From the Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801
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ABSTRACT |
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Eukaryotic chromosomal origins of replication are
best defined in Saccharomyces cerevisiae. Previous analysis
of yeast origins suggests that they are relatively simple structures
comprised of three or four small DNA sequence elements contained within approximately 100-200-base pair regions (Gilbert, D. M. (1998) Curr. Opin. Genet. Dev. 8, 194-199). In contrast, the
sequence elements that may comprise origins in multicellular eukaryotes are largely unknown. The yeast HMR-E region is both a
chromosomal origin of replication and a silencer that represses
transcription of adjacent genes through a position effect. The analysis
presented here indicated that HMR-E had a novel DNA
structure that was more complex than defined for other yeast origins,
and thus revealed that there is variation in the structural complexity
of yeast origins. In contrast to "simple" yeast origins, the origin
at HMR-E consisted of at least three independent subregions
that had the capacity to initiate replication. We have termed
HMR-E a compound origin to reflect its structural
complexity. Furthermore, only one origin within the compound origin was
a silencer.
The regulation of DNA replication ensures that duplication of
eukaryotic genomes occurs once per cell cycle. Initiation of replication is a primary control point in the regulation of replication (1-3). The ORC (origin recognition
complex), Mcm2-7p (minichromosome maintenance), and Cdc6p (cell
division cycle) proteins, which were identified
in Saccharomyces cerevisiae and are required for initiation
of replication, are conserved among eukaryotes, suggesting that at
least some of the underlying mechanisms of regulating initiation are
conserved. However, other features of replication do not appear to be
conserved; in particular, the DNA structure of origins appears to
differ among eukaryotes (4).
Eukaryotic origins of replication are best characterized in S. cerevisiae. In S. cerevisiae it is possible to define
the elements that comprise origins by combining techniques that map
origins to specific chromosomal regions with site-directed mutagenesis of those regions. This type of analysis, in addition to analysis of
plasmid origins (ARS elements; autonomous
replicating sequence), has led to the model
that yeast origins are relatively simple structures comprised of an
"A" element and two to three "B" elements that direct
initiation to a single site (5-11). The A element corresponds to the
11-base pair (bp)1 ACS
(ARS consensus sequence), which
binds ORC (12, 13). Some B elements are binding sites for
transcriptional activator proteins, whereas other B elements may
correspond to regions of easily unwound DNA (11, 14-19). In yeast, ORC
is bound to origins throughout the cell cycle and is required for
the regulated assembly and disassembly of an Mcm2-7p/Cdc6p-containing
complex that limits initiation to once per cell cycle (1-3).
Some features of yeast origins appear to be conserved among eukaryotes.
In particular, in Schizosaccharomyces pombe,
Drosophila melanogaster, Sciara coprophila,
Tetrahymena thermophila, and mammalian cell lines, sites of
initiation map to defined chromosomal regions, suggesting that
sequences within these regions are required for initiation of
replication (20-31). In cases where it has been possible to dissect
these regions genetically, specific sequences have been shown to be
required for initiation. For example, at least four S. pombe
ARS elements contain distinct DNA regions that, when deleted, result in
loss of initiation (24, 32-35). Additionally, recent genetic
dissection of one mammalian origin, the human Other features of yeast origins do not appear to be conserved among
eukaryotes. For instance, origins in S. cerevisiae are approximately 100-200 bp in length, whereas those in S. pombe are 500 bp to 2.0 kb in size, and some origins in
multicellular eukaryotes are several kilobases in length (32, 35-37).
