1 Secção de Genética, Departamento de Botânica e
Engenharia Biológica, Instituto Superior de Agronomia, Tapada da Ajuda,
1349-017 Lisboa, Portugal
2 Departamento de Ciências Biológicas e Naturais, Universidade
Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-042
Lisboa, Portugal
3 Departamento de Biologia Vegetal, Faculdade de Ciências de Lisboa, Bloco
C2, Campo Grande, 1749-016 Lisboa, Portugal
* Author for correspondence (e-mail: anadelaunay{at}isa.utl.pt )
Accepted 7 May 2002
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Summary |
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Key words: Homologous gene expression, rDNA topology, Chromosome structure, Telocentric chromosomes, Rye
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Introduction |
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A number of studies have examined the organisation of active and inactive
genes of the same locus during interphase, using ribosomal chromatin as an
experimental model (Junera et al.,
1997; Leitch et al.,
1992
; Rawlins and Shaw,
1990
; Robert-Fortel et al.,
1993
; Roussel et al.,
1996
). The rRNA genes occur in tandem arrays at multimegabase loci
known as the nucleolar organizer regions (NORs). The active ribosomal genes,
which usually give rise to a visible secondary constriction on metaphase
chromosomes, can be detected by a specific silver staining technique
(Ag-staining), which allows the visualization of Ag-NORs that were transcribed
in the previous interphase (Hubbell,
1985
; Jimenez et al.,
1988
; Morais-Cecilio et al.,
2000
; Robert-Fortel et al.,
1993
). This technique is based on the detection of a set of
argyrophilic protein markers associated with active ribosomal genes that
reduce the silver under acidic conditions and are found in the nucleoli during
interphase (Goodpasture and Bloom,
1975
; Roussel et al.,
1996
; Zurita et al.,
1998
). In plants, the usual pattern of active NORs detected by
FISH is described as `intranucleolar labelled structures that emanate from
perinucleolar heterochromatin sites'
(Delgado et al., 1995
;
Leitch et al., 1992
;
Rawlins and Shaw, 1990
;
Shaw et al., 1993
). This
pattern is in agreement with the current model of NOR chromatin organisation,
which proposes that a subset of rRNA genes is packaged into heterochromatin,
and thus is inaccessible to the transcription machinery, and that another
fraction is euchromatic and transcribed
(Carmo-Fonseca et al.,
2000
).
In actively dividing cells of most plant species the number of copies of
rRNA genes is several times higher than in most animals, although the numbers
of active genes inside the nucleolus are similar in both cell types
(González-Melendi et al.,
2001). Furthermore, in most plant species the number of active
rRNA genes is very small, as recently demonstrated in Pisum sativum,
where only about 5% of the units are transcribed
(González-Melendi et al.,
2001
). Thus, a diminutive fraction of rRNA genes from only one
locus appears to be sufficient to supply all the ribosomal machinery of a
cell. Even so, homologous NORs usually have identical behaviour in terms of
their expression, and consequently both are either transcribed or silenced.
Several attempts, using in situ hybridization with rDNA probes in various
diploid species with only one pair of homologous NORs, have shown a direct
correlation between the number of ribosomal cistrons in each NOR and its level
of transcription (Zurita et al.,
1997
; Zurita et al.,
1998
; Zurita et al.,
1999
). However, the relative activity of homologous loci with
similar numbers of ribosomal cistrons has not yet been investigated. This is
probably due to methodological constraints, as the evaluation of rRNA gene
activity is usually performed in highly condensed metaphase NORs.
The aim of the present study was to investigate the expression and organization patterns of homologous NORs in rye with equivalent numbers of rRNA genes. Expression patterns were characterized by silver staining in distended chromosomes that allowed the discrimination between dominant and under-dominant homologous rDNA loci. A structural variant rye line carrying an intact 1R chromosome and two telocentric 1R chromosomes (short and long arms), was also used to disclose any possible effect of parental imprinting on inter-homologous NOR dominance. The effects of nucleotypic changes, in terms of the chromosome structural variant, were also analysed. This was done using FISH with the pTa71 probe to better understand the modulation of RNA gene expression patterns between homologous loci.
