding and electroelution. HeLa cells (grown in sus-ment D (DBH, DHH, DHE).pension in S-MEM / 5% fetal calf serum) were washed in PBS,

encapsulated (107 cells/ml) in 0.5% agarose beads [34], grown againfor 2 h, washed in 100 mM Sorensen buffer (SB; Na/K phosphatebuffer, pH 7.3), and incubated (5 min, 377C) in 20 mM SB. This

rabbit (10 mg/ml; Southern Biotechnology). The slides were mountedhypotonic treatment disperses primarily the granular component ofin Citifluor. Photographs were taken with a conventional epifluores-nucleoli while leaving the dense fibrillar component and the fibrillarcence microscope (Leica) or recorded using a confocal laser scanningcenters behind [35, 36]. Encapsulated cells were permeabilized withmicroscope (Bio-Rad MRC 600).0.2% Triton X-100 (10 min on ice) in a ‘‘physiological buffer’’ (PB; pH

Electron microscopy. The three different samples were also fixed7.4, contains 22 mM Na/, 130 mM K/, 1 mM Mg2/, õ0.3 mM freein 2.5% formaldehyde with 0.5% glutaraldehyde in 0.1 M SB, dehy-Ca2/, 67 mM Cl0, 65 mM CH3COO0, 11 mM phosphate, 1 mM ATP,drated, and embedded in LR White. Thin sections were contrasted1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride;with uranyl acetate and lead citrate and photographed with a Jeolafter [34]) and washed four times in ice-cold PB. For permeabilization1200 electron microscope.of hypotonically treated cells, 30% (v/v) PB was used. Cells were then

incubated (20 min, 337C) with EcoRI (2500 u/ml) and HaeIII (500 u/ml) to cut chromatin into Ç10-kb pieces and subjected to electropho- RESULTSresis (4 V/cm, 3.5 h, 47C) in PB to remove Ç95% of chromatin [15].Hypotonically treated cells were electroeluted in 40% (v/v) PB. Sam-

Nucleolar Morphology after the Treatmentples were recovered and fixed in 4% paraformaldehyde in PB of ap-propriate ionic strength. All solutions were prepared with water We compared the nucleolar ultrastructure in un-treated with diethyl pyrocarbonate to minimize RNase activity [37];

treated HeLa cells and in cells after the hypotonichuman placental ribonuclease inhibitor (Amersham) was also addedduring permeabilization (2.5 u/ml), enzymatic digestion (25 u/ml), treatment, detergent permeabilization, DNA cuttingand electroelution (0.25 u/ml). with restriction enzymes, and subsequent electroelu-

In situ hybridization. For subsequent DNA hybridization in situ, tion. The untreated HeLa cell nuclei had the expectedthree samples of cells embedded in agarose beads were produced: (1) nucleolar morphology in electron microscopic sectionscells lysed without any hypotonic pretreatment, (2) cells lysed after

(Fig. 2A): several fibrillar centers, surrounded and con-the spreading of the granular component, and (3) cells lysed afternected with the dense fibrillar component, and boththe spreading of the granular component and electroelution. The

cells in agarose beads were postfixed in methanol and dropped onto embedded in the granular component. In contrast, theglass slides. The same hybridization protocol as in [38] was employed. hypotonic treatment dispersed the granular componentSlides were incubated in RNase A for 1 h. A modification was the of nucleoli, and the subsequent electroelution of cutuse of 0.1% pepsin in 0.01 M HCl for 5 min instead of proteinase K.

DNA fragments then removed Ç95% of chromatin inAfterward, the slides were fixed in 4% paraformaldehyde. After thea typical preparation. The resulting structure consistsapplication of the probe, the specimens were denatured (807C, 10

min) and then hybridized (377C, overnight). Three hybridization of a fibrillar network—the nucleoskeleton, associatedprobes (kindly provided by Dr. J. Sylvester) were used: first, the nuclear bodies, and nucleolar remnants—attached toEcoRI A-fragment of the rDNA gene (containing the 3 * half of 18S this skeleton [17, 24]. Figure 2B shows a typical sectionand most of 28S rDNA, the entire 5.8S rDNA, plus the two internal

through such a nucleus. The nucleolar area can be rec-transcribed spacers); second, the EcoRI B-fragment (containing theognized by the remnants of the granular component.entire 5* external transcribed spacer and the 5* half of 18S rDNA);

and third, a cocktail of several smaller DNA fragments (DBH, DHH, The other two nucleolar components, the fibrillar cen-DHE, and C) stretching over the intergenic spacer (see Fig. 1 for ters and the dense fibrillar component, persist and candetails). All three probes were labeled with digoxigenin by nick trans- be recognized by their typical ultrastructure.lation (Boehringer). The stringent washes were done in 50% for-mamide/SSC (three times 10 min at 427C).

