1 Department of Cell Biology, School of Life Sciences, Peking University,
100871, China
2 Institute of Genetics and Cytology, Northeast Normal University, Changchun,
Jiling, 130024, China
* Authors for correspondence (e-mails: weitao{at}pubms.pku.edu.cn; zhaizh{at}plum.lsc.pku.edu.cn)
Accepted 16 December 2002
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Summary |
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Key words: Nucleolar ultrastructure, DNA specific staining, Ag-NOR protein, rDNA transcription, Immunocytochemistry, Plant nucleolus
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Introduction |
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One nucleolar structure-function relationship that needs to be clarified is
that of rDNA arrangement in the nucleolus and its relationship with the
subnucleolar structure region (Biggiogera
et al., 2001). Although Miller's early spread technique revealed
the `christmas tree' structure for the active transcription of rRNA genes
(Miller et al., 1969), this structure in the nucleolus of living cells is in a
topological constraint state. It is very difficult to analyze the relationship
between the nucleolar structures and their functions by observing the
`christmas tree' structure in situ (Shaw et al., 1995;
Scheer et al., 1997
). In spite
of this, Miller's spread technique did reveal an important characteristic of
the rRNA gene, that is, rDNA is of a non-nucleosomal configuration. In the
nucleolus, the rRNA gene is in a highly decondensed state
(Derenzini et al., 1983
;
Medina et al., 2000
;
Biggiogera et al., 2001
).
The first objective of our study was, by employing the cytochemical
technique of DNA-specific staining NAMA-Ur
(Testillano et al., 1991), to
display the extended rDNA in situ in the nucleolus with the fibrillar center
(FC) still recognizable after DNA staining, thus creating a favorable premise
for direct analysis of the relationship between the rDNA and structures of the
subnucleolar region. Our second objective was to explore the arrangement and
the transcription sites of rDNA linked to the ultrastructure. On this basis,
we analyze and discuss the structure-function relationship between the
subnucleolar structural regions and the synthesis of rRNA.
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Materials and Methods |
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Conventional electron microscopy
Root tips were carefully excised and fixed in 2.5% glutaraldehyde in 0.1 M
phosphate buffer saline (PBS) pH 7.4 for 2 hours at room temperature. After
rinsing in double-distilled water for 20 minutes, they were postfixed in 1%
osmium tetroxide for 60 minutes. Samples were dehydrated in an ethanol-acetone
series and embedded in Epon 812. Ultra-thin sections of 60-80 nm were stained
with uranyl acetate and lead citrate and examined under a Hitachi-600
transmission electron microscope.
NAMA-Ur procedure for DNA-specific staining
To study the distribution of DNA in the nucleolus, we employed the NAMA-Ur
method reported by Testillano (Testillano
et al., 1991). Briefly, samples were fixed in 3% glutaraldehyde
and 4% formaldehyde in 0.1 M PBS for 1 hour at 4°C. After washing in 0.1 M
PBS, specimens were immersed in 0.5 N NaOH in 4% formaldehyde overnight (NA)
and then rinsed in double-distilled water three times for 10 minutes each,
followed by 1% acetic acid three times for 10 minutes each, and finally in
double-distilled water again for three times for 10 minutes each. After that,
specimens were treated with a freshly prepared methanol:acetic anhydride (5:1,
v:v) mixture at 25°C for 18-24 hours until the samples were bleached.
Specimens were dehydrated in a methanol series and embedded in Epon 812.
Semi-thin sections were stained with 2% aqueous uranyl acetate for 70 minutes
at 60°C. After washing in double-distilled water and drying at 25°C,
they were examined under a Hitachi EM 600-2 at 75 KV.
Electron microscopic silver staining
Root tips were immediately fixed in 4% paraformaldehyde and 2.5%
glutaraldehyde in PBS pH 7.4 for 2 hours at 4°C. After a treatment with
AgNO3 (50%) solution for 24-48 hours at 50°C, the root tips
were washed in 5% sodium thiosulfate, dehydrated and embedded in Epon 812.
