WO1998037206A1 - Utilisation de la petite sous-unite de l'acetolactate synthase d'une plante permettant la decouverte d'un nouvel herbicide - Google Patents

Utilisation de la petite sous-unite de l'acetolactate synthase d'une plante permettant la decouverte d'un nouvel herbicide Download PDF

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WO1998037206A1
WO1998037206A1 PCT/US1998/003506 US9803506W WO9837206A1 WO 1998037206 A1 WO1998037206 A1 WO 1998037206A1 US 9803506 W US9803506 W US 9803506W WO 9837206 A1 WO9837206 A1 WO 9837206A1
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als
acetolactate synthase
leu
val
seq
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PCT/US1998/003506
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Lynn Marie Abell
Howard Paul Hershey
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E.I. Du Pont De Nemours And Company
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Priority to AU66641/98A priority Critical patent/AU6664198A/en
Publication of WO1998037206A1 publication Critical patent/WO1998037206A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase

Definitions

  • This invention is in the field of herbicide discovery. Specifically, this invention pertains to nucleic acid fragments encoding the small subunit of plant acetolactate synthase, and use of the encoded protein to aid in the discovery of new herbicides that inhibit plant acetolactate synthase activity.
  • Acetolactate synthase (ALS; EC 4.1.3.18), also known as acetohydroxy acid synthase, is the first committed step in branched chain amino acid biosynthesis in plants and bacteria.
  • the enzyme is known to be the site of action of the several diverse classes of herbicides including the sulfonylureas [Chaleff, R.S. & Mauvais, C.J., Science 224:1443-1445 (1984); LaRossa, R. A. & Schloss, J.V., J. Biol. Chem. 259:8753-8757 (1984); Ray, T.B., Plant Physiol.
  • Bacterial ALS has been extensively characterized and is known to exist as three isozymes in E. coli. Each isozyme is a tetramer composed of two identical large subunits of approximately 60,000 Da molecular weight and two identical smaller subunits ranging in molecular weight between 9,000 Da and 17,000 Da depending on the isozyme of ALS [Chipman et al., in Biosynthesis of Branched Chain Amino Acids, eds. Barak, Z., Chipman, D.M. & Schloss, J.N.
  • the enzyme from plants is much less well characterized. Attempts to purify the enzyme from plant extracts have been hampered by the extreme lability of the enzyme and its low abundance. All attempts to purify the enzyme from plant sources have produced only a single major band on an SDS-PAGE gel which varies in molecular weight from 58,000 Da [Durner, J. & Boger, P. Z. Naturforsch 43c: 850-856 (1988)] to 65,000-66,000 Da [Muhitch et al., Plant Physiol. 83: 451-456 (1987)]. Attempts to immunoprecipitate the enzyme from plant extracts also resulted in the isolation of a single band at 65,000-66,000 Da molecular weight [Singh, B.K.
  • a cDNA clone has now been discovered and identified as the small subunit of plant ALS based upon the sequence identity shared between the peptide encoded by the clone and various bacterial ALS small subunits.
  • a full length clone was obtained from Nicotiana plumbaginifolia, expressed in E. coli, and partially purified. Mixing the putative small subunit from Nicotiana with various large subunits from several sources (Arabidopsis and Nicotiana) increased the specific activity of the large subunit 4-15 fold. This trend is similar to that observed with the bacterial enzyme and confirms, functionally, the identification of the cDNA clone as the small subunit of plant ALS.
  • the present invention comprises a nucleic acid fragment encoding the small subunit of plant ALS.
  • the invention also comprises a method for expression and purification of the small subunit, its use in preparing plant ALS holoenzyme and the use of the holoenzyme to screen for potentially herbicidal compounds based upon holoenzyme inhibition.
  • this invention pertains to an isolated nucleic acid fragment encoding the small subunit of a plant acetolactate synthase, the fragment comprising a member selected from the group consisting of (a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4; and (b) a nucleotide sequence essentially similar to the nucleotide sequence of (a).
  • a a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4
  • a nucleotide sequence essentially similar to the nucleotide sequence of (a) Preferred is an isolated nucleic acid fragment wherein the nucleotide sequence is set forth in SEQ ID NO:4.
  • Another embodiment of the instant invention is a plasmid vector comprising a nucleic acid fragment encoding the small subunit of a plant acetolactate synthase operably linked to at least one suitable regulatory sequence and a transformed host cell comprising the aforementioned plasmid vector.
  • the instant invention pertains to a method for evaluating at least one compound for its ability to inhibit acetolactate synthase activity, the method comprising the steps of: (a) transforming a host cell with the plasmid vector comprising a nucleic acid fragment encoding the small subunit of a plant acetolactate synthase operably linked to at least one suitable regulatory sequence; (b) facilitating expression of the nucleic acid fragment encoding the small subunit of a plant acetolactate synthase; (c) purifying the small subunit of a plant acetolactate synthase expressed by the transformed host cell; (d) mixing the purified small subunit with the large subunit of a plant acetolactate synthase in a suitable container, thereby forming a plant acetolactate synthase holoenzyme; (e) treating the holoenzyme with a compound to be tested; and (f) comparing the acetolactate synthas
  • Figure 2 shows the map of the expression plasmid pGEX-SSU used to express the Nicotiana plumbaginifolia acetolactate synthase small subunit polypeptide.
  • SEQ ID NO:l represents the nucleotide sequence of the cDNA insert encoding the small subunit of corn acetolactate synthase found in the plasmid clone ml5.12.bl2.sk20.
  • SEQ ID NO:2 is the sequence of oligodeoxynucleotide primer SU5R used to prime first strand cDNA synthesis of Nicotiana plumbaginifolia ALS small subunit.
  • SEQ ID NO: 3 is the sequence of oligodeoxynucleotide primer SU4R used for PCR amplification of the single stranded cDNA representing Nicotiana plumbaginifolia ALS small subunit.
  • SEQ ID NO: 4 is the full-length cDNA sequence of the Nicotiana plumbaginifolia ALS small subunit contained in the plasmid pSSU.NPl.
  • SEQ ID NO:5 and SEQ ID NO:6 are oligodeoxynucleotides (pTrx linkerl and pTrx linker2, respectively) that were used to aid construction of the plasmid vector pTrx-Bstl l07.
  • SEQ ID NO:7 and SEQ ID NO:8 are oligodeoxynucleotides (HIS-TAG5 and HIS- TAG3, respectively) that were used to aid construction of the plasmid vector pTrx-HIS.
  • SEQ ID NO:9 and SEQ ID NO: 10 are PCR primers (SSU-PCR1 and SSU-PCR2, respectively) used for amplification of the insert in cDNA clone SSU.NP1.
  • SEQ ID NO:l 1 and SEQ ID NO:12 are oligonucleotides (CAM19 and CAM20, respectively) used to modify the plasmid vector pGEX-2T to create the plasmid vector pGEX-2TM.
  • SEQ ID NO:13 and SEQ ID NO:14 are oligodeoxynucleotides (SSU oligo 9 and ' SSU oligo 10, respectively) that were used to aid construction of the plasmid vector pGEX-SSU.
