NOTES
Configuration of nucleolar
DNA in situ in nucleolus of
Allium cepa cells
TAO Wei1,2, HE Jie2, JIAO Mingda2, HE Mengyuan2
& HAO Shui 2
1. Department of Cell Biology, College of Life Sciences, Peking University, Beijing 100871, China;
2. Institute of Genetics and Cytology, Northeast Normal University,
Changchun 130024, China
Correspondence should be addressed to Hao Shui (e-mail:
[email protected])
Abstract The location and configuration of nucleolar DNA
have not been determined for a long time. In this paper, we
have observed the nucleolar ultrastructure and the character
of nucleolar DNA in Allium cepa cells by conventional electron microscopy and the cytochemical NAMA-Ur DNA specific staining method. Furthermore, we have properly improved the NAMA-Ur method so as to analyze the location
and configuration of nucleolar DNA in situ. Our results indicated that the nucleolar DNA in Allium cepa cells is mainly
located at the border between fibrillar centers and dense
fibrillar component, especially distributed in the configuration of encircling the fibrillar centers.
Keywords: nucleolus, NAMA-Ur method, configuration of nucleolar
DNA, Allium cepa.
Nucleolus is a subnuclear structure where the rRNA
genes are transcribed, rRNA transcript is processed and
pre-ribosome forms in eukaryote cells. The ultrastructure
of nucleolus are composed of fibrillar centers (FC), dense
fibrillar component (DFC) and granular component (G).
One of the most important problems about the nucleolar
structure is the nucleolar DNA location and its distribution
in nucleolus, for a thorough understanding of nucleolar
structure and the rRNA genes transcriptional sites, it is
very essential to elucidate this problem. Up to now, it is
still unknown whether the location of nucleolar DNA is at
DFC or at FC[1]. Although some models about the configuration of nucleolar DNA have already been mentioned,
they are just hypothesis, and need to be verified by direct
experimental evidence[2].
The experimental methods for the investigation of
nucleolar DNA location include osmium- ammine DNA
specific staining[3], DNA antibody labeling[4] and the incorporation of labeled dUTP[5]. Comparatively the osmium-ammine method is more visible since it makes nucleolar DNA be directly observed at the ultrastructure
level. However, since the osmium-ammine method is
comparatively complex and hard to operate a new cytochemistry DNA specific staining method is found, that is
64
NAMA-Ur[6]. Unfortunately, after treating the nucleolus by
either the early osmium-ammine method or the present
NAMA-Ur method, all of the subnucleolar components
except DNA are bleached, so researchers cannot trace the
accurate position of nucleolar DNA in situ, to say nothing
of analyzing nucleolar DNA configuration. Thereby, after
testing the present NAMA-Ur method, we further improved it properly. Thus, when the nucleolus was treated
by the improved NAMA-Ur method, nucleolar DNA was
specifically stained to the state with a high electron density, but FC and DFC were also reserved as the weak
background, this made us directly observe the location and
configuration of nucleolar DNA in situ. Our results
provide very important evidence for clarifying the structure of nucleolus and the transcription sites of rRNA
genes.
1 Materials and methods
( ) Materials. Root tip meristem of Allium cepa
was used as the material. It was obtained from onion bulbs
grown under standard conditions at 15 .
( ) Methods. Conventional specimen preparation:
The root tips were carefully excised and fixed with 2.5%
glutaraldehyde in 0.1 mol/L in phosphate buffer saline
(PBS, pH 7.4) for 2 h at room temperature. After rinsing in
bidistilled water for 20 min, they were postfixed in 1%
osmium tetroxide for 60 min. Samples were dehydrated in
an ethanol-acetone series and embedded in Epon 812.
Ultra-thin sections of 60 80 nm thick were stained with
uranyl acetate and lead citrate, then observed in a Hitachi600B transmission electron microscope.
NAMA-Ur procedure: The unmodified NAMA-Ur
DNA specific staining method is referred to ref. [6]. Modified experiment process is briefly described in the following: Samples were fixed in 3% glutaraldehyde and 4%
formaldehyde in 0.1 mol/L PBS for 3 h at 4 . After
washing in 0.1 mol/L PBS, specimens were immersed in
NA solution (0.5 mol/L NaOH, 4% formaldehyde) for 8 h,
then rinsed in bidistilled water three times for 10 min each,
then in 1% acetic acid three times for 10 min each, and in
bidistilled water again, three times for 10 min each. After
that, they were treated with a freshly prepared methanol︰
acetic anhydride (4.5︰1, v︰v) mixture at 25 for 18
24 h until the samples were properly bleached. They were
dehydrated in a methanol series and embedded in Epon
812. Semi-thin sections were stained with 2% aqueous
uranyl acetate for 70 min at 60 . After cooled down to
room temperature, sections were washed in bidistilled
water and dried at 25 , then observed in a Hitachti 600B
transmission electron microscope at 75 kV.
