From the Institute of Biological Chemistry, and the Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-6340
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ABSTRACT |
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Common sage (Salvia officinalis)
produces an extremely broad range of cyclic monoterpenes bearing
diverse carbon skeletons, including members of the
p-menthane (1,8-cineole), pinane (- and
-pinene),
thujane (isothujone), camphane (camphene), and bornane (camphor)
families. An homology-based polymerase chain reaction cloning strategy
was developed and used to isolate the cDNAs encoding three
multiproduct monoterpene synthases from this species that were
functionally expressed in Escherichia coli. The
heterologously expressed synthases produce (+)-bornyl diphosphate, 1,8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces
significant amounts of (+)-
-pinene, (+)-camphene, and (±)-limonene.
The 1,8-cineole synthase produces significant amounts of (+)- and
(
)-
-pinene, (+)- and (
)-
-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of
-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in
plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be
distinct enzymes.
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INTRODUCTION |
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The cyclization of the universal precursor geranyl diphosphate to
form monocyclic and bicyclic monoterpenes is catalyzed by a group of
enzymes termed monoterpene synthases (or cyclases). The biochemical
transformation of geranyl diphosphate to cyclic products has been
investigated using enzymes from a variety of plants, including both
angiosperms (1) and gymnosperms (2-4), and a mechanistic paradigm for
these transformations (Scheme 1) is well
established (1, 5). Thus, geranyl diphosphate is initially ionized and
isomerized to form either (3R)- or (3S)-linalyl diphosphate, depending on the particular enzyme. This step permits rotation about the C2-C3 single bond of the bound allylic isomer to
the cisoid conformer which, upon subsequent ionization, promotes electrophilic attack by C1 on the C6-C7 double bond, resulting in the
formation of the -terpinyl cation as a central intermediate. Further
transformations of this reactive intermediate may be effected by
additional intramolecular electrophilic additions, hydride shifts, or
other rearrangements before termination of the sequence by
deprotonation of the final cation or capture by an external nucleophile, such as a hydroxyl ion or the diphosphate group. Although
the fate of the substrate has been well characterized in numerous
monoterpene cyclization reactions, the molecular mechanisms by which
the enzymes effect these transformations is still poorly understood.
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Culinary sage (Salvia officinalis) produces a number of
monoterpenes, including (+)- and ()-
-pinene, (+)- and
(
)-
-pinene, (+)- and (
)-camphene, (+)-sabinene, (+)- and
(
)-limonene, myrcene, 1,8-cineole, and (+)-bornyl diphosphate (Scheme
1) (1). Because sage produces this broad range of acyclic, monocyclic,
and bicyclic monoterpenes, including several olefin isomers, a cyclic
ether and a diphosphate ester, this plant has provided an ideal system for the study of a variety of synthases, all of which utilize the same
substrate but produce different products by variations on a single
reaction mechanism (1, 5). These include (+)-bornyl diphosphate
synthase (the enzyme producing the precursor of (+)-camphor) (6, 7),
1,8-cineole synthase (8), (+)-sabinene synthase (the enzyme producing
the precursor of (
)-3-isothujone) (9, 10), and several pinene
synthases (11-14). As is typical of monoterpene cyclases (5, 15), many
of these enzymes from sage appear to generate multiple products from
geranyl diphosphate. Investigations with the partially purified native
enzymes have suggested that a single enzyme, termed (+)-pinene synthase
(cyclase I), is responsible for the synthesis of both (+)-
-pinene
and (+)-camphene, with lesser amounts of (+)-limonene and myrcene,
whereas a second enzyme, (
)-pinene synthase (cyclase II), has been
shown to produce (
)-
-pinene, (
)-
-pinene, and (
)-camphene,
with minor amounts of (
)-limonene, terpinolene, and myrcene (11, 12).
More recently, a third synthase from sage, termed cyclase III, has been
described which produces a mixture of (+)-
-pinene and
(+)-
-pinene, along with minor amounts of myrcene (13, 14). Evidence
that these reactions are catalyzed by individual multifunctional
enzymes is provided by co-purification and differential inhibition
studies (12), as well as by isotopically sensitive branching
experiments (13, 15, 16). Despite considerable effort, the (+)-pinene
synthase has never been chromatographically separated from the
aforementioned (+)-bornyl diphosphate synthase (17), nor has the
(
)-pinene synthase been fully resolved from 1,8-cineole synthase,
although stereochemical considerations indicate that the latter two are probably distinct enzyme species (8, 18).