  • SEQ ID NO: 15 and SEQ ID NO: 16 are oligodeoxynucleotides (SSU oligo 5 and SSU oligo 6, respectively) that were used to aid construction of the plasmid vector pGEX-SSU.
  • SEQ ID NO: 17 and SEQ ID NO: 18 are oligodeoxynucleotides (mt704+ and mt800-, respectively) that were used to aid construction of the plasmid vector pMTDRALS.
  • SEQ ID NO: 19 is the full-length cDNA sequence of the Nicotiana plumbaginifolia ALS large subunit contained in the plasmid pALSlO.
  • BIOLOGICAL DEPOSITS The following plasmid has been deposited under the terms of the Budapest Treaty at American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, and bears the following accession number:
  • the present invention provides a cDNA sequence encoding the small subunit of plant acetolactate synthase ("ALS").
  • ALS plant acetolactate synthase
  • SSU plant ALS small subunit protein
  • LSU plant acetolactate synthase
  • the resulting reconstituted ALS holoenzyme shows both increased catalytic efficiency and solution stability when compared to the large subunit alone.
  • the beneficial effects of reconstituting native ALS with this small subunit is not species-specific as shown by the ability of the N. plumbaginifolia small subunit to form an active complex with large subunits from other plant species.
  • ALS small subunit genes that encode the same functional protein as that encoded by the cDNA clone described herein. Therefore, it is expected that the invention can also be accomplished using ALS small subunit sequences from other plant species, both monocotyledonous and dicotyledonous. Indeed, based upon the work presented here, it is expected that the invention may be accomplished by mixing small and large ALS subunits from divergent species.
  • single pass DNA sequence analysis was performed on individual clones from a cDNA library made using RNA from corn embryos that were harvested 15 days post pollination.
  • sequences were compared to known sequences in the GenBank database until a clone was identified as the small subunit of plant ALS by the similarity of its coding region with those of various bacterial ALS small subunits.
  • a DNA fragment comprising the coding region from this clone was isolated and operably linked to suitable bacterial regulatory sequences to create plasmids capable of directing the expression of plant ALS small subunit in E. coli as thioredoxin (TRX) and glutathione-S-transferase (GST) fusion proteins.
  • TRX thioredoxin
  • GST glutathione-S-transferase
  • the recombinant ALS large and small subunits may be produced using any number of methods by those skilled in the art. Such methods include, but are not limited to, expression in bacteria, eukaryotic cell culture, inplanta, and using viral expression systems in suitably infected organisms or cell lines. Large and small ALS subunits may be expressed separately as mature proteins, or may be co-expressed in E. coli or another suitable expression background.
  • large and small subunits may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags.
  • Common fusion protein partners include glutathione S-transferase ("GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide ("(His)6").
  • GST glutathione S-transferase
  • Trx thioredoxin
  • maltose binding protein and C- and/or N-terminal hexahistidine polypeptide
  • the fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature large or small subunit. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cle
  • Purification of the ALS subunits or the holoenzyme may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor.
  • the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed large or small subunit or an affinity resin containing ligands which are specific for the small subunit.
  • small subunit of ALS is expressed as a fusion protein coupled to the C-terminus of thioredoxin.
  • a (His) ⁇ peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification.
  • the preferred purification protocol embodied in the examples involves the use of arsene oxide affinity chromatography with the commercially available ThioBondTM resin (Invitrogen Corporation, San Diego, CA) which has affinity for thioredoxin.
  • Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B.
  • the thioredoxin-SSU fusion protein is eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include ⁇ -mercaptoethanol or other reduced thiol.
  • the eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired.
  • Proteolytic cleavage of the thioredoxin fusion protein and the ALS small subunit to yield a complete small subunit may be accomplished after the fusion protein is purified, while the protein is still bound to the ThioBondTM affinity resin or other resin, or in the presence of an ALS large subunit.
  • the generality of this procedure is demonstrated in a preferred embodiment of the invention whereby the small subunit protein is expressed and purified as a fusion to GST.
  • the preferred purification protocol for this fusion protein embodied in the examples involves the use of a glutathione-containing Sepharose- or agarose-based affinity resin which has affinity for GST.
  • Other suitable affinity resins could be synthesized by linking the appropriate ligand to a suitable resin.
  • the GST- SSU is eluted using glutathione at pH 9.0; however, elution may be accomplished using other reagents and conditions of pH and ionic strength which serve to weaken the interaction between GST and the glutathione-containing resin.
  • the Arabidopsis ALS large subunit is expressed and purified as a fusion protein with glutathione-S-transferase; however, large subunit may be purified by a number of different means as stated above.
  • Another preferred embodiment of the invention comprises expression of the Nicotiana plumbaginifolia ALS-LSU as a mature enzyme containing a partial chloroplast transit peptide.
  • this enzyme is purified using conventional protein purification procedures. Variations on these procedures using different column materials and conditions can easily be envisioned by the skilled artisan.
  • This enzyme may also be expressed as a fusion protein and affinity-purified as described above.
  • Acetolactate synthase holoenzyme which is defined as a combination of large and small subunits, may be prepared by (i) mixing partially or completely purified large and small subunits, (ii) co-purification of the holoenzyme which is either prepared by co- expression or by mixing cell extracts containing individually expressed subunits either as fusion proteins or as mature subunits, or (iii) by mixing purified or partially purified fusion proteins.
  • the fusion protein of the small subunit is mixed either with a fusion protein of the large subunit or a mature form of the large subunit. In both cases, increases in specific activity were observed upon incubation compared to the large subunit by itself.
  • Activity is further enhanced by the addition of a specific protease, in this case thrombin, to cleave the fusion protein affinity tag to produce mature small and in some cases large subunits.
  • a specific protease in this case thrombin
  • thrombin a specific protease
  • Bovine serum albumin did not produce the same effect as the presence of the small subunit.
  • phosphate buffered saline at pH 8 was used. Buffers in the range of pH 6.5 to 9 are preferred with buffers in the range of pH 7 to 7.6 being optimal.
  • Cofactors required for ALS activity may also be present in the holoenzyme reconstitution such as flavin adenine dinucleotide, thiamine pyrophosphate and divalent metal ions. Stabilizing agents such as dithiothreitol and glycerol may also be used.
  • Acetolactate synthase holoenzyme is sensitive to inhibition by herbicidal compounds known to inhibit ALS in vivo as their mode of action.
  • the ALS holoenzyme is useful in screening for novel crop protection chemicals.
  • nucleic acid refers to a large molecule which can be single-stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, phosphate and either a purine or pyrimidine.
  • a “nucleic acid fragment” is a fraction of a given nucleic acid molecule.
  • deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of the information in DNA into proteins.
  • RNA ribonucleic acid
  • a “genome” is the entire body of genetic material contained in each cell of an organism.
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine can also be expected to produce a biologically equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein.
  • Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
  • "essentially similar" sequences encompassed by this invention are also defined by their ability to hybridize, under relatively stringent conditions (2X SSC, 0.1% SDS, 55°C), with the sequences exemplified herein.
  • Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3' non- coding) the coding region.
  • “Native” gene refers to the gene as found in nature with its own regulatory sequences.
  • “Chimeric” gene refers to a gene comprising heterogeneous regulatory and coding sequences.
  • “Endogenous” gene refers to the native gene nominally found in its natural location in the genome.
  • a “foreign” gene refers to a gene not nominally found in the host organism but that is introduced by gene transfer.
  • Coding sequence refers to a DNA sequence that codes for a specific protein and excludes the non-coding sequences.
  • “Initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). "Open reading frame” refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.
  • the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript.
  • RNA refers to RNA that can be translated into protein by the cell.
  • cDNA refers to a double-stranded DNA, one strand of which is complementary to and derived from mRNA by reverse transcription.
  • Sense RNA refers to RNA transcript that includes the mRNA.
  • regulatory sequences refer to nucleotide sequences located upstream (5'), within, and/or downstream (3') to a coding sequence, which control the transcription and/or expression of the coding sequences, potentially in conjunction with the protein biosynthetic apparatus of the cell. These regulatory sequences include promoters, translation-leader sequences, transcription-termination sequences, and polyadenylylation sequences.
  • Promoter refers to a DNA sequence in a gene, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • a promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. It may also contain enhancer elements.
  • operably linked refers to nucleic acid sequences on a single nucleic acid molecule which are associated so that the function of one is affected by the other.
  • a promoter is operably linked with a structural gene (i.e., a gene encoding aspartokinase that is lysine-insensitive as given herein) when it is capable of affecting the expression of that structural gene (i.e., that the structural gene is under the transcriptional control of the promoter).
  • a structural gene i.e., a gene encoding aspartokinase that is lysine-insensitive as given herein
  • expression is intended to mean the production of the protein product encoded by a gene. More particularly, “expression” refers to the transcription and stable accumulation of the sense (mRNA) or antisense RNA derived from the nucleic acid fragment(s) of the invention that, in conjunction with the protein apparatus of the cell, results in the accumulation of the encoded protein product.
  • mRNA sense
  • antisense RNA derived from the nucleic acid fragment(s) of the invention
  • “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. "Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
  • the "3' non-coding sequences” refers to the DNA sequence portion of a gene that contains a polyadenylation signal and any other regulatory signal capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
  • “Mature” protein refers to a post-translationally processed polypeptide wherein a number of amino acids at the N- and/or C-terminus of the primary translation product has been removed by proteolysis.
  • Precursor protein refers to the primary product of translation of mRNA.
  • a “chloroplast targeting signal” is an amino acid sequence which is translated in conjunction with a protein and directs it to the chloroplast.
  • Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast targeting signal.
  • Fusion protein refers to a polypeptide that is produced with additional amino acids at either its N-or C-terminus to aid in its expression and/or purification.
  • Transformation herein refers to the transfer of a foreign gene into a host organism and its genetically stable inheritance.
  • Home cell means the cell that is transformed with the introduced genetic material.
  • “Inhibition” refers to a decrease in the catalytic activity of an enzyme or holoenzyme complex by reversible or irreversible binding of a chemical to the enzyme.
  • Holoenzyme is defined as an intact enzyme containing all of the subunits and cofactors required for full activity. In the case of a plant acetolactate synthase these include a small subunit comprising a protein of approximately 45,000 Da, a large subunit comprising a protein of approximately 65,000 Da, and cofactors flavin adenine dinucleotide, thiamine pyrophosphate and divalent metal ions.
  • Specific activity is the number of enzyme units per mg protein. "Units” is defined as the micromoles of product produced per minute of reaction time.
  • the library was plated at low density and individual plaques were randomly selected and picked into SM (50 mM Tris-HCl pH 7.5, 10 mM MgSO 4 , 7H 2 0, 10 mM NaCl, 0.01 % gelatin). After elution of the phage from the agar plug, the resulting phage stocks were diluted 1:10, and cDNA insert was amplified from the phage using the polymerase chain reaction (PCR) using vector-directed primers (T3/T7).
  • PCR polymerase chain reaction
  • PCR products were purified using Qiagen PCR purification kit and an aliquot of the purified PCR product was checked by agarose gel electrophoresis for DNA integrity.
  • the DNA was then sequenced using an Applied Biosystems Inc. (Foster City, CA) Model 373 DNA sequencer and ABI dye terminator sequencing kit.
  • the resulting single pass cDNA sequence was compared to sequences in GenBank using NCBI Blast facility, and in this way, a clone designated ml5.12.bl2.sk20 was identified by the sequence similarity of its coding region to various bacterial small subunit of acetolactate synthase (SEQ ID NO:l).
  • Nicotiana plumbaginifolia was grown to the 5-leaf stage in Metromix 350 at 26°C in a growth chamber maintained at 75% relative humidity using a 16 hr/8 hr day/night cycle. Plants were removed from soil with roots, rinsed in 0.5 X Hoagland's solution to remove as much adhering soil as possible and frozen in liquid nitrogen. Total RNA was prepared from frozen tissue using guanidinium thiocyanate extraction and CsCl purification as described by Colbert et al. [Proc. Natl. Acad. Sci. USA 45 1703-1708 (1983)]. Total RNA was sent to Clontech Laboratories. Inc. (Palo Alto, CA) where a directional cDNA library in the vector lambda ZAP IITM was made using their custom cDNA library service.
  • a total of 3x10 6 phage were plated on 25x25 cm square NZY plates (10 g NZ amine, 5 g yeast extract, 2 g MgSO ⁇ at a density of 2.5xl0 5 plaques/plate.
  • 2.5x10 5 phage were mixed with 5 mL of an overnight culture of E. coli XL-1 Blue grown in NZY containing 0.2% maltose and cells were incubated at 37°C for 15 rnin.
  • the infected culture was mixed with 40 mL of NZY top agar (ZY media containing 0.7% agarose) and pored onto the plate. After the top agarose hardened, plates were incubated at 37°C for 6-8 h and then stored at 4°C.
  • Phage lifts were then performed by layering dry MagnaGraphTM nylon transfer membranes (Micron Separations Inc., Westborough, MA) on top of the phage plates for 5 min. Membranes were transferred with their DNA side facing upward onto Whatman 3MM paper saturated with 0.5 M NaOH, 1.5 M NaCl. After 5 min, membranes were transferred onto Whatman 3MM paper saturated with 0.5 M Tris-HCl pH 7.5, 1.5 M NaCl and incubated for an additional 5 min.