2 Results and discussion
( ) Location of nucleolar DNA. By conventional
electronic microscopy, the three basic components of nu-
Chinese Science Bulletin Vol. 46 No. 1 January 2001
NOTES
cleolus on ultra-thin sections were recognized. They included fibrillar centers (FC) with the lowest electronic
density, dense fibrillar component (DFC) around FC with
high electronic density, and granular component (G) distributed at the edge of nucleolus and between DFC. The
vacuole has also been observed inside the nucleolus (fig.
1(a)). Under the conventional staining condition, RNA,
DNA and proteins in nucleolus were all stained equally, so
we could not distinguish their respective distribution in
nucleolus directly.
After nucleolus was treated by unmodified NAMAUr method, all of the subnucleolar components were
bleached except DNA, which were specifically stained.
Under this condition, although nucleolar DNA could be
observed directly, we were unable to decide its accurate
location in situ in nucleolus. However, after nucleolus was
treated by the modified NAMA-Ur method, nucleolar
DNA could be recognized since they still had high electronic density, and at the same time, FC and DFC were
reserved as a background, thus we could ascertain nucleolar DNA location in situ (fig. 1(c), (d); fig. 2(a) (d)).
Our observation indicates that there were three kinds of
situations related to nucleolar DNA location: nucleolar
DNA located at FC (fig. 1(c)), at DFC (fig. 1(d)) and at
the border between FC and DFC (fig. 2(a)). On different
nucleolus sections, nucleolar DNA appeared at different
positions, while all of nucleolar DNA in different positions
possessed two kinds of modality structure: condensation
and uncondensation.
At present, the opinions about the DNA location in
nucleolus are diverse. Generally speaking, there are two
kinds of viewpoints: one is that nucleolar DNA are localized at DFC; the other at FC (see ref. [7]). In resent years,
a new concept about the subarea in nucleolus appeared,
which says that it is at the border between FC and DFC.
This has drawn the researchers’ attention, because some
resent results showed that there exist DNA components
(some reports describe the region as the periphery of FC or
DFC closed to FC)[1, 8, 9]. Thus, present controversial
Chinese Science Bulletin Vol. 46 No. 1 January 2001
problem about nucleolar DNA is whether the location of
nucleolar DNA is at DFC or at FC except for the border
between FC and DFC.
Our results revealed that nucleolar DNA is located at
DFC, FC and the border between DFC and FC, but on
different sections, DNA are located at different positions
(fig. 1(c), (d); fig. 2(a)). The former results of others are
obviously different from ours. The former results showed
that nucleolar DNA was at DFC or at FC. However, our
results indicated that nucleolar DNA exist at both FC and
DFC on different sections. The reasons of divergent results
are not clear at present. But we suggest that one reason is
the different experimental materials used, according to our
research results (unpublished data) and others’ conclusions,
the structure of plant cell nucleolus is very different from
the animal cell’s[2]. Another possible reason is the different
methods. Nucleolar DNA should be distributed in a threedimensional and continuous way in nucleolus, so the different place of nucleolus section showed different locations of nucleolar DNA. The strongpoint of our experimental method in this note lies in directly observing the
location of nucleolar DNA in situ and more convenient for
analyzing the nucleolar DNA configuration. Whereas
former studies about the location of nucleolar DNA were
limited by their experimental method, this leads to their
results lopsided.
( ) Configuration of nucleolar DNA. We made a
further analysis on nucleolar DNA configuration by the
improved NAMA-Ur method. It is commonly accepted
that nucleolar DNA comes from nucleolus-associated
chromatin. In this work, we have directly observed the
nucleolus-associated chromatin extend into nucleolus (fig.