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Materials and Methods |
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Seed treatments
Seeds were germinated in water for 3 days at 24°C. Root-tips from
cultivar Imperial were then cold treated (30 hours at 0°C), colchicine
treated in a 0.1 mg/ml solution during 4 hours at 22°C, or placed in a 1
ng/ml oryzalin solution for 3 hours at 22°C. From each numbered seedling,
three root-tips were excised so that at least one root-tip per seedling was
used for each of the three c-mitotic pre-treatments. For the
1R1RS1RL line, only colchicine was used. All root-tips
were fixed in 3:1 (v/v) absolute ethanol/glacial acetic acid for 24 hours at
room temperature and stored at -20°C.
Silver staining
The fixed material was transferred to a fresh solution of FAA (50%
ethanol:37% formaldehyde:glacial acetic acid, 18:1:1, v/v) for 3 days at
-20°C. Chromosome preparations were produced by enzymatic digestion, as
described previously (Schwarzacher et al.,
1989), and squashes were made in 45% acetic acid. Silver staining
followed a technique described elsewhere
(Neves et al., 1995
).
Flourescence in situ hybridisation (FISH) and imaging
Sequential FISH was performed on the same chromosome preparations from
cells previously silver stained, without removing the silver labelling. The
FISH procedure followed that described previously
(Morais-Cecílio et al.,
2000), using the DNA probe pTa71. This probe is a 9 kb
EcoRI fragment of the ribosomal DNA from wheat (Triticum
aestivum), containing the 5.8S, 18S, 25S and non-transcribed spacer
sequences. DNA was counterstained with DAPI (4',6 diamino-2-phenylindole
dihydrochloride). Preparations were observed by epifluorescence microscopy
(Leitz Biomed), with the appropriate filters. Images were collected using an
AxionCam digital camera (Zeiss) controlled by AxioVision 3.0, and processed
using the Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA).
Confocal microscopy and imaging
To analyse rDNA organization at interphase, root-tips were digested using
the same enzymatic mixture as before, followed by dissection in 45% acetic
acid under a stereomicroscope. The root-cap and non-dividing parts of the root
were discarded, and the remaining (mainly meristematic) cells were then
pipetted onto ethanol-cleaned eight-well glass slides (ICN Biochemicals), and
left to air dry. This procedure avoids squashing and preserves the
architecture of the nucleus. Following in situ hybridisation interphase cells
were imaged with an MRC-600 confocal scanning laser microscope equipped with a
15 mW krypton-argon laser (Bio-Rad Microscience, Hemel Hempstead, UK). The
microscopy data were transferred to NIH image (a public domain program for
Macintosh available via ftp from
ftp://zippy.nimh.nih.gov
) and processed using the Adobe Photoshop 5.0 (Adobe Systems).
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Results |
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To evaluate the level of expression of each rDNA locus, the length of each NOR was compared with the length of the satellite (chromosome region located on the telomeric side of the secondary constriction). At the first level of discrimination for NOR size we considered that a secondary constriction is condensed (Fig. 1) when it is smaller or has the same length as the satellite, and that it is distended (Fig. 2) when it is longer than the satellite. As shown in Table 1, there is a difference in NOR condensation according to the treatment applied. In the cold-treated root tips the overall frequency of condensed NORs is high, whereas using colchicine and oryzalin at 22°C, the majority of NORs were of the distended type (Table 1). No apparent difference was obtained with oryzalin or colchicine, and colchicine was chosen as the preferred treatment thereafter.
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|
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Homologous NORs with equivalent numbers of rRNA genes show
differential expression
In situ hybridisation with the rDNA probe pTa71 was used to estimate the
number of ribosomal cistrons present in a NOR, based on the strength of the
hybridisation signal, an approach followed by several authors
(Leitch and Heslop-Harrison,
1992; Mandrioli et al.,
1999
; Mellink et al.,
1994
; Suzuki et al.,
1990
; Zurita et al.,
1998
; Zurita et al.,
1999
). In metaphase cells from cold-treated root-tips of cultivar
Imperial the pTa71 detected by FISH showed equivalent signals (in size and
intensity) in homologous NOR loci of the condensed type. This result indicates
that homologous rDNA loci have similar numbers of genes. When silver staining
and FISH results were simultaneously observed in condensed NORs, the signals
were found to be largely overlapping (Fig.
1).
A comparison of homologous Ag-NOR sizes was performed in each cell, by observing Ag-NORs with similar lengths (Figs 1, 2) or with distinct dimensions (Fig. 3). Ag-NORs were considered differently sized (i.e. heteromorphic, resulting from differential rDNA expression) when differing by at least 25% in length. The frequency of cells with heteromorphic Ag-NORs was much higher after chemical treatment compared with cold treatment (Table 1). In addition, the structural resolution of the NOR loci was improved on metaphase chromosomes with distended secondary constrictions. Sequential silver staining and FISH with pTa71 revealed a centromere-proximal condensed NOR region, only detected by FISH, whereas the overlapping silver and in situ signals are more distally located in the NOR region that decondenses progressively towards the satellite (Fig. 3).
