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• W2134222431 abstract "Article1 June 2001free access Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli Gareth J. Sullivan Gareth J. Sullivan Biomedical Research Centre, University of Dundee, Ninewells Hospital, Dundee, DD1 9SY UK Search for more papers by this author Joanna M. Bridger Joanna M. Bridger Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH UK Search for more papers by this author Andrew P. Cuthbert Andrew P. Cuthbert Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH UK Present address: Division of Medical and Molecular Genetics, Guy‘s, King's and St Thomas' School of Medicine, London, SE1 9RT UK Search for more papers by this author Robert F. Newbold Robert F. Newbold Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH UK Search for more papers by this author Wendy A. Bickmore Wendy A. Bickmore MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU UK Search for more papers by this author Brian McStay Corresponding Author Brian McStay Biomedical Research Centre, University of Dundee, Ninewells Hospital, Dundee, DD1 9SY UK Search for more papers by this author Gareth J. Sullivan Gareth J. Sullivan Biomedical Research Centre, University of Dundee, Ninewells Hospital, Dundee, DD1 9SY UK Search for more papers by this author Joanna M. Bridger Joanna M. Bridger Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH UK Search for more papers by this author Andrew P. Cuthbert Andrew P. Cuthbert Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH UK Present address: Division of Medical and Molecular Genetics, Guy‘s, King's and St Thomas' School of Medicine, London, SE1 9RT UK Search for more papers by this author Robert F. Newbold Robert F. Newbold Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH UK Search for more papers by this author Wendy A. Bickmore Wendy A. Bickmore MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU UK Search for more papers by this author Brian McStay Corresponding Author Brian McStay Biomedical Research Centre, University of Dundee, Ninewells Hospital, Dundee, DD1 9SY UK Search for more papers by this author Author Information Gareth J. Sullivan1, Joanna M. Bridger2, Andrew P. Cuthbert2,3, Robert F. Newbold2, Wendy A. Bickmore4 and Brian McStay 1 1Biomedical Research Centre, University of Dundee, Ninewells Hospital, Dundee, DD1 9SY UK 2Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH UK 3Present address: Division of Medical and Molecular Genetics, Guy‘s, King's and St Thomas' School of Medicine, London, SE1 9RT UK 4MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2867-2877https://doi.org/10.1093/emboj/20.11.2867 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno-FISH) on three-dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs. Introduction The eukaryotic nucleus is functionally compartmentalized, and nowhere is this illustrated more clearly than at the nucleolus, where multiple loci from different chromosomes contribute to the formation of a functional nuclear compartment visible by light microscopy (Bridger and Bickmore, 1998; Matera, 1999). Nucleoli are the sites of rDNA transcription, rRNA processing and the assembly of ribosomes. These stages of ribosome biogenesis can be observed at the structural level (Scheer and Weisenberger, 1994; Scheer and Hock, 1999). The nucleolus can participate in many other aspects of gene expression and nuclear function (Pederson, 1998; Carmo-Fonseca et al., 2000; Olson et al., 2000). In the human, the five chromosomal loci that encode the ∼360 copies of ribosomal genes are termed nucleolar organizer regions (NORs). NORs are located on the short arms of acrocentric chromosomes (HSA13, 14, 15, 21 and 22) (Henderson et al., 1972). Each ribosomal gene cluster is on average 3 Mb in length (80 copies of a 43 kb repeat) (Sakai et al., 1995). This represents between a quarter and a third of the DNA present on the short arms of each of the acrocentric chromosomes. The remaining DNA is largely devoid of transcribed sequences and is composed of arrays of tandem repeated satellite DNA (Waye and Willard, 1989; Choo et al., 1990; Tagarro et al., 1994; Shiels et al., 1997b). NORs can be identified as secondary constrictions on metaphase chromosomes and can be visualized by silver staining, due to the abundance of associated argyophilic proteins (Goodpasture and Bloom, 1975). However, not all NORs form secondary constrictions or can be silver stained during metaphase. The number of silver-positive NORs varies between four and 10 (Babu and Verma, 1985). In dividing HeLa cells, usually six out of 10 NORs can be silver stained (Roussel et al., 1996; G.Sullivan, data not shown). The term secondary