Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea
* Author for correspondence (e-mail: kooh{at}yonsei.ac.kr)
Accepted 18 February 2004
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SUMMARY |
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Key words: Werner syndrome, Disease model, Aging, DNA damage, Checkpoint
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Introduction |
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Several defects at the cellular level have been detected in WS. Cells
cultured from WS patients have a reduced life span, an extended S phase, and
reduced RNA transcription by RNA polymerases I and II
(Martin et al., 1970;
Salk et al., 1985
;
Balajee et al., 1999
;
Shiratori et al., 2002
).
Although no direct role of WRN has been established in telomere metabolism, WS
fibroblasts expressing a transfected human telomerase (TERT) gene
have an increased life span and can be immortalized
(Wyllie et al., 2000
). These
results suggest that a telomerase-dependent pathway is involved in the
accelerated cellular senescence caused by the absence of WRN. WS cells are
also hypersensitive to certain DNA damaging agents, including the chemical
carcinogen 4-NQO (Ogburn et al.,
1997
), camptothecin (Poot et
al., 1999
), and DNA cross-linking agents
(Poot et al., 2001
).
WRN has been shown to form complexes with proteins involved in cellular
responses to DNA damage and in DNA replication. The identification of a
functional interaction between WRN and the p53 tumor suppressor protein has
emphasized the role of the RecQ family in maintaining genomic stability
(Spillare et al., 1999). WRN
also dramatically stimulates the cleavage reaction catalyzed by the human
5' flap endonuclease/5'
3' exonuclease FEN1
(Brosh et al., 2001
), a DNA
structure-specific nuclease implicated in DNA replication and repair
(Lieber, 1997
). The ability of
replication protein A (RP-A; RPA1 Human Gene Nomenclature Database) to
stimulate the unwinding of long stretches of DNA duplex by WRN helicase
suggests that WRN may function in replication, a notion supported by
interaction of WRN with other replication proteins. Recent evidence points to
a direct protein interaction between WRN and the Ku80/70 heterodimer
implicated in non-homologous end-joining of double-strand breaks
(Cooper et al., 2000
). In
addition to these functional interactions, WRN has been reported to physically
interact with human polymerase delta
(Szekely et al., 2000
), PCNA
and DNA topoisomerase I (Label et al.,
1999
). These interactions suggest that WRN is a central player in
a macromolecular complex essential for DNA replication or repair.
In C. elegans, four RecQ family proteins are predicted from the genomic DNA sequence. Of these proteins, the one encoded by the open reading frame (ORF) F18C5.2 is most homologous to human WRN. To understand the role of this C. elegans WRN homolog (WRN-1), we localized the protein in C. elegans and investigated the phenotypes arising from inhibited expression.
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Materials and methods |
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Cloning of a 5' cDNA fragment of C. elegans wrn-1 by RT-PCR and construction of full-length cDNA
The EST clone yk41c3 of the F18C5.2 ORF lacked the first exon (115
nucleotides; nt) of the predicted ORF of 16 exons. Therefore, to obtain a
5'-terminal cDNA clone, we isolated C. elegans total RNA using
an RNeasy kit (Qiagen). cDNA synthesis progressed in a reaction mixture (50
µl) containing C. elegans total RNA (3 µg), a primer (10
pmoles) of sequence 5'-GTGGACATAAGAACAAATTGGTC-3' (nt 752-729 in
the ORF) from exon 3, and Superscript reverse transcriptase II (200 units,
Stratagene), at 42°C for 1 hour. First cDNA strand synthesis was
terminated by heating at 70°C, and then template RNA was degraded by RNase
H (2 units, Takara). A cDNA fragment was amplified from the first cDNA strand
by PCR (polymerase chain reaction) using the SL1 primer
(5'-GGTTTAATTACCCAAGTTTGAG-3') and a primer of sequence
5'-CATTTCTGACAACATCCCACTG-3' (nt 715-694 in the ORF) from exon 3.
The amplified cDNA fragment was cloned into pGEM-T vector (Promega) and
sequenced with an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit
(Perkin-Elmer).