In this report, we describe the homology-based cloning, and subsequent sequencing and heterologous expression, of three monoterpene synthase cDNA genes from sage, the recombinant enzymes from which produce three different major types of cyclic monoterpene products, (+)-sabinene (a bicyclic olefin), 1,8-cineole (a bicyclic ether), and (+)-bornyl diphosphate (a bicyclic diphosphate ester), respectively (Scheme 1). Comparison of the sizes, subunit architectures, and product distributions of these multiple-product enzymes clarifies the assignment of specific catalytic functions to defined monoterpene synthases, and the deduced sequences provide information on the relatedness of these enzymes within the species and to other terpenoid synthases of plant origin. Additionally, comparison of the primary structures of these mechanistically different monoterpene synthases from the same plant, the first examples of this type thus far available, allows preliminary assessment of active site interactions and provides the foundation for more detailed study of structure-function relationships.
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EXPERIMENTAL PROCEDURES |
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Plant Materials, Substrate, and Reagents-- Sage plants (S. officinalis L.) were grown from seed as described previously (19). [1-3H]Geranyl diphosphate (250 Ci/mol) was prepared by an established method (8). Terpenoid standards were from our own collection. Unless otherwise stated, all reagents were obtained from Sigma or Aldrich Chemical Co. DNA sequences were assembled and analyzed using GCG software (20).
cDNA Library Construction,
PCR1-based Probe Generation,
and Library Screening--
Approximately 15 g of emerging sage
leaves (shoot tips) from 3-week-old plants were ground to a fine powder
in liquid nitrogen and extracted into buffer composed of 200 mM Tris-HCl (pH 8.5), 300 mM LiCl, 5 mM thiourea, 1 mM aurintricarboxylic acid, 10 mM dithiothreitol, and 10 mM EDTA, and
containing 1% (w/v) polyvinylpyrrolidone (Mr
~40,000). Total RNA thus extracted was prepared by precipitation with
isopropyl alcohol, followed by CsCl density gradient centrifugation, as
described previously (21). Poly(A)+ mRNA was isolated
by chromatography on oligo(dT)-cellulose (Qiagen) and 6.3 µg of the
resulting mRNA was used to construct a ZAPII cDNA library
according to the manufacturer's instructions (Stratagene).
cDNA Expression in E. coli--
The Bluescript plasmids
expressed in E. coli strain XL1-Blue were grown in 5 ml of
LB medium (26), supplemented with 100 µg of ampicillin/ml, to an
A600 = 0.5 at 37 °C with constant shaking, then induced with 1 to 3 mM
isopropyl-1-thio--D-galactopyranoside. The cells were
allowed an additional 4 h growth at 37 °C before harvesting by
centrifugation (2000 × g, 10 min) and lysis by
sonication (Braun-Sonic 2000 with microprobe at maximum power for
15 s), on ice, in 50 mM Mopso buffer containing 10%
glycerol, 10 mM MgCl2, and 5 mM
dithiothreitol (either pH 6.5 or 7.1, as appropriate for the assays
described below). The sonicates were cleared by centrifugation
(18,000 × g, 10 min) and the resulting supernatant was
used as the enzyme source. The pGEX constructs in E. coli XL1-Blue-MFR' cells were similarly grown at 37 °C to
A600 = 1.0 to 1.5, then induced with 1 mM isopropyl-1-thio-
-D-galactopyranoside and
incubated overnight at 20 °C with constant shaking. The cells were
then harvested and lysed, and the soluble supernatant prepared as
before. Purification of the resulting fusion proteins was attempted using the glutathione-Sepharose affinity column according to the manufacturer's instructions (Pharmacia). Of the three expressed monoterpene synthases (see below), only one (SBS) bound to the matrix
but, even in this case, affinity-based purification proved to be
unreliable. Therefore, partial purification of the heterologously expressed synthases was achieved by ion-exchange chromatography on
O-diethylaminoethyl-cellulose (Whatman DE-52) using a 0-400 mM NaCl gradient. The partially purified preparations were
desalted by repeated ultrafiltration and dilution using an Amicon
Centriprep 30 concentrator (30 kDa cutoff) and the appropriate assay
buffer. The pGEX-expressed fusion proteins were also subjected to gel permeation chromatography (Pharmacia FPLC system) using a Pharmacia XY
16 × 70 column packed with Superdex S-200 and equilibrated with
the appropriate 50 mM Mopso buffer system. The column was developed at a flow rate of 0.3 ml/min and calibrated using the Sigma
MW-GF-200 molecular weight marker kit. Kav
values of the recombinant enzymes were compared with the calibration
standards to establish molecular weights (29), which were then
corrected for the engineered fusion and transit peptide to estimate the molecular weight of the corresponding native form.
