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• W4234578412 abstract "Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (−)-limonene and (−)-α- and (−)-β-pinene are prominent components; (−)-limonene and (−)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression inEscherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (−)-limonene synthase, a myrcene synthase, and a (−)-pinene synthase that produces both α-pinene and β-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products. Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (−)-limonene and (−)-α- and (−)-β-pinene are prominent components; (−)-limonene and (−)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression inEscherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (−)-limonene synthase, a myrcene synthase, and a (−)-pinene synthase that produces both α-pinene and β-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products. Chemical defense of conifer trees against bark beetles and their associated fungal pathogens relies primarily upon constitutive and inducible oleoresin biosynthesis (1Johnson M.A. Croteau R. ACS Symp. Ser. 1987; 325: 76-91Crossref Google Scholar, 2Gijzen M. Lewinsohn E. Savage T.J. Croteau R.B. ACS Symp. Ser. 1993; 525: 8-22Crossref Google Scholar). This defensive secretion is a complex mixture of monoterpene and sesquiterpene olefins (turpentine) and diterpene resin acids (rosin) that is synthesized constitutively in the epithelial cells of specialized structures, such as resin ducts and blisters or, in the case of induced oleoresin formation, in undifferentiated cells surrounding wound sites (3Lewinsohn E. Gijzen M. Savage T.J. Croteau R. Plant Physiol. ( Bethesda ). 1991; 96: 38-43Crossref PubMed Scopus (111) Google Scholar). The volatile fraction of conifer oleoresin, which is toxic to both bark beetles and their fungal associates (4Raffa K.F. Berryman A.A. Simasko J. Teal W. Wong B.L. Environ. Entomol. 1985; 14: 552-556Crossref Scopus (104) Google Scholar), may consist of up to 30 different monoterpenes (5Lewinsohn E. Savage T.J. Gijzen M. Croteau R. Phytochem. Anal. 1993; 4: 220-225Crossref Scopus (83) Google Scholar), including acyclic types (e.g.myrcene), monocyclic types (e.g. limonene), and bicyclic types (e.g. pinenes) (Fig. 1). Although the oleoresin is toxic, many bark beetle species nevertheless employ turpentine volatiles in host selection and can convert various monoterpene components into aggregation or sex pheromones to promote coordinated mass attack of the host (2Gijzen M. Lewinsohn E. Savage T.J. Croteau R.B. ACS Symp. Ser. 1993; 525: 8-22Crossref Google Scholar, 6Byers J.A. Cardé R.T. Bell W.J. Chemical Ecology of Insects 2. Chapman & Hall, New York1995: 154-213Crossref Google Scholar). In grand fir (Abies grandis), increased formation of oleoresin monoterpenes, sesquiterpenes, and diterpenes is induced by bark beetle attack (3Lewinsohn E. Gijzen M. Savage T.J. Croteau R. Plant Physiol. ( Bethesda ). 1991; 96: 38-43Crossref PubMed Scopus (111) Google Scholar, 7Raffa K.F. Berryman A.A. Can. Entomol. 1982; 114: 797-810Crossref Scopus (97) Google Scholar,8Lewinsohn E. Gijzen M. Croteau R. Plant Physiol. ( Bethesda ). 1991; 96: 44-49Crossref PubMed Scopus (184) Google Scholar), and this inducible defense response is mimicked by mechanically wounding sapling stems (3Lewinsohn E. Gijzen M. Savage T.J. Croteau R. Plant Physiol. ( Bethesda ). 1991; 96: 38-43Crossref PubMed Scopus (111) Google Scholar, 8Lewinsohn E. Gijzen M. Croteau R. Plant Physiol. ( Bethesda ). 1991; 96: 44-49Crossref PubMed Scopus (184) Google Scholar, 9Funk C. Lewinsohn E. Stofer-Vogel B. Steele C. Croteau R. Plant Physiol. ( Bethesda ). 