constriction is somewhat of a misnomer since the chromatin in a silver-stained NOR is 10-fold less compact than the remainder of the metaphase chromosome and is organized in a distinct twisted loop (Heliot et al., 1997). The identity of the proteins responsible for this distinct chromatin structure is uncertain, but likely candidates include components of the RNA polymerase I (pol I) transcription machinery. The human pol I transcription machinery is comprised of upstream binding factor (UBF), selectivity factor 1 (SL1) and pol I with its associated factors (TIF IA and TIF IC) (Grummt, 1999). SL1 is a complex of the TATA-binding protein (TBP) and three TBP-associated factors (TAFI110, TAFI63 and TAFI48) (Comai et al., 1992). SL1 binds to the core element of the ribosomal gene promoter and is the key component of the pre-initiation complex (Bell et al., 1988). There is some debate as to the role of UBF in pol I transcription. UBF was originally defined as a factor that bound to ribosomal gene promoter sequences and facilitated the interaction of SL1 (Bell et al., 1988). The abundance of UBF (up to 106 molecules per cell, see Figure 2), its relaxed DNA sequence specificity and its remarkable ability to bend and loop target DNA now lead to the view that it is performing a more generalized structural role in ribosomal gene chromatin (Reeder et al., 1995). As cells enter mitosis, transcription by all three classes of RNA polymerase ceases or is at least greatly decreased (reviewed in Sirri et al., 2000). In the case of pol I, it appears that mitotic repression results from changes in the phosphorylation status of UBF and SL1 (Kuhn et al., 1998; Voit et al., 1999; Sirri et al., 2000). Cdc2–cyclin B kinase is primarily responsible for this repression (Sirri et al., 2000). UBF, SL1 and at least a fraction of pol I remain associated with the metaphase NOR (Weisenberger and Scheer, 1995; Jordan et al., 1996; Roussel et al., 1996). The pol I transcription machinery is only found associated with acrocentric chromosomes that exhibit a secondary constriction and silver stain. Retention of the transcription machinery on the mitotic NOR may be responsible for its unique structure. It is generally agreed (but unproven) that acrocentric chromosomes devoid of a secondary constriction are transcriptionally silent with respect to ribosomal genes. Some human NORs contain less than the expected amount of rDNA (Shiels et al., 1997a). This raises the possibility that some NORs may be transcriptionally active despite the lack of visible silver staining. As cells exit mitosis, nucleoli only form around transcriptionally active NORs (Ochs et al., 1985; Benavente et al., 1987; Jimenez-Garcia et al., 1994). Associated pol I transcription machinery may seed nucleolar reformation (Dousset et al., 2000). As cells progress through the cycle, the multiple small nucleoli that form around individual active NORs appear to fuse into one or a few large nucleoli, a phenomenon commonly referred to as nucleolar fusion (Anastassova-Kristeva, 1977). A consequence of this process is that multiple NORs can be found within a single nucleolus (Babu and Verma, 1985). The underlying mechanisms involved in this major dynamic nuclear reorganization involving multiple chromosome territories are not at all understood. The popular view that this phenomenon can be explained by an inherent affinity of nucleoli for each other has not been proven experimentally. A prediction of this model is that silent NORs are excluded from the nucleolus. Alternatively, one could hypothesize that human acrocentric chromosomes self-associate independently of NOR activity status. In this model, silent NORs associate with nucleoli. In support of this model, it has been shown recently in mice that large nucleoli contain apparently inactive, methylated rDNA (Akhmanova et al., 2000). Here we have exploited the phenomenon of species specificity of ribosomal gene transcription to discriminate between these two alternatives. Considerable divergence between vertebrates has been observed in both the sequence of the ribosomal gene repeat and components of the pol I transcription machinery. Mouse and human ribosomal genes cannot be transcribed in vivo (Miller et al., 1976) or in vitro by the other species' transcription machinery (reviewed in Heix and Grummt, 1995). This species specificity resides solely in the transcription factor SL1. Because of this species specificity, somatic cell hybrids provide a valuable model system to study the structure and subnuclear localization of NORs on intact human chromosomes in the certain knowledge that they are transcriptionally silent. Using a panel of monochromosomal somatic cell