Enzyme Assay-- Monoterpene synthase activities were assayed by methods previously described (6, 8, 12, 30). Briefly, an aliquot of the bacterial cell lysate, appropriate column fractions, or partially purified and desalted enzyme preparation, in 0.5 or 1.0 ml of 50 mM Mopso buffer (pH 6.1 for SBS, and 7.1 for SCS and SSS) containing 10 mM MgCl2, 5 mM dithiothreitol, and 10% (v/v) glycerol, was transferred to a 7-ml glass Teflon sealed, screw-capped tube, and the mixture was overlaid with 1 ml of pentane to trap volatile products. The reaction was initiated by the addition of 4.5 µM [1-3H]geranyl diphosphate (1.3 µCi), with incubation at 31 °C with gentle shaking for 0.5 to 3.0 h. The pentane layer and an additional pentane extract (2 × 1 ml) were passed over a short column of silica gel surmounted by anhydrous MgSO4 (in a Pasteur pipette) to afford the monoterpene olefin fraction. Subsequent extraction of the remaining aqueous phase with diethyl either (2 × 1 ml), and passage of this extract through the same column, yielded the oxygenated monoterpene fraction. The residual aqueous phase was then treated with excess potato apyrase and wheat germ acid phosphatase to hydrolyze monoterpenol diphosphate esters (6, 30). The liberated alcohols were then extracted into diethyl ether (2 × 1 ml) and the combined extract dried over anhydrous MgSO4. Radioactivity in the various fractions was determined by liquid scintillation counting of aliquots (Packard 460 CD with external standard quench correction) and the remaining material was concentrated for radio-GC and GC-MS analysis.
Kinetic analyses were carried out with the partially purified, recombinant pGEX fusion proteins by determination of initial reaction rates at a minimum of 10 substrate concentrations ranging from 0.45 to 45 µM [1-3H]geranyl diphosphate, at saturating levels of the divalent metal ion cofactor. The results were analyzed by non-linear regression of the Michaelis-Menten equation using the curve-fitting capabilities of Sigma-Plot (Jandel Corp.).Product Identification-- Radio-GC was performed on a Gow-Mac 550P gas chromatograph with thermal conductivity detector directly coupled to a Nuclear-Chicago 8731 gas proportional counter (31). An AT-1000 packed column (Alltech) was used with helium as carrier at 30 ml/min and with temperature programming from 70 to 200 °C (at 5 °C/min) for analysis of monoterpene olefins, and from 100 to 200 °C (at 5 °C/min) for analysis of oxygenated monoterpenes. Authentic standards (10-20 µg/component) were included with each injection in order to correlate the retention times determined by mass and radioactivity detectors.
GC-MS was performed on a Hewlett-Packard 6890 GC-quadrupole mass selective detector system interfaced with a Hewlett-Packard Chemstation for data analysis. An Alltech AT-1000 fused silica capillary column (30 m × 0.25 mm inner diameter) was employed. Inleting was done by cool, on-column injection at 40 °C, with oven programming from 40 °C (50 °C/min) to 50 °C (5 min hold) then to 160 °C (10 °C/min), under a constant flow of 0.7 ml of helium/min. Full spectra were recorded for major reaction products which were identified by comparison of retention times to authentic standards and by comparison of spectra to those of the NSB75K library using the G1033A NIST probability based matching algorithm. Chiral phase separations were performed on a Hewlett-Packard 5890 GC by split injection (80:1) on a 30-m cyclodex-B capillary column (J & W Scientific) using H2 as carrier at 0.6 ml/min and temperature programming from 70 to 200 °C at 10 °C/min with flame ionization detection. Compound identification was based on retention time identity with the authentic standard. ![]() |
RESULTS AND DISCUSSION |
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Similarity-based Cloning of Monoterpene Synthases from S. officinalis-- Because S. officinalis (culinary or common sage) produces such a broad structural variety of monoterpenes, this species has been utilized extensively for studies on the enzymology, stereochemistry, and mechanism of monoterpene cyclization reactions (1, 5). Structural analyses of the responsible monoterpene synthases, and more detailed study of the cyclization mechanisms, require the isolation of cDNA species encoding these target enzymes. Protein purification from sage, as the basis for cDNA isolation, has been of limited success (17) because of the number of synthases present and their similarity in physical properties (32), and thus far has not permitted a reverse genetic approach to cloning of any of the monoterpene synthases from this species.