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The structure of nucleolar chromosomes affects differential
expression between homologous NORs and discloses non-parental imprinting
We investigated the possible influence of NOR bearing chromosome structure
on the expression of homologous NORs using the double monotelosomic hybrid rye
line (1R1RS1RL). In this line, discrimination between
rDNA loci of parental origin is easily achieved, through clear NOR-bearing
chromosome identification based on chromosome morphology at metaphase. The
root-tip cells used were obtained from three different individuals, where
cultivar Imperial was the female progenitor in two of them, while the third
was produced using cultivar Imperial as the male parent. In situ hybridisation
with pTa71 showed that both rDNA loci have similar numbers of rRNA genes
(Fig. 4). In colchicine-treated
cells, the frequency of metaphases with Ag-NOR heteromorphism is reduced
almost to a half (58%) in the telosomic compared with the normal line cultivar
Imperial (94%). Furthermore, a random NOR dominance was detected (either the
1R-NOR or the 1RS-NOR dominates in an exact 1:1 proportion;
Table 2). There is no
preferential expression of a particular NOR over its homologue, neither is
there any differential parental effect. To link interphase organisation of
rDNA and Ag-NOR heteromorphism, the relative dimensions of the two nucleoli of
each nucleus were compared, considering them as heteromorphic when their area
differed at least 25% (Fig. 5).
A marked reduction in nucleoli heteromorphism was observed in the telosomic
line (52%) in comparison with cultivar Imperial (71%;
Table 2). It was noticeable
that no genotype influence was detected on the relative average frequency of
cells with one nucleolus, which was of 80% for the 1R1R line, and 81% for
1R1RS1RL.
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|
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Organisation and relative positioning patterns of homologous rDNA
loci at interphase are modified by changes in NOR-bearing chromosome
structure
Patterns of rDNA chromatin organization in interphase nuclei were
characterized through in situ hybridisation with the pTa71 probe, followed by
confocal analysis. Table 3
gives the frequencies of individual rDNA organization patterns classified into
three types. In seedlings of cultivar Imperial (either untreated or with
colchicine treatment) a few cells showed large perinucleolar knobs without
further traces of labelled chromatin within the nucleolus (Type I;
Fig. 6a). In most of the cells
the pattern is the same as that described previously
(Delgado et al., 1995;
Leitch et al., 1992
), where
nuclei exhibited two condensed perinucleolar knobs from which thin rDNA
filaments emerge towards the interior of the nucleolus (Type II;
Fig. 6b). The perinucleolar
rDNA chromatin knobs are localized on the nucleolar surface, positioned in the
nuclear pole where DAPI staining shows the chromatin to be more concentrated
(Figs 6,
7). At the opposite nucleolar
surface, the characteristic blocks of heterochromatin from rye telomeric
regions are clearly visible, but knobs of perinucleolar ribosomal chromatin
were usually undetectable. No major difference was observed in the frequencies
of rDNA organisation patterns in untreated and colchicine-treated root-tips of
the cultivar Imperial. Therefore, this c-mitotic treatment has no significant
influence in the rDNA topology at interphase. In the structural variant line
(1R1RS1RL), although the majority of rDNA loci belong to
Type II, a considerable frequency of NOR loci was detected exhibiting large
perinucleolar rDNA knobs and condensed intranucleolar spots (Type III;
Fig. 6c). rDNA loci with this
type of interphase organization, where thin threads of chromatin link
intranucleolar rDNA spots, are usually absent in normal rye lines.
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The rDNA loci in interphase root tip cells were also investigated to characterize relative patterns of ribosomal chromatin distribution between the standard cultivar and in its chromosome structural variant. Interphase cells with a single nucleolus, and without intra-nucleolar condensed spots (Types I and II), were selectively observed to ensure a precise comparison. Separated (Fig. 6a), adjacent (Fig. 7a) and fused (Fig. 7b) rDNA knobs were considered. The results are presented in Table 4. While in cultivar Imperial only 5% of interphase cells show both rDNA loci undistinguishable, 24% of cells in the 1R1RS1RL line exhibit fused rDNA loci, as only one larger single hybridization spot could be observed. The state of the genome, with respect to its nucleolar chromosomes, influences the relative domains of these chromosomes within the nucleus and indicates positional interaction.
