From the Laboratory of Cell Regulation and Carcinogenesis, NCI,
National Institutes of Health, Bethesda, Maryland 20892-5055, and the
Cancer Research Center, Seoul National University College
of Medicine, Seoul, Korea
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ABSTRACT |
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A 2.5-kilobase cDNA clone that encodes a
371-amino acid novel transcription factor was isolated from a human
placenta cDNA library using a yeast one-hybrid system. The novel
ets-related transcription factor
(ERT) showed a homology with the ETS DNA-binding domain. Using
constructs of the transforming growth factor- (TGF-
) type II
receptor (RII) promoter linked to the luciferase gene, we have
demonstrated that ERT activates transcription of the TGF-
RII gene
through the 5
-TTTCCTGTTTCC-3
response element spanning nucleotides
+13 to +24 and multiple additional ETS binding sites between
1816 and
82 of the TGF-
RII promoter. A specific interaction between ERT
and the ETS binding sites was also demonstrated using an
electrophoretic mobility shift assay. Deletion mapping of ERT protein
suggests that the transactivation domain resides in the amino terminus
while the DNA-binding domain is localized to the carboxyl-terminal
region. Our results suggest that ERT might be a major transcription
factor involved in the transcriptional regulation of the TGF-
RII
gene.
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INTRODUCTION |
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Transforming growth factor-
(TGF-
)1 plays a critical
role in many cellular processes, including regulation of the cell
cycle, cell differentiation, and extracellular matrix synthesis (1, 2).
Aberrant TGF-
function has been implicated in the pathogenesis of
many diseases, and it has also been suggested that diminished responsiveness to TGF-
may contribute to the process of malignant transformation (1). This decreased responsiveness to TGF-
could be
caused by defects not only in TGF-
expression or activation but also
by defects in the regulation of TGF-
receptors (3-6).
Much work has recently been directed toward characterizing the TGF-
receptors and their intracellular signaling pathways. TGF-
type II
and type I receptors (TGF-
RII and RI, respectively) are
transmembrane serine/threonine kinases that together are sufficient for
signal transduction (7). Association between the type I and type II
receptors is essential for signaling responses (8). It has been
repeatedly demonstrated that a genetic alteration of either RI or RII
resulting in dominant negative or loss of function can lead to loss of
responsiveness (3-6, 9-12).
In a previous study, our laboratory described a series of gastric
cancer cell lines in which resistance to TGF- is correlated with
gross structural mutations in the TGF-
RII gene (3). We have now
studied several additional TGF-
-resistant cell lines in which
Southern analysis failed to show gross deletions or rearrangements, yet
in which no TGF-
RII protein or mRNA was produced. This
suggested that abnormalities in transcriptional regulation of the type
II receptor might also be found to underlie certain instances of escape
from TGF-
-mediated growth inhibition.
We have recently cloned and sequenced the promoter region of the
TGF- RII gene, identified several positive and negative transcriptional regulatory elements, and reported the relevant target
sequences for three putative novel transcriptional factor complexes
(13, 14). Basal levels of transcription are determined by the core
promoter element in cooperation with both PRE1 and PRE2 (positive
regulatory elements 1 and 2). PRE1, consisting of nucleotides
219 to
172, contains two discrete target sequences that bind an
AP1/CREB-like transciption factor in addition to an unidentified novel
transcription factor complex. PRE2 is located between +1 and +35 and
contains two overlapping target sequences, both of which appear to bind
novel transcription factor complexes.