As a possible alternative to protein-based cloning of terpene synthases, a homology-based PCR strategy was recently proposed (22) that was developed by comparison of the deduced amino acid sequences of cDNAs encoding a monoterpene synthase (23), a sesquiterpene synthase (24), and a diterpene synthase (25) of phylogenetically distant angiosperm species. Three conserved regions of sequence were identified that appeared to be useful for the design of degenerate PCR primers. Two of these primers ultimately amplified a 600-bp fragment using cDNA from a sage leaf library as template. Cloning and sequencing showed the amplified products to correspond to two distinct sequence groups, both of which showed similarity to sequences of cloned terpene synthases, but only one of which hybridized strongly to a 2-kilobase target upon Northern blot analysis of sage leaf mRNA. This more efficient probe was utilized to screen the sage leaf cDNA library, from which 77 positive phagemids were purified. Size selection of the purified and in vivo excised clones yielded a subset of 44 with insert size greater than 1.6 kilobases, and these were expressed in E. coli XL1-Blue cells and the resulting extracts were assayed for functional monoterpene synthase activity by monitoring the conversion of [1-3H]geranyl diphosphate to monoterpene olefins, oxygenated monoterpenes, and monoterpenyl diphosphate esters. Nine functionally active clones were identified by this means, four types of which showed unique product profiles. Two cDNA clones, of which the clone designated 3C6 was most active in expression, yielded an enzyme in the corresponding bacterial extracts that produced principally bornyl diphosphate from geranyl diphosphate. This recombinant enzyme, designated SBS (sage bornyl diphosphate synthase), was presumed to represent the native (+)-bornyl diphosphate synthase of sage, one of the prominent enzymes of oil gland extracts of this species (6) that produces the first dedicated intermediate in (+)-camphor biosynthesis (7, 33, 34). Four clones, of which clone 3B5 yielded the highest activity, expressed a synthase in bacterial extracts that converted geranyl diphosphate to 1,8-cineole as the major product. This acquisition was designated SCS (sage 1,8-cineole synthase) and considered to represent the native 1,8-cineole synthase, an enzyme for which the mechanism of cyclization has been studied in detail (8, 35). Two additional clones, of which clone 3F25 was seemingly the most active in expression, yielded E. coli extracts capable of transforming geranyl diphosphate to sabinene as the dominant olefin product. This acquisition was named SSS (sage sabinene synthase), with correspondence assigned to the native (+)-sabinene synthase which catalyzes the cyclization to the bicyclic olefin precursor of (
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Product Profiles of Recombinant Synthases--
Since the formation
of multiple products from geranyl diphosphate is a common, if unusual,
feature of the monoterpene synthases (1, 5), the product profiles of
the recombinant enzymes were examined in detail by radio-GC and GC-MS.
Recombinant sabinene synthase (SSS) produces exclusively monoterpene
olefins, which by radio-GC analysis (Fig.
2) were identified as sabinene (63%), -terpinene (21%), terpinolene (7.0%), limonene (6.5%), and
myrcene (2.5%). The major products of this enzyme (sabinene and
-terpinene) are formed by a cyclization mechanism involving a
1,2-hydride shift in the
-terpinyl cation intermediate (Scheme 1).
Identification of sabinene as the major component was confirmed by
retention time identity with an authentic standard on GC-MS and by
comparison of the mass spectrum to the library standard: m/z
136 (P+, 14%), 94 (P+-42, 14%), 93 (P+-43, base peak), 91 (P+-45, 40%), 80 (P+-56, 10%), 79 (P+-57, 26%), 77 (P+-59, 36%), and 69 (P+-67, 9%). Chiral
phase capillary GC analysis demonstrated the biosynthetic sabinene to
be coincident with authentic (+)-sabinene (data not shown); however,
the (
)-enantiomer was not available for analysis to confirm the
absolute configuration of this product. Previous studies have shown
that cell-free extracts from sage produce only the (+)-antipode of
sabinene from geranyl diphosphate (9, 10), supporting the assignment of
the (+)-stereoisomer in this case; the other principal olefinic
products of SSS are achiral.