1994; 106: 999-1005Crossref PubMed Scopus (45) Google Scholar). Therefore, grand fir has been developed as a model system to study the biochemical and molecular genetic regulation of constitutive and inducible terpene biosynthesis in conifers (10Steele C. Lewinsohn E. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4164-4168Crossref PubMed Scopus (63) Google Scholar). Most monoterpenes are derived from geranyl diphosphate, the ubiquitous C10 intermediate of the isoprenoid pathway, by synthases that catalyze the divalent metal ion-dependent ionization (to 1) and isomerization of this substrate to enzyme-bound linalyl diphosphate which, following rotation about C2–C3, undergoes a second ionization (to 2) followed by cyclization to the α-terpinyl cation, the first cyclic intermediate en route to both monocyclic and bicyclic products (11Croteau R. Cane D.E. Methods Enzymol. 1985; 110: 383-405Crossref Google Scholar, 12Croteau R. Chem. Rev. 1987; 87: 929-954Crossref Scopus (478) Google Scholar) (Fig. 1). Acyclic monoterpenes, such as myrcene, may arise by deprotonation of carbocations 1 or 2, whereas the isomerization step to linalyl diphosphate is required in the case of cyclic types, such as limonene and pinenes, which cannot be derived from geranyl diphosphate directly because of the geometric impediment of thetrans-double bond at C2–C3 (11Croteau R. Cane D.E. Methods Enzymol. 1985; 110: 383-405Crossref Google Scholar, 12Croteau R. Chem. Rev. 1987; 87: 929-954Crossref Scopus (478) Google Scholar). Many monoterpene synthases catalyze the formation of multiple products, including acyclic, monocyclic, and bicyclic types, by variations on this basic mechanism (13Gambliel H. Croteau R. J. Biol. Chem. 1984; 259: 740-748Abstract Full Text PDF PubMed Google Scholar, 14Croteau R. Satterwhite D.M. Cane D.E. Chang C.C. J. Biol. Chem. 1988; 263: 10063-10071Abstract Full Text PDF PubMed Google Scholar, 15Croteau R. Satterwhite D.M. J. Biol. Chem. 1989; 264: 15309-15315Abstract Full Text PDF PubMed Google Scholar). For example, (−)-limonene synthase, the principal monoterpene synthase of spearmint (Mentha spicata) and peppermint (Mentha × piperita), produces small amounts of myrcene, (−)-α-pinene and (−)-β-pinene in addition to the monocyclic product (16Rajaonarivony J.I.M. Gershenzon J. Croteau R. Arch. Biochem. Biophys. 1992; 296: 49-57Crossref PubMed Scopus (108) Google Scholar, 17Colby S.M. Alonso W.R. Katahira E.J. McGarvey D.J. Croteau R. J. Biol. Chem. 1993; 268: 23016-23024Abstract Full Text PDF PubMed Google Scholar). Conversely, six different inducible monoterpene synthase activities have been demonstrated in extracts of wounded grand fir stem (18Gijzen M. Lewinsohn E. Croteau R. Arch. Biochem. Biophys. 1991; 289: 267-273Crossref PubMed Scopus (45) Google Scholar) indicating that formation of acyclic, monocyclic, and bicyclic monoterpenes in this species involves several genes encoding distinct catalysts. The inducible (−)-pinene synthase has been purified (19Lewinsohn E. Gijzen M. Croteau R. Arch. Biochem. Biophys. 1992; 293: 167-173Crossref PubMed Scopus (59) Google Scholar) and isotopically sensitive branching experiments employed to demonstrate that this enzyme synthesizes both (−)-α- and (−)-β-pinene (20Wagschal K. Savage T.J. Croteau R. Tetrahedron. 