To identify potential transcriptional activators of the TGF- RII
gene, we adapted the yeast one-hybrid system (15) to find proteins that
recognize the PRE2 of the TGF-
type II receptor gene. Screening a
human placenta cDNA library fused to the GAL4 activation domain, we
isolated a cDNA clone that induced greater LacZ activity. DNA
sequencing analysis of a corresponding plasmid, pACT2ERT, revealed that
the encoded gene belongs to a novel member of the ets
transcription factor family (16-22). Comparison of the nucleotide
sequence of ERT to the recently reported epithelial specific
ets-family member, ESX/ESE-1 (23, 24) showed it to be
identical, but the ERT cDNA revealed an additional 524 nucleotides in the 3
-UTR. Further, we demonstrate that the ERT protein
specifically binds to the PRE2 region of the TGF-
RII gene and
activates its transcription.
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MATERIALS AND METHODS |
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Reporter Constructs for Library Screen--
The following
oligonucleotides, 5-GAGGAGTTTCCTGTTTTCCCCCGC-3
and
5
-GCGGGGGAAACAGGTAAACTCCTC-3
, containing the previously described PRE2 binding site were synthesized and annealed (13). The
PRE2 mutant oligonucleotides were constructed by replacing the
underlined sequences with 5
-AAGTG-3
and 5
-CACTT-3
, respectively. The oligonucleotides were ligated and subcloned into the
BamHI site of pUC18. A fragment corresponding to a four
tandem repeat was subcloned into the yeast reporter plasmids, pHlSi
and pLacZi (CLONTECH). The reporter constructs
were subsequently integrated into the yeast strain YM4271 yielding
YM4271::PRE2::His3 (or lacZ) and YM4271::PRE2M::His3 (or
lacZ). These yeast strains were used as host strains for the
library screen.
Bacterial and Yeast Strains--
Saccharomyces
cerevisiae YM4271 (MATa, ura3-52, his3-200, ade2-101,
lys2-801, leu2-3, 112, trp1-903, tyr1-501) was purchased from
CLONTECH and used for yeast transformation.
Escherichia coli strains DH5 and DH10B (Life
Technologies, Inc.) were used for subcloning and electroporation
experiments.
Screening of the cDNA Library--
The histidine yeast
reporter strain YM4271-PRE2-His3 was transformed with a
MATCHMAKER human placenta cDNA library
(CLONTECH) by the LiAc/polyethylene glycol method.
Approximately 5 × 104 transformants were plated per
150-mm dish containing hisleu
minimal
selective medium supplemented with 45 mM 3-aminotriazole. Approximately 2 × 106 cDNA plasmids were screened
in three different transformations. Based on large colony size and
rapid growth, a total of 30 histidine positive clones were selected.
Plasmids were recovered and electroporated into the E. coli
strain DH10B. Plasmids were rescreened by transforming YM4271-PRE2-lacZ and plated on leu
ura
minimal medium. The filter replica method using X-gal
(40 µg/ml) was used to confirm
-galactosidase activities. One
plasmid, pACT2ERT, showed the strongest blue color. The specific DNA
binding of pACT2ERT was confirmed by comparing the
-galactosidase
activities of wild-type and mutant reporter strains using both the
filter replica method and O-nitrophenyl
-D-galactopyranoside (ONPG) liquid method
(CLONTECH).
Plasmid Constructions--
The plasmid pcDNA3.1-ERT was
generated by subcloning a 2.5-kb EcoRI-HindIII
fragment containing the entire ERT coding sequence into the
EcoRI-HindIII sites of pcDNA3.1()
(Invitrogen). The plasmid pGL2-pro derivatives including wild-type and
mutant PRE2 sequences were constructed by inserting the
KpnI-XbaI fragment of pUC18/PRE2 and pUC18/PRE2M
into the KpnI-NheI site of pGL2-pro (Promega).
The construction of plasmid
219/+35pTBPII-luc is explained elsewhere
(13). TGF-
RII promoter-luciferase constructs were generated by
polymerase chain amplification using genomic DNA containing the
5
-untranslated region of TGF-
RII as a template. Amplified DNA
fragments were cloned into a promoterless luciferase expression
plasmid, pGL2 (Promega) using BglII and SstI
restriction sites built into the oligonucleotides used for
amplification. The sequences of the polymerase chain reaction-generated
portions of all constructs were verified by DNA sequencing.