In addition, unlike the single site of initiation at yeast origins,
multiple sites of initiation have been identified at origins in
multicellular eukaryotes (30, 38-41). The human This study focused, in part, on the possibility that chromosomal
origins in yeast may vary in complexity. In particular, we analyzed
initiation of replication at the HMR silent mating-type locus of S. cerevisiae. The HMR-E silencer is a
control region that is required for heterochromatin-mediated silencing
of the mating-type genes at HMR. In addition to its role in
gene silencing, an origin of replication maps to the HMR-E
silencer. Furthermore, the ACS element of the silencer contributes to
both silencing and initiation of chromosomal replication (42). Previous
analysis also indicates that two distinct segments of HMR,
each of which contains a different part of the HMR-E
silencer, have ARS activity (43). Although only some ARS elements are
origins in their native chromosomal context, this observation raises
the possibility that HMR-E may be more complex than other
yeast origins. In this report, we investigated whether HMR-E
contained more than one origin. The work presented here revealed that
HMR-E is a compound origin that contains three regions which
can independently function as chromosomal and plasmid origins of
replication. This result suggests that some S. cerevisiae
origins are more complex than the "simple" origins described
previously. Furthermore, one of the three independent origins was a
silencer, whereas the other two were not.
Construction of a Plasmid to Measure ARS Activity and Mitotic
Stability--
Prior analysis indicated that HMR-E had a
property referred to as centromere antagonism, which can interfere with
mitotic stability assays. Centromere antagonism is experimentally
defined as the reduced mitotic stability of a plasmid containing both HMR-E and a centromere relative to one containing only
HMR-E (44, 45). Although its molecular basis is not clear,
we hypothesize that centromere antagonism may result from local action
of the silencer interfering with centromere function. Previous plasmids used in mitotic stability assays contained HMR-E silencer
sequences adjacent to centromere and marker sequences. In this study, a new test plasmid was constructed to minimize centromere antagonism, so
that mitotic stability values would accurately reflect ARS activity.
This plasmid, pDR1149, contained one polylinker for the insertion of
potential ARS elements, and a second polylinker, on the opposite side
of the plasmid, which contained URA3 and CEN6.
This placement maximized the distance between ARS elements and
CEN6. To determine whether pDR1149 was free of centromere antagonism, a 2.2-kb HMR-E fragment was inserted into the
polylinker and its mitotic stability was determined. This plasmid was
3-fold more stable than a derivative that contained HMR-E,
but not CEN6. Therefore, centromere antagonism was either
minimized or eliminated in pDR1149.
The sequence of pDR1149 has been submitted to GenBank (AF106619) and,
hence, is only briefly described here. The polylinker of pUC18 was
replaced by the Bluescript SK+ polylinker, and a second polylinker,
comprised of PmeI, PacI, AscI, and
FseI restriction sites, was inserted opposite to it using
the following oligonucleotides: 5'-GGCCGGCCGACTGGCGCGCCTCAGTTAATTAAGCATGTTTAAACGGAACGAAAACTCACGTTAAGGG-3' and
5'-GTTTAAACATGCTTAATTAACTGAGGCGCGCCAGTCGGCCGGCCACTGAGCGTCAGACCCCGTAG-3'. PacI and PmeI sites were added to URA3
by PCR amplification of pJJ244 (46) using the oligonucleotide primers
5'-GCCTTAATTAAGCTTTTCAATTCATCTTTTTTTTTTTTGTTC-3' and
5'-CGGGTTTAAACGGGTAATAACTGATATAATTAAATTGAAGCTC-3' to allow cloning of URA3 into the second polylinker. Similarly,
AscI and PacI sites were added to CEN6
by PCR amplification of pRS415 (47) using the oligonucleotide primers
5'-CTTGGCGCGCCCCGAAAAGTGCCACCTGGG-3' and
5'-ACCTTAATTAAGTTGGCGATCCCCCTAGAG-3' to allow cloning of
CEN6 adjacent to URA3. A polylinker comprised of
SfiI, EcoNI, EcoRI, and
SgrAI restriction sites was inserted between
EcoO109I and AatII sites using oligonucleotide
primers
5'-GGAGTCAGGGCCTCTAGTGGCCACTGAGGCCTAGTCCTAGATCAGGTCACGAATTCGTCACACCGGTGACTGGACGTCAGTC-3' and
5'-GGCCGGCCGACTGGCGCGCCTCAGTTAATTAAGCATGTTTTAACGGAACGAAAACTCACGTTAAGGGG-3'. The resulting plasmid, pDR1149, was used to test for ARS activity and to measure mitotic stability. Alleles of HMR-E that
lacked native spacing, described in detail below, were cloned into
pDR1149 as EcoRI-FspI fragments that contained
1356 bp of DNA flanking HMR-E. Alleles of HMR-E
that maintained native spacing were cloned into pDR1149 as
EcoRI-HindIII fragments that contained 2841 bp of
flanking DNA. Expand Polymerase (Boehringer Mannheim) was used in all
PCR reactions according to manufacturer's recommended conditions.