1(b)). But according to our observation, although nucleolar DNA in different sections was located at different positions, it was more universal that nucleolar DNA is associated with the border between FC and DFC in Allium
cepa cells (the results of statistical analysis indicate that
the number ratio of nucleolar DNA at the border between
65
NOTES
Fig. 1. (a) Electron micrograph of an Epon 812-embeded nucleolus, stained with uranyl acetate and lead citrate, showing the ultrastructure of Allium cepa cell nucleolus. FC, Fibrillar centers; DFC, dense fibrillar component; G, granular component; NV, nucleolar vacuole.
(b) Nucleolus stained with unmodified NAMA-Ur method. All of the nucleolar components were bleached except DNA. Extranucleolar
chromatin extended into nucleolus (arrows). (c) and (d) Nucleolus stained with modified NAMA-Ur method. Nucleolar DNA located at
FC (c) and at DFC (d). Arrows indicate DNA components. Nu, nucleolus. 20 000.
FC and DFC is 67%, which includes the number of nuclethe number ratio of nucleolar DNA only at FC is 11%;
only at DFC is 21%). Moreover, nucleolar DNA at the
border is distributed in the configuration of encircling FC
(fig. 2(b) (d)). Nucleolar DNA encircling FC is composed of DNA mini-conglomerations, DNA fibril
stretched out from the edge of each DNA conglomeration
(fig. 2(b), (c)). In addition, we have also found that nucleolar DNA passes through FC sometimes in the course
of encircling FC (fig. 2(d)).
Although more and more researchers describe the
border between FC and DFC as the possible core position
of nucleolar DNA[1], this suggestion is devoid of experimental evidence, it is also not clear how the nucleolar
66
DNA arranged in the core position. Our results directly
showed that nucleolar DNA at the border between FC and
DFC was in the configuration of encircling FC, this kind
of DNA configuration has not been reported before. Our
results also showed that there were DNA components at
both FC and DFC, we suggested that these DNA components were probably in the midway of the progress that
nucleolus-associated chromatin entered into nucleolus and
distributed in the form of encircling FC. We speculated
that rDNA should be transcribed under this kind of configuration. Although some reports indicated that transcription sites of rRNA genes existed at the border between
DFC and FC[10,11], there were also some evidence showing
that rRNA transcription occurred at DFC [12,13]. Further-
Chinese Science Bulletin Vol. 46 No. 1 January 2001
NOTES
more, we also found that DNA fibril coming from the
nucleolar DNA conglomeration surrounded FC at the border extended into DFC, thus we infer that the transcription
site of rRNA genes is probably located at the DFC and the
border between FC and DFC in Allium cepa cell nucleolus.
Fig. 2. Nucleolus stained with modified NAMA-Ur method. (a) Showing the nucleolar DNA located at the border between FC and
DFC. (b) and (c) The nucleolar DNA is located at the border region and distributed in the configuration of encircling FC. (d) Showing the nucleolar DNA passed through FC. Arrows indicate DNA components; Nu, nucleolus; FC, fibrillar centers. 20 000.
( ) Model of nucleolus structure. At present, there
are two models about rRNA genes configuration in nucleolus: helix model (rDNA arranged at DFC spirally) and
radial loop model (rDNA arranged at DFC radially)[2]. But
the two models are hypothetical and need to be further
verified. At present, it looks as if the helix model is more
coincident with our observation results. But the helix
model still has difference from our results. The helix model suggested that the spirally distributed rDNA constituted
DFC region, namely the transcribed rDNA takes up all the
DFC, whereas our observation showed that nucleolar
DNA is mainly located at the border in the form of encircling FC, not taking up all the DFC. Moreover, helix
model indicated that there were no DNA components at
Chinese Science Bulletin Vol. 46 No. 1 January 2001
the interior of FC, but our results manifest that the nucleolar DNA encircled FC sometimes passed through FC
(fig. 2(d)).
Raska et al.[14] pointed out that FC and DFC form a
single functional domain, they play different roles in
rRNA gene transcription activity. Concerning about our
findings that DNA is distributed in the configuration of
encircling FC, we infer that the border between DFC and
FC is probably the best place for FC and DFC to perform
their respective functions. As for the questions of whether
this configuration is fit for other cell systems and whether
it is related to the mechanism of highly efficient rDNA
transcription, further study is needed.
Acknowledgements This work was supported by the National Natural
67
NOTES
Science Foundation of China (Grant No. 39770367).
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(Received April 12, 2000)
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Chinese Science Bulletin Vol. 46 No. 1 January 2001