1991; 47: 5933-5944Crossref Scopus (56) Google Scholar). Deciphering the molecular genetic control of oleoresinosis and examining structure-function relationships among the monoterpene synthases of grand fir require isolation of the cDNA species encoding these key enzymes. Although a protein-based cloning strategy was recently employed to acquire a cDNA for the major wound-inducible diterpene synthase from grand fir, abietadiene synthase (9Funk C. Lewinsohn E. Stofer-Vogel B. Steele C. Croteau R. Plant Physiol. ( Bethesda ). 1994; 106: 999-1005Crossref PubMed Scopus (45) Google Scholar, 21LaFever R.E. Stofer-Vogel B. Croteau R. Arch. Biochem. Biophys. 1994; 313: 139-149Crossref PubMed Scopus (70) Google Scholar, 22Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), all attempts at the reverse genetic approach to cloning of grand fir monoterpene synthases have failed (10Steele C. Lewinsohn E. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4164-4168Crossref PubMed Scopus (63) Google Scholar). As an alternative, a similarity based PCR 1The abbreviations used are: PCR, polymerase chain reaction; GLC, gas liquid chromatography; MS, mass spectrum/spectrometry; bp, base pair(s); nt, nucleotide(s); ORF, open reading frame; I, inosine.strategy was developed (10Steele C. Lewinsohn E. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4164-4168Crossref PubMed Scopus (63) Google Scholar) that employed sequence information from terpene synthases of angiosperm origin, namely a monoterpene synthase, (−)-4S-limonene synthase, from spearmint (M. spicata, Lamiaceae) (17Colby S.M. Alonso W.R. Katahira E.J. McGarvey D.J. Croteau R. J. Biol. Chem. 1993; 268: 23016-23024Abstract Full Text PDF PubMed Google Scholar), a sesquiterpene synthase, 5-epi-aristolochene synthase, from tobacco (Nicotiana tabacum, Solanaceae) (23Facchini P.J. Chappell J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11088-11092Crossref PubMed Scopus (216) Google Scholar), and a diterpene synthase, casbene synthase, from castor bean (Ricinus communis, Euphorbiaceae) (24Mau C.J.D. West C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8497-8501Crossref PubMed Scopus (117) Google Scholar). In this paper, we describe the successful application of this strategy to the amplification of specific hybridization probes and their use in the isolation of six new “terpene synthase-like” cDNAs. Three of the full-length clones were functionally expressed inEscherichia coli and thereby identified as myrcene synthase, (−)-limonene synthase, and a pinene synthase that produces both (−)-α- and (−)-β-pinene (Fig. 1). This is the first report of the isolation of any cDNA encoding a monoterpene synthase from a gymnosperm, and the first report to describe the cloning of several different monoterpene synthases (for acyclic, monocyclic, and bicyclic products) from a single plant species. Sequence comparison revealed significantly greater conservation between the grand fir monoterpene synthases and other gymnosperm terpene synthases than with angiosperm terpene synthases, and targeted a number of highly conserved amino acid residues for further study. Additionally, Northern hybridization analysis demonstrated that induced oleoresinosis in grand fir is regulated at the level of monoterpene synthase RNA accumulation. [1-3H]Geranyl diphosphate (250 Ci/mol) (25Croteau R. Alonso W.R. Koepp A.E. Johnson M.A. Arch. Biochem. Biophys. 1994; 309: 184-192Crossref PubMed Scopus (89) Google Scholar), [1-3H]farnesyl diphosphate (125 Ci/mol) (26Dehal S.S. Croteau R. Arch. Biochem. Biophys. 1988; 261: 346-356Crossref PubMed Scopus (56) Google Scholar), and [1-3H]geranylgeranyl diphosphate (120 Ci/mol) (21LaFever R.E. Stofer-Vogel B. Croteau R. Arch. Biochem. Biophys. 