  • Membranes were then rinsed with 2X SSPE (20X SSPE is 3 M NaCl, 0.1 M Na 2 HPO 4 , 20 mM EDTA, pH 7.4), air dried for 10 min and DNA was crosslinked to the membrane using a StratalinkerTM UV Crosslinker (Stratagene; La Jolla, CA) in the "Auto Cross Link" mode by following the manufacturer's protocol. Membranes were stored at 4°C in sealed polyethylene bags. The cDNA insert from 25 ⁇ g of the ml5.12.bl2.sk20 plasmid was digested with 2X SSPE (20X SSPE is 3 M NaCl, 0.1 M Na 2 HPO 4 , 20 mM EDTA, pH 7.4), air dried for 10 min and DNA was crosslinked to the membrane using a StratalinkerTM UV Crosslinker (Stratagene; La Jolla, CA) in the "Auto Cross Link" mode by following the manufacturer's protocol. Membranes were stored at 4°C in sealed
  • Phage lift membranes were prehybridized for 4 h at 65°C in 6X SSPE, 0.5% SDS, 1 mM EDTA, 5X Denhardt's solution, 100 ⁇ g denatured and sonicated calf thymus
  • Membranes were then hybridized overnight with the ml5.12.bl2.sk20 probe in 6X SSPE, 0.5% SDS, 1 mM EDTA, 2X Denhardt's solution, 100 ⁇ g denatured and sonicated calf thymus DNA/mL.
  • the membranes were washed twice at room temperature with 2X SSPE, 0.1% SDS, twice with 2X SSPE, 0.1% SDS at 55°C, air dried and exposed to Kodak X-OMAT XAR-5 film overnight at -80°C using a single intensifying screen.
  • SM Tris- HCl pH 7.5, 0.1 M NaCl, 10 mM MgSO 4 , 0.01% gelatin
  • Plaque purification of phage was performed by serially diluting eluted phage with SM, infecting 100 uL cultures of E. coli XL-1 Blue with 100 uL aliquots of the dilutions and growing the infected bacteria overnight at 37°C on NZY plates.
  • Plasmid DNA was obtained from each pure phage isolate as follows. Overnight cultures of E. coli XL-1 Blue and E. coli SOLR were grown up in NZY and LB media, respectively. XL-1 Blue cells were diluted to an A ⁇ QQ of 1.0 with NZY and 200 uL of this dilution was infected with 100 uL of pure phage stock and 1 uL of a 10 10 pfu/mL stock of ExAssistTM helper phage (Stratagene). Following a 15 min incubation at 37°C, cells were diluted with 3 mL of 2X YT media and incubated with shaking at 37°C for 2 hr.
  • the culture was heated to 70°C for 20 min and then centrifuged at 10,000Xg for 5 min at 4°C. The supernatant was transferred to a fresh tube and 2 uL of this supernatant was mixed with 200 uL of SOLR cells from the overnight culture. Following a 15 min incubation at 37°C, 10 and 100 uL aliquots of the cultures were spread onto LB plates supplemented with 100 ⁇ g ampicillin/mL and the plates were incubated at 37°C overnight. Plasmids from individual antibiotic resistant colonies were analyzed for cDNA inserts by digestion with Eco RI and Xho I.
  • RNA prepared as described above was used as the target for RACE following the manufacturer's recommendation using oligodeoxynucleotide SU5R of the sequence
  • the RACE products were separated by electrophoresis in a 2% agarose gel. A region of the gel containing 400-500 bp DNA fragments was excised and the DNA is purified using a QIAquickTM gel extraction kit (Qiagen, Inc., Chatsworth, CA). The purified RACE product was ligated into the vector pGEM-T (Promega Corp., Madison, WI) using conditions described earlier, 1 uL of the ligation reaction was used to transform competent E. coli JM109.
  • oligodeoxynucleotides pTrx linker 1 and pTrx linker2 were phosphorylated separately for 30 min at 37°C in 100 uL of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10 mM dithiothreitol, 1 mM ATP and 100 units of T 4 polynucleotide kinase.
  • 3.2 uL aliquots of each phosphorylated primer were then combined, diluted to 50 uL with H 2 O and heated to 70°C for 20 min and allowed to cool to ambient temperature.
  • the bacterial expression vector pTrxFUS (Invitrogen) was digested to completion with Kpn I and the resulting 3' overhang was removed by incubation of the DNA with the Klenow fragment of DNA polymerase I for 1 h at 25°C in a buffer consisting of 50 mM Tris-HCl, 10 mM MgCl 2 , 0.1 mM each of dATP, dCTP, dGTP, and dTTP, and 1 unit of Klenow/ ⁇ g of DNA.
  • the DNA was then extracted sequentially with equal volumes of phenol:CHCl 3 :isoamyl alcohol (25:24:1) and CHC1 3 and precipitated on dry ice for 20 min after adding 0.1 volume of 3 M sodium acetate pH 6 and 2 volumes of ethanol. DNA was recovered by centrifugation at 14,000 X g for 10 min. The DNA was digested to completion with Sal I and dephosphorylated with calf intestinal alkaline phosphatase.
  • a polymerase chain reaction is performed using plasmid pTrxFUS as a target and the oligodeoxynucleotides HIS-TAG5 and HIS-TAG3 as primers.
  • HIS-TAG3 5 ' -CCTGTACGATTACTGCAGGTC-3 ⁇ (SEQ ID NO:8)
  • the reaction mixture was assembled in a total volume of 500 uL containing 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.001% gelatin, 0.2 mM each of dATP, dCTP, dGTP, dTTP, 800 ng plasmid pTrxFUS, 150 pmoles each of HIS-TAG5 and HIS-TAG3, and 12.5 units of AmpliTaq polymerase (Perkin Elmer Cetus, Norwalk,
  • the mixture was divided into 5 100 ⁇ L aliquots and the five tubes were subjected to 18 cycles of a temperature profile of: 95°C for 1 min, 55°C for 1 min, 72°C for 1 min. A final cycle of 95°C for 1 min, 55°C for 1 min, 72°C for 5 min completed the reaction.
  • the five aliquots were pooled and extracted sequentially with equal volumes of phenol : CHC1 3 : isoamyl alcohol (1:1:1) and CHCI3.
  • the DNA was precipitated by addition of 0.1 volume of 3 M sodium acetate pH 6 and 2 volumes of ethanol followed by a 20 min incubation on dry ice.
  • DNA was recovered by centrifugation at 14,000 x g for 10 min, dried in vacuo and dissolved in 100 uL of 10 mM Tris-HCl pH 7.5, 1 mM EDTA (TE). One half of the PCR product was digested to completion with Nde I and Rsr II.
  • the digest was extracted sequentially with equal volumes of phenokCHCLj ⁇ soamyl alcohol (25:24:1) and CHC1 3 .
  • the DNA was precipitated by addition of 0.1 volume of 3 M sodium acetate pH 6 and 2 volumes of ethanol followed by 20 min on dry ice. DNA was recovered by centrifugation at 14,000 X g for 10 min and the pellet was dried in vacuo and dissolved in 15 uL of TE.
  • Nde I/Rsr II digested PCR product was ligated with 1 ⁇ g of the vector pTrx-Bstl 107 that had been digested to completion with Nde I and Rsr II and dephosphorylated with calf intestinal alkaline phosphatase in a 10 uL ligation reaction using conditions described above.
