Department of Cell Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom
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Abstract |
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We have determined the relationship between overall nuclear architecture, chromosome territories, and transcription sites within the nucleus, using three-dimensional confocal microscopy of well preserved tissue sections of wheat roots. Chromosome territories were visualized by GISH using rye genomic probe in wheat/rye translocation and addition lines. The chromosomes appeared as elongated regions and showed a clear centromere-telomere polarization, with the two visualized chromosomes lying approximately parallel to one another across the nucleus. Labeling with probes to telomeres and centromeres confirmed a striking Rabl configuration in all cells, with a clear clustering of the centromeres, and cell files often maintained a common polarity through several division cycles. Transcription sites were detected by BrUTP incorporation in unfixed tissue sections and revealed a pattern of numerous foci uniformly distributed throughout the nucleoplasm, as well as more intensely labeled foci in the nucleoli. It has been suggested that the gene-rich regions in wheat chromosomes are clustered towards the telomeres. However, we found no indication of a difference in concentration of transcription sites between telomere and centromere poles of the nucleus. Neither could we detect any evidence that the transcription sites were preferentially localized with respect to the chromosome territorial boundaries.
Key words: BrUTP; transcription sites; chromosome territory; nuclear architecture; plant ![]() |
Introduction |
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THERE is accumulating evidence that interphase chromosomes occupy spatially distinct regions of the nucleus, often referred to as chromosome territories,
separated by interchromosomal channels (for reviews see
Strouboulis and Wolffe, 1996; Lamond and Earnshaw, 1998
). Early evidence for this organization was provided
by Cremer et al. (1982)
who irradiated interphase nuclei
with UV and showed that damage was localized to only a
few chromosomes. The use of in situ hybridization with
chromosome-specific DNA paints confirmed the arrangement of interphase chromosomes in distinct, non-overlapping territories (Cremer et al., 1988
; Lichter et al., 1988
).
Since then, a territorial organization of chromosomes has
been demonstrated in an increasing number of animal and
plant species (e.g., Heslop-Harrison and Bennett, 1990
).
However, the way the interphase chromosomes are arranged within the nucleus and with respect to each other seems to vary from species to species. Many years ago,
Rabl proposed that the centromere-telomere orientation
established at mitotic anaphase would continue throughout the cell cycle (Rabl, 1885
). This configuration, since referred to as the Rabl configuration, would imply that centromeres and telomeres were positioned at opposite poles
of the nucleus. The Rabl configuration has been demonstrated in some species, including Drosophila polytene nuclei (Hochstrasser et al., 1986
), Trypanosoma (Chung et
al., 1990
), fission yeast (Funabiki et al., 1993
), and some
plants (Heslop-Harrison et al., 1993
; Noguchi and Fukui,
1995
). However there is no evidence for a Rabl configuration in somatic cells of other studied species, such as humans and other mammals.
It has been proposed that the compartmentalization of
the nucleus into chromosome territories and interchromosome channels is reflected in the spatial organization of
the functional protein complexes responsible for nuclear
processes such as transcription, splicing, replication, and
repair (Cremer et al., 1993, 1995
). Thus it has been postulated that transcription takes place at the surfaces of the
chromosome territories, with transcript processing and export being directed through the three-dimensional (3D)1
network of interchromosome channels; interchromosome
channels ending at a nuclear pore would provide an efficient exit route from the nucleus for mRNA complexes
(Kurz et al., 1996
). However, the experimental evidence
for this hypothesis is based on the in situ localization of a
few genes, and to our knowledge there has so far been no
systematic study of the relation between all nuclear transcription sites and chromosome territorial organization.
Various groups have shown that BrUTP incorporation
accurately localizes transcription sites in the nucleus
(Jackson et al., 1993; Wansink et al., 1993
, 1994
). These
studies have revealed several hundred distinct, punctate
sites of labeling, showing that RNA polymerase II transcription takes place in numerous small domains dispersed
throughout the nucleus. The labeled sites remained after
most of the chromatin was digested away by nucleases,
suggesting the transcription sites are attached to a resistant nuclear matrix. A similar pattern has been observed
in plant cell nuclei (Straatman et al., 1996
) and nucleoli
(Hozak et al., 1994
; Thompson et al., 1997
). In confirmation of this distribution of transcription sites, RNA polymerase II and transcription factors were found distributed throughout the nucleoplasm in numerous small domains
(Grande et al., 1997
).
