From the Division of Endocrinology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048
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ABSTRACT |
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This study demonstrates that the transcriptional repressor sequence of the rat vasoactive intestinal peptide receptor (VIPR) gene constitutes a 42-base pair core element that is the binding site for a nuclear protein. We showed that this element was able to confer transcriptional repression to a heterologous promoter and that deletion or point mutations within this element resulted in loss of transcriptional repression. Southwestern blot analysis indicated that the VIPR repressor element interacts specifically with a nuclear protein of about 72 kDa. By screening a rat lung expression library coupled with rapid amplification of cDNA ends polymerase chain reactions, we isolated a cDNA clone (designated as VIPR-RP) that contains an open reading frame of 656 amino acids. VIPR-RP is 78% identical to a previously characterized protein, differentiation-specific element-binding protein, which is a member of a family of proteins including components of the DNA replication factor C complex. However, VIPR-RP cDNA encodes for a much smaller protein than differentiation-specific element-binding protein because of a frameshift. VIPR-RP mRNA is expressed in multiple tissues, including lung, liver, brain, heart, kidney, spleen, and testis. VIPR-RP protein specifically interacts with the VIPR repressor element as demonstrated by gel shift assays. Transfection of VIP-RP expression vector into Cos cells resulted in transcriptional repression of a reporter construct containing multiple copies of the VIPR repressor element. These results indicate that VIPR-RP is a novel transcriptional repressor protein that regulates VIPR expression.
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INTRODUCTION |
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Transcriptional repression plays a critical role in regulating
gene expression. Different repressors are known to function in a wide
variety of biological settings (1-4). Repressors bind DNA and inhibit
transcription by interference with 1) activator DNA binding, 2) the
activity of DNA-bound activators, and 3) the general transcription
machinery. Eukaryotic repressors that work by competitive DNA binding
have been uncovered in the regulation of genes involved in pattern
formation in fly embryos (5). Examples of quenching include
transcriptional repression of mouse proliferin gene, where AP-1 and
glucocorticoid receptor bind DNA together in a manner that prevents
transcriptional activation by either one of the proteins (6).
Similarly, glucocorticoid receptor and NF-B can cancel each other
out in the regulation of cytokine transcription (7). Most eukaryotic
repressors, however, seem to act directly on the general transcription
machinery. For example, the fly even-skipped protein, the mouse MSX-1
protein, the unligand human thyroid receptor, and the adenovirus
E1B-55K protein have all been shown to repress basal transcription
(8-12).
The rat type 1 VIP1 receptor
(VIPR) gene has recently been characterized (13, 14). Transcriptional
activation of VIPR gene is regulated by multiple factors, including Sp1
and glucocorticoid receptor (13, 15). Previous work in this lab
demonstrated that a segment of VIPR gene, located between 859 and
488 bp, represses VIPR promoter activity in lung cells (13, 15). In this study, we identified a 42-bp element in the 5'-flanking region of
the VIPR gene that is required for transcriptional repression of the
VIPR gene and confers transcriptional repression to a heterologous promoter. We have isolated a cDNA clone (designated VIPR-RP)
encoding a 72-kDa novel protein that interacts specifically with the
VIPR repressor element. Expression of VIPR-RP led to transcriptional repression. These results indicate that positive and negative factors
interact with VIPR promoter to regulate its expression.
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MATERIALS AND METHODS |
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Plasmids Constructions--
Construction of the VIPR promoter
sequential deletions and luciferase fusion plasmids was described
previously (13, 15). To generate DNA constructs containing the VIP
receptor repressor element linked to a heterologous promoter, an
oligonucleotide containing DNA sequence between 815 and
773 bp of
the VIP receptor promoter with BamHI restriction site
attached to the end was synthesized. After annealing to the
complementary strand, the double-stranded DNA was ligated, and a
ligation product containing four copies of the oligonucleotide was
isolated by electrophoresis and cloned in front of a minimal thymidine
kinase promoter linked to luciferase reporter gene. This plasmid was
designated 4FTKLUC.
