EP1090135A2 - Procedes de criblage de composes herbicides utilisant air synthetase d'arabidopsis thaliana - Google Patents

Procedes de criblage de composes herbicides utilisant air synthetase d'arabidopsis thaliana

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Publication number
EP1090135A2
EP1090135A2 EP99936458A EP99936458A EP1090135A2 EP 1090135 A2 EP1090135 A2 EP 1090135A2 EP 99936458 A EP99936458 A EP 99936458A EP 99936458 A EP99936458 A EP 99936458A EP 1090135 A2 EP1090135 A2 EP 1090135A2
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EP
European Patent Office
Prior art keywords
plant
enzyme
air synthetase
air
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99936458A
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German (de)
English (en)
Inventor
Eric Russell Ward
Charles David Guyer
Sharon Lee Potter
Venkiteswaran Subramanian
Eric Walters
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Syngenta Participations AG
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Syngenta Participations AG
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Publication of EP1090135A2 publication Critical patent/EP1090135A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the invention relates to methods for screening herbicidal compounds which inhibit the enzymatic activity of 5'-phosphor ⁇ bosyl-5-am ⁇ no ⁇ m ⁇ dazole (AIR) synthetase, an enzyme involved in de novo purine biosynthesis.
  • AIR 5'-phosphor ⁇ bosyl-5-am ⁇ no ⁇ m ⁇ dazole
  • the invention also relates to the use of thereby identified herbicidal chemicals to control the growth of undesired vegetati ⁇ n
  • the invention may also be applied to the development of herbicide tolerance in plants, plant tissues, plant seeds, and plant cells.
  • the AIR synthetase is an enzymatic step in the de novo purine biosynthesis pathway, which leads to the synthesis of the purine nucleotides IMP, AMP and GMP.
  • De novo purine biosynthesis plays a central role in the nitrogen assimilation pathway and is conserved among bacteria, yeast, Drosophila and mammals (Schnorr et al. (1994) The Plant Journal, 6: 1 13-121 ).
  • the AIR synthetase enzymatic activity corresponds to the fifth step in the pathway and catalyzes the conversion of 5'-phosphor ⁇ bosyl-N- formylglycinamidine (FGAM) to 5'-phosphor ⁇ bosyl-5-am ⁇ no ⁇ m ⁇ dazole (AIR).
  • FGAM 5'-phosphor ⁇ bosyl-N- formylglycinamidine
  • AIR 5'-phosphor ⁇ bosyl-5-am ⁇ no ⁇ m ⁇ dazole
  • this step is carried out by a protein encoded by the purM gene.
  • an Arabidopsis c-DNA encoding an enzyme having AIR synthetase activity has been cloned and its sequence has been determined (Senecoff and Meagher (1993) Plant Physiol. 102. 387-399; Schnorr et al (1994) The Plant Journal, 6: 1 13-121 ).
  • Herbicides that exhibit greater potency, broader weed spectrum, and more rapid degradation in soil can also, unfortunately, have greater crop phytotoxicity.
  • One solution applied to this problem has been to develop crops that are resistant or tolerant to herbicides. Crop hybrids or varieties tolerant to the herbicides allow t ⁇ r the use of the herbicides to kill weeds without attendant risk of damage to the crop. Development of tolerance can allow application of a herbicide to a crop where its use was previously precluded or limited (e.g. to pre-emergence use) due to sensitivity of the crop to the herbicide.
  • U.S. Patent No. 4,761 ,373 to Anderson ef al. is directed to plants resistant to various imidazolinone or sulfonamide herbicides.
  • U.S. Patent No. 4,975,374 to Goodman et al. relates to plant cells and plants containing a gene encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that were known to inhibit GS, e.g. phosphinothricin and methionine sulfoximine.
  • GS glutamine synthetase
  • U.S. Patent No. 5,013,659 to Bedbrook et al. is directed to plants expressing a mutant acetolactate synthase that renders the plants resistant to inhibition by sulfonylurea herbicides.
  • One object of the present invention is to provide methods for identifying new or improved herbicides. Another object of the invention is to provide methods for using such new or improved herbicides to suppress the growth of plants such as weeds. Still another object of the invention is to provide improved crop plants that are tolerant to such new or improved herbicides.
