Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria
* Author for correspondence (e-mail: josef.loidl{at}univie.ac.at )
Accepted 9 December 2001
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Summary |
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Key words: Yeast, Chromosomes, Nucleus, Nuclear architecture, GFP, Pairing, FISH, Mitosis
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Introduction |
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Since in most cell types it is impossible to trace individual chromosomes
during interphase, fluorescence in situ hybridization has been used to label
chromosomes or parts thereof to study their positions inside nuclei. As a
means to investigate chromosome distribution and behaviour in living cells of
various organisms, the lacO/LacI-GFP system was introduced
(Straight et al., 1996;
Belmont and Straight, 1998
;
Belmont, 2001
). Also, the
similar tetO/TetR-GFP system was used to study the segregation
behaviour of chromosomes in live yeast cells
(Michaelis et al., 1997
). Both
systems are based on the transgenic expression of a bacterial regulating
protein fused to green fluorescent protein (GFP). The fusion protein then
binds to a target DNA sequence, of which many copies are tandemly integrated
into a specified chromosomal region, at which the GFP tags produce
microscopically visible fluorescence.
When we attempted to adapt the tetO/TetR-GFP system to study various aspects of chromosomal organization within S. cerevisiae interphase nuclei, we observed that integration of tetO repeats into chromosomes promotes the association of the target loci. Here, we describe the nature of these associations and their effect on the architecture of interphase nuclei. We also discuss whether mechanisms similar to those by which tetO repeats associate, could play a role in various nonrandom chromosomal interactions with putative functions in DNA repair and epigenetic regulation of gene expression.
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Materials and Methods |
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These strains were backcrossed at least five times to strain SK1
(Kane and Roth, 1974) by using
a haploid derivative of SK1 (NKY857, kindly provided by Nancy Kleckner,
Harvard University, Cambridge, MA); diploid strains FKY806, FKY1012 and
FKY1024 with homozygous and heterozygous centromere-near and telomere-near
tetO integrations (Table
1; Fig. 1B) were
constructed by crossing. The strains were microscopically checked for the
presence of chromosomal GFP dots. Strains SLY1662 and SLY1663 carry
heterozygous CEN and TEL tetO inserts and homozygous TEL
tetO inserts, respectively, but they are missing the tetR
construct. Therefore they do not show chromosomal GFP dots and tetO
loci have to be detected by FISH (see below). Strains SLY1662 and SLY1663 were
derived from leu2 spores of strains FKY1012 and FKY1024 that were
crossed to wild-type haploids. leu2 tetO segregants were selected and
the presence of tetO sequences at the desired locations was tested by
PCR.
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Culture conditions and cytological preparation
Yeast cells were grown in liquid YPD medium. For some experiments, cells
were arrested in S-phase by treatment with hydroxyurea (10 mg/ml) for 3 hours.
Arrest was confirmed by microscopic examination of bud formation and by
immunolabelling of the spindle (see below).
Living cells were collected by centrifugation and resuspended in YPD:
glycerol 3:1 on a side for microscopy. For the semi-spreading procedure, which
is described in detail elsewhere (Jin et
al., 2000), samples of 3-5 ml were taken and formaldehyde was
added directly to the culture medium at a final concentration of 4%. Fixation
was performed at room temperature for 30-60 min. After rinsing twice in 2%
KAc, cell pellets were resuspended in 500 µl 2% KAc, and 10 µl 0.5 M
dithiotreitol and 14 µl of a Zymolyase 100T (Seikagaku Co., Tokyo) stock
solution (10 mg/ml) were added. After the digestion of the cell walls for 20
minutes at 37°C, the cells were washed in 2% KAc and resuspended in
suitable volumes of 2% KAc. 20 µl of this cell suspension were put on a
slide and mixed with 80 µl detergent (1% aqueous solution of Lipsol; LIP
Ltd, Shipley, UK) and 120 µl fixative (4% paraformaldehyde and 3.4% sucrose
in distilled water). The mixture was then spread out with a glass rod and left
to solidify in a chemical hood. For microscopy, the slides were washed in
water (5 minutes), air dried and nuclei were stained with DAPI (1
µg/ml).