hybrids, each containing an intact human acrocentric chromosome, we confirm that human ribosomal genes are transcriptionally silent within the murine cell environment. We demonstrate that mUBF can interact with human NORs to a degree similar to that observed on active murine NORs. Most surprising of all, we observe that in every case, human acrocentric chromosomes are associated with a mouse nucleolus in vivo. This is irrespective of their UBF loading status. We further demonstrate that human NORs co-purify with nucleoli isolated from hybrid cells. Thus we conclude that localization of human NORs to nucleoli can be uncoupled from ribosomal gene transcription and UBF loading. Additionally, we show that sequences from the long arms of acrocentric chromosomes co-purify with nucleoli. In the light of these findings, we favour a model in which nucleolar fusion results from the ability of NOR-containing chromosomes to associate independently of ribosomal gene transcription. Results Human ribosomal genes are transcriptionally silent in a mouse background In order to study the relationship between subnuclear localization of individual human NORs and ribosomal gene transcription, we have utilized a panel of mouse> human somatic cell hybrids (Cuthbert et al., 1995). In this panel, mouse A9 cells contain individual human chromosomes 13, 14, 15, 21 and 22 (HSA13, 14, 15, 21 and 22, respectively), tagged with a selectable hygromycin resistance marker (HyTK) to maintain the stability of the hybrid. Hybrids containing the human chromosome 2 or X (HSA2 and X) served as non-acrocentric controls. Extensive characterization of these hybrids has shown that they each contain only a single intact human chromosome (Cuthbert et al., 1995). Southern blotting analysis confirms the presence of the expected amount of human rDNA in hybrids containing HSA13, 14, 15 and 21 (Figure 1A). As has been reported previously, HSA22 contains approximately one-tenth of the human rDNA observed on other human acrocentric rDNAs (Shiels et al., 1997a). Southern blots with probes derived from elsewhere in the human rDNA repeat show no detectable reorganization of the human rDNA content in hybrid cells compared with normal human cells (data not shown). Figure 1.Human ribosomal genes are transcriptionally silent in hybrid cell lines. (A) HindIII-digested high molecular weight DNA from hybrid, human (HeLa) and mouse (A9) cells was probed with a human rDNA-specific hybridization probe (see Materials and methods). The source of the DNA is shown above each lane and molecular weight markers are shown on the left. (B) Total RNA samples (10 μg each) from HeLa, mouse A9 and A9HyTK hybrid cells were used in S1 nuclease protection assays with probes designed to detect the 5′ ends of mouse and human 47S precursor rRNAs (Mouse ETS and Human ETS, respectively) and a probe designed specifically to detect human 28S rRNA. RNA samples from untreated and TPA-treated cells are designated − and +, respectively. The human chromosome present in each of the hybrid cell lines is shown above and the probe used is shown on the left of the appropriate panel. Download figure Download PowerPoint Figure 2.Characterization of anti-UBF human autoantibodies. Human 1BR.2 primary fibroblasts were lysed directly into SDS–PAGE loading buffer at 104 cells/μl. A 10 μl aliquot of this cell lysate (equivalent to 105 cells) was electrophoresed on an 8% polyacrylamide gel alongside increasing amounts (shown above gel) of an equimolar mixture of pure recombinant hUBF1 and 2. A western blot of the gel was probed with anti-UBF human autoantibody (see Materials and methods). Note that the lower pair of bands (∼70 kDa) apparent in the first lane are proteolytic breakdown products of UBF. Download figure Download PowerPoint Previously it has been demonstrated that in rodent> human cell hybrids, human ribosomal genes are transcriptionally silent (Miller et al., 1976). In order to demonstrate that this was the case in the hybrids used here, we determined the transcriptional status of human ribosomal genes using an S1 nuclease protection assay that detects the 5′ most 40 nucleotides of the 47S rRNA precursor. The first 3656 nucleotides of the 47S precursor specify the external transcribed spacer, which is processed and degraded rapidly in vivo. Consequently, signal generated with this probe can be considered a measure of ongoing transcription. Whereas this probe readily detected 47S precursor in HeLa cell RNA, no signal was observed with equivalent amounts of RNA prepared from A9HyTK-13, 14, 15, 21 and 22 hybrid cells or mouse A9 cells (Figure 1B, middle panel). A similar probe prepared from mouse rDNA detected mouse 47S precursor in RNA from