  • the inventors of the present invention have demonstrated that the 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase activity is essential in plants. This implies that chemicals which inhibit AIR synthetase in plants are likely to have detrimental effects on plants and are potentially good herbicide candidates.
  • the present invention therefore provides methods of using a purified AIR synthetase to identify inhibitors thereof, which can then be used as herbicides to suppress the growth of undesirable vegetation, e.g.
  • crops in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • the present invention discloses for the first time the correct nucleotide sequence of the Arabidopsis AIR synthetase gene.
  • the nucleotide sequence encodingJhe pre-protein is set forth in SEQ ID NO:1 and the nucleotide sequence encoding the putative mature protein is set forth in SEQ ID NO:3.
  • the correct amino acid sequence of the Arabidopsis AIR synthetase pre-protein is set forth in SEQ ID NO:2 and of the correct amino acid sequence of the putative mature Arabidopsis AIR synthetase is set forth in SEQ ID NO:4.
  • the present invention also encompasses isolated enzymes having AIR synthetase activity and comprising an amino acid sequence that is identical or substantially similar to the amino acid sequences set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the amino acid sequence is derived from a plant.
  • the present invention also encompasses an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the nucleotide sequence is SEQ ID NO:1 or SEQ ID NO:3.
  • the nucleotide sequence is deposited in E. coli strain DH ⁇ apASM designated as NRRL accession number B-21976.
  • a chimeric gene comprising a heterologous promoter sequence operatively linked to the nucleic acid molecule of the invention; a recombinant vector comprising such a chimeric gene; and a host cell comprising such a chimeric gene.
  • the host cell is a bacterial cell, a yeast cell, or a plant cell.
  • the present invention also encompasses a plant comprising a plant cell of the invention and seed from such a plant.
  • the present invention describes a method of identifying chemicals having the ability to inhibit plant growth or viability, comprising:(a) combining an enzyme having AIR synthetase activity in a first reaction mixture with a substrate of AIR synthetase under conditions in which the enzyme is capable of catalyzing the synthesis of AIR; (b) combining the chemical to be tested and the enzyme in a second reaction mixture with a substrate of AIR synthetase under the same conditions and for the same period of time as in the first reaction mixture; (c) determining and comparing the activity of the enzyme in the first and second reaction mixtures; wherein less, desirably significantly less, enzyme activity in the second reaction mixture than in the first reaction mixture indicates that the chemical of (b) has the ability to inhibit plant growth or viability.
  • the substrate of AIR synthetase is 5'-phosphoribosyl-N-formylglycinamidine (FGAM) and in a further preferred embodiment, the substrate of AIR synthetase is b-FGAM.
  • the enzyme having AIR synthetase activity is derived from a plant and more preferably, is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:- " or SEQ ID NO:3.
  • the AIR synthetase enzyme is encoded by a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • the AIR synthetase enzyme has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the chemical is capable of inhibiting the growth or viability of a plant by inhibiting the activity of AIR synthetase in the plant.
  • the activity of the enzyme is determined by measuring the AIR produced in the reaction mixture.
  • the activity of the enzyme is determined by measuring the ADP derived from ATP in the reaction mixture.
  • the present invention describes a method of identifying chemicals having the ability to inhibit plant growth or viability, comprising: (a) combining an enzyme having 5'-phosphoribosyl-N-formylglycinamidine (FGAM) synthetase activity and an enzyme having AIR synthetase activity in a first reaction mixture with a substrate of FGAM synthetase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of AIR; (b) combining a chemical to be tested and the enzymes in a second reaction mixture with a substrate of FGAM synthetase under the same conditions and the same period of time as in the first reaction mixture; and (c) determining and comparing the activity of the enzyme having AIR synthetase activity in the first and second reaction mixtures; wherein less, preferably significantly less, AIR synthetase enzyme activity in the second reaction mixture than in the first reaction mixture indicates that the chemical of (b) has the ability to inhibit plant growth or
  • the substrate of FGAM synthetase is 5'-phosphoribosyl-N-formylglycinamide (FGAR) and in a further preferred embodiment, the substrate of FGAM synthetase is b- FGAR.
  • the enzyme having AIR synthetase activity is derived from a plant and more preferably, is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.
  • the AIR synthetase enzyme is encoded by a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • the AIR synthetase enzyme has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the chemical is capable of inhibiting the growth or viability of a plant by inhibiting the activity of AIR synthetase in the plant.