For testing whether the tetO sequence repeat number is
sufficiently stable during mitotic growth, a single colony of strain FKY806
was transferred from a plate into 1 litre of liquid medium and grown to
stationary phase. The number of cells in the culture was found to be
1.5x1011 cells, which means that cells have on average gone
through 37 mitotic cycles. 10 µl of the suspension
(1.5x106 cells) were used to inoculate 10 ml of
presporulation medium, grown for another seven generations (to a density of
2x107 cells/ml) and then transferred to sporulation
medium. Cells were fixed as described
(Williamson et al., 1983
) and
the number of GFP signals per pachytene nucleus was determined.
Fluorescence in situ hybridization (FISH) and immunostaining
To obtain FISH probes for the tetO integration regions, either
immediately adjacent chromosomal sequences or the tetO sequence
itself were PCR-amplified. FISH probes on chromosome V were produced
by PCR using the Expand Long Template PCR System (Roche Diagnostics GmbH,
Mannheim, Germany) according to the manufacturer's instructions. Appropriate
primers were designed based on the published yeast genomic sequence
(Saccharomyces Genome Database
http://genome-www.stanford.edu/Saccharomyces
). For the amplification of products of around 10 kb the following conditions
were applied: 2 minutes at 94°C; 10 cycles with 10 seconds at 94°C, 30
seconds at 58°C, 8 minutes at 68°C; 20 cycles with 10 seconds at
94°C, 30 seconds at 58°C, 8 minutes at 68°C with an increment of
20 seconds per cycle and a final extension step of 10 minutes. The amplified
PCR products were purified using the Gel Extraction Kit QIAEX II (Qiagen,
Valencia, CA) and subsequently labelled by nick translation either with
Cy3-dUTP (red; Amersham, Little Chalfont, UK) or Fluorescein-12-dUTP (green;
Roche Diagnostics GmbH, Mannheim, Germany) as described previously
(Loidl et al., 1998). The
chromosomal localizations of FISH probes are shown in
Fig. 1C. For generating FISH
probes for the tetO repeats themselves, the sequence was
PCR-amplified from the plasmid pCM189 containing tetO palindromic
units (Gari et al., 1997
) and
simultaneously labelled with Cy3-dUTP.
Labelled probes were dissolved in hybridization solution (50% formamide, 10% dextran sulfate, 2x SSC) to a final concentration of approximately 30 ng/µl. After 5 minutes of denaturation at 95°C, the probes were dropped onto slides, denatured for 10 minutes at 80°C and hybridized for 48 hours at 37°C. Post-hybridization washes were performed in 50% formamide/2x SSC (37°C), 2x SSC (37°C) and 1x SSC (room temperature) for 5 minutes each. Finally, slides were mounted in Vectashield anti-fading medium (Vector Laboratories, Burlingame, CA) supplemented with 0.5 µg/ml DAPI (4'6-diamidino-2-phenylindole) as DNA-specific counterstain.
Microtubules and the spindle pole body (SPB) were immunolabelled according
to a standard protocol (Pringle et al.,
1991) with the monoclonal rat anti-yeast tubulin antibody YOL1/34
(Kilmartin et al., 1982
)
purchased from Serotec, Kidlington, UK). Slides were washed twice for 3
minutes in 1x PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM
NaH2PO4, pH 7.5), excessive liquid was drained and the
slides were incubated with a drop of primary antibody (diluted 1:200 in
1x PBS) under a coverslip at 4°C overnight. After three 3 minute
washes in 1x PBS, slides were incubated with FITC- or TRITC-conjugated
secondary antibody for 120 minutes at room temperature. The slides were then
washed three times for 3 minutes in 1x PBS. Cells were postfixed for 10
minutes in paraformaldehyde fixative (see above), washed for 3 minutes in
distilled water and either mounted under a coverslip in Vectashield
supplemented with DAPI or airdried and subjected to the standard FISH
procedure (see above).