In Vitro Transcription and Translation--
One µg of DNA of
pcDNA3.1()-ERT constructs was used as the DNA template for
in vitro RNA transcription using T7 RNA polymerase (Promega). The RNAs were translated in vitro using rabbit
reticulocyte lysate (Promega) and [35S]methonine.
Expressed proteins were electrophoresed on a 4-20% SDS-polyacrylamide
gradient gel, dried, and autoradiographed.
Electrophoretic Mobility Shift Assay (EMSA)-- In vitro translated protein-DNA complexes were formed by incubating at room temperature for 20 min with 10,000 cpm of 32P-labeled probe, 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothretiol, 1 mM EDTA, 5% glycerol, bovine serum albumin (300 µg/ml) in 20 µl of binding mixture.
Transient Transfection and Luciferase Assays--
HepG2 human
hepatoblastoma cell line was maintained in minimal essential medium
supplemented with 10% fetal bovine serum. For the transient expression
assays, cells were transfected using the Lipofectin-mediated
transfection method (Life Technologies, Inc.). Following incubation
with Lipofectin for 15 min, cells were incubated for 48 h. The
cells were then harvested, and luciferase activity was measured.
-Galactosidase activity was used to correct for transfection
efficiencies.
Northern Blot analysis-- SNU620 human gastric cancer cells, HeLa229 human cervical cancer cells, and SK-BR3 breast cancer cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Total RNA was isolated from cells using guanidium isothiocyanate/phenol/chloroform. 10 µg of RNA was eletrophoresed on a 1.0% agarose gel containing 0.66 M formaldehyde, transferred to a Duralon-UV membrane, and cross-linked with a UV Stratalinker (Stratagene). Blots were prehybridized and hybridized in 1% bovine serum albumin, 7% (w/v) SDS, 0.5 M sodium phosphate, 1 mM EDTA at 65 °C. RNA blots were hybridized with 32P-labeled cDNA probes for ERT/ESX/ESE-1 (23, 24). A probe for glyceraldehyde-3-phosphate dehydrogenase was used to assess sample loading.
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RESULTS |
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Isolation of the cDNA Clone Encoding a TGF- RII
Promoter-binding Protein--
We are interested in isolating novel
transcription factors that regulate expression of the TGF-
RII gene.
The "yeast one-hybrid" strategy (15) was used to screen for human
placenta cDNAs encoding for proteins which bind to the TGF-
RII
receptor promoter PRE2. Four tandem copies of the PRE2 were ligated
together and subcloned into the upstream region of the minimal promoter
of either the pHISi or pLacZi reporter plasmids and integrated into the
yeast genome of YM4271 (Fig.
1A). The nucleotide sequence
of the ligated PRE2 was verified by DNA sequencing. With the strategy
described under "Materials and Methods," we isolated several
plasmids that induced greater LacZ activity in YM4271 (PRE2wt). One
cDNA clone, pACT2ERT, showed the strongest blue color on
-galactosidase assay utilizing the filter replica method. Liquid
assay of
-galatosidase activity using ONPG as a substrate confirmed
the specific transcriptional activation of pACT2ERT. While the presence
of the pACT2ERT plasmid doubled
-galactosidase activity in the
wild-type strain, it did not increase enzymatic activity in the mutant
strain (Fig. 1B).
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Deduced Amino Acid Sequences of ERT and Sequence Comparison in the
ETS Domain between ERT and Other ets Family Members--
The complete
sequence of a 2.5-kb cDNA insert was determined by the dideoxy
sequencing method. The 2.5-kb ERT cDNA contains a
1116-base pair open reading frame that encodes a 371-amino acid protein
with a predicted molecular mass of 41,000 daltons. Comparison of the
nucleotide sequence of ERT to the recently reported epithelial specific
ets-family member, ESX/ESE-1 (23, 24), was identical, but
the ERT cDNA revealed an additional 524 nucleotides in the 3-UTR.