  • Competent E. coli GI 724 cells were prepared by following the protocol supplied by Invitrogen. A 4 uL aliquot of the ligation mixture was used to transform 110 uL of competent E. coli GI 724 cells using the chemical transformation protocol supplied by Invitrogen, Inc. Aliquots of the transformation mixture were spread on RMG plates [6 g/L Na 2 HPO 4 , 3 g/L K 2 HPO 4 , 0.5 g/L NaCl, 1 g/L NH 4 C1, 2% amicase (acid casein hydrolysate; Sigma Chemical Co., St.
  • a polymerase chain reaction was performed using cDNA clone SSU.NP1 as a target and the oligodeoxynucleotides SSU-PCR1 and SSU-PCR3 as primers.
  • PCR was performed using the conditions described above with 150 pmoles each of SSU-PCR1 and SSU-PCR3, and 800 ng of SSU.NP1 in the reaction.
  • the PCR reaction was then extracted, precipitated, and redissolved in 100 uL of TE as described earlier.
  • One half of the PCR product was digested to completion with Eco RV and Bam HI.
  • the digest was then extracted sequentially with equal volumes of phenokCHC j ⁇ soamyl alcohol (1 :1:1) and CHCI3.
  • the DNA was precipitated by addition of 0.1 volume of 3 M sodium acetate pH 6 and 2 volumes of ethanol followed by 20 min on dry ice. DNA was recovered by centrifugation at 14,000 x g for 10 min.
  • the pellet was dried in vacuo and dissolved in 15 uL of TE.
  • Approximately 250 ng of Eco RV/Bam HI digested PCR product was ligated with 1 ⁇ g of the vector pTrx-HIS that had been digested to completion with Bstl 1071 and Bam HI and dephosphorylated with calf intestinal alkaline phosphatase in a volume of 10 uL.
  • a 4 uL aliquot of the ligation mixture was used to transform competent E. coli GI724 cells as described above and aliquots of the transformation mixture were spread on RMG plates and incubated at 30°C overnight.
  • Plasmids from individual antibiotic resistant colonies were analyzed for inserts by digestion with Nde I and Xba I until a colony was found that contains a plasmid showing a 1.7 kbp Nde I/Xba I insert. This plasmid was designated pTrx-HIS/SSU.
  • pGEX-2T protein expression vector pGEX-2T (Pharmacia Biotech, Uppsala, Sweden).
  • pGEX-2T (10 ⁇ g) was digested to completion with BamH I and EcoRI and the digestion products were separated by electrophoresis using a 1% agarose gel. The 4.95 kbp band was excised from the gel and the DNA was recovered using a QIAquickTM Gel Extraction Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions.
  • Sodium acetate (pH 5.2) was added to the final column eluant to give a concentration of 0.3 M; 10 ⁇ g of tRNA and 2 volumes of cold ethanol were then added and the DNA was recovered by centrifugation. The pellet was washed with 70% ethanol, air dried at room temp, dissolved in 10 ⁇ L TE and quantified by running a 1 ⁇ L aliquot on a 1% agarose gel and comparing the band intensity with that of a commercial mass ladder standard. Approximately 50 ng of digested plasmid were mixed with 70 ng each of phosphorylated CAM 19 and CAM20 oligonucleotides and the mixture was incubated at 45°C for 5 minutes and cooled on ice. The ligated oligonucleotides create the following sites: BamH I, Ncol, Sail, Xhol, PinAI and EcoRI.
  • T4 DNA ligase buffer and 1 unit of T4 DNA ligase were added and the mixture was incubated at 37° C for 1 hour.
  • the ligation mix was then transformed into DH5 Max Efficiency Competent E. coli (GibcoBRL) using a standard heat shock protocol (Sambrook) and spread onto LB plates containing 100 ⁇ g carbenicillin/mL.
  • Ncol sites was selected and designated pGEX-2TM.
  • oligodeoxynucleotides SSU oligo 9 and SSU oligo 10 were phosphorylated separately for 30 min at 37°C in 100 ⁇ L of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2j 10 mM dithiothreitol, 1 mM ATP and 100 units of T 4 polynucleotide kinase.
  • the plasmid pNPl 5/6 was digested to completion with Kas I and Bam HI and the digestion products were separated by electrophoresis using a 6.5% agarose gel. The 1.3 kbp Kas I/Bam HI fragment was recovered from the gel. 400 ng of this DNA was ligated with 1 ⁇ g of the plasmid pSSU 9/10 that had been digested to completion with Kas I and Bam HI and dephosphorylated as described above. The ligation reaction was diluted five fold with H 2 O and used to transform competent Max Efficiency E. coli HB101 as described above.
  • Plasmids from individual antibiotic resistant colonies were analyzed by digestion with Bbs I and Xho I until a clone found was found that contained the desired 1.3 kbp insert. This plasmid was designated pSSUBbs/Bam.
  • the plasmid pSSU Bbs/Bam was digested to completion with Bbs I and Xho I and the digestion products were separated by electrophoresis using a 6.5% agarose gel and the 1.35 kbp Bbs I/Xho I fragment was recovered from the gel.
  • Three hundred ng of this DNA was ligated with 1 ⁇ g of the plasmid pGEX-2TM that had been digested to completion with BamH I and Xho I and dephosphorylated as described above.
  • the ligation reaction was diluted five fold with H 2 O and used to transform competent Max Efficiency E. coli DH5 ⁇ as described above. Plasmids from individual antibiotic resistant colonies were analyzed by restriction endonuclease digestion until a clone found was found that contained the desired ALS small subunit coding region. This plasmid was designated pGEX-SSU.
  • the strain GI724 harboring the plasmid pTrx-HIS/SSU (designated GI724/Trx- HIS/SSU) was struck onto a fresh RMG plate containing 100 ⁇ g/mL ampicillin and grown overnight at 30°C. The next day, a single colony was then used to inoculate a 20 mL culture of RM (6 g/L Na 2 HPO 4 , 3 g/L K 2 HPO 4 , 0.5 g/L NaCl, 1 g/L NH 4 C1, 20 g/L amicase (acid casein hydrolysate; Sigma Chemical Co., St.
  • a single colony of BL21 transformed with pGEX-SSU from an LB/ Amp (100 ⁇ g/mL) plate was used to inoculate 10 mL of LB containing 100 ⁇ g/mL ampicillin. These cultures were allowed to incubate overnight with shaking at 28°C. Two milliliters of the overnight culture was used inoculate each liter of LB containing 100 ⁇ g/mL ampicillin. The cultures were grown at 28°C until the absorbency at 600 nm was equal to 0.6. At this stage, 1 mL of 0.1 g/mL IPTG was added to each culture.
  • the cultures were allowed to remain at 28°C for 5 h with shaking after which time the cultures were placed on ice and the cells were harvested by centrifugation at 8000 rpm in a GS3 rotor for 15 min. The cells were stored frozen at -80°C until use.
  • the small subunit of plant ALS can be purified, using the appropriate affinity resin, as a fusion protein with a variety of affinity tags including but not exclusive to thioredoxin, hexahistidine and glutathione-S-transferase.