Cell Cultures and Transfection-- Rat L2 and monkey kidney Cos-7 cell lines were grown in F-12K and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum. Cells were transfected with 5 µg of DNA by calcium phosphate precipitation (13). After 24 h incubation, cells were assayed for luciferase and CAT activities. Each construct was transfected using triplicate plates for each experiment, and each construct was tested in at least three independent experiments. In all experiments, cells were co-transfected with 1 µg of TKCAT (thymidine kinase promoter and CAT fusion gene) to monitor transfection efficiency. A negative control plasmid containing a promoterless luciferase gene (pA3) and a positive control plasmid containing Rous sarcoma virus 3' long terminal repeat promoter fused to luciferase were included in all experiments. Luciferase assays and CAT assays were performed as described previously (15).
Gel Mobility Shift Assays--
Nuclear extract was prepared from
cultured cell as described by Dignam (16). To generate the probe, an
oligonucleotide containing DNA sequence between 815 and
773 bp of
the VIP receptor promoter was synthesized. After annealing to the
complementary strand, the double-stranded DNA was end labeled using
[
-32P]ATP and T4 polynucleotide kinase. 10,000 cpm of
the probe was used in each reaction. Binding reactions were performed
in a total volume of 20 µl containing 20 mM Tris, pH 7.5, 1 mM EDTA, 2 mM MgCl2, 50 mM NaCl, 20 mM dithiothreitol, 10% glycerol,
and 1 µg of poly(dI-dC), with 15-µg nuclear extracts. Competitor
DNA was mixed with the probe prior to receptor addition. Binding was
for 20 min at room temperature. The anti-RXR antibody (1 µg) (Santa Cruz Biotechnology) was added, and the binding was continued on ice for
an additional 30 min. The reactions were electrophoresed on 4%
nondenaturing polyacrylamide gel, dried, and exposed to x-ray film for
3 h.
Southwestern Blot Analysis-- Cell nuclear extracts (50 µg) were separated on a 10% polyacrylamide-SDS gel next to rainbow protein standards (Amersham Pharmacia Biotech). The protein was transferred onto nitrocellulose membrane, treated with decreasing concentrations of guanidine HCl (6, 3, 1.5, 0.75, 0.375, and 0.1875 M) and pre-bound in 10 mM NaPO4, pH 7.4, 5% nonfat milk, 150 mM NaCl, 1% bovine serum albumin, 2.5% PVP-40, 0.1% Triton X-100, at 4 °C for 1 h. Binding was performed at 4 °C overnight in a buffer containing 10 mM Tris, pH 7.5, 0.5% nonfat milk, 0.5% bovine serum albumin, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 2 mM dithiothreitol, 0.1% Triton X-100, 5% glycerol, and 10 µg/ml salmon sperm DNA. 106 cpm/ml of the above probe used for gel shift analysis was used. The membrane was washed twice for 30 min in the binding buffer without salmon sperm DNA at 4 °C, and exposed to x-ray films for 2 h.
Expression Screening of gt11 Library--
A rat lung
5'-stretch plus cDNA
gt11 library was purchased from
CLONTECH. The library was screened using a end
labeled double-stranded oligonucleotide containing DNA sequence between
773 and
815 bp of the VIP receptor promoter. An oligonucleotide
containing point mutations within this region (see above) was used as a
negative control. Approximately 106 plaque-forming units
were screened using the protocol described by Singh et al.
(17). Clones encoding sequence-specific DNA-binding proteins were
identified and plague-purified. Crude cell extracts from recombinant
phage lysogens were prepared using the method by Singh et
al. (17) and were used in gel mobility shift assays to test the
specific interaction between the fusion proteins and the DNA-binding
sequence. The cDNA encoding the sequence-specific DNA binding was
isolated by digesting phage DNA with EcoRI and was cloned
into pGEM3Z plasmid vector for restriction mapping and DNA sequencing.