In the present work, we have examined the organization of transcription sites in relation to the arrangement of chromosomes in wheat root tissue. For this study we used well-preserved, intact tissue for whole-mount in situ hybridization and for BrUTP incorporation, and analyzed the labeling using 3D confocal microscopy. It has not so far been possible to produce chromosome paints to label individual wheat chromosomes, or indeed those of any other plant. For this reason we used a wheat line containing an extra pair of chromosomes from rye, as well as a translocation line containing a single pair of rye chromosome arms. The rye chromosomes and chromosome arms were detected using total rye genomic DNA as a probe for in situ hybridization. The arrangement of the chromosomes was confirmed by in situ hybridization with centromere and telomere probes. To study the pattern of distribution of transcription sites in the wheat nucleus we used BrUTP incorporation in unfixed root sections. By combining BrUTP incorporation with in situ hybridization we visualized transcription sites in relation to chromosome territories, and to telomeres and centromeres.
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Materials and Methods |
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Root Sections
Seeds of Triticum aestivum (cv Chinese Spring/1R disomic addition and
Chinese Spring 1A/1R translocation) were germinated on water-soaked
filter paper. Roots were excised 3 d after germination. For in situ studies,
root tips were fixed in 4% (wt/vol) PFA in PEM (50 mM Pipes, 5 mM
EGTA, 5 mM MgSO4, pH 6.9, with KOH) for 1 h, followed by washing for 10 min in TBS (10 mM Tris, 140 mM NaCl, pH 7.4, with HCl). Root tips were
sectioned under water into 30-µm-thick sections using a Vibratome Series
1000 (TAAB Laboratories Equipment Ltd., Aldermaston, UK). Sections
were placed immediately on multi-well slides (ICN Biomedicals Inc., Costa Mesa, CA) coated with glutaraldehyde-activated -aminopropyl triethoxy silane (APTES; Sigma Chemical Co., St. Louis, MO) and left to air dry.
BrUTP Incorporation into Tissue Sections
For transcription studies, the method described by Thompson et al. (1997)
was adapted. To improve nuclear transcription as opposed to nucleolar
transcription, 1% BSA was added to the modified physiological buffer
(MPB: 100 mM KAc, 20 mM KCl, 20 mM Hepes-KOH, pH 7.4, 1 mM
MgCl2, 1 mM ATP in 50 mM Tris, pH 8, 1% thiodiglycol, 2 mg/ml aprotenin, 0.5 mM PMSF), and the permeabilization step with Triton X-100 was
replaced by a very short (10 s) treatment with 0.05% Tween 20 in MPB. In
vitro transcription was allowed to continue for 5 min.
In Situ Hybridization with Centromeric and Telomeric Probes
Tissue sections on slides were treated with 2% (wt/vol) cellulase (Onuzuka R-10) in TBS for 1 h at room temperature and washed in TBS followed by 0.1× SSC (SSC: 150 mM NaCl, 15 mM sodium citrate). Denaturation of both target DNA and probe was done in 0.1× SSC at 98°C for 5 min, followed by 5 min in ice-cold 0.1× SSC. The surface liquid was blotted off slides and 10 µl of ice-cold hybridization mixture was added. The
hybridization mixture comprised 50% deionized formamide, 10% 100 mM
Pipes/10 mM EDTA, pH 8, 20% dextran sulphate, 10% 3 M NaCl, 100 ng
centromeric or telomeric probe, 50× excess blocking salmon sperm DNA.
Probes were produced as described by Aragón-Alcaide et al. (1997). Hybridization was carried out overnight in a humid chamber at 37°C. Post-hybridization washes were performed in 0.1× SSC at 50°C for 1.5 h with
two changes.