In situ hybridization
The rDNA probe was obtained from the pTA71 plasmid, which contains the rDNA
repeat unit of wheat cloned in pUC18. A 3.6 kb fragment of pTA71 containing
18S and 25S rDNA were excised by BamHI digestion, isolated by agarose
gel electrophoresis and labeled by nick translation in the presence of
bio-16-dUTP (Bionic, Gibco). The probe was purified by a Spin G-25 column,
precipitated by ethanol, dissolved in sterile water and stored at
20°C.
The root tips treated with the NAMA-Ur method were sectioned and directly subjected to in situ hybridization without the final step of uranyl acetate staining. In situ hybridization was performed according to the non-radioactive in situ hybridization application manual (Boehringer Mannheim). Briefly, ultra-thin sections were first treated with proteinase K (10 µg/ml) for 15 minutes at 37°C. The sections were then treated for 10 minutes at 80°C in 75% deionized formamide in 2xSSC and then placed in 100°C denatured probe solution (50% formamide, 5xSSC, 10% dextran sulfate, 10 mmol Tris-HCl, 0.5% sodium dodecyl sulphate, 250 µg/ml salmon sperm DNA and 5 mg/ml sodium pyrophosphate) and hybridized at 42°C in a moist chamber for 18 hours. Sections were floated three times for 10 minutes on drops of PBS and incubated with Streptavidin-conjugated 10 nm gold particles (Sigma). After washing and drying, sections were stained with 2% aqueous uranyl acetate for 20 minutes at 60°C. After washing in double-distilled water and drying at 25°C, they were observed under a Hitachi EM 600-2 at 75 KV.
Immunocytochemistry
The anti-DNA/RNA hybrid antibody was kindly provided by B. D. Stollar
(Rudkin et al., 1997). The anti-fibrillarin antibody was purchased from Santa
Cruz. Lowicryl K4M was obtained from Chemische Werke Lowi GMBH
& Co., Germany. Protein A conjugated to 10 or 15 nm colloidal gold
particles was purchased from Sigma.
Processing for Lowicryl K4M embedding
Samples were fixed in 3% glutaraldehyde and 4% formaldehyde in PBS for 2
hours. After washing in double-distilled water three times, for 30 minutes
each, they were dehydrated in an ethanol series and permeated by 100% ethanol:
K4M (1:1) mixture for 12 hours at 0°C, 100% ethanol: K4M (1:2)
mixture for 1 hour at 10°C, and 100% K4M for 60 hours at
30°C. After that specimens were embedded in Lowicryl K4M
at 30°C under UV irradiation for over 24 hours, and then irradiated
again for 2-3 days at room temperature.
Immunogold labeling of an anti-fibrillarin antibody and an
anti-DNA/RNA hybrid antibody
Immunogold labeling of an anti-fibrillarin antibody and DNA/RNA hybrid
antibody were carried out as described previously
(Testillano et al., 1994).
Briefly, ultra-thin Lowicryl sections were mounted on Formvar nickel grids and
washed in PBS three times for 1 minute each, and in 5% BSA (PBS, 0.05% Triton
X-100) for 10 minutes. Then the grids were incubated with the anti-fibrillarin
antibody or anti-DNA/RNA hybrid antibody diluted 1:300 in PBS for 1 hour at
room temperature. After several washings in PBS, they were floated in protein
A conjugated to 15 nm colloidal gold particles diluted 1:25 in PBS for 45
minutes at room temperature. They were washed in PBS and in double-distilled
water. After drying, some sections were stained with 5% uranyl acetate, and
others were subjected to the NAMA-Ur method and observed under a Hitachi EM
H-600-2 at 75 KV. Controls were done by replacing the primary antibody with
diluents.
Nucleolar component measurement
The areas of components in sections were measured using an IBAS image
analysis system from Germany. 30 micrographs were chosen from different grids.
Among the 30 micrographs, 15 were taken from NAMA-Ur-stained sections. For a
more accurate measurement of the areas, micrographs of the subnucleolar
regions were enlarged to as high a magnification as 120,000x.