1994; 313: 139-149Crossref PubMed Scopus (70) Google Scholar) were prepared as described previously. Terpenoid standards were from our own collection. All other biochemicals and reagents were purchased from Sigma or Aldrich, unless otherwise noted. Construction of the λZAP II cDNA library, using mRNA isolated from wounded grand fir sapling stems, was described previously (22Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Based on comparison of sequences of limonene synthase from spearmint (17Colby S.M. Alonso W.R. Katahira E.J. McGarvey D.J. Croteau R. J. Biol. Chem. 1993; 268: 23016-23024Abstract Full Text PDF PubMed Google Scholar), 5-epi-aristolochene synthase from tobacco (23Facchini P.J. Chappell J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11088-11092Crossref PubMed Scopus (216) Google Scholar), and casbene synthase from castor bean (24Mau C.J.D. West C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8497-8501Crossref PubMed Scopus (117) Google Scholar), four conserved regions were identified for which a set of consensus degenerate primers (primers A–D) were synthesized. Primers A–C have been described previously (10Steele C. Lewinsohn E. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4164-4168Crossref PubMed Scopus (63) Google Scholar); primer D (see Fig. 2) was designed based on the conserved amino acid sequence motif DD(T/I)(I/Y/F)D(A/V)Y(A/G) of the above noted terpene synthases (17Colby S.M. Alonso W.R. Katahira E.J. McGarvey D.J. Croteau R. J. Biol. Chem. 1993; 268: 23016-23024Abstract Full Text PDF PubMed Google Scholar, 23Facchini P.J. Chappell J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11088-11092Crossref PubMed Scopus (216) Google Scholar, 24Mau C.J.D. West C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8497-8501Crossref PubMed Scopus (117) Google Scholar). The sequence of sense primer D was 5′-GA(C/T) GA(C/T) III T(T/A)(T/C) GA(C/T) GCI (C/T)A(C/T) GG-3′. Each of the sense primers, A, B, and D, was used for PCR in combination with antisense primer C by employing a broad range of amplification conditions. PCR was performed in a total volume of 50 μl containing 20 mm Tris/HCl (pH 8.4), 50 mmKCl, 5 mm MgCl2, 200 μm each dNTP, 1–5 μm each primer, 2.5 units of Taqpolymerase (Life Technologies, Inc.), and 5 μl of purified grand fir stem cDNA library phage as template (1.5 × 109plaque-forming units/ml). Analysis of the PCR reaction products by agarose gel electrophoresis (27Sambrock J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) revealed that only the combination of primers C and D generated a specific PCR product of approximately 110 bp. This PCR product was gel-purified, ligated into pT7Blue (Novagen), and transformed into E. coli XL1-Blue cells. Plasmid DNA was prepared from 41 individual transformants, and the inserts were sequenced (DyeDeoxy Terminator Cycle Sequencing, Applied Biosystems). Four different insert sequences were identified and were designated as probes 1, 2, 4, and 5. Subsequent isolation of four new cDNA species, encoding terpene synthases from grand fir corresponding to these probes, allowed the identification of three additional conserved sequence elements which were used to design a set of three new PCR primers. Degenerate primer E (designed to conserved element GE(K/T)(V/I)M(E/D)EA (see Fig. 2)) and degenerate primer F (designed to conserved element Q(F/Y/D)(I/L)(T/L/R)RWW) were based on comparison of the sequences of five cloned terpene synthases from grand fir as follows: a monoterpene synthase corresponding to probe 2, two sesquiterpene synthases 2C. L. Steele, J. Bohlmann, J. E. Crock, and R. Croteau, submitted for publication. corresponding to probe 4 and probe 5, respectively, a previously