Vol. 43 No.3
SCIENCE IN CHINA (Series C)
June 2000
Structures of nucleolus and transcription sites of rRNA
genes in rat liver cells
TAO Wei (陶 伟)* JIAO Mingda (焦明大) HE Jie (赫 杰)
HE Mengyuan (何孟元) & HAO Shui (郝 水)
Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China
Correspondence should be addressed to Hao Shui
*Current address: School of Life Science, Peking University, Beijing 100871, China
Received November 30, 1999
Abstract We observed the ultrastructure of nucleolus in rat liver cells by conventional electron
microscopy, and employed cytochemistry NAMA-Ur DNA specific stain method to analyze the distribution and position of nucleolar DNA in situ. The results showed that nucleolar DNA of rat liver
cells comes from nucleolus-associated chromatin, and continuously extends in the dense fibrillar
component (DFC) of nucleolus, localizes at the periphery of fibrillar center (FC) and in DFC. Furthermore, by employing anti-DNA/RNA hybrid antibodies, we directly and selectively labeled transcription sites of rRNA genes and testified that localization of transcription sites not only to DFC but
also to the periphery of FC.
Keywords:
nucleolus, NAMA-Ur method, location of nucleolar DNA in situ, transcription sites of rRNA genes, rat liver
cell.
Basic structure of nucleolus in eukaryote cells can be divided into fibrillar centers (FC), dense fibrillar component (DFC) and granular component (G). At the end of the 1960s, Miller and
others directly observed the structure of Christmas tree formed when rDNA was transcribed,
which caused the most interests of researchers to localize transcription sites of rRNA genes in
nucleolus. Early autoradiography results showed that the transcription sites of rRNA genes in
nucleolus were located at the DFC[1], but it has been suspected since the 1980s. The results of the
RNA polymerase I antibody immunogold labeling and labeled UTP tracking experiments showed
that the transcription sites of rRNA genes in nucleolus were located at FC and on the edge of FC,
but not DFC[2,3]. Up to now, there are no definite conclusions about the location of DNA and transcription site of rRNA genes in nucleolus[4]. We directly observed the distribution and location of
DNA in nucleolus in situ, and employed a new selective direct labeling method to locate the happening transcription activity of rRNA genes in nucleolus. We report here the results of our studies
on the structures of nucleolus and transcription sites of rRNA genes in rat liver cells.
1 Materials and methods
1.1 Cells and tissues
Hepatocytes from mouse liver were used as animal tissue. Small fragments of liver were ob-
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tained by dissection of 2-month-old mice.
1.2
Conventional specimen preparation for electron microscope
Samples were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffered saline (PBS)
pH 7.4 for 2 h; after rinsing in bidistilled water for 20 min, they were dehydrated in an ethanolacetone series and embedded in Epon812. Ultra-thin sections of 60
80 nm thick were stained
with uranyl acetate and lead citrate, and observed in a Hitachi EM 600 at 75 kV.
1.3
NAMA-Ur method for DNA performed EN Bloc
DNA specific stain method was according to ref. [9]. As far as the distribution of DNA in
nucleolus was concerned, we employed the NAMA-Ur method reported by Testillano et al.[9] and
made certain modification. The modified experiment processing is briefly described as follows:
samples were fixed in 3% glutaraldehyde and 4% formaldehyde in 0.1 mol/L PBS for 1 h at 4 .
After washing in 0.1 mol/L PBS, they were immersed in 0.5 mol/L NaOH in 4% formaldehyde
overnight (NA), and then rinsed in (i) bidistilled water, three times for 10 min each, (ii) 1% acetic
acid three times for 10 min each, and (iii) bidistilled water again, three times for 10 min each.
After that they were treated with a freshly prepared methanol: acetic anhydride (5:1, v : v) mixture
at
25
for 18 24 h until samples were bleached. They were dehydrated in a methanol series
and embedded in Epon812. Semi-thin sections were stained with 2% aqueous uranyl acetate for 70
min at 60 . After washing in bidistilled water and drying at 25 , they were observed in a Hitachi EM 600-2 at 75 kV.