Centromere probes were detected using conjugated sheep anti-digoxigenin antibody-FITC (Boehringer Mannheim Corp., Indianapolis, IN) and the signal was sequentially amplified with rabbit anti-sheep-FITC (DAKO Corp., Carpinteria, CA) and sheep anti-rabbit-FITC (Sigma Chemical Co.). Telomere probes were detected using extravidin-Cy3 (Sigma Chemical Co.). Antibodies were diluted in TBS/3% BSA. Antibody incubations were carried out in a damp chamber for 45 min at room temperature and TBS washes were carried out between antibody incubations. Slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.) (1 µg/ml) for 5 min, and then mounted in antifade solution (Vectashield; Vector Laboratories Inc., Burlingame, CA).
In Situ Hybridization with Rye Total Genomic Probe
Sections were treated with cellulase as described above. Probe preparation and hybridization procedures followed those described by Schwarzacher et al. (1992) with the following modifications: the hybridization mixture was denatured at 95°C for 5 min before addition to the preparations,
and denaturation of slides with probe was performed at 78°C for 10 min
using a modified thermocycler (Omnislide; Hybaid Ltd., Long Island, NY).
Combined BrUTP Incorporation and In Situ Hybridization on Root Sections
BrUTP incorporation was performed on sections as described above. After the antibody detection of BrUTP incorporation, the sections were fixed a second time with 4% PFA for 30 min. Then the sections were washed in TBS and in situ hybridization was performed as described above except in the case of centromere in situ labeling where the denaturation of sections was done for 5 min at 80°C in 70% formamide 2× SSC.
Microscopy, Photography, and Image Processing
Specimens were surveyed using a Zeiss Universal (Carl Zeiss, Inc.,
Oberkochen, Germany) or a Nikon Eclipse 800 (Nikon Corp., Tokyo, Japan) fluorescence microscope. Confocal optical section stacks were collected using a Biorad MRC-600 or a Biorad MRC-1,000 UV confocal
scanning microscope (Bio-Rad Laboratories, Hercules, CA) as described
previously (Beven et al., 1996). Images were transferred to a PC or a Macintosh computer and assembled into composite images using Photoshop
(Adobe Systems Inc., Mountain View, CA) and NIH Image, a public domain program for the Macintosh available via anonymous ftp from
. Images were printed on a Pictrography P3000 printer.
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Results |
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Chromosome Arrangement in the Wheat Interphase Nucleus
Wheat lines containing the addition of a pair of rye chromosomes (1R) or a 1A/1R translocation line, where one arm of wheat chromosome 1A is replaced by one arm of rye chromosome 1R, were used to visualize individual chromosome territories. In both lines the rye chromosomes or chromosome arms were labeled by in situ hybridization using fragmented total rye DNA into which digoxigenin had been incorporated. In the interphase nuclei the rye chromosomes appeared as elongated regions generally traversing the nucleus from one side to the other and they were usually parallel to each other (Fig. 1). Often, the labeled chromosomes in several nuclei in a cell file were in the same orientation (see Fig. 1). In the addition line, the two chromosome arms always lay alongside each other. Often the two arms were so close that only a single labeled region was seen; sometimes the two arms were distinguishable (Fig. 1, arrow). A similar pattern of chromosome labeling was seen, irrespective of the size of the nucleus, and thus the presumed phase of the cell cycle. We examined these specimens for evidence of somatic association of the homologues. We considered the two homologous chromosomes or chromosome arms to be associated if only one region of labeling, with no intervening space, could be seen for the two chromosomes or arms. In all, 20 out of 84 nuclei from the addition line, and 9 out of 99 nuclei from the translocation line showed evidence of homologous pairing (24% and 9%, respectively). We consider that this is probably not significant, although a detailed statistical analysis is beyond the scope of this paper.
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Centromeres and Telomeres Are Located at Opposite Poles of the Nucleus
Double fluorescence in situ hybridization was carried out
on root sections, using centromeric and telomeric probes.
As shown in Fig. 2 a, there was a strong polarization of the
sites labeled in the interphase nuclei, with the centromeres
clustered at one side of the nucleus and the telomeres located at the opposite sidea clear Rabl configuration. Fig.