A9, A9HyTK-13, 14, 15, 21 and 22 cells but not from HeLa cells (Figure 1B, top panel). To look at accumulated rRNA, we used a probe that can specifically detect human 28S rRNA. This oligonucleotide probe is complementary to nucleotides 3326–3369 of human 28S rRNA. This sequence represents an expansion region that is absent from murine 28S rRNA. Mature 28S rRNA is stable and provides a highly sensitive marker for accumulated human ribosomal gene transcripts. Using this probe, we readily detected human 28S rRNA in HeLa cells but not in hybrid or A9 cells (Figure 1B, bottom panel). In mixing experiments, we can detect <1 part in 1000 of human 28S rRNA (data not shown). In more complex hybrids containing multiple human chromosomes, human ribosomal gene transcription can be reactivated by treatment with either SV40 T antigen or phorbol esters (Soprano and Baserga, 1980). The most reasonable interpretation of these experiments is that the hybrids used contain not only human acrocentric chromosomes but also human chromosomes that carry the species-specific component(s) of ribosomal gene transcription. It is envisaged that these species-specific components are able to reprogramme the mouse pol I transcription machinery to recognize and transcribe human rDNA. However, it is also formally possible that treatment of hybrids with T antigen or phorbol esters could relax the specificity of the mouse pol I transcription machinery such that it can now transcribe human rDNA. Treatment of the monochromosomal hybrids used in this study with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), does not result in transcriptional reactivation as determined by S1 nuclease protection assays with either human probe (Figure 1B). Thus we conclude that in A9HyTK-13, 14, 15, 21 and 22 cells, human ribosomal genes are transcriptionally silent. UBF can associate with human NORs in hybrid cells UBF is highly conserved and functionally interchangeable between human and mouse (Grummt, 1999). In principal, mUBF is capable of recognizing a human NOR. It was of interest, therefore, to determine whether mUBF does bind to human NORs in hybrid cells. This would provide evidence that human rDNA could be recognized as an NOR despite its transcriptional silence. Association of UBF with individual chromosomes/NORs can be determined conveniently by combined immunofluorescence and in situ hybridization (immuno-FISH) performed on metaphase chromosome spreads. In order to visualize UBF, we have utilized human autoantibodies. Antibodies against UBF also known as NOR90 are common in patients with autoimmune disorders and can readily detect UBF on metaphase chromosomes (Chan et al., 1991; Roussel et al., 1993). The anti-UBF autoantibodies used here were identified using an enzyme-linked immunosorbent assay (ELISA)-based screen of human sera against recombinant hUBF (A.Gibson and B.McStay, unpublished). These autoantibodies are highly specific for UBF as demonstrated by western blotting of a human cell lysate with recombinant hUBF1 and 2 controls (Figure 2). Quantitation of this blot revealed that human primary fibroblasts contain between 5 × 105 and 1 × 106 molecules of UBF/cell. We observe similar levels of UBF in A9 and hybrid cells (data not shown). Metaphase spreads prepared for A9HyTK-13, 14, 15, 21 and 22 cells were subjected to immuno-FISH. UBF was first visualized with anti-UBF autoantibodies and fluorescein isothiocyanate (FITC)-labelled secondary antibodies (green). Following fixation of bound antibodies, human chromosomes were identified by FISH using labelled human Cot-1 (red) as a probe (Figure 3A). These experiments clearly demonstrate that HSA14 and 15 have associated UBF in a mouse background. Furthermore, the level of UBF observed on HSA15 is comparable to that observed on mouse chromosomes in the same cells (Figure 3A). No UBF appears to be associated with HSA13, 21 and 22 in A9HyTK-13, 21 and 22 cells, respectively. Figure 3.UBF associates with human NORs in hybrid cells. (A) Metaphase chromosome spreads (counterstained with DAPI) from A9HyTK-13, 14, 15, 21 and 22 were subject to combined immunofluorescence using anti-UBF human autoantibody 9386 (FITC/green) and FISH with Spectrum red-labelled human Cot-1 DNA as probe. The identity of the human chromosome is indicated in the top left corner of each panel. Panels on the left show UBF and DAPI staining. In the centre panels, the signal from the human Cot-1 probe has been merged. The right hand panels show an enlarged version of the merged signal over the human chromosome. (B) Metaphase chromosome spreads (counterstained with DAPI) from A9HyTK-13 were subject to combined immunofluorescence using anti-UBF human autoantibody 