DNA sequencing was performed on both strands using internal
primers.
Rapid Amplification of cDNA Ends (RACE)-- The full-length cDNA was obtained by 5' and 3' RACE reactions using a Marathon cDNA amplification kit (CLONTECH) according to manufacturer's instructions. Rat lung poly(A)+ RNA (CLONTECH) was used for cDNA synthesis. The 5' RACE gene-specific primer was: 5'-AGCTGCTTGGCGATGGCCTCATCA-3', and the 3' RACE gene-specific primer was: 5'-CAAAAAATCCAAGTACGAAATCGCTGC-3'. The PCR reactions include: 1) denaturing at 94 °C for 1 min; 2) 5 cycles of 94 °C for 30 s and 72 °C for 4 min; 3) 5 cycles or 94 °C for 30 s and 70 °C for 4 min; and 4) 25 cycles of 94 °C for 20 s and 68 °C for 4 min in a total volume of 50 µl. After the cycling was completed, 10 µl of the PCR products was analyzed on agarose gel. The PCR products were clone into TA vector (Invitrogen) for sequencing. The entire coding region of the cDNA was amplified by PCR using the following primers: 5'-GGCTGCGATGGACATTCGGAAATTCTTTGGG-3' and 5'-TATCGCTCGCATTTAGTTCCACATAACTGT-3'.
The PCR reaction was performed in a total volume of 50 µl using the following program: 94 °C for 1 min, followed by 25 cycles of 94 °C for 30 s and 68 °C for 4 min. The PCR product was cloned into eukaryotic TA vector (Invitrogen) for further analysis, and cotransfection experiments were conducted.Northern Blot Analysis--
A rat multiple tissue Northern blot
was purchased from CLONTECH. Approximately 2 µg
of poly(A)+ RNA/lane from eight different rat tissues was
run on a denaturing formaldehyde 1.2% agarose gel, transferred to
nylon membrane, and UV-cross-linked. The membrane was first hybridized
to the full-length cDNA probe and then was stripped and
rehybridized to a human -actin cDNA control probe. Hybridization
was performed at 60 °C for 1 h in ExpressHyb hybridization
solution (CLONTECH). Washing was twice for 15 min
at room temperature in 2× SSC, 0.05% SDS and twice for 15 min at
65 °C in 0.1% SSC, 0.1% SDS.
In Vitro Transcription and Translation-- VIPR-RP was transcribed from T7 promoter and translated in reticulocyte lysate using transcription- and translation-coupled reticulocyte lysate system (Promega). A typical reaction contains 25 µl of rabbit reticulocyte lysate, 2 µl of reaction buffer, 20 mM amino acid mixture, 1 µg of DNA template, 40 units of ribonuclease inhibitor, and 10 units of T7 RNA polymerase in a total volume of 50 µl. The reactions were carried out at 30 °C for 1 h. 5 µl of the reaction was used in gel shift assays.
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RESULTS |
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A Lung Cell Nuclear Protein Interacts with a 42-bp Sequence in the
VIPR 5'-Flanking Region--
Previous transfection studies in rat lung
cells indicated that a potential transcriptional repressor sequence is
present between 488 and
859 bp in the VIPR 5'-flanking region. To
characterize this repressor sequence further, we first sought to
determine whether nuclear proteins in lung cells interact with a
specific sequence in this region. As shown in Fig.