Construction of HMR-E Alleles--
A polylinker containing the
restriction sites NotI, KpnI, BamHI,
and XhoI was inserted at HMR in place of the
868-bp region of HMR-E using oligonucleotides
5'-CCGGATCGTCGACGCGGCCGCTTGGGTACCCGTGGATCCTCCCTCGAGCGCCG- 3' and
5'-CGGCGCTCGAGGGAGGATCCACGGGTACCCAAGCGGCCGCGTCGACGATCCGG-3'. Each
of the three subregions of HMR-E was cloned into pDR1149 by
addition of restriction sites using PCR amplification. All alleles of
HMR-E were confirmed by DNA sequence analysis. The oligonucleotides used to amplify the HMR-E alleles were:
5'GCACGCGCGGCCGCATAGGCTAGATCGTAATCCACTACG-3' and
5'-GCACGCGGTACCAAAAAATCAAACATTGTTTAATAAA-3' (L subregion), 5'-GCACGCGGATCCCTTAATCTTCCATAAAAATATTTGA-3' and
5'-GCACGCCTCGAGTATGACGATAAAATTTTTGTTTTTC-3' (R subregion). The
138-bp silencer was isolated from pDR291 (43) as a
KpnI-BamHI restriction fragment. The
oligonucleotides used to amplify sections of LYS2 DNA for
the substitution analysis were
5'-GAATGCGGCCGCGTTTCTTATTTCGAAGTTAAATCAA-3' and
5'-CGGCCATGGATGTTGGCTCATTGTCGGCATT-3' (L subregion),
5'-CGGGGTACCATCACATTTATGGTCCCAAGATTTGA-3' and
5'-GCAGGATCCGAAAGTCCTGATGTCCTTGGATAAAA-3' (138-bp silencer), and
5'-GCGGGATCCACTTTCTTAAAGAAAAGATTGGCTAG-3' and
5'-GCCCTCGAGACCATAAGTCTCTAACCTCGCGCTCA (R subregion).
HMR-E alleles were integrated into the genome as described
previously (48). Allelic replacement at HMR-E was confirmed
by DNA blot analysis.
Mitotic Stability Assays--
Mitotic stability assays were as
described previously (45) with the following modifications; cultures
were diluted into liquid casamino acid medium supplemented with uracil,
and each sample was analyzed at a minimum of six time points during
logarithmic growth. The rate of plasmid loss per generation was
calculated as L = 1 Two-dimensional Analysis of Chromosomal Replication
Intermediates--
Replication intermediates were isolated and
analyzed as described previously (42). Briefly, genomic DNA was
isolated from 1010 log-phase cells in an asynchronous
culture and digested to completion with HindIII. Replication
intermediates were enriched by benzoylated napthoylated DEAE cellulose
chromatography (Sigma) and then subjected to two-dimensional
electrophoresis (50). Intermediates were then transferred to Zeta-probe
membrane (Bio-Rad) and hybridized as described previously (42).
Media, Strains, and Genetic Manipulations--
Rich medium (YPD)
and casamino acid medium were as described previously (51). Mating
assays were performed as described previously using DRY1226 as the
tester lawn (52). Transformation was by a modified lithium-acetate
method (53). All strains were isogenic to DRY434 (MAT Previous characterization of the DNA sequence elements that
comprise the HMR-E origin and silencer focused primarily on
the identification of the minimal set of elements required for
silencing and for initiation of replication (42-45). Deletion of an
868-bp region of HMR, referred to here as HMR-E,
removes both silencer and origin function (42-45, 48). Replacement of
HMR-E with a 138-bp subregion of HMR-E,
containing a perfect match to the ACS and two transcription factor
binding sites, restores silencing and initiation of replication (42,
45). The 138-bp subregion can, therefore, restore silencer and origin
function independent of other HMR-E regions.