1.4 Immunogold labeling of anti-DNA/RNA hybrid antibody
The anti-DNA/RNA hybrid[5] antibody was kindly provided by Prof. Stollar. Lowicryl K4M
was bought from Chemische Werke Lowi GMBH & Co in Germany. Protein A conjugated to
colloidal gold particles was bought from Sigma.
Processing for Lowicryl K4M embedding. Samples were fixed in 3% glutaraldehyde and 4%
formaldehyde in PBS for 2 h. After washing in bidistilled water for three times, 30 min each, they
were dehydrated in an ethanol series and permeated by 100% ethanol: K4M (1:1) mixture for 12 h
at 0
,
100% ethanol:K4M (1:2) mixture for 1 h at −10
, and 100% K4M for 60 h at
−30
. After that they were embedded in Lowicryl K4M at −30
under UV irradiation over 24 h,
then irradiated again for 2 3 days at room temperature.
Immunogold labeling of anti-DNA/RNA hybrid antibody. Ultra-thin Lowicryl sections from
rat liver cells were mounted on formvar nickel grids and washed in 2 SSC (1
SSC=0.15 mol/L
sodium chloride, 0.015 mol/L sodium citrate) for 5 min, then floated on 50% formamide in 2
SSC for 10 min at 25 . PBS three times for 1 min each; and 5% BSA (PBS, 0.05% Triton X-100)
for 10 min. Then the grids were incubated in the goat anti-DNA/RNA hybrid antibodies diluted at
1:300 in PBS for 1 h at room temperature. After several washings in PBS, they were floated in
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protein A conjugated to 10-nm colloidal gold particles diluted at 1:25 in PBS for 45 min at room
temperature. They were subsequently washed in PBS and bidistilled water. Finally, the sections
were counterstained with 5 % uranyl acetate. Sections were observed in a Hitachi EM H-600-2 at
75 kV.
Controls were done, replacing the goat anti-DNA/RNA hybrid antibodies by antibody diluent.
2 Results
2.1
Ultrastructure of nucleolus
Using conventional electron microscopy, we found that the nucleolus of rat liver was composed of fibrillar centers (FC), dense fibrillar component (DFC) and granular component (G). FC
has the lowest electronic density, and more or less was filled with certain substance. DFC has the
highest electronic density, and surrounds FC. G was located on the borders of the nucleolus and
between DFC. There was nucleolus-associated chromatin (NAC) arranged on the outer edge of
nucleolus. Nucleolus is usually located under the nuclear membrane, because the telomere DNA
sequence has high affinity to Lamin B[6] (fig. 1(a), (b)).
2.2
Distribution of DNA in nucleolus
In order to study the distribution of DNA in nucleolus, we used NAMA-Ur cytochemistry
DNA specific stain method to treat the rat liver cells. Cytoplasm, interchromatin and most part of
nucleolus in rat liver cells were bleached, whereas DNA in nucleoplasm and nucleolus was easily
recognized since it had high electronic density by specifically staining (fig. 1(c), (d)). The abovementioned observation showed that NAMA-Ur cytochemistry method is very effective on DNA
specific staining.
From nucleolus treated with NAMA-Ur technique for DNA, we could obviously observe a
large amount of DNA arranged around the edge of nucleolus, in the form of enveloping nucleolus.
We can usually find dump-like or line-like DNA clumps arranged in the center or on the edge of
nucleolus in the form of condensation or uncondensation, and DNA fibril stretching out from the
edge of DNA clumps (fig. 2(a), (b)). We could also observe a large amount of DNA in some nucleoli, which mingled together, continuously extended, and occupied most part of region of nucleolus (fig. 2(c)). From fig. 2(d), we could find DNA in NAC entering into nucleolus in the form
of line. In some parts it was in the form of condensation, while in most parts it was in the form of
uncondensation. The mentioned results testified that DNA in nucleolus comes from NAC, and the
structure of the nucleolar DNA is variable (fig. 2(d)).