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Results |
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To demonstrate and locate nucleolar DNA components, we first employed the NAMA-Ur DNA-specific staining technique to treat the cells and then observed them under the electron microscope after semi-thin sectioning. We found that the semi-thin sections produced the better contrast of the DNA components than the ultra-thin sections, and this allowed a clearer visualization. The cytoplasm was basically bleached; the DNA in the nucleoplasm was intensely and specifically stained and mainly present as high-density clumps. In the nucleolus, although the background of the medium electronic density was retained, the specifically stained high electronic density component of DNA can still be seen. We found that the DNA components in the nucleolus existed in two forms, one was the decondensed DNA fiber, which was distinctly different from the DNA component in nucleoplasm (Fig. 1B,D), and the other was the condensed DNA clumps, which appeared to be composed of tightly coiled DNA filaments and surround by DNA fibers (Fig. 1C, arrows). In addition, the DNA clumps were only located in the regions with low electronic density in the nucleolus. These low-density regions seemed to correspond to FCs. Since fibrillarin protein is the marker protein of DFC, the anti-fibrillarin antibody immunolabeling experiment was carried out to prove that these low-density regions were FCs. The labeling signals were distributed at the periphery of the regions with the lowest electronic density in nucleolus, whereas the regions with the lowest electronic density were devoid of labeling, indicating that after the treatment with DNA-specific staining the regions with the lowest electronic density in the nucleolus were FCs. Moreover, we also found that the region of fibrillarin labeling was bigger than that occupied by DNA fibers, suggesting that not all DFC contained DNA (Fig. 2).
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To find out if the DNA component in the nucleolus is an rRNA gene, we used the biotin-labeled rDNA fragments containing 25S and 18S as probes, as well as the biotin antibody conjugated with 10 nm colloid gold as a labeling signal to perform in situ hybridization. After hybridization, the sections underwent the final step of specific staining. The labeling signals of gold particles appeared mainly on the extended DNA fibers, and none appeared on DNA within FC (Fig. 3). This result clearly shows that the DNA fibers distributed around the FC represent the sites of rRNA genes, whereas the DNA clumps located within the FC were not rRNA genes.
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After determining that the fibrillar DNA component in the nucleolus was an rRNA gene, we further investigated its location and arrangement and found that the highly extended rDNA fibers were mainly arranged and distributed around the FC, with their initial positions at the boundary of FC. The DNA in the FC was kept in a condensed state (Fig. 4). In general, rDNA was arranged in a circular configuration with each FC as its center (Fig. 4, arrows).
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Our observations also showed that rDNA was always arranged in an identical configuration, with FC as the center, regardless of its size and quantity (Fig. 5A). In some sections of the nucleolus, parts of the DFC region at the periphery of the FC were not surrounded by rDNA, whereas others were completely devoid of rDNA (Fig. 5B), indicating that the arrangement of rDNA in the DFC was not uniform. Besides, in some sections of nucleolus, the rDNA in the nucleolus was connected to the extranucleolar DNA at many sites, as shown in Fig. 5D.
|
When viewed under a high magnification
(Fig. 6A,B), we observed some
DNA fibers that stretched out of the aggregated DNA clumps within FC while
still connected to DNA clumps with the extended rDNA in DFC regions. The rDNA
fibers surrounding the FC were variable in diameter. This variation may be
ascribed to the presence of many `arrow-like structures' along the rDNA fibers
(Fig. 7A, arrows). An enlarged
image of FC and its surrounding rDNA shown in
Fig. 7A enabled us to take a
closer look at these structures (Fig.
7B), and it can be seen that the arrow-like structures were
arranged along the rDNA fibers with a certain distance interval. Owing to the
variable angles of sectioning, the appearance of these structures varied. The
maximum length of the arrow-like structures measured about 200 nm
(Fig. 7C,D). Fine fibers 6
nm in diameter extending laterally from these structures can occasionally be
detected (Fig. 7D, arrows). The
thinner rDNA fibers were located in between the arrow-like structures.
Meanwhile, transversal views of the arrow-like structures were also found on
these sections (Fig. 7E). On
the basis of these images and measurements, we postulate that these structures
represent in situ rDNA transcription units, equivalent to the tightly packed
in situ Christmas tree structures (Scheer
et al., 1997
; Gonzalez-Melendi
et al., 2001
). This phenomenon may reflect the morphology of
elongating rRNA in transcribing regions, even though the transcripts were
tightly packed with rDNA.
|
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Early studies found that some argyrophilic proteins specifically appeared
in the metaphase NOR and interphase nucleolus (Goodpasture et al., 1975).