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Discussion |
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Structural and functional domains within NORs at metaphase correspond
to rDNA interphase organization
In plants, it has been clearly established that interphase patterns of rDNA
loci organization detected by FISH are correlated with their activity
(Delgado et al., 1995;
Leitch et al., 1992
;
Shaw et al., 1993
;
Wallace and Langridge, 1971
).
rRNA gene activity can be revealed by a silver staining technique that allows
the detection of metaphase Ag-NORs that were actively transcribed during the
previous interphase (Hubbell,
1985
; Jímenez et al.,
1988
; Zurita et al.,
1998
). We reported here that, after sequential silver staining and
FISH with the rDNA probe pTa71, distended NORs show two different structural
and functional domains. These domains involve a condensed subset of rDNA
chromatin in the centromere proximal region, without silver staining; and a
more distal subset of distended rDNA chromatin co-localised with silver
staining. In metaphase NORs, different structural and functional domains have
not previously been described, since only condensed NORs have been analysed
before, where Ag-staining and labelling with the FISH probe pTa71 are
superimposed. Previous studies on rDNA chromatin organization and NOR
structure propose that the NOR may include a stronger (darker) stained
heterochromatic knob (i.e. a chromomere) in the centromere-proximal region
(McClintock, 1934
). This knob,
after further interpretation (Wallace and
Langridge, 1971
), was thought to be composed mainly of inactive
ribosomal RNA genes, and the secondary constriction represented the active,
transcribed ones. Our experimental approach shows that not only is
heterochromatic rDNA always present at the centromere proximal end of the NOR,
but it also allowed us to find a correlation between the centromere-proximal
rDNA knobs detected in metaphase and the rDNA perinucleolar knobs observed in
interphase. These knobs are usually located at the nuclear pole where the
centromeres are clustered (Abranches et
al., 1998
; Anamthawat-Jonsson and Heslop-Harrisson, 1990;
Leitch, 2000
). Other
approaches have shown that only a fraction of rRNA genes at interphase are in
an accessible (presumably active) chromatin configuration
(Banditt et al., 1999
;
Conconi et al., 1989
; Dammann
et al., 1995; González-Melendi et
al., 2001
). At the whole NOR level, we can conclude that rRNA gene
silencing in rye is not fully randomised, as distended metaphase NORs always
show the condensed inactive rDNA chromatin in the centromere-proximal region.
The absence of silver staining in this region indicates that these genes
remain largely inactive during the previous interphase, probably due to
protein-protein interactions between nearby heterochromatin domains
(Carmo-Fonseca et al., 2000
;
Pluta et al., 1995
). C-banding
in rye has revealed a heterochromatic band adjacent to the NOR at the
centromere-proximal region (Sybenga,
1983
), which may therefore influence the organization of the
neighbouring rDNA chromatin.
Homologous NORs with equivalent numbers of rRNA genes display
different expression patterns
At metaphase, Ag-NOR size and intensity of the silver staining have been
correlated with rRNA gene activity in the previous interphase
(Hernandez-Verdun, 1991;
Morais-Cecílio et al.,
2000
; Robert-Fortel et al.,
1993
; Zurita et al.,
1997
; Zurita et al.,
1998
; Zurita et al.,
1999
). A correlation between the size of the hybridisation signal
and the number of rRNA genes present at a NOR has also been described in
homologous (Zurita et al.,
1998
) or non-homologous loci
(Leitch and Heslop-Harrison,
1992
; Mellink et al.,
1994
; Suzuki et al.,
1990
). Using these approaches we demonstrate that homologous NORs
with equivalent numbers of genes per locus display differential
expression.