Slides were evaluated with a ZEISS Axioplan II epifluorescence microscope equipped with appropriate filter combinations for FITC, Cy3 and DAPI. Images were captured separately for the different fluorochromes using a computer-controlled cooled CCD camera (Photometrics, Tucson AZ), and pseudocolored and merged with the help of the IPLab image analysis software (Scanalytics, Fairfax, VA). GFP or FISH signals were classified as associated if they were lying side-by-side and touching, or if they were merged to form a single spot.
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Results |
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Strains with tetO repeats at two allelic positions showed a single
GFP signal in 67.2±6.1% (CEN/CEN) and 74.2±4.6% (TEL/TEL)
(n=600 each) of living cells. Comparable high levels of GFP signal
associations were observed in cells treated according to the semi-spreading
procedure (61.8±7.1% for CEN/CEN and 71.1±6.8% for TEL/TEL,
n=1400 each; Fig. 2A;
Fig. 3). To test whether the
association of signals reflects the normal relative positions of these
chromosomal loci or if they are promoted by tetO inserts, the
corresponding chromosomal regions were labelled by FISH (probes 1 and 2,
Fig. 1C) in a yeast strain
without inserted tetO repeats and the frequencies of signal
associations were counted. Centromeric regions were associated in
33.7±4.3% and telomeric regions in 26.3±3.1% of the 250 nuclei
scored. The association of homologous centromeres is elevated compared with
other regions because of the general clustering of centromeres
(Jin et al., 1998) but still
less frequent than in the presence of tetO repeats. The average
association frequency for other homologous chromosome regions (13 sites on 8
different chromosomes tested) was found to be about 18% (Q. Jin, J.F. and
J.L., unpublished).
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Also in a strain with the tetO arrays at non-allelic positions on
the two chromosomes V (trans-CEN/TEL), and in a haploid strain
containing both tetO tracts on the same chromosome (cis-CEN/TEL),
associations of tetO sites were frequent (51.4±2.7% and
55.9±7.2%; n=1400 each)
(Fig. 3). This is remarkable
because in yeast interphase nuclei there exists a roughly parallel orientation
of chromosome arms with all centromeres clustered at one pole and the
chromosome ends assembling near the opposite pole
(Jin et al., 1998;
Jin et al., 2000
).
Since the false impression of association or fusion of GFP signals could be generated by signal loss caused by mitotic intrachromosomal recombination, we checked the stability of inserted tetO repeats. Cultures of the trans-CEN/TEL strain (FKY806), which had been grown for an average of at least 40 mitotic generations, were sporulated and the number of signals was determined in pachytene nuclei. Homologous synapsis at pachytene relocates the non-allelic regions with inserted tetO repeats to different regions on the bivalent and thus suppresses their fusion (J.L., unpublished). Of 100 arbitrarily selected pachytene nuclei, 91% showed two separate CEN-GFP/TEL-GFP signals and 9% showed a single signal. There was no nucleus without any signal, and in nuclei with one signal this was strong, suggesting that it was produced by two fused tetO repeats (presumably due to the looping back of the bivalent on itself). Thus there was no notable loss of signals during extended vegetative growth and we can safely assume that the presence of a single signal per nucleus in the experiments, which were all done on fresh cultures, is not normally due to the loss of tet operator sequences and reduced GFP signal intensity.
The association of tetO repeats depends on the presence of
TetR molecules
To study whether the association of tetO sequences reflects an
intrinsic property of tandem repeats or if it is caused by the binding of TetR
molecules, tetO association was evaluated in strains with
tetO tracts at the centromere- and the telomerenear loci but lacking
the transgenic tetR construct responsible for TetR-GFP expression.
The frequency of associations was tested by FISH with probes specific for
regions close to the integration sites of the tetO repeats (probes 1
and 2) as well as with a probe specific for the tetO sequence (probe
3, see Fig. 1C). The haploid
cis-CEN/TEL w/o tetR strain (SLY1664; see
Table 1) showed
18.7±6.3% of associated tetO regions (n=300), which
is similar to the frequency of associations of the corresponding chromosome
regions in the haploid wildtype strain NKY857 (16.0±4.0%;
n=300) (Fig. 4). The
diploid TEL/TEL w/o tetR strain SLY1663 showed a single FISH signal in
24.8±1.2% of the 200 nuclei inspected, which is comparable with the
frequency of the association of the corresponding region in the wild-type
strain (SK1) without tetO sites (26.3±3.1%; n=250).