To obtain a full-length cDNA construct of wrn-1, PCR was carried out using two primers: 5'-CGCGGATCCATGATAAGTGATGATGACGATC-3', containing nt 1-22 of the ORF, and a BamHI recognition sequence (underlined); and 5'-CATTTCTGACAACATCCCACTG-3', corresponding to nt 715-694 of the ORF. The amplified DNA product was digested with BamHI and HindIII (nt 641-646 in the ORF) restriction enzymes, electrophoresed on a 1% agarose gel, and then eluted from the gel using a DNA extraction kit (Intron Biotechnology, Korea). The purified cDNA fragment was inserted into plasmid yk41c3, previously digested with BamHI and HindIII, to yield pCeWRN.
Antibody preparation
A 5'-terminal cDNA fragment was amplified from pCeWRN by PCR using
primers: nt 1-22 of the ORF with a BamHI recognition sequence; and nt
627-609 with a SalI recognition sequence (underlined),
5'-CAGCTGCGTCATTGATGCCCACTTC-3'. The cDNA fragment was
cloned into pGEM-T, excised from the recombinant T-vector, and then inserted
between the BamH1 and SalI sites of the pMAL-c2
overexpression vector (New England Biolabs). E. coli XL1-Blue cells
harboring pMAL/CeWRN were cultured at 37°C to O.D.600nm 0.5,
and isopropyl-thio-ß-D-galactoside (Calbiochem) was added to 1 mM. After
further incubation for 3 hours, the cells were harvested and sonicated in 10
ml PBS with 10 mM Na2HPO4, 2 mM
KH2PO4 (pH 7.4), 137 mM NaCl and 2.7 mM KCl. Cell lysate
was microcentrifuged at 14,000 rpm for 5 minutes, and the supernatant
electrophoresed on a 7% SDS polyacrylamide gel. The overexpressed protein band
was excised, crushed in PBS, mixed with Freund's adjuvant, and then injected
into Balb/c mice four times at weekly intervals (200 µg protein per
injection).
Immunostaining
C. elegans embryos were immunostained by a slightly modified
version of the procedure of Crittenden and Kimble
(Crittenden and Kimble, 1999).
Embryos were freeze-cracked, fixed, incubated with polyclonal mouse antiserum
against the N-terminal 209 amino acids of WRN-1 (1:25 dilution), and then with
FITC-conjugated goat anti-mouse immunoglobulin G (1:500 dilution, Santa Cruz
Biotechnology) pre-treated with C. elegans acetone powder. After
being stained with DAPI (4,6-diamidino-2-phenylindole, 1 µg/ml), specimens
were observed with a fluorescence microscope (DMR HC, Leica). Gonads and
intestines were extruded by decapitating adult C. elegans, fixed in
3% paraformaldehyde, then immunostained as described
(Jones et al., 1996
).
Whole-worm staining was carried out by the collagenase method of Nonet et al.
(Nonet et al., 1993
). After
fixation in 4% paraformaldehyde, worms were incubated in a reducing solution
[5% ß-mercaptoethanol, 1% Triton X-100, 0.1 M Tris-Cl (pH 6.9)] at
37°C overnight, and then reacted with collagenase (1000 units/ml, Sigma)
in buffer [0.1 M Tris-Cl (pH 7.5), 1 mM CaCl2] at 37°C for 5
hours. Subsequent reactions with primary and secondary antibodies were as
described above for embryos.
Inhibition of wrn-1 expression by double-stranded RNA microinjection
The pCeWRN recombinant plasmid was linearized with BamHI and
ApaI restriction enzymes at its multicloning site to prepare
antisense and sense transcripts of wrn-1, respectively. Antisense RNA
was synthesized using BamHI-digested plasmid DNA (2 µg), T7 RNA
polymerase (5 units, MBI), ribonucleoside triphosphates (rNTPs, 0.4 mM each)
and RNase inhibitor (5 units, Takara) in buffer [40 mM Tris-Cl (pH 8.0), 8 mM
MgCl2, 2 mM spermidine, 50 mM NaCl, 18 mM DTT; total 50 µl], at
37°C for 2 hours. Sense RNA was synthesized under the same reaction
conditions as described for antisense RNA, except for the use of
ApaI-treated DNA (2 µl) and T3 RNA polymerase (5 units, MBI).