Several previous studies on the regulation of rDNA transcription, at both
the intra- and interspecific level, point to a model where competition between
rDNA loci for essential transcription factors in limiting concentrations seems
to be the more consensual explanation for differential activity between
different NORs (reviewed by Pikaard,
1999; Pikaard,
2000
; Zurita et al.,
1998
; Zurita et al.,
1999
). This model implies distinct abilities of different NORs to
recruit these transcription factors, based mainly on the number of the rRNA
gene units present at an NOR. However, this hypothesis cannot explain our
results in rye, because both rDNA loci have equivalent numbers of rDNA units,
as shown by the FISH results. This suggests that an alternative hypothesis is
required for the mechanism to explain differential NOR expression. In the
chromosome structural variant line we observed a significant reduction in the
frequency of interphase cells with heteromorphic nucleoli, which is a direct
measure of rDNA loci transcription level
(Martini and Flavell, 1985
),
together with a significant decrease in the frequency of metaphase cells with
Ag-NOR heteromorphism in comparison with cultivar Imperial. Thus a minor
modification in the genomic structure is sufficient to promote significant
changes in the relative expression of homologous rDNA loci. It should be
stressed that the frequency of heteromorphic NORs is higher than the frequency
of heteromorphic nucleoli in each genotype analysed. This apparent discrepancy
can be readily explained, since heteromorphic nucleoli were assessed early in
the cell cycle (G1) where nuclei display two nucleoli. Observations
of metaphase NOR heteromorphism come from rDNA loci expression during all
interphase stages (i.e. G1, S, G2).
Differential expression of homologous rDNA loci also raises the question as
to the existence or not of any preferential activity of one particular locus
over its homologue. Through the use of the 1R1RS1RL
line, where the parental origin of the NOR could be clearly identified by
chromosome morphology, we were able to detect random differential silencing.
There is evidence that each particular NOR retains its relative transcription
level between cell divisions (Roussel et
al., 1996), although a single rDNA transcription unit may exhibit
different expression patterns throughout cell divisions
(Damman et al., 1995
). The
random inter-homologous NOR dominance observed in this work can either result
from differential patterns established after each cell division, or from
patterns imposed early on in development and then maintained, recalling the
cell mosaicism described previously
(Platero et al., 1998
).
Relative positioning of homologous rDNA loci is correlated with their
differential activity
In a wide range of organisms many types of gene silencing processes appear
to involve DNA-DNA interactions between genes at allelic and non-allelic sites
(Henikoff, 1997;
Kooter et al., 1999
;
Matzke and Matzke, 1998
;
Matzke et al., 1994
;
Wolffe and Matzke, 1999
).
Here, we show that by modifying the NOR-bearing chromosome structure, the
frequency of interphase cells with fused perinucleolar knobs is fivefold
higher than in the standard rye cultivar Imperial. Moreover, in 26% of the
interphase cells of the variant structural line we also observed ribosomal
intra-nucleolar condensed spots in addition to the already known perinucleolar
knobs. Intra-nucleolar condensed spots of rDNA seem to be characteristic of
some species, such as hexaploid wheat
(Leitch et al., 1992
;
Morais-Cecilio et al., 2000
),
and rare in other species, such as rye
(Delgado et al., 1995
;
Leitch et al., 1992
). These
spots, recently referred to as ribosomal heterochromatin
(Carmo-Fonseca et al., 2000
),
were previously interpreted as unexpressed condensed regions that separate
transcribing rRNA genes (Leitch et al.,
1992
).
The observations on the telosomic variant line clearly show a greater proximity of the NORs from 1R and 1RS, and fusion between homologous rDNA loci, indicating that rDNA chromatin disposition is modified by this alteration in chromosome structure. Moreover, condensed intra-nucleolar knobs of rDNA chromatin were also observed. This analysis allows us to establish a correlation between NOR disposition and organization patterns, and their differential expression, since Ag-heteromorphism was reduced in the telosomic variant line.
This greater proximity of homologous rDNA loci can either be due to a more
direct recognition between homologous chromosome arms in the nucleus, or
derive from modifications to the relative disposition of chromosomes in the
haploid complement, due to the presence of telocentric chromosomes. The
correlation between NOR closeness and reduction of differential expression
between homologous rDNA loci could be explained by differences in the
localization of NORs in nuclear domains where transcription factors are
unevenly distributed. This hypothesis would imply that when NORs colocalize
(as in the variant line) only then are they exposed to the same pool of
transcription factors. However, it still remains unclear whether or not
transcription factors have any preferential nuclear domains of localization.
It is known for example, that transcription sites are not correlated with
chromosome territories in wheat nuclei, and show a uniform distribution
throughout the nuclear volume (Abranches et
al., 1998). Although we cannot discriminate trans-interactions
between genes of homologous loci, or make conclusions about regulatory
processes, the greater frequency in the intranucleolar rDNA chromatin
condensations that we observe in the variant line, where NORs are in closer
proximity, may suggest a raised level of `sensing of homologous sequences'
between rDNA loci.
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Acknowledgments |
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References |
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