described diterpene synthase (22Vogel B.S. Wildung M.R. Vogel G. Croteau R. J. Biol. Chem. 1996; 271: 23262-23268Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), and a truncated terpene synthase 3J. Bohlmann, J. E. Crock, R. Jetter, and R. Croteau, manuscript in preparation. corresponding to probe 1. The sequence of sense primer E was 5′-GGI GA(A/G) A(A/C)(A/G) (A/G)TI ATG GA(A/G) GA(A/G) GC-3′ and of sense primer F was 5′-GA(A/G) (C/T)TI CA(G/A) (C/T)TI (A/C/T)(C/G/T)I (A/C)GI TGG TGG-3′. Degenerate primer G (see Fig. 2) was designed according to the amino acid sequence DVIKG(F/L)NW obtained from a peptide generated by trypsin digestion of purified (−)-pinene synthase from grand fir. 4C. L. Steele, E. Lewinsohn, and R. Croteau, unpublished results. The sequence of antisense primer G was 5′-CCA (A/G)TT IA(A/G) ICC (C/T)TT IAC (A/G)TC-3′. Primers E and F were independently used for PCR amplification in combination with primer G, with grand fir stem cDNA library as template. The combination of primers E and G yielded a specific PCR product of approximately 1020 bp. This PCR product was ligated into pT7Blue and transformed into E. coli XL1-Blue. Plasmid DNA was prepared from 20 individual transformants, and inserts were sequenced from both ends. The sequence of this 1022-bp insert was identical for all 20 plasmids and was designated as probe 3. For library screening, 100 ng of each probe (1 through 5) was amplified by PCR, gel purified, randomly labeled with [α-32P]dATP (28Feinberg A.P. Vogelstein B. Anal. Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5190) Google Scholar), and used individually to screen replica filters of 105 plaques of the wound-induced grand fir stem cDNA library plated on E. coli LE392. Hybridization with probes 1, 2, 4, and 5 was performed for 14 h at 65 °C in 3 × SSPE and 0.1% SDS. Filters were washed three times for 10 min at 55 °C in 3 × SSPE with 0.1% SDS and exposed for 12 h to Kodak XAR film at −70 °C (27Sambrock J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). All of the λZAPII clones yielding positive signals were purified through a second round of hybridization (probe 1 gave 25 positives, probe 2 gave 16 positives, probe 4 gave 49 positives, and probe 5 gave 12 positives). Hybridization with probe 3 was performed as before, but the filters were washed three times for 10 min at 65 °C in 3 × SSPE and 0.1% SDS before exposure. Approximately 400 λZAPII clones yielded strong positive signals, and 34 of these were purified through a second round of hybridization at 65 °C. Approximately 400 additional clones yielded weak positive signals with probe 3, and 18 of these were purified through a second round of hybridization for 20 h at 45 °C. Purified λZAP II clones isolated using all five probes were in vivo excised as Bluescript II SK− phagemids and transformed into E. coli XLOLR according to the manufacturer's instructions (Stratagene). The size of each cDNA insert was determined by PCR using T3 and T7 promoter primers, and selected inserts (>1.5 kb) were partially sequenced from both ends. Except for cDNA clones pAG3.18 and pAG3.48, all of the partially sequenced inserts were either truncated at the 5′-end, or were out of frame, or bore premature stop codons upstream of the presumptive methionine start codon. For the purpose of functional expression, a 2001-bp insert fragment from plasmid pAG2.2 and a 1903-bp insert fragment from pAG3.18 were subcloned in frame into pGEX vectors (Pharmacia Biotech Inc.). A 2046-bp insert fragment from pAG10 was subcloned in frame into the pSBETa vector (29Schenk P.M. Baumann S. Mattes R. Steinbiss H.