After RNA synthesis, RNase-free DNase I (2 units) was added to degrade
template DNA, and then phenol (pH 4.5) extraction and ethanol precipitation
were carried out. An equivalent amount of sense and antisense RNAs were mixed
to a total concentration of 1 µg/µl, and then microinjected into the
intestines of young adult N2 worms. The worms were placed on an NGM plate with
an E. coli OP50 lawn, and were transferred to new plates after 12
hours.
Measurement of life span
Twelve hours after microinjection, the worms were allowed to lay embryos
for 6 hours. F1 progeny (>100) were grown at 20°C or 25°C, and
transferred to fresh plates every one or two days. Death was scored by absence
of any movement after several light pokes with a platinum wire.
Phenotypic analysis
F1 progeny of the microinjected worms, designated Ce-wrn-1(RNAi)
worms, were grown at 20°C or 25°C. Over 500 F1 worms were examined
daily, with a stereomicroscope or with Nomarski optics (DMR HC, Leica), from
the L1 stage. Worms with abnormal phenotypes were counted to 8 days old. The
wild-type N2 strain was used as a control instead of a mock-RNAi strain, as
the phenotypes produced by microinjection of dsRNA derived from a
5'-upstream DNA sequence were the same as wild type.
C. elegans sensitivity to DNA damage
F1 larvae at the L1 stage, derived from the microinjected P0 worms, were
-irradiated with a 137Cs source (IBL 437C, CIS
Biointernational) at 60 Gray (Gy). After being kept at 20°C for 3 days,
over 200 worms were examined under a stereomicroscope or with Nomarski optics.
Wild-type N2 worms were also
-irradiated and their phenotypes examined
as a
-irradiation control. In order to measure larval growth rate at
20°C, over 150 L1 stage worms were
-irradiated at 0, 10 or 20 Gy.
Subsequently, worms reaching the L4 stage, as defined by vulva shape, were
scored every 12 hours.
Aging phenotypes induced by bacteria-mediated RNAi of Ce-wrn-1
The EST clone yk41c3 of the F18C5.2 ORF was digested with NotI and
ApaI enzymes, and inserted into the pPD129.36(L4440) vector
(Timmons and Fire, 1998),
which contains two convergent T7 polymerase promoters in opposite
orientations, separated by the multicloning site. Plasmid DNA was transformed
into E. coli HT115(DE3) (W3110, rnc14::
Tn10) cells by
electroporation (Invitrogen). Cells harboring plasmid DNA were directly
applied onto agar plates, composed of standard NGM/agar medium supplemented
with 100 µg/ml ampicillin, 12.5 µg/ml tetracycline and 0.4 mM IPTG, and
then cultured overnight at room temperature. L4 stage N2 worms were grown to
adults on the plate covered with E. coli cells producing dsRNA of
wrn-1, allowed to lay embryos for 1 hour, and then removed from the
plate. The embryos were incubated at 20°C until they reached the L4 larval
stage, followed by a temperature shift to 25°C. Twenty-four hours later,
autofluorescence of adult worms was photographed using a fluorescence
microscope (525 nm filter), and their heads were observed with Nomarski optics
for 7 days. Control worms were fed with non-transformed HT115(DE3) E.
coli cells.
Time-lapse microscopy of embryonic cell divisions
The EST clone yk1302e07 of chk-1 (Y39H10A.7) was digested with
XhoI and inserted into the pPD129.36(L4440) vector. On the
feeding-plate covered with E. coli cells producing dsRNA of
wrn-1, chk-1, or both, wild-type N2 or div-1(or148ts) worms
at the L4 stage were placed and grown to adults at 25°C. The adult worms
were dissected to isolate 2-cell-stage F1 embryos, which were then observed
microscopically with Nomarski optics at appropriate intervals. In order to
measure the duration of S and M phase in the early embryos, embryos were
photographed every 10 seconds by time-lapse microscopy (Leica IM 1000).