2 b shows a diagrammatic interpretation of the labeling
pattern seen. It was striking that a common nuclear orientation was maintained for many adjacent cells in a given
cell file, suggesting a strong conservation of overall chromosome order during several rounds of cell division, and
confirming the previous observations of the orientation of the labeled chromosomes.
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Transcription Foci Are Scattered throughout the Nucleus
Transcription sites were visualized by incubating unfixed
vibratome sections from wheat roots with a transcription
mix containing BrUTP, fixing with formaldehyde, and
then detecting incorporated BrUTP by immunofluorescence labeling. It was necessary to use very short permeabilization times (10 s) with 0.05% Tween to allow the
transcription mix into the nuclei, while preserving nuclear transcriptional activity. Longer incubation with detergent
gave solely nucleolar incorporation of BrUTP (Thompson
et al., 1997), possibly because of disruption or inactivation
of RNA polymerase II. All procedures were carried out at
room temperature, and transcription was allowed to proceed for 5 min. Published methods for animal cells specify
4°C for permeabilization; we found that treatment at 4°C
inactivated transcription in plant cells, and that it did not
recover subsequently at room temperature. In control experiments, BrUTP was omitted from the transcription
reaction, or actinomycin D, an inhibitor of RNA polymerases, was added. In these control experiments no fluorescence in the nucleus or nucleolus was seen.
Fig. 3 shows an example of BrUTP incorporation (Fig. 3 a, red), compared with DAPI counterstain (Fig. 3 b, blue). The nucleoli are clearly visible in the DAPI-stained images as dark holes in the nuclei, and the nucleolar transcription sites are dispersed through a sub-region of the nucleoli. The nucleolar labeling is stronger than that in the nucleoplasm, and in these images the nucleolar labeling has been overexposed so as to show the fainter nucleoplasmic transcription sites. These are seen as many small foci, distributed fairly evenly throughout the nucleoplasm. Some sites are significantly brighter than the others. In this species the chromatin is arranged in a reticulate network, and there is some tendency for the transcription sites to be located in the darker interchromatin regions rather than in the bright DAPI-stained regions, but the pattern of transcription sites does not match the reticulate pattern of DAPI staining very closely (Fig. 3 c).
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The Distribution of Transcription Sites in Interphase Nuclei Is Not Polarized
There is evidence that in the physical map of wheat chromosomes, the telomeric regions of all the chromosomes
are significantly more gene-rich than the centromeric regions (Gill et al., 1996). Since we have shown above that
wheat nuclei are highly polarized, with the centromeres
clustered at one side of the nucleus, we might expect that
transcription sites would be concentrated in the opposite
half of the nucleus, and that the region of the nucleus near
the centromeres would be relatively depleted in transcription sites. We therefore carried out double-labeling experiments to show transcription sites by BrUTP incorporation, followed by fluorescence in situ hybridization with
centromere probe. Fig. 4 shows a confocal optical section
through a pair of nuclei in G1. Transcription sites are
shown in red in the left-hand panels, centromeres in green
(clearly clustered on opposite sides of the sister nuclei) in
the central panels, and the two probes superimposed in the right hand panels. There is no evidence for any polarization in the distribution of transcription sites; there seems
to be as high a density near the centromeres as at the opposite pole of the nuclei.
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Transcription Sites Are Not Preferentially Located at Chromosome Territory Boundaries
To compare the distribution of transcription sites with chromosome territories, we used a wheat line with an additional pair of rye chromosomes (1R addition), and a line containing a translocated rye chromosome arm (1A/1R translocation). Transcription was detected by BrUTP incorporation, and this was then followed by genomic in situ hybridization using total genomic rye probe. Three consecutive confocal optical sections from the translocation line are shown in Fig. 5; BrUTP incorporation is shown in red in the left-hand panels, the rye chromosomes in green in the central panels, and the two probes superimposed in the right hand panels. There is no correlation between the region occupied by the rye chromosome arms and the transcription sites. In fact, transcription sites are seen throughout the labeled chromosome territory. Two particularly strong transcription sites within this territory are indicated by an arrow. There is no sign that the interior regions of these chromosome territories are devoid of transcription sites. It should be noted that in places, including the position of the strong arrowed transcription sites, the labeled chromosome territories are at least 2-µm wide. Thus location of the transcription sites, which range in size from 0.5 µm to sizes at or below the resolution limit (0.25 µm), to sub-regions of the chromosome territory would be well within the resolution limit of this technique.