The Transcription Unit and the Intergenic SpacerImmunofluorescence microscopy. The slides were treated with 5%Differ in Attachmentsnonfat dry milk in 41 SSC with 0.05% Tween 20 (20 min). The probes

were visualized by three layers of rhodamine-conjugated antibodiesThe hybridizations were repeated three times; thediluted in the dry milk/SSC solution: sheep anti-digoxigenin (20 mg/

ml; Boehringer), rabbit anti-sheep (20 mg/ml; SAR), and goat anti- results obtained were very consistent. In control cells

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376 WEIPOLTSHAMMER ET AL.

in the case of the A-fragment DNA probe, a slight blur-ring around the intense dots occurs; in general, how-ever, there are no notable differences. In both controlsand hypotonically treated cells, the percentage of in-tensely labeled cells was close to 100%. In contrast,when the hybridization was performed after the DNAdigestion and removal of unattached fragments, thehybridization signals were different with the threeprobes used (Figs. 3G–3I). The B-fragment probe sig-nal did not differ from the signal obtained in the controlor in the hypotonically treated cells. The A-fragmentprobe hybridized to dots in nucleoli as in the hypotoni-cally treated cells—however, these dots were smallerin diameter. The intergenic rDNA probe hybridizationusually did not produce any nucleolar labeling at all;however, a weak signal distributed throughout the nu-cleoplasm was seen. In a small fraction of cells in someexperiments, some residual dot-like signal of variousintensity was seen in the nucleolus.

DISCUSSION

The ApproachOur approach has several advantages when com-

pared with other protocols used so far. First, thenucleoli are very dense structures. Interpretations ofexperiments, relying on a sufficient accessibility of mo-lecular probes, can be therefore problematic. We avoidthis difficulty by the brief hypotonic treatment of non-permeabilized cells that leaves essentially only the fi-brillar centers and the dense fibrillar component inplace of nucleoli; the granular component and hetero-chromatin are largely dispersed. As a result, the intra-nucleolar structures are directly exposed to restrictionenzymes allowing the electroelution to easily removeall unattached DNA fragments. Similarly, the intra-FIG. 2. Electron microscopic comparison of nucleolar morphology

in control and treated HeLa cells. (A) Control HeLa cells embedded nucleolar structures are easily accessible to the DNAin agarose beads. Nucleoli have a typical morphology including the probes used in the subsequent in situ hybridization.fibrillar centers (arrow), the dense fibrillar component (d), and the Second, our approach allows us to test the attachmentgranular component (g). (B) HeLa cells were embedded in agarose

of various DNA sequences directly in situ. Biochemicalbeads, the granular component of nucleoli was dispersed, DNA wascut with restriction enzymes, unattached chromatin fragments were experiments are typically used for such analyses em-electroeluted, and the cells were then processed for electron micros- ploying multiple isolation steps and nonphysiologicalcopy. The nucleolar area (arrowheads) can be recognized by the rem- ionic concentration. These disruptive conditions havenants of the granular component, while the fibrillar nucleolar compo-

been used in most experiments without any structuralnents were preserved in such preparations. Bar, 2 mm.control, and, most important, have been shown to pro-duce artificial aggregations of DNA with thenucleoskeleton [4]. Third, the conditions used in this

fixed without any pretreatment, the hybridization sig- study preserve, under optimal conditions, most tran-nals obtained with the three DNA probes were compa- scriptional and replicational activity of the living cellrable. Depending on the level of optical section, various up to fixation [17, 24, 34]. This provides an additionalnumbers of intense dots in nucleoli were seen (Figs. assurance that structures analyzed are not generated3A–3C). The dots are usually discrete, occasionally artifactually.they are interconnected by bridges of lower intensity.

The Transcription Unit of rDNA Is Attached to aFollowing the brief hypotonic treatment, the resultsNucleoskeletonare similar to the control and the hybridization signals

with all three probes are very intense (Figs. 3D–3F). Detected hybridization signals in untreated cellsgenerally agree with the known focal distribution ofIn some cells, distances between dots are increased and

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377ATTACHMENT SITES IN RIBOSOMAL GENES

FIG. 3. The transcribed and non-transcribed parts of ribosomal gene repeat differ in attachments. Signals obtained after DNA in situhybridization with (A, D, G), the A-fragment probe, (B, E, H) the B-fragment probe, and (C, F, I) a mixture of DNA probes for sequencesstretching over the intergenic spacer. Cells used for in situ hybridization were permeabilized (A–C), permeabilized after the hypotonicspreading of the granular component (D–F), or hybridized following the hypotonic treatment, DNA cutting, and removal of unattachedchromatin fragments (G, H, I). The intergenic rDNA probe hybridization essentially did not produce any nucleolar labeling at all whenhybridized after the electroelution (I). Bar, 5 mm.

rDNA in nucleoli—in the fibrillar centers and/or in scribed intergenic spacer can be rarely detected afterthe electroelution.the dense fibrillar component [38–43]. Comparison of

control and hypotonically treated cells shows that dis- This difference can be explained by three possibilit-ies: First, by differences in number of restriction sitespersion of the granular component did not significantly

change the localization of rDNA. The presented differ- in rDNA due to various DNA sequences; second, bydifferences in accessibility of rDNA to the cutting en-ences observed after the removal of unattached chro-

matin fragments therefore clearly show that the tran- zymes; and third, by differences in attachments of cutfragments. Restriction maps do not support the firstscribed and the non-transcribed parts of the ribosomal

genes differ in attachment to a supporting structure in hypothesis: cutting of rDNA by EcoRI and HaeIII re-sults in small fragments along the entire rDNA. Fornucleoli. While the transcribed portions of the rDNA

are retained in electroeluted nuclei, the non-tran- example, human 28S rRNA gene (source: GBPRI:hum-