1, when lung cell nuclear extracts were
incubated with an oligonucleotide containing 42-bp VIPR 5' sequence
between
773 and
815, a mobility shifted band was observed (Fig. 1,
lane 2). This band was not competed by an oligonucleotide
with unrelated sequences (Fig. 1, lane 3) but was competed
by the unlabeled oligonucleotide of the same sequence (Fig. 1,
lane 4). Because a sequence similar to the half-site for RXR
(GGTGA) is present within this 42-bp sequence, an anti-RXR antibody was
included in the reaction to test whether the mobility shifted band was
a result of RXR interaction with this sequence. Fig. 1, lane
5 shows that addition of anti-RXR antibody has no effect on the
mobility shifted band. These results indicate that a nuclear protein in
lung cells specifically interacts with VIPR 5' sequence between
773
and
815 bp and that this protein is not RXR.
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The GGTGA Motif Is Required for the Nuclear Protein Binding to the 42-bp Sequence-- The above results showed that RXR does not bind to the 42-bp sequence despite the presence of a half-site. To test whether the GGTGA motif is important for DNA-protein interaction, point mutations were made to change this sequence to tactg. As shown in Fig. 2, an oligonucleotide containing these mutations could not compete for binding of the lung nuclear protein with the wild-type sequence (Fig. 2, lanes 4 and 5). When the mutant oligonucleotide was used as the probe, no mobility shifted band was present (Fig. 2, lanes 7-10). These results indicate that the GGTGA motif is required for interaction of the lung cell nuclear protein with the 42-bp sequence.
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Deletion of the 42-bp Sequence from VIPR Promoter Results in Loss
of Transcriptional Repression--
To test whether the 42-bp nuclear
protein-binding site mediates transcriptional repression of the VIP
receptor gene, this sequence was deleted from the VIPR-luciferase
fusion construct. As shown in Fig.
3A, luciferase activity of the
fusion construct containing 859 bp of the VIPR 5' sequence (859LUC)
is about 8-fold lower than that of
489LUC, whereas a construct
containing deletion between
773 and
815 bp (
859
LUC) does not
show significant reduction in luciferase activity compared with the
luciferase activity of
489LUC. These results indicate that the 42-bp
sequence is required for transcriptional repression of the VIPR
gene.
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The 42-bp Sequence Confers Transcriptional Repression to a Heterologous Promoter-- To test whether the 42-bp VIPR 5'-flanking sequence is able to repress transcription of a heterologous promoter, oligonucleotide containing either the wild-type or point mutation of this sequence was multimerized and cloned in front of a minimal thymidine kinase promoter linked to luciferase (TKLUC). Fig. 3B shows that the luciferase activity of the construct containing four copies of the wild-type sequence (4FTKLUC) is about 4-fold lower than that of TKLUC, whereas there is no significant difference in luciferase activity between the construct containing four copies of the mutant sequence (m4FTKLUC) and TKLUC. These results indicate that the 42-bp sequence is capable of conferring transcriptional repression to a heterologous promoter.
The VIPR Repressor Element Interacts with a 72-kDa Nuclear Protein-- To characterize further the nuclear protein that interacts with the VIPR repressor element, Southwestern blot analysis was performed. Fig. 4 shows that the VIPR repressor element detected a protein of about 72 kDa from lung cell nuclear extracts (Fig. 4, lane 1). To determine whether this protein was specific to lung cells, nuclear extracts from a neuroblastoma and pituitary tumor cell lines were also included. As shown in Fig. 4 (lanes 2 and 3), the 72-kDa protein is also present in nuclear extracts from both cell lines. When an oligonucleotide containing point mutations within the repressor element was used to probe the same blot, no specific protein band was detected. These results indicate that the VIPR repressor elements interact specifically with a 72-kDa nuclear protein that is not only expressed in lung cells but also in other cell types.