Hydroxyurea treatment of the germ line
On the feeding-plate covered with E. coli cells producing dsRNA of
wrn-1, wild-type N2 worms were grown from L1 to L4 stages at
25°C. L4 stage worms were transferred to a new feeding-plate containing 25
mM hydroxyurea and were dissected to isolate the gonads 12 hour later. After
staining with DAPI, the gonads were observed using a fluorescence
microscope.
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Results |
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WRN-1 localization in C. elegans
WRN-1 was immunolocalized in C. elegans at various developmental
stages (Fig. 2). The protein
was non-uniformly distributed in nuclei from the early embryonic stage
throughout embryogenesis. In early embryos, the protein level was higher in
mitotic cells than in interphase cells, as is clearly shown in 6- and 8-cell
stage embryos (mitotic cells marked with arrowheads or an arrow). At
metaphase, WRN-1 had an unusual location, overlapping with the periphery of
equatorially aligned condensed chromosomes facing the spindle poles (see the
cell marked with an arrow in Fig.
2A, and the metaphase cell in
Fig. 2D), and also, less
frequently, overlapping with the mitotic spindles
(Fig. 2A, arrow). This
localization to one side of a mitotic sister chromatid was observed for CENP-A
and CENP-C homologs in C. elegans embryos, which are
centromere-binding proteins of holocentric C. elegans chromosomes
(Moore and Roth, 2001), and
also for SAN-1, which is involved in the spindle checkpoint
(Nystul et al., 2003
).
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Life span is reduced in wrn-1(RNAi) worms
To assess the in vivo function of WRN-1, RNA interference (RNAi) was
carried out by microinjecting double-stranded RNA (dsRNA) of the
wrn-1 gene into young adult worms (P0). When the progeny (F1)
wrn-1(RNAi) worms were grown at 20°C, their life span was not
affected: wild type and wrn-1(RNAi) worms lived for 17.1
(±0.2) and 16.8 (±0.2) days after birth, respectively (J.-S.Y.,
unpublished). By contrast, RNAi significantly reduced their life span at
25°C: the life span was 11.0 (±0.2) days for wrn-1(RNAi)
worms and 13.6 (±0.1) days for wild-type worms (P<0.001;
Fig. 3). In addition, the brood
size of wrn-1(RNAi) worms was reduced to 94% of wild type at
20°C, and to 84% at 25°C (J.-S.Y., unpublished), whereas embryonic
hatching was unaffected at either temperature.
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The RNAi phenotypes of wrn-1 were enhanced by ionizing radiation
L1 stage larvae were -irradiated and their growth to adults was
examined after 3 days. As shown in Fig.
5D,
-radiation greatly increased the frequency of the
phenotypes listed in Fig. 5A
even at 20°C, both in wild type and wrn-1(RNAi) strains. Among
these phenotypes, small body and bag of worms appeared in 40% and 35% of
wrn-1(RNAi) worms, respectively, much higher than the values (close
to 10% for each) for wild type. These large increases in the frequency of
abnormal phenotypes must be due to enhancement of the RNAi effects by
-radiation, given the much lower frequency (<2%) of these phenotypes
in the absence of ionizing radiation (at 20°C). This finding suggested
that WRN-1 participates in cellular responses to DNA damage and that the
exacerbated developmental defects in its absence probably resulted from
defective cellular signaling or/and DNA repair. The fact that the dumpy
phenotype, unlike the other phenotypes listed in
Fig. 5, was not significantly
increased by ionizing radiation is very intriguing, as it points to a
distinctive role of elevated metabolic rate at a higher temperature in
inducing dumpiness.
The rapid larval growth of wrn-1(RNAi) worms is not affected by ionizing radiation
As the enhancement of RNAi phenotypes by ionizing radiation suggested that
WRN-1 played a role in cellular responses to DNA damage, larval growth rate
was measured after irradiating L1 stage worms with lower doses of -ray
than those worms shown in Fig.