-H. BioTechniques. 1995; 19: 196-200PubMed Google Scholar). To introduce suitable restriction sites for subcloning, fragments were amplified by PCR using primer combinations 2.2-BamHI (5′-CAA AGG GAT CCA GAA TGG CTC TGG-3′) and 2.2-NotI (5′-AGT AAG CGG CCG CTT TTT AAT CAT ACC CAC-3′) with pAG2.2 as template, 3.18-EcoRI (5′-CTG CAG GAA TTC GGC ACG AGC-3′) and 3.18-SmaI (5′-CAT AGC CCC GGG CAT AGA TTT GAG CTG-3′) with pAG3.18, and 10-NdeI (5-GGC AGG AAC ATA TGG CTC TCC TTT CTA TCG-3′) and 10-BamHI (5′-TCT AGA ACT AGT GGATCC CCC GGG CTG CAG-3′ with pAG10. PCR reactions were performed in volumes of 50 μl containing 20 mmTris/HCl (pH 8.8), 10 mm KCl, 10 mm(NH4)2SO4, 2 mmMgSO4, 0.1% Triton X-100, 5 μg of bovine serum albumin, 200 μm each dNTP, 0.1 μm each primer, 2.5 units of recombinant Pfu polymerase (Stratagene), and 100 ng of plasmid DNA with the following program: denaturation at 94 °C, 1 min; annealing at 60 °C, 1 min; extension at 72 °C, 3.5 min; 35 cycles with final extension at 72 °C, 5 min. The PCR products were purified by agarose gel electrophoresis and used as template for a secondary PCR amplification with the identical conditions in total volumes of 250 μl each. Products from this secondary amplification were digested with the above indicated restriction enzymes, purified by ultrafiltration, and then ligated, respectively, intoBamHI/NotI-digested pGEX-4T-2 to yield plasmid pGAG2.2, into EcoRI/SmaI-digested pGEX-4T-3 to yield plasmid pGAG3.18, and intoNdeI/BamHI-digested pSBETa to yield plasmid pSBAG10; these plasmids were then transformed into E. coliXL1-Blue or E. coli BL21(DE3). For expression, bacterial strains E. coli XLOLR/pAG3.18,E. coli XLOLR/pAG3.48, E. coli XL1-Blue/pGAG2.2,E. coli XL1-Blue/pGAG3.18, and E. coliBL21(DE3)/pSBAG10 were grown to A600 = 0.5 at 37 °C in 5 ml of LB medium (27Sambrock J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) supplemented with 100 μg of ampicillin/ml or 30 μg of kanamycin/ml as determined by the vector. Cultures were then induced by addition of 1 mmisopropyl-1-thio-β-d-galactopyranoside and grown for another 12 h at 20 °C. Cells were harvested by centrifugation (2000 × g, 10 min) and resuspended in either 1 ml of monoterpene synthase assay buffer (50 mm Tris/HCl (pH 7.5), 500 mm KCl, 1 mm MnCl2, 5 mm dithiothreitol, 0.05% (w/v) NaHSO3, and 10% (v/v) glycerol), 1 ml of sesquiterpene synthase assay buffer (10 mm dibasic potassium phosphate, 1.8 mmmonobasic potassium phosphate (pH 7.3), 140 mm NaCl, 10 mm MgCl2, 5 mm dithiothreitol, 0.05% (w/v) NaHSO3, and 10% (v/v) glycerol), or 1 ml of diterpene synthase assay buffer (30 mm Hepes (pH 7.2), 7.5 mm MgCl2, 5 mm dithiothreitol, 10 μm MnCl2, 0.05% (w/v) NaHSO3, and 10% (v/v) glycerol). Cells were disrupted by sonication (Braun-Sonic 2000 with microprobe at maximum power for 15 s at 0–4 °C); the homogenates were cleared by centrifugation (18,000 × g, 10 min), and 1 ml of the resulting supernatant was assayed for monoterpene synthase activity with 2.5 μm [1-3H]geranyl diphosphate, for sesquiterpene synthase activity with 3.5 μm[1-3H]farnesyl diphosphate, or for diterpene synthase activity with 5 μm [1-3H]geranylgeranyl diphosphate following standard protocols (11Croteau R. Cane D.E. Methods Enzymol. 1985; 110: 383-405Crossref Google Scholar, 21LaFever R.E. Stofer-Vogel B. Croteau R. Arch. Biochem. Biophys. 1994; 313: 139-149Crossref PubMed Scopus (70) Google Scholar, 26Dehal S.S. Croteau R. Arch. Biochem. Biophys. 