In order to find the accurate location of DNA in nucleolus, we made some modification to
NAMA-Ur DNA specific stain method, so that nucleolus could reserve some backgrounds to
make clear the structure of FC and DFC after staining. This could help us localize DNA in nucleolus in situ (fig. 3(a), (b)). After DNA around nucleolus entered into it, DNA extended from
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Fig. 1. Electron micrographs showing the ultrastructure of nucleolus and nucleolar DNA in rat liver cells. (a)
Nucleus (N) and nucleolus (arrowhead) of mouse hepatocyte. Bar: 1 µm. (b) High magnification of nucleolus in (a)
indicated by arrowhead, showing fibrillar centers FC
dense fibrillar component DFC
granular component (G)
and nucleolus-associated chromatin (NAC). Cyt: cytoplasm. Bar: 0.5 µm. (c) Nucleus (N) of mouse hepatocyte
after being treated with NAMA-Ur technique for DNA. Cytoplasm (Cyt
interchromatin and most part of nucleo-
lus (arrowhead) were bleached. Chromatin in nucleoplasm and DNA in nucleolus could be recognized by specific
stain method. Bar: 1 µm. (d) High magnification of nucleolus in (c) indicated by arrowhead, showing nucleolar
DNA after NAMA-Ur technique (arrowhead). Bar: 0.5 µm.
DFC to FC, and showed a trend towards encircling FC (fig. 3(a)). DNA continuously extended
and moved in DFC. Sometimes DNA could reach or enter into the edge of FC, but it cannot enter
interior of FC (fig. 3(b)). The above-mentioned results indicated that DNA is located both in DFC
and on the edge of FC in nucleolus of rat liver cell.
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Fig. 2.
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Distribution and configuration of nucleolar DNA. (a) and (b) Nucleolus (Nu) after DNA specific staining.
There was a lot of nucleolus associated-chromatin DNA, which enveloped the nucleolus. Dump-like or line-like DNA
clumps are located in the center (a) or on the edge (b) of nucleolus in the form of condensation or uncondensation, and
we could find that DNA fibrils extend out on the edge of DNA. Bar: 0.5 µm. (c) and (d) Nucleolus (Nu) after DNA
specific staining. DNA was intertwined and constantly extended and occupied the relatively large regions in nucleolus
(c). DNA of NAC entered into nucleolus (arrowhead). Some were in the form of condensation, but most were in the
form of uncondensation (d). Bar: 0.5 µm.
2.3
Transcription sites of rRNA genes in nucleolus
During transcription, DNA could form temporary DNA/RNA hybrid double chain structure.
Under the help of anti-DNA/RNA hybrid antibodies, we could selectively and directly label transcription sites of rRNA genes. By the control experiment, we could find the good specificity of the
immunogold labeling. These results showed that in the nucleoplasm, some labeling signals of
DNA transcription activity appear at uncondensed chromatin or on the edge of condensed chro-
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matin. In nucleolus, however, the labeling signals of DNA transcription activity appear in DFC
and on the edge of FC. We have never found the labeling signals in NAC, G and inside FC. So the
transcription sites of rRNA genes in rat liver cell should be at the periphery of FC as well as in
DFC (fig. 3(c)).
Fig. 3. Localization of DNA and transcription sites in situ within nucleolus. (a) and (b) Nucleolus treated with the improved NAMA-Ur DNA specific stain method. We could distinguish FC and DFC in nucleolus. DNA on the edge of nucleolus entered into nucleolus, and extended from DFC to FC, distributed
around FC (a). DNA constantly extended in DFC. It could reach the edge of FC, but could not enter into
FC (b).
N, Nucleus. Bar 0.5 µm. (c) Nucleolus labeled by anti-DNA/RNA hybrid antibody. Signal
(arrowhead) of transcription activity is located on the edge of FC and in DFC. G, Granular component;
NAC, nucleolus associated-chromatin. Bar: 0.5 µm.