5D. Even in the absence of
-irradiation,
wrn-1(RNAi) larvae surprisingly grew faster than wild-type N2 larvae,
as shown in Fig. 6. Although
wrn-1(RNAi) larvae reached the adult stage about 6 hours earlier than
wild type at 20°C, this was much less than the difference of 2.6 days
(P<0.001) in life span that was observed between the two strains
at 25°C. Therefore, there remains a substantial reduction in life span
(Fig. 3) even when the
difference in larval growth rate is taken into account. Another striking
aspect of the growth rate of wrn-1(RNAi) larvae was its independence
from ionizing radiation, which contrasted with the growth retardation of
wild-type larvae with increasing
-ray dose
(Fig. 6). The fast growth of
wrn-1(RNAi) larvae and its insensitivity to DNA damage suggested that
the strain probably was defective in a cell cycle checkpoint responding to DNA
damage.
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Discussion |
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WRN-1 in C. elegans is distinct in subcellular localization from its human and mouse homologs
C. elegans WRN-1 is diffusely distributed in the nucleoplasm
during interphase as is mouse WRN, whereas human WRN is predominantly
localized to the nucleolus (Marciniak et
al., 1998; Suzuki et al.,
2001
). However, human WRN relocates from the nucleolus to
nucleoplasmic foci upon induction of DNA damage with UV, ionizing radiation,
camptothecin, etoposide or 4-nitroquinoline-1-oxide
(Gray et al., 1998
;
Sakamoto et al., 2001
;
Blander et al., 2002
). The WRN
foci overlap with the foci of RP-A almost completely, and overlap with those
of RAD51 partially, suggesting that human WRN cooperates with RP-A and RAD51
in response to DNA damage (Sakamoto et
al., 2001
). Similarly, interruption of DNA synthesis by depleting
deoxynucleotides with hydroxyurea results in the movement of WRN from nucleoli
to distinct nuclear foci that co-localize with RP-A
(Constantinou et al., 2000
).
However, the localization of WRN-1 to the poleward periphery of metaphase
chromosomes in the early C. elegans embryo has not been observed in
other organisms. This peculiar localization may be needed to ensure equal
partition of the protein into daughter cells, or may mean that WRN-1 functions
in a spindle checkpoint involving holocentric chromosomes during mitosis.
wrn-1(RNAi) phenotypes resemble the symptoms of human WRN syndrome
RNAi of C. elegans wrn-1 did not cause discernable phenotypes at
20°C, whereas at 25°C the worms had a reduced life span and showed
significant increases in such developmental abnormalities as small body, bag
of worms, ruptured body, dumpy shape, growth arrest and transparency
(Fig. 5). These developmental
abnormalities have been induced by deficiency of ATR and RAD51 homologs in
C. elegans, and their frequency was increased by ionizing radiation
(Aoki et al., 2000;
Rinaldo et al., 2002
).
Therefore, the developmental defects probably resulted from DNA damage during
development. Some of these phenotypes resemble the symptoms of human Werner
syndrome, such as short stature and premature aging. The enhanced phenotypes
of wrn-1(RNAi) at 25°C was very likely due to the fact that the
metabolic rate of C. elegans is 1.2 times higher at 25°C than at
20°C (Van Voorhies and Ward,
1999
), thus generating a greater oxidative stress. The slight
reduction in life span was probably due to premature aging, as was
demonstrated by faster lipofuscin accumulation in the C. elegans
intestine and tissue deterioration in the head, although other phenotypes such
as bag of worms, ruptured body and growth arrest certainly contributed to the
reduction. Many small C. elegans mutants are known to have defects in
the TGF-ß signaling pathway (Gumienny
and Padgett, 2003
), and others are mutated in the spectrin ß
chain (sma-7), a MAP kinase (sma-5), a fatty acid elongation
enzyme (elo-2) or a basement membrane protein (SPARC). Dumpiness in
C. elegans is generally due to mutation of genes participating in
collagen production or in dosage compensation. The phenotype bag of worms is
thought to be caused by defects in HSN or chemosensory neurons, or to partial
defects in other egg-laying systems, based on its sensitivity to serotonin. A
C. elegans line harboring human WRN cDNA linked to a strong C.
elegans promoter was prepared, but the occurrence of wrn-1(RNAi)
phenotypes was not reduced in the transgenic line (S.M.H., unpublished). The
failure to rescue the phenotypes could be due to non-equivalence of human and
C. elegans WRNs, or to inefficient expression of the exogenous
gene.