  • the activity of the enzyme is determined by measuring the AIR produced in the reaction mixture.
  • the activity of the enzyme is determined by measuring the ADP derived from ATP in the reaction mixture.
  • the present invention also further describes an assay comprising the steps of: (a) combining an enzyme having 5'-phosphoribosyl-N-formylglycinamidine (FGAM) synthetase activity and an enzyme having AIR synthetase activity in a first reaction mixture with a substrate of FGAM synthetase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of AIR; (b) combining a chemical and the enzymes in a second reaction mixture with a substrate of FGAM synthetase under the same conditions and for the same period of time as in the first reaction mixture; (c) determining the activity of the enzyme having AIR synthetase activity in the first and second reaction mixtures; wherein the chemical is capable of inhibiting the activity of the enzyme having AIR synthetase activity if the activity of the enzyme having AIR synthetase activity in the second reaction mixture is less, desirably significantly less, than the activity of the enzyme having AIR synth
  • the substrate of FGAM synthetase is 5'-phosphoribosyl-N-formylglycinamide (FGAR) and in a further preferred embodiment, the substrate of FGAM synthetase is b-FGAR.
  • the activity of the enzyme is determined by measuring the AIR produced in the reaction mixture.
  • the reaction mixture comprises ATP and the activity of the enzyme is determined by measuring the ADP derived from ATP in the reaction mixture.
  • the present invention describes a method for identifying chemicals having herbicidal activity that inhibit AIR synthetase activity in plants, comprising:(a) obtaining transgenic plants, plant tissue, plant seeds or plant cells comprising an isolated nucleotide sequence encoding an enzyme having AIR synthetase activity and capable of overexpressing an enzymatically active AIR synthetase; (b) applying a chemical to be tested to the transgenic plants, plant cells, tissues or parts and to the isogenic non-transformed plants, plant cells, tissues or parts; (c) determining the growth or viability of the transgenic and non-transformed plants, plant cells, tissues after application of the chemical; and (d) comparing the growth or viability of the transgenic and non- transformed plants, plant cells, tissues after application of the chemical, wherein suppression of the growth or viability of the non-transgenic plants, plant cells, tissues or parts, without significantly suppressing the growth or viability of the isogenic transgenic plants, plant cells, tissues or parts indicates that the chemical
  • the chemical suppresses the viability or growth of the non-transgenic plants, plant cells, tissues or parts, without significantly suppressing the growth of the viability or growth of the isogenic transgenic plants, plant cells, tissues or parts.
  • the enzyme having AIR synthetase activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.
  • the AIR synthetase enzyme is encoded by a nucleotide sequence capable of encoding the ammo acid sequence of SEQ ID NO:2 or SEQ ID NO.4.
  • the AIR synthetase enzyme has an ammo acid sequence identical or substantially similar to the ammo acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the present invention further embodies plants, plant tissues, plant seeds, and plant cells that have modified AIR synthetase activity and that are therefore tolerant to inhibition by a herbicide at levels normally inhibitory to naturally occurring AIR synthetase activity
  • Herbicide tolerant plants encompassed by the invention include those that would otherwise be potential targets for normally inhibiting herbicides, particularly the agronomically important crops mentioned above.
  • plants, plant tissue, plant seeds, or plant cells are transformed, preferably stably transformed, with a recombinant DNA molecule comprising a suitable promoter functional in plants operatively linked to a nucleotide coding sequence that encodes a modified AIR synthetase that is tolerant to inhibition by a herbicide at a concentration that would normally inhibit the activity of wild- type, unmodified AIR synthetase.
  • Modified AIR synthetase activity may also be conferred upon a plant by increasing expression of wild-type herbicide-sensitive AIR synthetase by providing multiple copies of wild-type AIR synthetase genes to the plant or by overexpression of wild-type AIR synthetase genes under control of a stronger-than-wild- type promoter
  • the transgenic plants, plant tissue, plant seeds, or plant cells thus created are then selected by conventional selection techniques, whereby herbicide tolerant lines are isolated, characterized, and developed Alternately, random or site-specific mutagenesis may be used to generate herbicide tolerant lines.
  • the present invention provides a plant, plant cell, plant seed, or plant tissue transformed with a DNA molecule comprising a nucleotide sequence isolated from a plant that encodes an enzyme having AIR synthetase activity, wherein the enzyme has AIR synthetase activity and wherein the DNA molecule confers upon the plant, plant cell, plant seed, or plant tissue tolerance to a herbicide in amounts that normally inhibits naturally occurring AIR synthetase activity.