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Molecular Cloning of the cDNA That Encodes for the 72-kDa
Protein--
To isolate the cDNA clones, a gt11 cDNA
expression library made from rat lung (CLONTECH)
was screened using the end labeled 42-p VIP repressor element as the
probe. About 106 plaques were screened according to Singh
et al. (17) and Vinson et al. (18). One positive
clone was obtained, and lysogens prepared from this phage clone were
used in gel shift assays to test its specific binding to the VIPR
repressor element. As shown in Fig. 5,
when the extracts of the phage lysogen were incubated with the 42-bp
VIPR repressor oligonucleotide, a mobility shifted band was observed
(Fig. 5, lane 2). This band was competed out by unlabeled wild-type VIPR repressor oligonucleotide (Fig. 5, lanes 3 and 4) but not by the mutant oligonucleotide (Fig. 5,
lane 5). These results indicate that the cDNA in this
phage clone encodes a protein that exhibits sequence-specific DNA
binding to the VIPR repressor element.
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VIPR-RP Exhibits Sequence-specific DNA Binding to the VIPR Repressor Element-- Examination of the VIPR-RP sequence did not reveal any obvious homology with any DNA-binding motifs such as zinc fingers and homeodomains. To test whether VIPR-RP specifically interacts with the VIPR repressor element, VIPR-RP was transcribed and translated in vitro. As shown in Fig. 8, when VIPR protein was incubated with the 42-bp VIPR repressor oligonucleotide, two mobility shifted bands were observed (Fig. 8, lane 2). The presence of the faster migrating band was likely to be the result of binding a partially transcribed and translated VIPR-RP protein to DNA. These bands were competed out by increasing amounts of the unlabeled wild-type VIPR repressor oligonucleotide (Fig. 8, lanes 3-6) but not by the mutant oligonucleotide (Fig. 8, lanes 7-10). Unlabeled oligonucleotide containing consensus binding sites for transcription factor Sp1 or AP2 also did not compete for VIPR-RP binding (Fig. 8, lanes 11 and 12). These results indicate that VIPR-RP exhibits sequence-specific DNA binding to the VIPR repressor element.
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Transcriptional Repression by VIPR-RP-- To assess the functional effect of VIPR-RP, VIPR-RP cDNA was inserted downstream of the cytomegalovirus promoter and cotransfected with reporter plasmids into Cos-7 cells. As shown in Fig. 9, co-transfection with the VIPR-RP expression vector resulted in 6-fold reduction in luciferase activity of the reporter plasmid containing four copies of the wild-type VIPR repressor element (Fig. 9, 4FTKLUC), whereas expression of VIPR-RP did not significantly affect luciferase activity of reporter plasmid containing four copies of the mutant VIPR repressor element (Fig. 9, m4FTKLUC) or the plasmid without the repressor element (Fig. 9, TKLUC). These results demonstrate that VIPR-RP acts as a sequence-specific transcriptional repressor in vivo.
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DISCUSSION |
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In previous work we demonstrated the presence of a negatively
acting element that regulates VIPR gene expression in lung cells (13,
15). In this report, we have restricted the biological activity of the
negatively acting element to a 42-bp DNA sequence located between 773
and
815 bp that contributes importantly to transcriptional repression
of the VIPR gene. We showed that deletion of this sequence results in
loss of transcriptional repression and that this sequence can repress
transcription of a heterologous promoter.
The VIPR repressor element interacts with a nuclear protein in lung cells as demonstrated by gel shift assays. Although a GGTGA motif similar to RXR-binding half-site is present within the repressor element, RXR does not appear to be the protein that binds to this element, for addition of anti-RXR antibody in gel shift assays did not change the pattern of the mobility shifted band. This motif, however, is essential for the nuclear protein-binding and -mediating transcriptional repression. Base substitutions within this motif resulted in loss of protein binding and transcriptional repression.
At least three categories of transcriptional regulation by negatively acting elements have been described (22): 1) repression via competition between repressors and activators for the same or overlapping binding sites, 2) neutralization of activators by interactions with a repressor protein, and 3) silencing through direct interaction of a repressor with the target site, regardless of distance from the promoter. The nuclear protein that interacts with the VIPR repressor element was characterized further by Southwestern blot analysis. The results showed that the repressor element detected a single protein of about 72 kDa in nuclear extracts of not only lung cells but also neuroblastoma and pituitary tumor cell lines. These results indicate that the VIPR repressor element interacts with a ubiquitously expressed nuclear protein and that transcriptional repression is likely mediated by direct interaction of the repressor protein with this element.