9386 (FITC/green) and FISH with Spectrum red-labelled 28S rDNA probe (see Materials and methods). The left, centre and right panels show UBF staining, rDNA hybridization and the merge of both signals, respectively. (C) Metaphase chromosome spreads (counterstained with DAPI) from A9HyTK-15 were subject to combined immunofluorescence using anti-UBF human autoantibody 9386 (FITC/green) and FISH with Spectrum red-labelled human-specific rDNA probe (see Materials and methods). The left, centre and right panels show UBF staining, rDNA hybridization and the merge of both signals, respectively. Arrowheads indicate UBF and human NOR co-localization. Download figure Download PowerPoint In order to demonstrate co-localization of UBF staining with mouse and human NORs, we performed immuno-FISH with a 28S rDNA and a human rDNA-specific probe (Figure 3B and C, respectively). The results in Figure 3B show clear co-localization of UBF (green) with mouse rDNA (red) on mouse chromosomes. The results in Figure 3C show clear co-localization of UBF (green) with human rDNA (red) on HSA15 in A9HyTK-15. For a given hybrid, all the cells in that metaphase preparation displayed similar loading of UBF on the human chromosome (data not shown). Thus the UBF loading status appears to be copied faithfully during cell division, as has been described previously in human cells (Roussel et al., 1993). We conclude from this experiment that despite their transcriptional inactivity, human NORs can be recognized by components of the mouse pol I transcription machinery. It is interesting to speculate that the heterogeneity of UBF loading on human NORs in hybrid cells reflects that observed in the parental line (1BR.2). Nucleolar association of human acrocentric chromosomes within the mouse nucleus In human cells, UBF localizes exclusively to the nucleolus (Chan et al., 1991; Roussel et al., 1993). The finding that mUBF can bind to human NORs in hybrid cells indicates that these NORs may associate physically with a mouse nucleolus. To address this question, we have analysed the subnuclear localization of HSA13, 14, 15, 21, 22 and X within the three-dimensionally preserved nuclei of hybrid cells (Figure 4). The position of the nucleolus in these analyses was determined by co-immunofluorescence with an anti-fibrillarin monoclonal antibody. The probes used in these experiments were q arm-specific chromosome paints for HSA13, 14, 15, 21 and 22. A whole chromosome paint (p and q) was used for HSAX (Guan et al., 1996). Probes were verified against metaphase spreads of the appropriate hybrid (data not shown). Figure 4.Human acrocentric chromosomes associate with nucleoli in hybrid cells. (A) Optical sections (a–h) were obtained by laser-scanning confocal microscopy performed on an individual interphase A9HyTK-13 cell in which the three-dimensional structure was preserved. Nucleoli were visualized with an anti-fibrillarin mAb (red) and HSA13 with a biotinylated long arm-specific paint (green). Note that endogenous biotin present in the cytoplasm defines the nuclear–cytoplasmic boundary. The scale bar represents 5 μm. (B) Mid-sections obtained as above from A9HyTK-14, 15, 21, 22 and X. HSA14, 15, 21 and 22 were visualized with the relevant long arm-specific paint. HSAX was visualized with paint derived from both chromosome arms. Note that for A9HyTK-21, two closely adjacent cells are shown. Download figure Download PowerPoint The three-dimensional nuclear positioning of the acrocentric and the X chromosome territories was assessed by optically sectioning nuclei using a confocal laser-scanning microscope. Galleries of the two-dimensional data (each optical section) were analysed. The gallery for A9HyTK-13 is shown in Figure 4A. Mid sections for A9HyTK-14, 15, 21, 22 and X control are shown in Figure 4B. A chromosome territory was scored as being co-localized with the nucleolar staining if the paint was found to be overlapping the nucleolar stain or immediately abutting it. In the sections d and e of HyTK-13 (Figure 4A), co-localization of HSA13 with the upper of the two nucleoli is particularly evident. Approximately 50 cells were analysed for each monochromosome hybrid, and it was found for A9HyTK-13, 14, 15, 21 and 22 that the human chromosomes were co-localized with the fibrillarin staining in 86, 84, 92, 82 and 82% of cells, respectively. In contrast, the X chromosome was only co-localized with nucleoli in 39% of cells. These numbers show that human acrocentric chromosomes co-localize with mouse nucleoli. Some of the cells that were scored negative had chromosomes extremely close to the nucleolus but, given our scoring criteria, were scored as not co-localizing. It should be noted that