By contrast, when the chromosome regions harbouring the tetO repeats
in the TetR-GFP-expressing strain FKY 1024 (TEL/TEL) were labelled by FISH,
they showed the expected high association of FISH signals (79.4±7.9%;
n=200).
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In another series of experiments, TetR-GFP binding to tetO was
inhibited by the addition of 12 µg/ml tetracycline to the cell cultures.
TetR has a high affinity to tetracycline so that nontoxic amounts can
effectively induce inactivation of the repressor
(Hillen and Berens, 1994).
Similarly, the association frequencies were reduced to the frequency of
associations in haploid wild-type cells without the tetO insertions
(Fig. 4).
The association of tetO repeats is cell cycle-dependent
In stationary cultures (cell density 2x108 cells/ml),
associations of tetO sites were more frequent than in cycling
cultures. In strains with the tetO repeats at allelic positions
(CEN/CEN and TEL/TEL) the association of the GFP signals reached almost 90%
and tetO repeats at non-allelic positions (CEN/TEL) were associated
in up to nearly 80% of nuclei (Fig.
3). To test whether associations in cycling cultures were cell
cycle-dependent, we arrested cells at S-phase by hydroxyurea. These cells
showed small buds and short bipolar spindles
(Cheeseman et al., 2001
).
Associations were greatly reduced for all pairs of tetO inserts
(CEN/CEN, 32.5±2.9%; TEL/TEL, 47.1±7.1%; trans-CEN/TEL,
7.1±1.0%, cis-CEN/TEL, 15.7±1.5%)
(Fig. 2B). (We can exclude that
the presence of two signals was caused by signal duplication at a single
replicated locus, as we never observed three or four signals. Also, we never
observed two signals in strains with a single tetO repeat.)
The disruption of associations in S-phase was partially restored prior to or during subsequent mitosis. For the identification of anaphase cells, spindles were immunolabelled with an antibody against tubulin. Anaphase cells possessed a long spindle and large buds, and had their elongated nucleus localized half-way through the bud-neck. It was found that CEN/CEN associations were maintained or re-established in both half nuclei in 74% of anaphase cells (n=100) (Fig. 2C) and TEL/TEL associations in 64% (n=100) (Fig. 2D). By contrast, only 40% (n=100) of half nuclei showed CEN/TEL associations (Fig. 2E). It is plausible to assume that the anaphase orientation, with the centromeres migrating with the spindle poles and the telomeres trailing, is detrimental to the re-association of centromere-near and telomere-near tetO sites but may promote re-association of allelic tetO sites. The highest levels of association of ectopically inserted tetO repeats are possibly reached during G1 when the anaphase orientation of chromosome arms is partially relaxed.
The association of tetO repeats perturbs normal chromosome
arrangement
Although the association of allelic tetO repeats caused the
alignment of flanking homologous chromosome regions, the global nuclear
organization with roughly parallel chromosome arms and clustered centromeres
around the SPB (Rabllike configuration)
(Jin et al., 2000), remained
unaffected. However, the association of tetO arrays at non-allelic
positions (trans-CEN/TEL and cis-CEN/TEL) would be expected to lead to the
deviation of chromosome V from this order. We measured the distances
from the SPB of FISH-labelled centromeric (probe 1) and telomeric (probe 2)
sites of chromosome V in the wild-type and in the trans-CEN/TEL
strain. As can be seen from Fig.
5, in the WT centromeric regions were near the SPB and the
telomeric regions away from it, as expected. In the trans-CEN/TEL strain,
always one of the two telomeric regions was as close to the SPB as the
centromere. (By GFP we confirmed that it was the tetO-carrying
telomeric region that was near the SPB; data not shown.) Therefore,
tetO associations cause the right arm of chromosome V to form a loop
within the nucleus.