Based on the specific interaction between VIPR repressor element and
the 72-kDa nuclear protein, we isolated a cDNA clone that encodes
for this protein (VIPR-RP). The deduced amino acid sequence of VIPR-RP
shares high identity (78%) to DSEB (19) and the large subunit of
murine activator 1 complex, A1p145 (23), which is also known as
replication factor C (RFC) (24). DSEB and A1p145/RFC are nearly
identical in amino acid sequence, except for the differences in three
amino acids. DSEB binds to an enhancer element in angiotensinogen gene
promoter that mediates the irreversible induction of transcriptional
activation during differentiation of 3T3-L1 adipoblasts to adipocytes
(19). A1p145/RFC is part of a heteropentameric protein complex
essential for DNA replication (25). Interestingly, the N-terminal 375 amino acids of VIPR-RP are almost identical to the human
-MHC-binding factor, which has unknown function. Our result also
showed that the mRNA tissue distribution of VIPR-RP and DSEB is
different. Although VIPR-RP mRNA is ubiquitously expressed, DSEB
mRNA shows more selected expression pattern. DSEB mRNA was not
detected in heart and skeletal muscle (19). These data indicate that
although VIPR-RP is closely related to DSEB/RFC, it is a distinct
gene.
Like DSEB and A1p145, VIPR-RP does not contain the well characterized DNA-binding motifs such as zinc fingers or homeodomain. Sequence-specific binding of DSEB and A1p145 was demonstrated by gel shift experiments in which recombinant DSEB and A1p145 preferentially bind sequences within the promoter of angiotensinogen (19) or collagen IV (23), respectively. Our results showed that in vitro translated VIPR-RP protein binds to the VIPR repressor element specifically and that a GGTGA motif within the repressor element is essential for VIPR-RP binding. A similar motif GGTAA is present within the binding site for DSEB (19), but the functional importance of this motif has not been tested.
VIPR-RP differs from DSEB and A1p145 in that it encodes a much smaller protein. A single base deletion at amino acid 581 in VIPR-RP results in a frameshift and a termination codon at amino acid 657. As a consequence, the C-terminal 75 amino acids of VIPR-RP show very little similarity to DSEB and A1p145. The precise functions of DSEB and A1p145 in DNA replication and transcriptional activation remain to be elucidated. DSEB was unable to transactivate reporter genes containing its binding sites when co-transfected into Cos-1 and NIH3T3 cells (19). Our results showed that VIPR-RP repressed transcription of a reporter plasmid containing four copies of its binding sequence when co-transfected into Cos-7 cells, suggesting that VIPR-RP not only binds to DNA but also mediates transcriptional repression. It is possible the C-terminal portion of VIPR-RP may possess functions that are distinct from DSEB and A1p145.
In summary, we have identified a negatively acting element in the VIPR promoter that is the binding site for a novel transcriptional repressor, VIPR-RP. Interaction of VIPR-RP with its target site results in transcriptional repression of the VIP receptor gene.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK-02346 and Research Grant RG-018-N from the American Lung Association.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) AF059678.
To whom correspondence should be addressed: Div. of Endocrinology,
Cedars-Sinai Medical Center, 8700 Beverly Blvd., D-3066, Los Angeles,
CA 90048. Tel.: 310-855-7682; Fax: 310-559-2357.
1 The abbreviations used are: VIP, vasoactive intestinal peptide; VIPR, VIP receptor; bp, base pair(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; RXR, retinoid X receptor; RACE, rapid amplification of cDNA ends; DSEB, differentiation-specific element-binding protein; MHC, myosin heavy chain; RFC, replication factor C.
2 E. Morkin, unpublished observation.
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REFERENCES |
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