Ionizing radiation increased the frequency of wrn-1(RNAi)
phenotypes synergistically, suggesting that C. elegans WRN-1 is
involved in the DNA damage response (Fig.
5D). WRN-1 may be involved in non-homologous end-joining by
interacting with Ku70/80 (Cooper et al.,
2000), or may be involved in recombinational repair. WRN-1 may
also play a role in DNA damage signaling, as in the control of p53-mediated
transcriptional activation by human WRN
(Spillare et al., 1999
).
Indeed, the fast larval growth of the wrn-1(RNAi) strain, especially
its insensitivity to ionizing radiation, strongly suggests that WRN-1 acts in
a DNA damage checkpoint pathway. In germ cells treated with hydroxyurea, WRN-1
was required to activate the DNA replication checkpoint, which agreed well
with the role of a Saccharomyces cerevisiae homolog, Sgs1, at the
same checkpoint (Frei and Gasser,
2000
; Myung and Kolodner,
2002
). In addition, S phase was accelerated in
wrn-1(RNAi) embryos, indicating a role of WRN-1 as a checkpoint
protein. Nevertheless, wrn-1(RNAi) was not as potent as
chk-1(RNAi) in reducing the extended S-phase of
div-1(or148ts) embryos, in which priming of Okazaki fragments is
inefficient. And, double RNAi of wrn-1 and chk-1 was no more
effective than single RNAi of chk-1. This suggests that WRN-1 works
in a sub-pathway that diverges from CHK-1 in the DNA replication checkpoint
pathway, and that is either up- or downstream of CHK-1. In
sgs1
S. cerevisiae cells, S phase proceeds faster
than in wild-type cells, but the termination stage of S phase takes longer, so
that the total length of S phase is unchanged
(Versini et al., 2003
). The
fast progression of S phase in S. cerevisiae and C. elegans
resulting from the absence of a RecQ homolog contrasts with the extended S
phase in human WRN cells
(Martin et al., 1970
;
Salk et al., 1985
). Recently,
human WRN cells were found to be defective in the
chromosomal decatenation checkpoint in G2 phase
(Franchitto et al., 2003
),
suggesting the possibility that C. elegans WRN-1 may also participate
in a DNA damage checkpoint during G2 phase.
There have been conflicting reports concerning the sensitivity of
wrn-deficient cells to DNA damaging agents. Human WS patient cells
were not hypersensitive to UV or X-ray
(Fujiwara et al., 1977), but
were sensitive to camptothecin, 4-nitroquinoline-N-oxide and DNA-crosslinking
agents (Poot et al., 1999
;
Poot et al., 2001
;
Poot et al., 2002
). The
embryonic stem cells of wrn-knockout mice are hypersensitive to
camptothecin and etoposide, which are inhibitors of DNA topoisomerases I and
II, respectively, but were not sensitive to
-radiation, UV or mitomycin
(Lebel and Leder, 1998
). WRN
mutants of the chicken B-cell line DT-40 were sensitive to various DNA
damaging agents, such as methylmethanesulfonate, 4-nitroquinoline-N-oxide,
etoposide and camptothecin (Imamura et
al., 2002
). The insensitivity of human and murine
WRN cells to ionizing radiation
(Fujiwara et al., 1977
;
Lebel and Leder, 1998
) is in
contrast to the enhancement of the C. elegans wrn-1(RNAi) phenotypes
by ionizing radiation demonstrated in this study.
Although a clear explanation for their pleiotropic phenotypes cannot be
provided, the similarity in phenotypes between wrn-1(RNAi) worms and
Werner syndrome patients suggests that the RNAi worm could be a useful model
for the syndrome. The fact that Wrn knockout mice did not
recapitulate Werner syndrome phenotypes such as premature aging, small
stature, developmental abnormalities and tumor formation
(Lebel and Leder, 1998;
Lombard et al., 2000
) further
emphasizes the importance of wrn-deficient C. elegans as a
potential model for Werner syndrome.
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ACKNOWLEDGMENTS |
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