Under acidic condition, these proteins can be stained specifically by
AgNO3 and readily visualized. Later it was found that the Ag-NOR
protein was the marker of active rRNA gene and that rDNA transcription did not
take place in the absence of Ag-NOR protein (Fakan et al., 1986;
Pession et al., 1991;
Derenzini et al., 2000). In our experiment, we observed the in situ
arrangement of rDNA. If this arrangement truly exists, then the distribution
of Ag-NOR proteins that are closely related to rDNA transcription will conform
with such an arrangement. To prove this assumption, we analyzed the
distribution of silver-stained proteins. After the intact cells were
silver-stained, the silver-stained proteins mainly appeared in the nucleolus
(Fig. 8B). Under a high
magnification, Ag-NOR proteins in the nucleolus could be seen in many circular
configurations. Each circle was a relatively independent unit, but these
circles were interconnected, and no silver-stained protein was present in the
center of the circles (Fig.
8A). Furthermore, we compared one rDNA configuration image with
that of Ag-NOR under the same magnification
(Fig. 9A,B). It can be seen
that the distributions of rDNA and Ag-NOR proteins were very similar, and the
result of statistical analysis demonstrated that the areas of the regions
occupied by the nucleolar Ag-NOR proteins and rDNA fibers were fundamentally
identical (Fig. 11). This
analysis suggested that our inference was correct. However, we also noticed
that there was a distinct difference between these two arrangement
configurations. The central portion of the rDNA configuration had DNA
components in an aggregated state, whereas the central portion of the circular
configuration had no silver-stained protein.
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When the rDNA was under transcription, the transient DNA/RNA hybrid double-stranded structure is sometimes formed. With the aid of an anti-DNA/RNA hybrid antibody, we were able to directly and selectively label the transcription sites of the rRNA gene. Comparative experiments proved that the antibody labeling system had a very good specificity (data not shown).
The result of the labeling study of the rRNA gene transcription sites showed that in the nucleus some signals indicating DNA transcription activities appeared on the decondensed chromatin or the boundary area of the condensed chromatin. However, in the nucleolus, the signals were located at the periphery of the FC and the DFC region near the FC. We noticed that the signals were never seen in the nucleolus-associated chromatin, granular component and the interior of the FC. Hence, it can be inferred that the transcription sites of rRNA in the nucleolus are located at the periphery of the FC and DFC regions near the FC (Fig. 10)
|
To estimate the areas of the DFC occupied by rDNA, we randomly selected 30 nucleolus sections, of which 15 nucleolus sections were treated with DNA-specific staining, and analyzed statistically the total area of the nucleolus, granular regions, FC, DFC and rDNA filaments as well as the area occupied by the Ag-NOR proteins (Fig. 11). The statistical results showed that the area occupied by the highly decondensed rDNA fibers accounted for about one-third of the total area of the DFC, implying that one-third of the DFC region near the FC (including the periphery of the FC) was the transcription domain for the rRNA gene.
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Discussion |
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Sirri et al. identified nucleolin and protein B23 as the two major Ag-NOR
proteins in the nucleolus (Sirri et al.,
2000). It was thought that Ag-NOR proteins were distributed in
both FCs and DFCs (Fakan et al., 1986). However, extensive studies on the
localization of nucleolin and B23 using electron microscopy have reached a
general agreement that nucleolin and B23 are located in the DFC around the FC,
whereas FC is devoid of these proteins
(Ginisty et al., 1999
;
Biggiogera et al., 1990
). In
this paper, after the nucleolus was treated with the procedure described in
Materials and Methods, it was found that the arrangement and distribution of
Ag-NOR proteins were very similar to those of rDNA, that is, Ag-NOR proteins
were present as circular configurations with hollow centers. The results of
statistical analysis showed that the distribution areas of Ag-NOR proteins and
rDNA were basically identical. Although we did not perform a labeling study of
the Ag-NOR protein distribution sites, on the basis of the distribution
pattern of Ag-NOR proteins and previous results, we conclude that locations of
Ag-NOR proteins were consistent with that of rDNA and are arranged and
distributed around the FC.