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Discussion |
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Somatic pairing as the cause of tetO associations would be plausible for tetO sequences integrated at allelic chromosome positions and on the condition that an appreciable degree of somatic pairing exists. However, here we showed that associations of chromosome regions with tetO repeats are much more frequent than associations in the absence of tetO inserts, and that this is true both for allelic and ectopic pairs of tetO sites.
Thus, association of chromosome regions is conferred by tetO
integrations. A previous study found that, in wheat, multiple transgene
integration sites were brought together at interphase in spite of their
considerable distances on a chromosome; the authors suggested that an ectopic
pairing mechanism might act between them
(Abranches et al., 2000). In a
study similar to ours, Aragon-Alcaide and Strunnikov integrated tetO
and lacO tandem repeats at allelic and ectopic chromosome regions and
detected them by the binding of GFP-fused repressor molecules
(Aragon-Alcaide and Strunnikov,
2000
). They found frequent associations between tetO and
lacO sites, respectively, and reached the conclusion that the
repeated arrangement of DNA sequences can promote and stabilize interactions
that are based on DNA sequence homology.
Here, we show that the association of tetO sequences requires TetR
and therefore does not reflect an inherent property of tandem repeats per se.
It is likely that TetR-GFP molecules bind to several tetO tracts,
which physically connects operator sequences and their flanking regions from
different chromosomal loci. It had been shown previously in vitro that
tetrameric Lac repressor (LacI) can bind two lac operators on
different DNA molecules simultaneously
(Kramer et al., 1987). In
addition, it was demonstrated in vivo that a tetramerizing GFP-Lac repressor
fusion could hold pairs of sister chromatids together by linking integrated
lac operator repeats (Straight et
al., 1996
). Other studies excluded the possibility that
associations are generated by the interaction between Lac repressor molecules
as they used constructs with the tetramerization domain deleted
(Aragon-Alcaide and Strunnikov,
2000
; Chen and Matthews,
1992
).
TetR binds to tetO as a dimer
(Hillen and Berens, 1994) but
unlike LacI, it does not possess a C-terminal tetramerizing domain, and a TetR
dimer can bind only a single tetO motif (W. Hillen, personal
communication). Therefore it must be assumed that oligomerization, which
enables TetR to link separate tetO loci, occurs under the special
conditions of ectopic expression or expression as a GFP fusion protein. It
might be speculated that, also in the case of non-tetramerizing LacI, it is
the modification of the protein by the GFP-fusion that causes the association
with more than one lac operator.
To explore the possibility that the fusion with GFP promotes TetR oligomerization, nuclear tetO distribution will be studied in cells that express untagged TetR. Furthermore, tetO repeat numbers could be increased to enhance a possible weak tendency of tandemly repeated chromosomal DNA sequences to associate autonomously in the absence of potentially linking regulatory proteins. If transgenes were found to associate, then it would be worth constructing tandem repeat arrays of yeast endogenous sequences to test whether they share this property.
Ectopic tetO associations interfere with the chromosomal
order within nuclei
In yeast interphase nuclei, chromosomes are oriented with their centromeres
to one pole and the ends towards the opposite pole
(Jin et al., 1998). This
arrangement is reminiscent of the Rabl-configuration found in various higher
eukaryotes. Consequently, loci that are the same distance from the centromere
occupy the same latitude of the nucleus with respect to the centromeric pole
(Jin et al., 1998
). This
causes allelic loci to be on average in closer proximity than two randomly
selected loci. In fact, FISH labelling of specific chromosomal loci has shown
that loci of the same centromeric distance are associated in as many as 24% of
nuclei if they are allelic and in slightly less (10%) if they are nonallelic
(J.F., Q. Lin, A.L. and J.L., unpublished). Previous studies observed an even
higher preference for allelic loci to associate and presented additional
evidence for their physical interaction, which were interpreted by a tendency
of homologous chromosomal regions to engage in transient somatic pairing
(Burgess et al., 1999
;
Burgess and Kleckner, 1999
).