1988; 261: 346-356Crossref PubMed Scopus (56) Google Scholar). In the case of the monoterpene synthase and sesquiterpene synthase assays, the incubation mixture was overlaid with 1 ml of pentane to trap volatile products. In all cases, after incubation at 31 °C for 2 h, the reaction mixture was extracted with pentane (3 × 1 ml), and the combined extract was passed through a 1.5-ml column of anhydrous MgSO4 and silica gel (Mallinckrodt 60 Å) to provide the terpene hydrocarbon fraction free of oxygenated metabolites. The columns were subsequently eluted with 3 × 1 ml of ether to collect any oxygenated products, and an aliquot of each fraction was taken for liquid scintillation counting to determine conversion rate. To obtain sufficient product for analysis by radio-GLC, chiral capillary GLC, and GLC-MS, preparative-scale enzyme incubations were carried out. Thus, the enzyme was prepared from 50 ml of cultured bacterial cells by extraction with 3 ml of assay buffer as above, and the extracts were incubated with excess substrate overnight at 31 °C. The hydrocarbon fraction was isolated by elution through MgSO4-silica gel as before, and the pentane eluate was concentrated for evaluation by capillary radio-GLC as described (30Croteau R. Satterwhite D.M. J. Chromatogr. 1990; 500: 349-354Crossref PubMed Scopus (16) Google Scholar), by chiral column capillary GLC (5Lewinsohn E. Savage T.J. Gijzen M. Croteau R. Phytochem. Anal. 1993; 4: 220-225Crossref Scopus (83) Google Scholar), and by combined GLC-MS (Hewlett-Packard 6890 GC-MSD with cool (40 °C) on-column injection, detection via electron impact ionization (70 eV), helium carrier at 0.7 p.s.i., column: 0.25-mm inner diameter × 30-m fused silica with 0.25-μm film of 5MS (Hewlett-Packard) programmed from 35 °C (5 min hold) to 230 °C at 5 °C/min). Inserts of all recombinant bluescript plasmids, pAG1.28, pAG2.2, pAG3.18, pAG3.48, pAG4.30, pAG5.9, and pAG10, and inserts of all recombinant pGEX plasmids, pGAG2.2, pGAG3.18, and pSBAG10, were completely sequenced on both strands via primer walking and nested deletions (27Sambrock J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using the DyeDeoxy Terminator Cycle Sequencing method (Applied Biosystems). Sequence analysis was done using the Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, WI. Grand fir sapling stem tissue was harvested prior to wounding or 2 days after wounding by a standard procedure (18Gijzen M. Lewinsohn E. Croteau R. Arch. Biochem. Biophys. 1991; 289: 267-273Crossref PubMed Scopus (45) Google Scholar). Total RNA was isolated (31Lewinsohn E. Steele C.L. Croteau R. Plant Mol. Biol. Rep. 1994; 12: 20-25Crossref Scopus (64) Google Scholar), and 20 μg of RNA per gel lane was separated under denaturing conditions (27Sambrock J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and transferred to nitrocellulose membranes (Schleicher and Schuell) according to the manufacturer's protocol. To prepare hybridization probes, cDNA fragments of 1.4–1.5 kb were amplified by PCR fromag2.2 with primer JB29 (5′-CTA CCA TTC CAA TAT CTG-3′) and primer 2–8 (5′-GTT GGA TCT TAG AAG TTC CC-3′), from ag3.18with primer 3–9 (5′-TTT CCA TTC CAA CCT CTG GG-3′) and primer 3–11 (5′-CGT AAT GGA AAG CTC TGG CG-3′), and from ag10 with primer 7–1 (5′-CCT TAC ACG CCT TTG GAT GG-3′) and primer 7–3 (5′-TCT GTT GAT CCA GGA TGG TC-3′). The probes were randomly labeled with [α-32P]dATP (28Feinberg A.P. Vogelstein B. Anal. Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5190) Google Scholar). Blots were hybridized for 24 h at 55 °C in" @default.
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• W4234578412 title "Monoterpene Synthases from Grand Fir (Abies grandis)" @default.
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