In addition to the perfect 11/11 bp ACS at the 138-bp silencer, several
9/11 or 10/11 bp near-matches to the ACS reside in the vicinity of the
silencer (44). Analysis of the 868-bp HMR-E region
identified 27 9/11 or 10/11 bp near-matches to the ACS, including nine
cases in which the near-matches overlapped by 5 or more base pairs
(data not shown). This observation led us to question whether regions
other than the 138-bp silencer contributed to initiation of replication
at HMR-E. For instance, some of the near-matches may be
components of additional, unidentified origins within HMR-E.
Alternatively, the near-matches may contribute to initiation at the
previously identified 138-bp silencer, much as B elements contribute to
initiation at other yeast origins. An additional possibility is that
the near-matches may play no role in initiation.
To determine whether regions of HMR-E other than the 138-bp
silencer contributed to initiation, HMR-E was divided into
three subsections: the previously defined 138-bp silencer
(HMR
INTRODUCTION
Top
Abstract
Introduction
References
-globin origin,
indicates that initiation is dependent upon particular sequences at
this locus (20). Thus, a requirement for specific sequences is a
conserved feature of at least some eukaryotic origins.
-globin locus, for
example, contains two to four sites of initiation within an 8.0-kb
region (20). Furthermore, studies of the DHFR locus
identified two potentially preferred sites of initiation, and several
additional sites of initiation spread throughout a 60-kb region (30,
39, 41). Collectively, these data suggest that, although the proteins that regulate initiation of replication are conserved, the structure and complexity of origins may vary among eukaryotes.
EXPERIMENTAL PROCEDURES
10m,
where L is the loss rate per generation (mitotic stability) and m is the change in the fraction of cells containing the
plasmid per cell division (49). Mitotic stability values reported were the average of values from three independent trials.
HMRa-
I ade2-1 his3-11, 15 leu2-3, 112 trp1-1 ura3-1),
which is an isogenic derivative of W303-1a that lacks the
HMR-I silencer.
RESULTS AND DISCUSSION
E::138), the 520-bp region to the left of
the 138-bp silencer, the L allele (HMR
E::L),
and the 207-bp region to the right of the 138-bp silencer, the R allele
(HMR
E::R). An additional construct included all of HMR-E except for the 138-bp silencer, the L+R allele
(HMR
E::L+R). To explore the possible role of
these subregions in replication, each was tested for the ability to
support autonomous replication of a plasmid (ARS activity), as measured
by high frequency transformation. Each HMR-E allele was
flanked by approximately 1.3 kb of HMR DNA to avoid any
potential influence from heterologous plasmid DNA. In this context, a
plasmid that lacked HMR-E (pDR1155) was not capable of high
frequency transformation, whereas one that contained the 138-bp
silencer (pDR1161) was (Fig.
1A). These data indicated that
the DNA flanking HMR-E did not contain an ARS element and confirmed previous observations that the 138-bp silencer is an ARS
element. To determine whether HMR-E contained an ARS element in addition to the 138-bp silencer, the L+R allele (pDR1167) was tested
for ARS activity. Similar to the 138-bp silencer, the L+R allele was
capable of high frequency transformation, which revealed that
HMR-E is comprised of at least two separable ARS elements. To map more precisely the additional ARS element(s), the L allele (pDR1163) and the R allele (pDR1165) were tested for ARS activity independent of one another. Both alleles were capable of high frequency
transformation (Fig. 1A). Thus, HMR-E contained
at least three separable ARS elements.
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Fig. 1.