Many reports indicated that the active transcription regions were located
at the periphery of the FC and DFC near the FC
(Testillano et al., 1994;
Meecak et al., 1996
;
Lazdins et al., 1997
; de
Carcer et al., 1999). Data presented in this paper indicate that in Allium
sativum cells, the distribution of rDNA was restricted to the limited
areas near to the FC, including the periphery of the FC
(Fig. 1C,
Fig. 4). Our results also
demonstrated that fibrillarin-labeled regions that were distal to FC were
devoid of rDNA (Fig. 2).
Moreover, the immunolabeling with anti-RNA/DNA antibody showed that no
transcription took place in DFC regions away from the FC
(Fig. 10). Hence, the result
of this paper supports the notion that only the DFC near the FC contains
active sites of rDNA transcription, and no transcription activity occurs at
DFCs far away from the FC. Thus, the next question is what is the proportion
of transcriptionally active DFC in the total DFC? At present, there are no
reports of the precise structural subdivisions of the DFC. Our statistical
analysis in this study is the first attempt, and we put forward the notion
that only about one-third of the DFC near the FC is distributed with rDNA and
that no transcriptional activities exist in the remaining two-thirds of the
DFC region. Data from early studies demonstrated that the DFC far away from
the FC is involved in the splicing and processing of the transcription
products of rRNA genes (Shaw et al., 1995).
It should be noted that the thickness of rDNA fibers revealed in our
experiments varied, and we reason that the cause of this variation was the
occurrence of the arrow-like structures, which we postulate to be the rDNA in
the form of tightly packed transcription units. We make this hypothesis on the
following grounds. (1) The arrow-like structures occurred only on rDNA fibers.
(2) They closely resembled the densely packed christmas tree structures in
morphology. (3) They measured 200 nm in length, close to the dimensions
of the in situ Christmas tree reported previously
(Scheer et al., 1997
;
Gonzalez-Melendi et al., 2001
).
(4) They were specifically distributed in DFC regions near to the FC. However,
the important question arising from this phenomenon is what caused the
occurrence of this structure. We postulated that the tightly compacted rDNA
transcription units contained RNA and protein that are tightly associated with
rDNA, and this prevented reagents from penetrating into the structures in
DNA-specific staining processes, resulting in an overall staining and
visualization of the transcription units by the final step of uranyl acetate
staining. This effect may be more prominent in our semi-thin sections.
Nevertheless, the in situ christmas tree structures revealed in our
preparations provided direct evidence that rDNA transcription units are
located only at DFC regions near to the FC and that native rDNA transcription
units are linear compacted christmas trees
(Gonzalez-Melendi et al.,
2001
). In our preparations, although the appearance of the in situ
christmas tree resulting from the elongation of rRNA could vaguely be
recognized, the structural details of the rRNA and rDNA fibers inside in situ
`christmas trees' were not clearly distinguished, presumably owing to the
tight association between rRNA and rDNA. The length of these rDNA
transcription units ranged between 100 nm and 200 nm, shorter than those
previously reported for plant rDNA transcription units
(Gonzalez-Melendi et al.,
2001
). One possible explanation is that the method we have used to
observe the in situ `christmas tree' is not the immunogold labeling technique
(Gonzalez-Melendi et al.,
2001
), which may result in an error owing to the size of gold
particles. However, our observations may imply a higher compaction ratio of
rDNA transcription units than expected
(Koberna et al., 2002
).
In plant cells, FCs may be divided into the heterogeneous FC and the
homogeneous FC; the former contains chromatin in a condensed state whereas the
latter contains chromatin in a dispersed state
(Risueno et al., 1982).
Recently, Biggiogera et al. used EFTEM (energy filtering transmission electron
microscopy) to detect that the DNA component within the nucleolar FC of a
murine P815 animal cell was distributed as `DNA cloud'. Biggiogera et al.
believed that the rDNA in the FC extended to the DFC to initiate transcription
activities (Biggiogera et al.,
2001
). The conclusion from their experiments is fundamentally
consistent with the results of this paper. However, we have found that all DNA
components within FC are in a tightly condensed state, or composed of tightly
coiled DNA fibers, and no any decondensed DNA components were observed in the
FC of the nucleolus. The reason for this phenomenon is still unclear to us.