Comparison of the amino acid sequences of ERT and other ets
family members revealed a high degree of homology in the ETS domain,
located at the carboxyl-terminal region of the molecule. Approximately
40% of the ETS domain amino acid sequence is shared by ERT and other
ets family members such as ets-1 (16),
ets-2 (16), ERGB (17), Spi-1 (18), E1A-F (19), ER81 (20), and ERM (21) in the ETS domain. There is no significant homology between ERT and any other ets family members outside the
carboxyl-terminal domain.
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Transcriptional Activation of the TGF- RII Promoter by
ERT--
Most of the ets family members are known to be
potent transcriptional activators when tested in transient transfection
assays (22). To analyze the ability of ERT to activate the TGF-
type II receptor promoter, the TGF-
RII promoter-luciferase constructs were cotransfected with an expression vector for ERT into HepG2 human
hepatoblastoma cells. While ERT did not induce luciferase expression in
the control reporter construct, we found ERT to be a potent
transcriptional activator of the TGF-
type II receptor promoter
construct. As seen in Fig. 3,
1,670/+36pTBPII-luc construct was
induced 5-fold by ERT. The
1,670/+36mtpTBPII-luc construct, in which
the second positive regulatory element was mutated, was also activated
more than 2.5-fold, suggesting that the fragment between
1,670 and +2
contains multiple ERT regulatory sequences (Fig.
3). In the previous study (13), we
reported that at least two distinct nuclear DNA binding proteins shared
a common recognition sequence from +11 to +29 in the PRE2 of the
TGF-
type II receptor promoter. This sequence contains two putative
target sequences for protein-binding of ets family members
in a reverse-orientation (5
-GGAAACAGGAAACT-3
). Competitive inhibition
for DNA binding to the +1/+50 sequence was abolished by mutation of
nucleotides +16 to +20 (see Fig. 6B).
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ERT Binds to the PRE2 of the TGF- RII Promoter--
To analyze
the ability of ERT to bind to the TGF-
RII promoter element PRE2,
in vitro translated ERT protein was made by subcloning the
EcoRI-BglII portion of the insert into the
EcoRI-BamHI site of pcDNA3.1. This 41-kDa
in vitro translated ERT protein was consistent with the
predicted size of the open reading frame and was used for the
electrophoretic mobility shift assay (Fig. 6A). A radiolabeled PRE2
(+1/+50) probe was incubated with in vitro translated ERT
protein in competition with unlabeled PRE2 and mutant PRE2
oligonucleotides. While specific unlabeled competitors competed for
labeled protein-DNA complexes (Fig. 6B, lane 4), oligonucleotides mutated in nucleotides +16 to +20 comprising the
ETS-binding site (lane 5) did not compete for binding to
ERT. Deletion of the carboxyl-terminal ETS-domain from the ERT protein produced a smaller protein of approximately 30 kDa (Fig. 6A,
lane 3) that failed to interact with the wtPRE2 (Fig.
6B, lane 6).
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ERT Binds to Functionally Important ETS-related Binding Sites in a
Variety of Genes--
To analyze the DNA sequence requirements for the
binding of ERT and the relative binding affinities, we designed
oligonucleotides encoding a whole spectrum of different functionally
relevant binding sites for ets-related factors, including
the site in the interleukin-2 receptor -chain gene (Fig.
7A). The relative binding
affinities of ERT for these sites were compared with its affinity for
the human TGF-
RII promoter ETS sites. Equivalent amounts of
wild-type labeled oligonucleotides were used as probes in EMSAs with
equal amounts of full-length ERT in vitro translated protein
(Fig. 7B). The in vitro translated ERT formed a
complex with all the ets-related binding site
oligonucleotides tested but with different affinities (Fig.