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Discussion |
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By applying fluorescence in situ hybridization combined
with confocal 3D imaging to well-preserved wheat root tissue we have shown that the nuclei in these cells have a remarkably well ordered and consistent 3D architecture. We
have shown previously that the in situ procedures we used
cause minimal cellular disruption, at least at optical resolution (Shaw et al., 1995), in contrast to standard in situ
methods which cause considerable structural distortion
because of squashing and harsh denaturation conditions. We have also previously shown that the distribution of nucleolar transcription sites visualized by BrUTP incorporation into plant root tissue agrees well with that determined
by in situ hybridization using an anti-sense probe to the
external transcribed spacer of the rRNA (Thompson et al.,
1997
). Furthermore, in the present study, the distribution
of transcription sites visualized by BrUTP incorporation
was not affected by subsequent in situ labeling.
The interphase chromosomes occupy elongated regions, usually stretching right across the nucleus. The chromosomes are approximately parallel to one another and their arms lie next to each other. Both centromeres and telomeres are located at the nuclear periphery. The centromeres are highly clustered in one region of the nuclear periphery, whereas the telomeres are more dispersed around the opposite side of the nuclear periphery. This suggests that both centromeres and telomeres interact with peripheral nuclear structures, possibly the nuclear pore-lamina complex or another nuclear matrix component, whereas more specific interactions are also involved in the organization of the centromeres into clusters. This strong Rabl configuration gives a polarity to the nuclei, and the direction of this polarity can be maintained through several rounds of cell division.
By using wheat lines containing the addition of pairs of
rye chromosomes or translocations of single rye arms, we
were able to visualize individual chromosome territories
by genomic in situ hybridization. This confirmed the
strong Rabl configuration in these nuclei, with all the chromosomes lying parallel to one another across the nucleus,
the two chromosome arms next to each other. We found
no evidence for significant association of homologues, in
contrast to previous observations on squashed preparations of nuclei in wheat and other plants (Avivi and Feldman, 1980). This confirms previous observations on pre-meiotic wheat nuclei (Aragón-Alcaide et al., 1997
), using
these and other similar addition lines. In the developmental stages leading up to meiosis in wheat anthers no association of the homologues was found until shortly before
meiosis. However, in the pre-meiotic interphase a high
level (90%) of homologue pairing occurred in both the
meiocytes and the surrounding somatic tapetal cells
(Aragón-Alcaide et al., 1997
). In some species, notably in
Drosophila polytene salivary gland nuclei (Hochstrasser et
al., 1986
) there is a very clear somatic association of all the
homologues. On the other hand, in most species, including
mammals, there is no evidence for association of homologues, except in meiotic prophase. It appears that wheat shows an intermediate behavior, with little or no homologous pairing in somatic cells until shortly before meiosis.
Whether the homologous pairing observed in wheat anthers should be regarded as part of the process of meiosis,
or as a switching on of a mechanism for somatic pairing in
the developmental pathway leading to meiocytes (and associated somatic cells) is still unknown.
The fact that wheat root nuclei, along with the other
wheat somatic cell types we have examined, show such a
high degree of structural organization makes them a very
good system to analyze the organization of transcription
sites, and to test previous hypotheses relating transcription
sites to chromosome territories. Such a well-ordered and
reproducible interphase chromosomal organization should
clearly reveal a systematic organization of transcription sites if the location of transcription sites is related in any obvious way to chromosome territories. BrUTP incorporation shows many small foci distributed in the nucleoplasm, in addition to the strong incorporation we previously showed in the nucleolus (Thompson et al., 1997).
This nuclear distribution of transcription foci is very similar to published images of human HeLa and T24 cultured
cells (Jackson et al., 1993
; Wansink et al., 1993
). It has
been shown previously that BrUTP incorporation faithfully represents transcription sites (e.g., Wansink et al.