Three subregions of HMR-E
were ARS elements and chromosomal origins. A,
HMR sequence diagrams represent the DNA present in each
plasmid or chromosomal allele; lines represent flanking
HMR DNA, open boxes the subregion(s) of HMR-E
present and gaps the subregions absent. Plasmids analyzed
were (top to bottom) pDR1149 (vector), pDR1159
(HMR-E), PDR1155 ( E), pDR1161 (
E::138 bp
silencer), pDR1167 (
E::L+R), pDR1163 (
E::L),
and pDR1165 (
E::R). High frequency transformation assays
(HFT) were scored as "
" for no transformants, each
"+" indicates approximately 1.0 × 104
transformants/µg of DNA, and N/A is not applicable.
Mitotic stability assays are as described under "Experimental
Procedures." B, two-dimensional gel analysis of
replication intermediates from isogenic strains DRY 1285 (
E::138 bp silencer), DRY1293 (
E::L+R),
DRY1287 (
E::L), and DRY1283 (
E::R). The
HMR-E fragments analyzed were 3995, 4557, 4373, and 4061 bp,
respectively. Arrows indicate the presence of bubble
arcs.
Origins of replication in yeast exhibit a wide range of initiation efficiency, with some origins initiating replication in greater than 85% of cell cycles and others in less than 10% of cell cycles (54). To assess the relative initiation efficiency of the three HMR-E ARS elements, the mitotic stability of plasmids containing each of the individual alleles was determined. Wild-type HMR-E (pDR1159) was the most efficient origin (Fig. 1A). The 138-bp (pDR1161) and L+R alleles (pDR1167) were comparable to one another and were slightly less efficient than wild-type. The L allele (pDR1163) was less efficient than the 138-bp silencer, and the R allele (pDR1165) was the least efficient plasmid-based origin (Fig. 1A). Taken together, these results indicated that the three HMR-E ARS elements vary in their efficiency of initiation.
Not all ARS elements are origins of replication in their native chromosomal contexts (55, 56). To establish if any of the HMR-E subregions could function as chromosomal origins, each of the above HMR-E alleles was integrated into the chromosome in place of the wild-type HMR-E locus and subjected to a two-dimensional gel electrophoresis method (50). The replication intermediates of genomic regions that contain an origin are bubble-shaped, whereas the replication intermediates of genomic regions that do not contain an origin are fork-shaped. Bubble- and fork-shaped replication intermediates each yield distinctive arc patterns when separated on two-dimensional agarose gels and subjected to DNA blot analysis. The presence of a bubble arc indicates that the chromosomal region examined contains an origin, whereas the absence of such an arc indicates that the chromosomal region does not contain an origin. As described above, yeast origins vary in the efficiency with which they initiate replication (54); consequently, regions that contain origins typically give rise to both bubble and fork arcs (50).
As reported previously, chromosome-derived restriction fragments from strains that contained either the wild-type HMR-E allele (DRY434) or the 138-bp silencer (DRY1285) gave rise to bubble arcs, confirming that both are chromosomal origins of replication (42) (Fig. 1B). Restriction fragments from strains that contained the L+R allele (DRY 1293) also gave rise to a bubble arc, which indicated the presence of at least two independent chromosomal origins within HMR-E (Fig. 1B). Furthermore, analysis of the individual L (DRY1287) and R alleles (DRY1283) indicated that each gave rise to a bubble arc. These data revealed that HMR-E is composed of at least three subregions that could function independently as chromosomal origins of replication.
In each of the HMR-E alleles described above, the spacing among the various HMR-E subregions and the flanking DNA varied. To determine whether each of the three subregions of HMR-E was an origin when native spacing was maintained relative to each other and the flanking DNA, each subregion was replaced with DNA from the LYS2 gene (Fig. 2A). This DNA lacked any near-matches to the ACS and was typical of the AT/GC content of the yeast genome. Furthermore, the LYS2 DNA was derived from the coding region of the gene and was, thus, unlikely to contain any cryptic regulatory elements.