These observations suggest that the nonrandom arrangement of chromosomes
within nuclei promotes the association of tetO inserts at allelic
chromosomal sites.
However, TetR not only links DNA tracts that occupy the same region of the
nucleus: if tetO repeats are inserted at non-allelic chromosomal
loci, TetR-mediated associations are also strong enough to perturb the
polarized chromosome arrangement. Whereas normally the roughly parallel
orientation of chromosome arms between the centromeric and the telomeric
nuclear pole prevails in interphase nuclei, a centromere-near and a
telomere-near tandem tetO repeat can bring together chromosomal
regions from the two opposite nuclear domains. We have shown for chromosome
V that the chromosome end loops back to the centromeric pole whereas
the centromere maintains its position (Fig.
5). This confirms that the centromeres are physically linked to
the SPB during interphase, as was proposed previously
(Jin et al., 2000), rather
than left assembled near the pole as a consequence of the preceding anaphase
orientation.
The tethering of centromeric and telomeric chromosome regions via
TetR-mediated tetO associations implies that these regions must at
least transiently contact each other, since a long-range interaction of
tetO sites is difficult to imagine. This suggests that, in spite of
the highly ordered nuclear architecture
(Jin et al., 2000), there is
sufficient stirring of the nuclear contents. As was shown by Marshall et al.,
there is a diffusion of chromatin in living cells, which is probably powered
by Brownian motion but confined in its extent by microtubules
(Marshall et al., 1997b
).
Furthermore, Heun et al. observed in time-lapse experiments dramatic movements
of GFP-tagged chromosomal sites over distances as large as a third of the
nuclear diameter, within seconds (Heun et
al., 2001
). This chromatin motion could have the capacity to lead
to the initial contacts of ectopic tetO inserts.
The possible contribution of protein-mediated associations to nuclear
architecture
There is an increasing number of cases known where protein-DNA interactions
appear to function in the association of specific chromosome regions. The
Drosophila protein zeste can self-associate and can thereby
possibly spatially link chromosomal loci to which it binds
(Bickel and Pirrotta, 1990). A
similar linking effect could be exerted by the sequence-specific DNA-binding
members of the mammalian Ikaros family of transcription factors that recognize
related DNA sequences and are capable of dimerizing with themselves and other
family members (Brown et al.,
1997
; Cobb et al.,
2000
; Perdomo et al.,
2000
). In addition, the ectopic pairing of heterochromatin in
Drosophila is possibly mediated by the self-association of
heterochromatin-binding proteins (Dernburg
et al., 1996
). The latter authors found that a heterochromatic
insertion at the brown locus caused its relocalization to the
centromeric pole of nuclei due to the physical association of the insert with
centromeric heterochromatin.
The question is whether protein-mediated associations of a similar kind
play a role in the establishment or maintenance of nonrandom interphase
chromosomal arrangement in yeast, and in particular the transient homologous
chromosome associations that have been reported to occur in S.
cerevisiae (Burgess and Kleckner,
1999; Burgess et al.,
1999
). In this context it may be interesting to note that, like
tetO associations, these somatic associations are disrupted during
S-phase (Burgess et al., 1999
).
The linkage of tetO repeats at ectopic chromosomal sites by TetR is
sufficiently strong to perturb the normal polarized orientation of chromosome
arms, demonstrating that protein-mediated specific chromosome associations can
be quite robust. However, in this case it is likely that the strength of
associations results from the tandemly repeated nature of tetO
inserts providing a large number of binding sites for TetR molecules. Unlike
in eukaryotes, tandem repeats are not common in the yeast genome. However, a
large number of weak associations along a pair of homologous chromosomes could
cause somatic pairing. Cook proposed that promoters, enhancers or other
elements of transcription regulation that are part of a DNA polymerizing
complex could bind to a homologous DNA molecule in trans
(Cook, 1997
). Such an event
would induce only a weak link between homologous DNAs, but interactions
between thousands of such `transcription factories' could act cooperatively to
lead to stable somatic pairing.
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Acknowledgments |
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