  • affinity tags including but not exclusive to thioredoxin, hexahistidine and glutathione-S-transferase.
  • the following example is for purification of the small subunit as a fusion protein with thioredoxin.
  • Approximately 11 g of GI724 cells harboring the plasmid pTrx-HIS/SSU were suspended in 33 mL of buffer containing 5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.9 and 0.5% Triton X-100.
  • the resuspended cells were sonicated using a half-inch horn on a Heat Systems Sonicator. Cells were sonicated, on ice, at full power using a 50% pulsed duty cycle for 15 s followed by 45 s rest. This sequence was repeated 19 times. The cell lysate was centrifuged at 10,000 rpm for 15 min in an SS34 rotor. The supernatant was transferred to a clean centrifuge tube and the centrifugation was repeated until the supernatant was clear. The lysate supernatant was added to 20 mL of ThioBondTM resin (Invitrogen). Binding of the fusion protein was allowed to proceed for 45 min by gently rocking the resin at room temperature.
  • ThioBondTM resin Invitrogen
  • the resin was allowed to settle and the liquid was decanted. Unbound protein was removed by washing the resin with 40 mL of buffer containing 50 mM MOPS, pH 7.0 and 1 mM EDTA. The resin was equilibrated with this buffer by gentle rocking and then allowed to settle and the supernatant was removed. This procedure was repeated twice.
  • the fusion protein was eluted by washing the resin with buffer containing 50 mM MOPS pH 7.0, 1 mM EDTA, 10 mM DTT and 10% glycerol. After equilibration, the supernatant was decanted and the procedure was repeated. The fusion protein was eluted with a total of 100 mL of buffer. The eluted protein was concentrated using an Amicon concentrator and a PM30 membrane. The sample was dialyzed into PBS buffer containing 10% glycerol and then frozen at -80°C for storage. Purification of GST-SSU
  • BL21 cells harboring the plasmid pGEX-SSU were suspended in 100 mL of 1 X PBS buffer.
  • the cell suspension was subjected to three passes through a microfluidizer.
  • a 10% Triton X-100 solution and a 20% sarkosyl solution were each added to the cell lysate to a final concentration of 1%.
  • the solution was allowed to equilibrate for 30 min at room temperature with gentle agitation.
  • the cell debris was then centrifuged at 10,000 rpm in a GSA rotor for 30 min. The supernatant was removed and added to 100 mL of glutathione agarose (Sigma).
  • the resin and lysate were allowed to equilibrate at room temperature for 30 minutes with gentle rocking.
  • the supernatant was removed by centrifugation at 500 x g (swinging bucket rotor) for 5 minutes.
  • the supernatant was decanted and the resin was washed with 200 mL of 1 X PBS.
  • the resin and PBS were allowed to equilibrate at room temperature for 10 minutes and with rocking and the supernatant was removed by centrifugation as above. The washing procedure was repeated a total of 4 times.
  • the bound GST-SSU was eluted by adding 200 mL of 25 mM Ches pH 9 containing 10 mM reduced glutathione.
  • the resin was equilibrated with the Ches buffer for 10 min with gentle rocking at room temperature. The supernatant was collected by centrifugation as above. The elution procedure was repeated a total of three times. The eluted protein was concentrated to 9 mL using a Millipore ultrafiltration apparatus. The protein was dialyzed extensively to remove the glutathione and then stored frozen at -80°C.
  • EXAMPLE 5 Expression and Purification of an Arabidopsis thaliana ALS large subunit
  • the ALS large subunit may also be cloned into a variety of expression vectors and purified as a fusion protein using numerous affinity chromatography strategies specific for, but not exclusive to, thioredoxin, glutathione-S-transferase and hexahistidine.
  • affinity chromatography strategies specific for, but not exclusive to, thioredoxin, glutathione-S-transferase and hexahistidine.
  • the procedure described below can be used for any ALS large subunit cloned into a pGEX expression vector ["GST Gene Fusion System” Pharmacia Biotech, 1996; Bernasconi et al., J. ofBiol. Chem. 270: 17381-17385 (1995)].
  • the plasmid pGATX was created by cloning the 5.8 kbp Xba I fragment of the genomic lambda phage clone 7 [Mazur et al. Plant Physiol. 55:1110-111 (1987)], into the plasmid vector pGEMl (Promega). This 5.8 kbp Xba I fragment contains a complete copy of the Arabidopsis thaliana ALS large subunit gene.
  • the plasmids pGEX-2TM (see Example 2) and pGATX were both digested to completion with Nco I and the digests then placed on Qiagen QIAquickTM columns and the Nucleotide Removal Kit protocol followed.
  • the final column eluant was adjusted to IX React 4 (Gibco BRL) and the DNAs were digested to completion with PinAI.
  • the pGEX-2TM digest was heated to 65° C for 10 minutes to destroy remaining PinAI activity and the DNA was dephosphorylated by incubating it at 37°C for 30 min. with 1 unit of calf intestinal alkaline phosphatase. Both pGEX-2TM and pGATX were subjected to electrophoresis using a 1% agarose gel.
  • the 2 kbp fragment from pGATX and the 4.9 kbp fragment from pGEX-2TM were removed from the gel and the DNAs were recovered using a QIAquickTM Gel Extraction Kit according to the manufacturer's instructions. Purified DNAs were precipitated, re-dissolved and quantified as above. Approximately 90 ng of 2.0 kbp pGATX fragment were mixed with 120 ng of the NcoI/PinAI digested pGEX-2TM and mixture was heated for 5 minutes at 45° C followed by cooling on ice. T4 DNA ligase buffer and 1 unit of T4 DNA ligase were mixed with the fragments and mixture was incubated for 4 hours at ambient temperature.
  • a single colony of BL21(DE3) transformed with pGEX-OCM2 (encoding a fusion protein designated GST- ALS, comprising the glutathione-S-transferase fused to the Arabidopsis thaliana large subunit of acetolactate synthase, including the chloroplast transit peptide) from an LB/Amp (100 ⁇ g/mL) plate is used to inoculate 10 mL of LB containing 100 ⁇ g/mL carbenicillin. These cultures were allowed to incubate overnight with shaking at 30°C. Two milliliters of the overnight culture was used inoculate each liter of LB containing 100 ⁇ g/mL carbenicillin.
  • the cultures were grown at 30°C until the absorbency at 600 nm was equal to 0.6. At this stage, 1 mL of 0.1 g/mL IPTG was added to each culture. The cultures were allowed to remain at 30°C for 4 h with shaking after which time the cultures were placed on ice and the cells were harvested by centrifugation at 8000 rpm in a GS3 rotor for 15 min. The cells were stored frozen at -80°C until use.
  • oligodeoxynucleotides mt704+ (SEQ ID NO: 17) and mt800- (SEQ ID NO: 18) were combined in 100 uL of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , heated briefly in a boiling water bath and slow cooled to 50°C. The resulting double stranded DNA fragment was digested to completion with Nde I and Eco0109I.