7B). EBS oligonucleotides in the TCR
enhancer T
2,
polyomavirus PEA3, and HTLV-1 LTR promoters bound to the ERT with
strong affinity, whereas ERT interacted with IL-2-receptor
chain,
2 integrin CD18, and HIV2 LTR ets binding site
oligonucleotides with weaker affinities. To determine if ERT binding
correlates to the transcriptional induction by ERT, we cotransfected
HepG2 cells with these oligonucleotides linked to a luciferase reporter
gene and an ERT expression vector or a control pGL2-pro. A direct
correlation was found between the relative strengths of the binding
affinities and the level of activity induced by ERT (Fig.
7C). PEA3 was induced 5-fold, TR
and HTLV-1 were both
induced 3-fold, while the other weak ERT affinity binding
oligonucleotides showed no appreciable induction, suggesting that
binding is necessary for ERT-mediated transcriptional induction.
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Mapping of Transactivation Domain--
Transcription factors often
display a modular structure with domains being responsible for DNA
binding, transactivation, or protein-protein interaction. To identify
potential transactivation domains within ERT, several truncations of
ERT were tested in transient transfection assays (Fig.
8). Deletion of the first 200 amino
acids led to a severe reduction of ERT-mediated transcription, implicating the existence of an amino-terminal transactivation domain.
Interestingly, the ERT DNA binding domain alone (200/371 or 301/371)
suppressed basal activity of TGF- type II receptor promoter,
suggesting that it acts as a dominant negative mutant.
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DISCUSSION |
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We have isolated a new member of the ets family of
transcription factors, which is a potent transcriptional activator of
the TGF- RII gene, by using a novel genetic approach based on the yeast one-hybrid system. Using this genetic selection to screen a human
placenta cDNA library for sequences encoding DNA-binding domains
that can recognize the PRE2 of the human TGF-
RII gene in yeast, we
isolated a human cDNA that codes for a protein, ERT, that binds to
the PRE2 of the TGF-
RII promoter in a sequence-specific manner and
activates transcription. The deduced amino acid sequence shows high
homology with the ETS-domain, the DNA binding region in the
ets family genes. Comparison of the nucleotide sequence of
ERT to the recently reported epithelial specific ets-family member, ESX/ESE-1 (23, 24) was identical, but the ERT cDNA revealed
an additional 524 nucleotides in the 3
-UTR.
In a previous study, we demonstrated that PRE2 contained at least one
nuclear protein recognition sequence from +11 to +29 (13). This region
contains two direct repeats of the purine-rich sequences (GGAAAC) in a
reverse-orientation. Competition for binding to ERT was abolished by
mutation of this sequence, suggesting that these purine-rich sequences
are the binding sites for ERT. Expression of exogenous ERT increases
the level of transcription from the TGF- RII promoter, implying an
activating role for ERT in TGF-
RII expression. We have detected
high-affinity binding sites for ERT in the regulatory regions of
various genes, and we have demonstrated that ERT can transactivate the
isolated ETS sites of these promoters.
Proteins of the ets gene family members have a conserved
DNA-binding domain (the ETS domain) and regulate transcriptional initiation from a variety of cellular and viral gene promoter and
enhancer elements, including the human interleukin-2 receptor -chain
gene promoter (25) and the human
2 intergrin CD18 promoter (26). A
combination of EMSA and methylation interference studies on the binding
of ETS proteins to the target sequences has shown that the GGAA purine
core is essential for the specific binding of ETS-related proteins. All
members of the ets family share a common recognition
sequence, whereas the flanking sequences are divergent for different
members of the ets family. Differences in ERT binding to and
ERT-mediated transactivation of a variety of genes containing EBS
examined in this study could therefore reflect the precise recognition
sequence for ERT. We identified multiple EBS-like sequences in the
region between
1816 and +36 by a computer analysis. The TGF-
RII
promoter PRE2 contains two copies of the consensus core sequence GGAA
located at +14 to +17 and +21 to +24 which are between +1 and +50. This
region is essential for TGF-
RII promoter and enhancer activities.