[1993] used microinjection of BrUTP precursors to verify
results using permeabilization of cells). In plants, we have
shown that BrUTP is incorporated into the same nucleolar
sites as are labeled by an in situ probe to nascent rRNA
transcripts (Thompson et al., 1997
). It was not possible to
quantify accurately the number of nucleoplasmic foci, but
most nuclei contained of the order of a few hundred. This is consistent with the numbers of sites observed in mammalian cells, and is significantly less than the estimated
number of active genes. This may mean that only the most
active genes were seen, with many other transcription sites
below the detection limit. An alternative explanation is
that each site represents transcription of several genes
either a group of smaller sites too close together to be resolved, or more than one gene being transcribed at a single
cluster of many polymerase molecules. Iborra et al. (1996)
have provided evidence that all the transcription sites are
in fact visualized in similar experiments in mammalian cells, and have suggested that each transcription site represents a "factory" where more than one gene is transcribed,
and where other nuclear activities such as DNA replication take place (Jackson and Cook, 1995
; Jackson, 1995
).
It has been reported that in the physical map of wheat
chromosomes, the distal, telomeric regions of all the chromosomes are gene-rich compared with the proximal, centromeric region (Gill et al., 1996, and references therein).
This was based on the analysis of a number of markers in
several series of deletion lines, and contrasts with the genetic map based on recombination frequencies, particularly in the proximal region. Given the chromosome arrangement we have shown for the wheat nucleus, we might
expect that there would be a significant polarity in the arrangement of transcription sites, the volume of the nucleus
nearer the telomeres containing a higher concentration of
transcription sites than that nearer the centromeres. We
did not observe this; in fact, the transcription foci were distributed fairly homogeneously throughout the volume of
the nucleoplasm. One possible explanation could be that
the fully condensed metaphase chromosomes decondense
unevenly on reinitiation of transcription, the gene-rich regions decondensing more than the gene-poor regions. This
would imply a gradient or polarity in chromatin decondensation levels along the chromosomes. We found no sign of
such a difference in chromatin density, on the basis of
staining intensity with DAPI. We stained with several different concentrations of DAPI to check this point, but did
not observe any polarity in DAPI staining intensity. A second possibility is that the limited number of markers used
by Gill et al. (1996)
in constructing their physical maps is
not representative of all the transcribed genes. In particular, the markers used in the physical maps may have been
biased towards non-conserved genes and away from conserved housekeeping genes, which may be more likely to
be located in recombination-poor chromosome regions such as the proximal, centromere regions. In addition to
this, it may be that the genes in these proximal regions,
even if they are sparsely distributed, may be highly expressed, as would be expected for housekeeping genes. Although we cannot exclude this possibility, it seems unlikely
in view of the comparison of wheat and rice chromosome
maps which show considerable synteny and confirm that
the distal halves of the wheat chromosome arms are gene-rich compared with the proximal halves (Kurata et al.,
1994
). A third possibility is that there is not a strict relationship between chromosomal location of a gene and the
site at which it is transcribed. In support of this possibility,
Toledo et al. (1992)
showed that two amplified markers
that alternated in multiple repeats on a single chromosome often clustered together in two distinct regions of the
interphase nucleus. This implied that the linear DNA carrying the successive copies of the two markers could be arranged in a complex way, with the successive gene copies
looping back and forth. There is also recent evidence that
long-range interactions can alter the nuclear positioning of
genes and lead to gene silencing. Dernburg et al. (1996)
have shown that the insertion of heterochromatin at the
brown locus in Drosophila caused this gene to associate specifically with the centromeric heterochromatin at a particular developmental stage. Brown et al. (1997)
have recently demonstrated that the transcriptional regulator protein ikaros is localized to domains containing centromeric
heterochromatin, and that inactive, but not active genes
are recruited to these domains. Thus there is accumulating
evidence that genes can be moved quite large distances through the nucleus depending on their transcriptional
state. Such an organization is consistent with the idea of
groups of genes being transcribed at factories (Jackson,
1995
), to which DNA loops carrying transcriptionally
active genes can move. In this way a gene could be
transcribed at a site some distance from its physical map
position along the chromosome. In this hypothesis, transcription factories might be assembled throughout the nucleus at locations on a nuclear matrix, rather than directly
organized on the interphase chromosomes. If this is the
case, it would appear that one of the organizing principles,
at least in this species, for such factories is a fairly homogeneous distribution throughout the nucleoplasm.