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3 Discussion
We have found many reports about the sites and arrange form of DNA in nucleolus. To study
DNA location in nucleolus, the researchers employed the osmium-ammine DNA specific stain
method, DNA antibody labeling method[7], in situ hybridization using electron microscopy[8] and
so on, but the results were quite different. The discussion focused on whether DNA is located in
FC or DFC. Although researchers have directly observed the distribution feature of DNA in nucleolus by using the osmium-ammine DNA specific stain method, this method is sophisticated,
and its employment is limited. In recent years, a new DNA specific stain method of NAMA-Ur
has gradually been set up[9]. At present, the application of this method is rare, but our result
showed that this experiment system had good capability for DNA specific staining. It could show
the feature of DNA distribution in nucleolus, and at the same time showed us that DNA in nucleolus came from NAC .
As far as DNA distribution in nucleolus sites is concerned, it is just as Jordan said: seeing is
believing[10]. By using DNA specific stain method (including the osmium-ammine method and
NAMA-Ur method), we found that all nucleolar components were bleached except DNA. So nucleolar DNA could hardly be used to distinguish DNA distribution in situ. We could only infer
DNA sites according to the probable region of DFC and FC. This result would be suspicious. In
order to solve this problem, we modified NAMA-Ur method to some extent, so that the direct
evidence of DNA distribution in nucleolus in situ was gained at the level of ultrastructure. And the
result clearly showed that DNA in nucleolus of rat liver cell was located on the edge of FC and in
DFC, whereas we could not find DNA inside FC. This is the first time that cytochemistry method
was used to directly observe DNA distribution in nucleolus in situ. Compared with rDNA molecule hybrid and DNA anti-body immunogold labeling method, our results are reliable.
The usual method to locate the transcription sites of rRNA genes in nucleolus is to incorporate the labeled UTP into various cells. Hao et al. used autoradiography method to provide labeling on transcription sites of rRNA genes in nucleolus of Allium cepa and got the results to support the traditional opinion that DFC is the transcription sites of rRNA genes in nucleolus of Allium cepa [11]. However, Thiry and Goessens used the same method to study the transcription sites
of rRNA genes in nucleolus in Ehrlich cancer cell, and they found that labeling signal concentrated on FC. So they thought that the transcription sites of rRNA gene were located at FC[3] . In recent years, a new way to locate transcription sites of rRNA gene is to draw Br-UTP into cells as
tracemark. Hozak et al. drew Br-UTP into HeLa cell. Their results showed that no matter how
long it was drawn, there was no exception for marked signal to appear in DFC, but there was no
marked signal inside FC[12]. Meecak did the same operation on protoplast from Allium cepa root
tip meristems, and drew Br-UTP into nucleolus at a certain time. The result indicated that marked
signal concentrates on the edge of FC[13].
To sum up, the results of studying transcription sites of rRNA genes in nucleolus are quite
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different and uncertain. One of the important reasons is that most of the present methods to study
transcription sites of rRNA gene still have disadvantages, and the results are suspected, which,
based on different process and uncertain time of incorporation, especially the quick extension of
transcription template, may cause the marked signal that was not the original transcription site[8].
Aiming at the disadvantages of the present methods, it is important to use anti-DNA-RNA
hybrid antibody to directly and selectively label rRNA genes, which are being transcribed. Some
researchers have already used RNA/DNA hybrid antibodies to mark the transcription sites of puffs
in polytene chromosomes by immunofluorescence[5]. Furthermore, to study transcription sites of
rDNA in nucleolus of rat liver cell, we used this method at the level of ultrastructure. Our results
showed that the transcription sites of rRNA genes in nucleolus of rat liver cell are located on the
edge of FC and in DFC. What’s more, the labeling of transcription sites of rRNA genes in nucleolus is at the same place as DNA in situ got from our observation by NAMA-Ur method.
In addition, some research reported that RNA polymerase I[14], RNA polymerase I transcription factor[15] and some protein molecules related to rRNA gene transcription activity are located
on the edge of FC and in DFC. These results are coincident with ours.
Acknowledgements
We are most grateful to Prof. Stollar (University of Tufts, USA) for kindly providing us
with anti-RNA/DNA hybrid antibody. This work was supported by the National Natural Science Foundation of China
(Grant No. 39770367).
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