It is noteworthy, however, that these ETS-binding sites by themselves
are not sufficient to fully activate the promoter since mutational
analysis reveals that the first positive regulatory element (PRE1) of
the TGF-
RII promoter cooperates with the second positive regulatory
element (PRE2) to sustain basal levels of promoter activity (13). Since the sequence between
1 and
1883 is also responsive to ERT, it is
possible that these ETS-binding sites functionally interact with the
first positive regulatory element to achieve the full promoter activity
of the TGF-
RII gene.
The expression patterns of different members of the ets gene
family vary between tissues (22). Many members of this family are
expressed in hematopoietic cells, suggesting a role for these members
of the ets family in hematopoietic cell growth and
differentiation (22). All the ets family genes, with the
exception of yan (32) and ERF (33), are known to
be potent transcriptional transactivators. Recently, Chang et
al. (23) and Oettgen et al. (24) reported the
identification of a new epithelium-restricted ETS, ESX/ESE-1, based on
a search of expressed sequence tags. The sequence of the ESX gene is
identical to the ERT nucleotide sequence; however, our ERT cDNA is
524 nucleotides longer in the 3-untranslated region and 150 nucleotides longer in the 5
-untranslated region compared with the
published ESX sequence (23, 24). Our preliminary results show that
other ets family members such as ETS-1 and ETS-2 also induce
TGF-
RII promoter activity, suggesting that ets family members may be one of the major transcription factors involved in
regulation of TGF-
RII gene expression. Since most cells express the
TGF-
receptors, it is possible that expression of the TGF-
RII
gene may be regulated by distinct ets family members in
different tissues.
TGF- plays a critical role in many cellular processes, including
regulation of cell cycle and cell differentiation, and it has now been
demonstrated that aberrant expression of TGF-
receptors may play a
role in a wide variety of human pathologies. We have recently
identified a subset of human gastric cancer cell lines that are
resistant to TGF-
and that lack TGF-
RII mRNA expression despite evidence of a normal gene, suggesting that transcriptional regulation may play an important role in controlling TGF-
RII expression. We demonstrate that there is a strong correlation between
expression patterns of TGF-
RII mRNA and ERT mRNA in human
gastric cancer cell lines.2
Examination of the mechanisms underlying loss of TGF-
RII expression in different human gastric cancer cell lines suggests a complex interplay between mutational events and transcriptional regulation. Given the importance of the ets family genes in regulating
TGF-
RII gene expression, it is quite likely that regulation of this family of transcription factors, including ERT, will emerge as a key
mechanism controlling cellular responsiveness to TGF-
.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Silvio Gutkind for pCEFL-AU5 expression vector and Anita B. Roberts for helpful discussion and critical review of the manuscript.
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FOOTNOTES |
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* This work was supported in part by HAN Project of Korean Ministry of Science and Technology (MOST 8-1-10).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF017307.
§ To whom reprint requests should be addressed: Laboratory of Cell Regulation and Carcinogenesis, NCI, Bldg. 41, Rm. B1106, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-8350; Fax: 301-496-8395.
1
The abbreviations used are: TGF-,
transforming growth factor-
; PRE, positive regulatory element; ESX,
epithelium-restricted with serine box; ESE-1, epithelium-specific ETS;
ERT, ets-related transcription factor; EBS, ETS binding
site; TGF-
RI and RII, TGF-
type I and II receptors,
respectively; UTR, untranslated region; X-gal,
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside; kb,
kilobase; TCR
, T-cell receptor
; EMSA, electrophoretic mobility shift assay; ONPG, O-nitrophenyl
-D-galactopyranoside.
2 S.-G. Choi, M. Kato, J. Chang, Y.-J. Bang, and S.-J. Kim, manuscript in preparation.
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REFERENCES |
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