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378 WEIPOLTSHAMMER ET AL.

rgna) is cut at 14 sites, 18S rRNA gene (source: nucleolar architecture as they are so numerous andrelatively strong—they survived all the experimentalGBPRI:humrge) at 15 sites, the intergenic spacer

(source: GBPRI:hsrspac) at 25 sites. The second hy- treatments. The attachments via polymerase com-plexes probably are complemented by additional typespothesis could provide a more plausible explanation.

Actively transcribed chromatin is generally character- of attachment sites. For example, the attachment siteslocated in non-transcribed parts of the ribosomal re-ized as DNase I sensitive, i.e., accessible. This should

be also true for the active ribosomal genes known to peat [26–29, 52, 53] were suggested to have supportingfunctions in organizing the non-transcribed sequencesbe highly extended and probably in a nonnucleosomal

form [27, 44]. However, in our experiments we saw the and/or other functions, related to modulation of tran-scription, initiation of DNA replication, or recombina-opposite: the transcribed rDNA was retained in nuclei

after digestion. Loading of active ribosomal genes with tion [53].polymerases and accessory proteins with attached na-scent transcripts could contribute to the ‘‘hiding’’ of The Fibrillar Centers: Primary Elements of Nucleolarsome potential restriction sites in rDNA and prevent Architecturethem from being cut. We therefore used such conditions

The fibrillar centers are the only nucleolar structurefor DNA restriction that ensured practically all accessi-remaining associated with rDNA when the nucleolusble sites within the ribosomal gene repeat were cutis disassembled during mitosis [54–56]; new nucleoli[33]. Therefore, there must be a mechanism preventingthen form around them. After removing the granularthe cut fragments from electroelution. The third hy-component, most of the dense fibrillar component, chro-pothesis suggests the most plausible explanation: thematin, and fibrillar centers remain at nodes in the nu-rDNA fragments are prevented from the electroelutioncle(ol)ar skeleton. This suggested an additional struc-by an attachment to some larger structure.tural role for the fibrillar centers, perhaps a centralcomponent nucleating the formation of the radial net-

Is the Transcription Complex Responsible for the work that connects the nucleolus with the nuclear lam-Attachment? ina [24]. Transcription of rDNA takes place on the sur-

face of the fibrillar centers and in the dense fibrillarTwo different principal models explain how genescomponent [for review, see 5]. Our results corroboratecan be attached to the nucleoskeleton. The first oneand extend these observations and imply that the fi-suggests a direct linkage between certain DNA se-brillar centers are indeed the ‘‘central’’ structures ofquences and structural proteins of the nucleoskeleton,nucleoli—they serve as a structural support for thedefined as matrix-attached regions or scaffold-attachedtranscriptional complexes of RNA polymerase I. Mostregions in mitotic chromosomes [for reviews, see 45–of the transcribed ribosomal units are associated with48]. The second view is that the nuclear matrix is con-the fibrillar centers and only a small part stretchesnected with the DNA via functional proteins, e.g., thebetween them [see also 43]. Therefore, the spatial dis-transcriptional complex [22, 49, 50]. This suggests atribution of the fibrillar centers (attached to themore direct involvement of nucleoskeleton in nuclearnucleoskeleton) apparently determines the morphologyfunctions. Several studies imply that a nucleoskeletonof nucleoli since the formation of the dense fibrillaris necessary for normal transcription to occur [reviewedcomponent and the granular component occurs aroundby 45]. For example, Blencowe et al. [21] demonstratedthe fibrillar centers.that antibodies raised against nuclear matrix immuno-

In conclusion, our results do not support the idea ofprecipitate the exon transcript complex and that somesequence-specific attachment sites for ribosomal chro-of them also inhibit pre-mRNA splicing. Our resultsmatin in interphase cells. They point rather to sometogether with the previously observed attachment offunctional attachments via the polymerase complexes.RNA polymerase I activity to the nucleoskeleton [33]Nucleolar fibrillar centers, embedded into the nucle(o-also point to a functional explanation of the attach-l)ar skeleton, provide structural support for these at-ments of ribosomal genes to the nucleoskeleton. In fact,tachments.the most probable conclusion would be that the tran-

scription complex is responsible for the attachment ofWe thank Dr. Kubınova for her help with confocal microscopy. Thisribosomal genes to the nucleoskeleton. This is also in

work was supported by the Grant Agency of the Academy of Sciencesline with the recently presented model of nucleolar of the Czech Republic (539402), the Grant Agency of the Czech Re-transcription defining the transcription complexes as public (304/94/0148), and the Welcome Trust.immobile entities fixed mostly on the surface of thefibrillar centers; the DNA template is then pulled

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Received April 9, 1996Revised version received June 12, 1996

AID ECR 3272 / 6i12$$$443 08-20-96 20:56:33 eca AP: Exp Cell