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The HMR-E allele in which all three subregions were replaced with LYS2 (HMR(LYS2)) DNA did not have ARS activity when inserted into a plasmid (pDR1198) (Fig. 2A). Similarly, chromosome-derived restriction fragments from strains that contained this allele integrated in place of the wild-type HMR-E locus (DRY1779) did not give rise to a bubble arc (Fig. 2B). These results indicated that the DNA used to maintain spacing did not function as an ARS element or a chromosomal origin. Both the 138-bp silencer (HMR(LYS2)::138) and the L+R allele (HMR(LYS2)::L+R) with native spacing were ARS elements when inserted into a plasmid (pDR1206 and pDR1202, respectively) (Fig. 2). Restriction fragments from strains containing either of these chromosomal alleles (DRY1781 and DRY1783, respectively) gave rise to bubble arcs. Hence, in the context of wild-type spacing, HMR-E was comprised of at least two separable origins. Both the L (HMR(LYS2)::L) and R (HMR(LYS2)::R) plasmid-based alleles with native spacing also had ARS activity. In addition, chromosomal restriction fragments containing either of these two alleles gave rise to bubble arcs (DRY1785 and DRY1787, respectively) (Fig. 2). HMR-E, therefore, contained a minimum of three subregions that were independent origins of replication in two plasmid contexts as well as two chromosomal contexts.
The R allele (DRY1787) in which spacing was maintained reproducibly gave rise to a weakly detectable bubble arc (Fig. 2B and data not shown), which suggested that the R subregion contained an inefficient origin. This idea was supported by mitotic stability assays, which indicated that the R allele (pDR1204) was a relatively inefficient origin of replication when contained on a plasmid (Fig. 2A). Similarly, mitotic stability assays of the L+R (pDR1202), 138-bp silencer (pDR1206), and L (pDR1196) alleles with native spacing indicated that each was a less efficient origin than wild-type HMR-E (pDR1159).
The observation that HMR-E contained three subregions that
could initiate replication raised the possibility that HMR-E
might also contain multiple subregions that could repress
transcription. The ability of the three HMR-E subregions to
function as silencers was assessed by a mating type assay. Haploid
MAT cells in which HMRa is silent
display the
mating phenotype, whereas haploid MAT
cells in which HMRa is not silent display the
non-mating phenotype. Among the series of chromosomal HMR-E alleles that either lacked or maintained wild-type spacing, only the
alleles that contained the 138-bp silencer could repress transcription (Fig. 3 and data not shown). Thus,
although the L and R subregions were origins, they were not silencers.
The HMR-E compound origin, therefore, contained only one
silencer element.
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The results presented here revealed that HMR-E contained at least three contiguous subregions that could direct initiation of replication. The organization of this origin region contrasts with previously defined yeast origins, which have "simple" structures and span approximately 100-200 bp (7-11). We refer to HMR-E as a compound origin to reflect its relatively large size and structural complexity. Our data indicated that origins in yeast may be simple or compound, and thus, yeast origins may vary in structural complexity. In addition, this work established that only one origin within the compound origin has the unique properties required for gene silencing.
At least two models can account for the structural complexity of the
compound origin. One model is that the compound HMR-E origin
consists of a cluster of three or more autonomous, simple origins. By
this model, each autonomous origin is likely to "compete" with the
others for initiation, similar to origins experimentally placed
adjacent to one another (57, 58). At the other extreme, the compound
origin at HMR-E could consist of a single complex origin
rather than a collection of simple origins. By this model, the various
subregions of HMR-E could interact, such that each contributes to the overall efficiency of initiation of that genomic region.
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ACKNOWLEDGEMENTS |
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We thank K. Replogle and J. Ekena for help with plasmid construction, K. Replogle for help with two-dimensional gel analysis, and Bonita Brewer and Byron Kemper for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grant GM52103 (to D. R.), by Basil O'Connor Starter Scholar Research Award Grant 5-FY96-0578 (to D. R.), and by NIH Predoctoral Training Grant 5T32-GM07283 (to S. H.).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) AF106619.
To whom correspondence should be addressed: Dept. of Cell and
Structural Biology, B107 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-244-0060; Fax: 217-244-1648;
E-mail: rivier{at}uiuc.edu.
The abbreviations used are: bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction.
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REFERENCES |
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