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Sequence Analysis--
Alignment of the deduced amino acid
sequences (Fig. 1) of SBS clone 3C6 (2025 bp, with an open reading
frame of 1794 nucleotides encoding 598 amino acids for a protein of
69.3 kDa and calculated pI of 6.06), SCS clone 3B5 (1968 bp, with an
open reading frame of 1773 nucleotides encoding 591 amino acids for a
protein of 69.4 kDa and calculated pI of 5.79), and SSS clone 3F25
(1911 bp, with an open reading frame of 1767 nucleotides encoding 589 amino acids for a protein of 68.9 kDa and calculated pI of 5.22), and
SUS truncated clone 3F5 (2022 bp), with the published sequences for
()-limonene synthase from Mentha spicata (spearmint) (23) and Perilla frutescens (42), and three monoterpene olefin
synthases from Abies grandis (grand fir) (43), illustrates
that there are several regions of conservation between these eight
monoterpene synthases of diverse origin. Comparison of these sequences
using the GCG GAP program (20) (Table I)
revealed the monoterpene synthases from sage to resemble each other and
the limonene synthases from related members of the Lamiaceae (50-70%
identity, 70-85% similarity) more closely than the monoterpene
synthases of the gymnosperm grand fir (~32% identity), or the
linalool synthase from Clarkia breweri (44)(~25%
identity).
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Properties of Recombinant Synthases--
Calibrated gel permeation
chromatography of the pGEX fusion form of SBS revealed a single peak of
activity at an elution volume corresponding to an
Mr ~200,000, indicating that the expressed fusion preprotein (corresponding to a molecular weight of about 2 × 96,300) was a functional dimer. Treatment of the SBS protein with
thrombin to remove the glutathione S-transferase fusion tag, followed by re-chromatography, indicated a decrease in molecular weight
to approximately 135,000, consistent with the loss of the 27-kDa
transferase peptide from each subunit at a calculated molecular weigth
of 69,300 for the preprotein. Further correction of the molecular mass
to account for the transit peptide would yield a dimer of about 120 kDa
which corresponds roughly to the native dimer molecular mass of both
(+)-bornyl diphosphate synthase and (+)-pinene synthase from sage (6,
12), two enzymes which have never been satisfactorily resolved as
distinct species. Although a dimeric quaternary structure is not unique
to these two synthases, the vast majority of the monoterpene synthases
characterized to date are monomeric (1, 5). The product profile of SBS
clone 3C6 is qualitatively similar to the combination of both of these enzymes (i.e. (+)-bornyl diphosphate and the (+)-series of
-pinene and related olefins) (6, 12), although the quantitative
distributions do not exactly match, and the stereochemistry of the
olefin products is anomalous. Thus, as indicated previously, (+)-bornyl
diphosphate, (+)-
-pinene, (+)-camphene, and (+)-limonene arise via
the same overall cyclization stereochemistry, and these enantiomers are produced exclusively from geranyl diphosphate by the native (+)-bornyl diphosphate and (+)-pinene synthase activities (18, 38-41). The small
amounts of (
)-limonene and (
)-camphene formed by the recombinant enzyme are attributed to antipodal cyclizations via abnormal, extended
substrate conformations, as the phenomenon has been described previously, especially when using neryl diphosphate (the
cis-analog of geranyl diphosphate) as an alternate substrate
(40, 64). The geranyl substrate, however, was verified as >99% pure,
thereby eliminating this possibility in the present instance and
suggesting that loss of stereochemical fidelity (to the extent of 5%
of the total product mixture) may be attributed to the presence of the glutathione S-transferase fusion peptide plus transit
peptide which may alter substrate binding directly, or indirectly by
compromising subunit assembly. To address this question, as well as the
correct identity of the SBS protein, will require the detailed
assessment of truncated enzymes that more closely resemble the native
form.
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ACKNOWLEDGEMENTS |
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We thank Gerhard Munske of the Washington State University Laboratory for Bioanalysis and Biotechnology for primer synthesis and nucleotide sequencing, and Joyce Tamura-Brown for typing the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM-31354 and Project 0268 from the Agricultural Research Center, Washington State University.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 sequences reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data banks under accession numbers AF051899 (1,8-cineole synthase), AF051900 (bornyl diphosphate synthase), and AF051901 (sabinene synthase).
Plant Biochemistry Research and Training Center Postdoctoral
Fellow.
§ Present address: Mendel Biotechnology, Inc., 21375 Cabot Blvd., Hayward, CA 94543.
¶ To whom correspondence should be addressed: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340. Tel.: 509-335--1790; Fax: 509-335-7643; E-mail: croteau{at}mail.wsu.edu.
1 The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); GC, gas chromatography; Mopso, 3-(N-morpholino)-2-hydroxypropane sulfonic acid; MS, mass spectrum/spectrometry; SBS, sage bornyl diphosphate synthase; SCS, sage 1,8-cineole synthase; SSS, sage sabinene synthase; SUS, sage unknown synthase.
2 J. Gershenzon, G. W. Turner, E. Nielsen, and R. Croteau, manuscript in preparation.
3 D. C. Williams, E. J. Katahira, D. J. McGarvey, and R. Croteau, submitted for publication.
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REFERENCES |
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