It has been suggested that nuclei contain a three-dimensional network of intra-chromosomal channels where the
machinery for transcription, splicing, and other essential
nuclear functions are located. According to this model, the
channels would be maintained between adjacent chromosome surfaces, possibly by repulsive electrostatic forces
between the chromosome surfaces, and would enable
transport of proteins and RNAs either by channeled diffusion or via matrix filaments. Kurz et al. (1996) provided
evidence that active and inactive genes were localized
preferentially at the periphery of chromosome territories,
whereas non-expressed fragments were randomly distributed or localized preferentially in the interior of the chromosome territory. However, an objection to this evidence is that only a few genes were examined, rather than the
full range of transcribed genes. Zirbel et al. (1993)
also
showed that viral RNA concentration was generally localized at the territory surface of the chromosome harboring
the viral genes. The latter data certainly supports the idea
that RNA processing or export is somehow related to the
chromosome territory surface, but does not clearly demonstrate where transcription takes place. Other observations that might support such a model are the differences
observed in the surface shape of active and inactive human
X interphase chromosomes (Eils et al., 1996
).
A prediction of this model would be that transcription
sites would be located at a series of surfaces bounding the
chromosome territories within the nucleus, and that the interior of the chromosome territories would be devoid of
transcription sites. We used both the addition line and the
translocation line to test this prediction in double labeling
experiments, and obtained very similar results. We present
the results from the translocation line here because they
are more unequivocal. The two chromosome arms lie next
to each other in the nuclei, and are almost invariably visualized as a single region. However, there is a territorial
boundary between the two arms. In the addition line, it
would be impossible to exclude the possibility that transcription sites inside the chromosome region visualized
were located at this inter-arm boundary. For this reason
we have shown results from the translocation line, where
only a single arm is labeled, and this problem does not arise. It is quite clear that the prediction is not borne out by our results. The rye chromosome territories are well
differentiated by genomic in situ hybridization, and there
are clear transcription foci throughout the volume of the
chromosome territories. There is no sign of chromosome-shaped regions devoid of transcriptional activity. This result also demonstrates that the rye chromosomes and
chromosome arms are transcriptionally active. Given the
highly organized structure of these wheat nuclei, with all
the chromosomes parallel to each other lying across the
nucleus in a Rabl configuration, we should expect to see
some clear indication of this in the distribution of transcription sites. In fact we could not detect any polarity, density gradient, or systematic departures from homogeneity in the distribution of BrUTP foci. Our conclusion is,
therefore, that the distribution of transcription sites in
wheat nuclei is not restricted to chromosome territorial
boundaries, at least as revealed by total chromosome in
situ labeling. It might be argued that the real chromosome
territory surfaces might be much more convoluted than
shown by the in situ labeling, and that the transcription
sites apparently in the chromosome interior in fact lie on
such a convoluted surface (e.g., Wansink et al., 1996). However, in our view, this reduces the strength of the territorial surface hypothesis to the extent that it makes no
testable predictions.
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Footnotes |
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Received for publication 6 July 1998 and in revised form 17 August 1998.
Address all correspondence to Peter J. Shaw, John Innes Centre, Colney,
Norwich NR4 7UH, UK. Tel.: (44) 1603-452571. Fax: (44) 1603-501771. E-mail: peter.shaw{at}bbsrc.ac.uk
This work was supported by the Biotechnology and Biological Sciences Research Council of the UK; by a fellowship from the Gulbenkian Foundation and Program PRAXIS XXI Portugal (R. Abranches), and by the John Innes Foundation (L. Aragón-Alcaide).
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Abbreviations used in this paper |
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BrUTP, bromouridine triphosphate; DAPI, 4',6-diamidino-2-phenylindole; MPB, modified physiological buffer; 3D, three-dimensional.
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