One possible explanation is that it may be a result of the different
experimental materials used.
As to the functions of FC, at present there are at least two hypotheses,
one is that FC is the anchoring site for the inactive rDNA storage, and the
other is that FC is the assembly site of the rRNA gene transcription machinery
(Medina et al., 2000). So far,
there has been clear evidence showing that RNA polymerase I is located in the
FC (Scheer et al., 1984). The reliability of this viewpoint cannot be verified
in this paper, but results from our experiments and many others show that FC
contains DNA that has no transcriptional activities
(Medina et al., 2000
;
Tao et al., 2001
). Our results
have also demonstrated that some extended DNA fibers stretch out of DNA clumps
in FC to connect with the extended rDNA. This has led us to believe that the
DNA within FC is the anchoring site for the rDNA special arrangement.
On the basis of the data from this paper and other researches, we have constructed a model for the nucleolus of plant cells (Fig. 12). This model mainly illustrates that FC contains DNA components in a highly condensed state, which may serve as the anchoring site for rDNA arrangement. The transcribing rRNA genes are present in a non-nucleosomal configuration in a highly decondensed state and are arranged in one-third of the DFC region, near the FC, which includes the periphery of FC and Ag-NOR proteins, as well as the protein molecules needed for transcription, such as RNA polymerase I transcription factor UBF, which are also distributed in this region (de Carcer et al., 1999).
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Acknowledgments |
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References |
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---|
Biggiogera, M., Burki, K., Kaufmann, S. H., Shaper, J. H., Gas, N., Amalric, F. and Fakans, S. (1990). Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy. Development 110,1263 -1270.[Abstract]
Biggiogera, M., Malatesta, M., Abolhassani-Dadras, S., Amalric, C., Rothblum, L. I. and Fakan, S. (2001). Revealing the unseen: the organizer region of the nucleolus. J. Cell Sci. 114,3119 -3205.
de Carcer, G. and Medina, F. J. (1999). Simultaneous localization of transcription and early processing markers allows dissection of functional domains in the plant cell nucleolus. J. Struct. Biol. 128,139 -151.[CrossRef][Medline]
Derenzini, M. (2000). The Ag-NORs. Micron 31,117 -120.[CrossRef][Medline]
Derenzini, M., Pession, A., Betts-Eusebi, C. M. and Novello, F. (1983). Relationship between extended, non-nucleosomal intra-nucleolar chromatin in situ and ribosomal RNA synthesis. Exp. Cell Res. 145,127 -143.[Medline]
Derenzini, M., Farabegoli, F. and Trere, D.
(1993). Localization of DNA in the fibrillar components of the
nucleolus: a cytochemical and morphometric study. J. Histochem.
Cytochem. 41,829
-836.
Fakan, S. and Hernandez-Verdun, D. (1986). The nucleolus and the nucleolar organizer region. Biol. Cell 56,189 -206.[Medline]
Ginisty, H, Sicard, H., Roger, B. and Bouvet, P.
(1999). Structure and functions of nucleolin. J. Cell
Sci. 112,761
-772.
Gonzalez-Melendi, P., Wells, B., Beven, A. F. and Shaw, P. J. (2001). Single ribosomal transcription units are linear, compacted Christmas tree in plant nucleoli. Plant J. 27,223 -233.[CrossRef][Medline]
Goodpasture, C. and Bloom, S. E. (1975). Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53, 37-50.[Medline]
Hozak, P. (1995). Catching RNA polymerase I in Fragranti: ribosomal genes are transcribed in the dense fibrillar component of the nucleolus. Exp. Cell Res. 216,285 -289.[CrossRef][Medline]
Jordan, E. G. and Rawlins, D. (1990). Three-dimensional localization of DNA in the nucleolus of Spirogyra by correlated optical tomography and serial ultra-thin sectioning. J. Cell Sci. 95,343 -352.[Abstract]
Koberna, K., Malinsky, J., Pliss, A., Masata, M., Vecerova, J.,
Fialova, M., Bednas, J. and Raska, I. (2002). Ribosomal genes
in focus: new transcripts label the dense fibrillar components and form
clusters indicative of "Christmas trees" in situ. J.