the p arms containing the rDNA were not painted, which may have resulted in us not visualizing all co-localizations. Both arms of the control HSAX were painted. We conclude from these experiments that each of the human acrocentrics co-localize with hybrid cell nucleoli irrespective of their UBF loading status and despite the transcriptional silence of the associated ribosomal genes. Characterization of purified nucleoli The above experiments demonstrate an association between human acrocentric chromosomes and nucleoli in intact cells. To demonstrate that hybrid cell nucleoli contain transcriptionally silent human NORs, we have performed analyses on purified nucleoli. Due to their mass, nucleoli can be purified conveniently by sonication and centrifugation in a high-density medium (Muramatsu et al., 1963; Maggio, 1966). Using this methodology, suspensions of essentially pure nucleoli can be prepared. We have verified nucleolar purification in our preparations by immunofluorescence staining with antibodies directed against known nucleolar antigens (UBF and nucleolin) and nuclear antigens that are excluded from the nucleolus (c-Jun and cyclin D1). As expected, UBF and nucleolin antibodies stain purified nucleoli whereas c-Jun and cyclin D1 antibodies do not (Figure 5). Figure 5.Verification of nucleolar purification. 1BR.2 cells and nucleoli purified from these cells (both counterstained with DAPI) were stained with antibodies against nucleolar antigens (nucleolin and UBF) and nuclear antigens (cyclin D1 and c-Jun) that are excluded from nucleoli. Panels on the left show antibody staining of 1BR.2 cells (green). Centre panels show DAPI staining of 1BR.2 cells (blue). Right panels show combined antibody (green) and DAPI (blue) staining of nucleoli purified from 1BR.2 cells. Note that the scale bars for cell staining and nucleolar staining are different (10 and 2 μm, respectively). Download figure Download PowerPoint Transcriptionally silent human NORs are present within purified nucleoli To determine whether human NORs are incorporated into nucleoli in hybrid cells, FISH was performed on isolated nucleoli (Figure 6). All NORs (mouse and human) were visualized using a 28S rDNA probe and human NORs were visualized using a probe derived from the human rDNA intergenic spacer. This experiment was performed on nucleoli isolated from A9HyTK-15 and 21. Note that the NOR on HSA15 has associated UBF while that on HSA21 does not (see Figure 3). A positive signal is observed over all nucleoli with the 28S probe (Figure 6A). The presence of multiple signals in the larger nucleoli is consistent with them containing multiple mouse NORs. Given that hybrid cells each contain on average three nucleoli (data not shown) and only a single human acrocentric chromosome (Cuthbert et al., 1995), we would expect 33% of purified nucleoli to contain human rDNA sequences. Indeed, we found that 20–40% (dependent on the identity of the hybrid) of purified nucleoli did contain a single hybridization signal when utilizing the human rDNA-specific probe. Typical positive-staining nucleoli are shown in Figure 6B. Figure 6.Hybrid cell nucleoli contain human NORs. (A) Nucleoli isolated from HSA15 and 21 were probed with a 28S rDNA probe that detects both human and mouse NORs. Left hand panels show DAPI staining of nucleoli, right hand panels show 28S rDNA signal (red). The identity of the human chromosome present in the hybrid is shown in the top left hand corner. The scale bar represents 1 μm. (B) Nucleoli isolated from HSA15 and 21 were probed with a human-specific rDNA. Left hand panels show DAPI staining of nucleoli, right hand panels show human rDNA signal (red). (C) Quantitative PCR was performed on both genomic DNA from hybrid cells and DNA isolated from their purified nucleoli (see Materials and methods for details). PCR products generated with primer pairs that amplify mouse and human rDNA promoter sequences are labelled M and H, respectively. The source of template DNA is shown above. In control reactions (C), PCR was performed in the absence of input DNA. Signals were quantified using phosphorimaging (Bio-Rad) and the ratio of H to M signal for each template is shown below. Download figure Download PowerPoint For a more quantitative comparison, DNAs extracted from A9HyTK-14, 15 and 21 nucleoli were used as templates in quantitative PCR (Figure 6C). A9 nucleoli provided a control. Primer pairs that specifically recognize either human or mouse ribosomal gene promoter seque" @default.
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• W2134222431 title "Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli" @default.
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