  • the plasmid ALS 10 that contains a full length cDNA copy of the Nicotiana plumbaginifolia ALS gene in the plasmid vector pBluescript was used as the source of the ALS coding region for pMTDRALS.
  • the sequence of this cDNA is shown in SEQ ID NO: 19.
  • ALS 10 was digested to completion with Xho I and EcoOl 091, and the digestion products were separated by agarose gel electrophoresis.
  • the 1.9 kbp Xho 1/ Eco0109I ALS 10 DNA fragment was excised from the gel and the DNA was recovered using a QIAquickTM Gel Extraction Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions.
  • the plasmid pET24a (Novagen, Inc., Madison, WI) was digested to completion with Nde I and Xho I and the DNA was dephosphorylated with calf intestinal alkaline phosphatase. Equimolar amounts of Nde I/Xho I digested and dephosphorylated pET24a and the 2.0 kbp Xho II Nde I digestion product were ligated together and an aliquot of the ligation mixture was used to transform competent E. coli DH5 ⁇ as described above. Aliquots of the transformation mixture were spread onto LB plates containing 100 ⁇ g/ml kanamycin.
  • pMTDRALS plasmids isolated from kanamycin-resistant bacterial colonies were analyzed for the presence of the insert encoding the ALS large subunit by double digestion with Nde I and Xho I.
  • One such plasmid, designated pMTDRALS was then transformed into E. coli. BL21(DE3) using methods known to those skilled in the art. This plasmid directs the expression of the Nicotiana plumbaginifolia ALS large subunit.
  • This overnight culture was in turn used to inoculate 1 liter of minimal M9 medium supplemented with 2 mL IM MgSO4, 1 ml CaCl2, 50 ⁇ L 1% vitamin Bl, 40 ml of 10% casamino acids and 30 ⁇ g/mL kanamycin.
  • the cells were grown until ODOOO as equal to 0.6 and expression was induced by the addition of IPTG to a final concentration of 0.5 mM.
  • the cells were harvested by centrifugation 5 hours later.
  • the tubes containing ALS activity were combined and the protein precipitated with 50% ammonium sulfate.
  • the precipitated protein was centrifuged at 11000 rpm (GSA rotor) and the pellet resuspended in Tris, pH 7.2 containing ImM EDTA, ImM DTT and 20 ⁇ M FAD. The solutions were kept dark.
  • the redissolved protein was desalted and fractionated using an S300 filtration column equilibrated with the FAD containing Tris buffer.
  • the tubes containing ALS activity were combined, concentrated and the material loaded onto a MonoQ column equilibrated with Tris, pH 7.2 containing ImM EDTA, ImM DTT and developed with a linear NaCl gradient to 0.5M.
  • the resulting enzyme was concentrated and washed with Tris buffer and stored as frozen aliquots in 10% glycerol. The exposure of solutions to light was minimized during this purification.
  • ALS holoenzyme As discussed above, one way to prepare the ALS holoenzyme is to mix purified or partially purified large subunit with purified or partially purified small subunit.
  • a typical subunit mixing experiment which demonstrates the effect of the small subunit on the specific activity of the LSU consists of the samples and the results shown in Table 1.
  • thaliana LSU expressed as a glutathione-S-transferase fusion protein from plasmid pGEX-OCM2.
  • Trx-HIS/SSU Expressed as units/mg LSU. Each sample contained 3 ⁇ l of 10X PBS and distilled, deionized water was added to each sample to bring the final volume to 30 ⁇ L. When small subunit was not added to the sample, an equivalent amount of BSA was added as a control protein.
  • ALS-LSU is a fusion between GST and the LSU of Arabidopsis ALS which contains the complete chloroplast transit peptide on the N-terminus of the LSU.
  • GST-LSU fusion is cleaved to give free GST and Arabidopsis LSU.
  • the other ALS-LSU (LSU) is from Nicotiana plumbaginifolia and contains only a partial chloroplast transit peptide on the N-terminus as described above and contains no thrombin cleavage site. Each sample was assayed for ALS activity (see below) prior to thrombin addition.
  • Table 1 show that the presence of the small subunit increases the specific activity of the LSU before and after thrombin cleavage of the fusion protein regardless of whether the LSU is from Arabidopsis or Nicotiana and that the SSU positively affects the stability of the LSU activity.
  • a comparison of the specific activity of sample 1 and 2 before thrombin addition shows that when Trx-HIS/SSU is added to GST-LSU from Arabidopsis there is a 3-fold increase in specific activity even with fusion proteins attached to both large and small subunits.
  • a comparison of the specific activities of samples 3 and 4 prior to thrombin addition shows that the presence of the Trx-HIS/SSU increases the specific activity of the Nicotiana LSU by a factor of 5.
  • the specific activity of the Arabidopsis LSU decreases from 0.3 to 0.04 units/mg LSU.
  • the specific activity of the Arabidopsis LSU increases slightly compared to the same sample in the absence of thrombin (sample 2) to 1.1 units/mg LSU representing more than a 20 fold increase in specific activity compared to the Arabidopsis LSU alone.
  • sample 3 the addition of thrombin does not cleave the Nicotiana large subunit, but the enzyme loses some activity during the incubation period.
  • ALS assays were conducted using the following reaction mixture containing 100 mM sodium phosphate pH 7.6, 0.5 mM dithiothreitol, 1 mM MgCl 2 , 100 ⁇ M thiamine pyrophosphate, and 100 ⁇ M flavin adenine dinucleotide.
  • the assay was be conducted in a microtiter plate wherein each well contained 80 ⁇ L of assay mix and 10 ⁇ L of 500 mM sodium pyruvate. The mixture was allowed to equilibrate for 5 min at 37°C. The desired amount of enzyme to be assayed was then added to the well and mixed by gentle shaking. The plate was incubated for the desired reaction time at 37°C.
  • the reaction was quenched by the addition of 10 ⁇ L 3 M H 2 SO 4 to each well.
  • the contents of the plate were mixed well with gentle shaking and incubated at 60°C for 15 min.
  • the amount of acetoin produced was detected by the rapid addition of 100 ⁇ L of 0.5% creatine and 100 ⁇ L of 5% ⁇ -naphthol in 2.5 M NaOH.
  • the contents of the plate were mixed and the plate was incubated for 15 min at 60°C uncovered.
  • the plate was cooled to room temperature for 5 min with constant gentle shaking and the absorbency at 530 nm was read.
  • the specific activity can be calculated based upon the concentration of the large subunit given that 1 ⁇ mole of acetoin produces an absorbency of 0.35 under these conditions.
  • EXAMPLE 8 A holoenzyme mixture was prepared containing 50 ⁇ L of N. plumbaginifolia LSU (0.7 mg/mL; encoded by pMTDRALS), 60 ⁇ L GST-SSU (1.22 mg/mL; encoded by pGEX-SSU)), 10 ⁇ L distilled, deionized water, and 180 ⁇ L of a buffer/cofactor mixture containing 100 mM phosphate, pH 7.6, 10% glycerol, 0.5 mM DTT, 2 mM MgCl 2 , 200 ⁇ M FAD and 200 ⁇ M TPP.