Cell Biol. 157,743
-748.
Lazdins. I. B., Delannoy, M. and Sollner-Webb, B. (1997). Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization. Chromosoma 105,481 -495.[CrossRef][Medline]
Medina, F. J., Cerdido, A. and Garcer, de G. (2000). The functional organization of the nucleolus in proliferating plant cells. Eur. J. Histochem. 44,117 -131.[Medline]
Meecak, I., Risueno, M. C. and Raska, I. (1996). Ultrastructural nonisotopic mapping of nucleolar transcription sites in onion protoplasts. J. Struct. Biol. 116,253 -263.[CrossRef][Medline]
Miller, O. L. and Beatty, R. R. (1969). Visualization of nucleolar genes. Science 164,955 -957.[Medline]
Olson, M. O. J., Dundr, M. and Szebeni, A. (2000). The nucleolus: an old factory with unexpected capabilities. Trends Cell Biol. 10,189 -196.[CrossRef][Medline]
Pession, A., Farabegoli, D., Trere, F., Novello, L., Montanaro, S., Sperti, F. and Derenzini, M. (1991). The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli. Chromosoma 100,242 -250.[Medline]
Puvion-Dutilleul, F., Bachellerie, J. and Puvion, E. (1991). Nucleolar organization of HeLa cells studied in situ hybridization. Chromosoma, 100,395 -409.[Medline]
Risueno, M. C., Medina, F. J. and Moreno Dzde la Espina, S. (1982). Nucleolar fibrillar centers in plant meristematic cells: ultrastructure, cytochemistry and autoradiography. J. Cell Sci. 58,313 -329.[Abstract]
Rudkin, G. T. and Stollar, B. D. (1977). High resolution detection of DNA/RNA hybrids in situ by indirect immunofluorescence. Nature 265,472 -473.[Medline]
Scheer, U. and Rose, M. K. (1984). Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immuno-cytochemistry. Proc. Natl. Acad. Sci. 81,1431 -1435.[Abstract]
Scheer, U., Xia, B. Y., Merkert, H. and Weisenberger, D. (1997). Looking at Christmas trees in the nucleolus. Chromosoma 105,470 -480.[CrossRef][Medline]
Shaw, P. J. and Jordan, G. E. (1995). The nucleolus. Annu. Rev. Cell Dev. Biol. 11, 93-121.[CrossRef][Medline]
Sirri, V., Roussel, P. and Hernandez-Verdun, D. (2000). The AgNOR proteins: qualitative and quantitative changes during the cell cycle. Micron 31,121 -126.[CrossRef][Medline]
Tao, W., Xu, W., Valdivia, M. M., Hao, S. and Zhai, Z. H. (2001). Distribution and transcription activity of nucleolar DNA in higher plant cells. Cell Biol. Intl. 25,1167 -1171.
Testillano, P. S., Sanchez-Pina, M. A., Olmedilia, I. A. and Risueno, M. C. (1991). A specific ultrastructural method to reveal DNA: the NAMA-Ur. J. Histochem. Cytochem. 39,1427 -1438.[Abstract]
Testillano, P. S., Gorab, N. and Risueno, M. C. (1994). A new approach to map transcription sites at the ultrastructural level. J. Histochem. Cytochem. 1, 1-10.
Thiry, M. and Thiry-Blaise, L. (1991). Locating transcribed and nontranscribed rDNA spacer sequences within the nucleolus by in situ hybridization and immunoelectron microscopy. Nucleic Acid Res. 19,11 -19.[Abstract]
Thiry, M. and Goessens, G. (1996). The Nucleolus During Cell Cycle. Berlin: Springer-Verlag.
Wachtler, F., Hartung, M., Devictor, M., Wiegant, J., Stahl, A. and Schwarzacher, H. G. (1989). Ribosomal DNA is located and transcribed in the dense fibrillar component of human Sertoli cell nucleoli. Exp. Cell Res. 184,61 -71.[Medline]