  • a buffer/cofactor mixture containing 100 mM phosphate, pH 7.6, 10% glycerol, 0.5 mM DTT, 2 mM MgCl 2 , 200 ⁇ M FAD and 200 ⁇ M TPP.
  • the fusion proteins were cleaved by the addition of 1 ⁇ L of Thrombin (0.8 units/mL; Novagen) to the mixture and the solution was allowed to incubate at 25°C for several hours.
  • a microtiter plate was prepared in which each well contains 80 ⁇ L of assay mix (see ALS assay procedure above) and 10 ⁇ L of a stock solution containing the desired concentration of the inhibitor to be tested. The plate was incubated at 37°C for 5 min and then 1 ⁇ L of the holoenzyme mixture was added to each well. The reaction was started by the immediate addition of 10 ⁇ L of 500 mM sodium pyruvate to each well.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTGGCTCCTT GGATAGATCT 20
  • AGG GCA AAA ATT GTG GAT ATA TCT GAT CAA TCT CTG ACT ATT GAG GTA 612 Arg Ala Lys He Val Asp He Ser Asp Gin Ser Leu Thr He Glu Val 130 135 140 ACT GGA GAT CCA GGG AAG ATG GTG GCT GTT CAG AGG AAC TTA AGT AAA 660
  • GAA AAA ATG GGG GAA TCT GCT CCT TTT TGG CGG TTT TCA GCA GCA TCA 756
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 15: TGCGGAATGT GTGCGAACGT GGATGACTGA ATGGATCCGG TAC 43
  • MOLECULE TYPE other nucleic acid
  • TTC ACC ATC TCC AAT GTC ATT TCC ACT ACC CAA AAA GTT TCC GAG ACC 722 Phe Thr He Ser Asn Val He Ser Thr Thr Gin Lys Val Ser Glu Thr 65 70 75
  • GCT GTT GGA AGA CCG GAT 2066 Gly Phe Gly Leu Pro Ala Ala He Gly Ala Ala Val Gly Arg Pro Asp 510 515 520 525

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Abstract

La présente invention concerne une séquence d'ADNc codant la petite sous-unité de l'acétolactate synthase ('ALS') d'une plante. Lorsque la protéine de la petite sous-unité de l'ALS d'une plante est mélangée avec le produit génique, que certains appellent acétolactate synthase de la plante (et qui est ici dénommé grande sous-unité de l'ALS), l'holoenzyme d'ALS reconstitué résultant présente à la fois une efficacité catalytique et une stabilité dans la solution accrues par rapport à celles de la grande sous-unité seule. L'holoenzyme de l'acétolactate synthase est sensible à l'inhibition par les composés herbicides dont le mode d'action est connu pour inhiber l'ALS in vivo. Ainsi, l'holoenzyme de l'ALS est utilisé dans le criblage en vue de trouver de nouveaux composés chimiques de protection des cultures.
PCT/US1998/003506 1997-02-24 1998-02-23 Utilisation de la petite sous-unite de l'acetolactate synthase d'une plante permettant la decouverte d'un nouvel herbicide WO1998037206A1 (fr)

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AU66641/98A AU6664198A (en) 1997-02-24 1998-02-23 Use of the small subunit of plant acetolactate synthase for new herbicide discovery

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Cited By (4)

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WO2000026390A2 (fr) 1998-10-29 2000-05-11 American Cyanamid Company Genes et vecteurs servant a conferer une resistance aux herbicides aux plantes
WO2002010210A2 (fr) * 2001-08-28 2002-02-07 Bayer Cropscience Ag Polypeptides permettant d'identifier de nouveaux composes herbicides actifs
FR2832421A1 (fr) * 2001-11-20 2003-05-23 Hoquet Michel Sequence nucleotidique codant pour une enzyme ayant une activite acetolactate synthase, cellule vegetale et plante la contenant, et procede de desherbage de plantes
EP1776462A2 (fr) * 2004-08-04 2007-04-25 BASF Plant Science GmbH Séquences ahass de monocotylédone et leurs méthodes d'utilisation

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EP0608722A1 (fr) * 1993-01-29 1994-08-03 American Cyanamid Company Procédés de dépistage pour la détection des herbicides
US5356789A (en) * 1993-05-28 1994-10-18 American Cyanamid Company Methods for detecting acetohydroxyacid synthase inhibitors

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EP0608722A1 (fr) * 1993-01-29 1994-08-03 American Cyanamid Company Procédés de dépistage pour la détection des herbicides
US5356789A (en) * 1993-05-28 1994-10-18 American Cyanamid Company Methods for detecting acetohydroxyacid synthase inhibitors

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* Cited by examiner, † Cited by third party
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US7351880B2 (en) 1998-10-29 2008-04-01 Basf Aktiengesellschaft Genes and vectors for conferring herbicide resistance in plants
WO2000026390A3 (fr) * 1998-10-29 2000-08-31 American Cyanamid Co Genes et vecteurs servant a conferer une resistance aux herbicides aux plantes
US6348643B1 (en) 1998-10-29 2002-02-19 American Cyanamid Company DNA sequences encoding the arabidopsis acetohydroxy-acid synthase small subunit and methods of use
JP2010263901A (ja) * 1998-10-29 2010-11-25 Wyeth Holdings Corp 植物に除草剤抵抗性を付与するための遺伝子およびベクター
WO2000026390A2 (fr) 1998-10-29 2000-05-11 American Cyanamid Company Genes et vecteurs servant a conferer une resistance aux herbicides aux plantes
AU770361B2 (en) * 1998-10-29 2004-02-19 American Cyanamid Company Genes and vectors for conferring herbicide resistance in plants
US6825399B2 (en) * 1998-10-29 2004-11-30 Basf Aktiengesellschaft Genes and vectors for conferring herbicide resistance in plants
US7498429B2 (en) 1998-10-29 2009-03-03 Basf Se AHAS small subunit promoter
WO2002010210A2 (fr) * 2001-08-28 2002-02-07 Bayer Cropscience Ag Polypeptides permettant d'identifier de nouveaux composes herbicides actifs
WO2002010210A3 (fr) * 2001-08-28 2003-02-20 Bayer Ag Polypeptides permettant d'identifier de nouveaux composes herbicides actifs
WO2003044200A1 (fr) * 2001-11-20 2003-05-30 Hoquet, Michel Sequence nucleotidique codant pour une enzyme ayant une activite acetolactate synthase de cichorium
FR2832421A1 (fr) * 2001-11-20 2003-05-23 Hoquet Michel Sequence nucleotidique codant pour une enzyme ayant une activite acetolactate synthase, cellule vegetale et plante la contenant, et procede de desherbage de plantes
EP1776462A2 (fr) * 2004-08-04 2007-04-25 BASF Plant Science GmbH Séquences ahass de monocotylédone et leurs méthodes d'utilisation
EP1776462A4 (fr) * 2004-08-04 2010-03-10 Basf Plant Science Gmbh Séquences ahass de monocotylédone et leurs méthodes d'utilisation

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