  • the enzyme having AIR synthetase activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NOrt-or SEQ ID NO:3, or has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the invention also provides a method for suppressing the growth of a plant comprising the step of applying to the plant a chemical that inhibits the naturally occurring AIR synthetase activity in the plant.
  • the present invention is directed to a method for selectively suppressing the growth of weeds in a field containing a crop of planted crop seeds or plants, comprising the steps of: (a) planting herbicide tolerant crops or crop seeds, which are plants or plant seeds that are tolerant to a herbicide that inhibits the naturally occurring AIR synthetase activity; and (b) applying to the crops or crop seeds and the weeds in the field a herbicide in amounts that inhibit naturally occurring AIR synthetase activity, wherein the herbicide suppresses the growth of the weeds without significantly suppressing the growth of the crops.
  • the present invention further provides a method for forming a mutagenized DNA molecule encoding an enzyme having AIR synthetase activity from a template DNA molecule encoding an enzyme having AIR synthetase activity, wherein said template DNA molecule has been cleaved into double-stranded-random fragments, comprising the steps of: (a) adding to the resultant population of double-stranded-random fragments at least one single-stranded or double-stranded oligonucleotide, wherein said oligonucleotide comprises an area of identity and an area of heterology to the template DNA molecule; (b) denaturing the resultant mixture of double-stranded-random fragments and oligonucleotides into single- stranded molecules; (c) incubating the resultant population of single-stranded molecules with a polymerase under conditions which result in the annealing of said single-stranded molecules at said areas of identity to form pairs of annealed
  • a mutagenized DNA molecule encoding an enzyme having AIR synthetase activity obtained by the above method, wherein said mutagenized DNA molecule encodes an AIR synthetase enzyme having enhanced tolerance to a herbicide which inhibits the AIR synthetase activity encoded by said template DNA molecule.
  • the present invention also provides a method for forming a mutagenized DNA molecule encoding an enzyme having AIR synthetase activity from at least two non-identical template DNA molecules encoding enzymes having AIR synthetase activity, comprising the steps of: (a) adding to the template DNA molecules at least one oligonucleotide comprising an area of identity to each of the template DNA molecule; (b) denaturing the resultant mixture into single-stranded molecules; (c) incubating the resultant population of single- stranded molecules with a polymerase under conditions which result in the annealing of the oligonucleotides to the template DNA molecules, wherein the conditions for polymerization by the polymerase are such that polymerization products corresponding to a portion of the template DNA molecules are obtained; (d) repeating the second and third steps for at least two further cycles, wherein the extension products obtained in the third step are able to switch template DNA molecule for polymerization in the next cycle, thereby forming
  • a mutagenized DNA molecule encoding an enzyme having AIR synthetase activity obtained by the above method, wherein said mutagenized DNA molecule encodes an AIR synthetase enzyme having enhanced tolerance to a herbicide which inhibits the AIR synthetase activity encoded by said template DNA molecule.
  • At least one template DNA molecule is derived from a eukaryote. More preferably, said eukaryote is a plant. Still more preferably, said plant is Arabidopsis thaliana. Most preferably, said species of template DNA molecule is identical or substantially similar to the SEQ ID NO:1 or SEQ ID NO:3. In another embodiment of either of the above two methods, at least one template DNA molecule is derived from a prokaryote.
  • Activatable DNA Sequence a DNA sequence that regulates the expression of genes in a genome, desirably the genome of a plant.
  • the activatable DNA sequence is complementary to a target gene endogenous in the genome. When the activatable DNA sequence is introduced and expressed in a cell, it inhibits expression of the target gene.
  • An activatable DNA sequence useful in conjunction with the present invention includes those encoding or acting as dominant inhibitors, such as a translatable or untranslatable sense sequence capable of disrupting gene function in stably transformed plants to positively identify one or more genes essential for normal growth and development of a plant.
  • a preferred activatable DNA sequence is an antisense DNA sequence.
  • the target gene preferably encodes a protein, such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the plant.
  • the target gene encodes an enzyme having AIR synthetase activity.
  • Activatable DNA Construct a recombinant DNA construct comprising a synthetic promoter operatively linked to the activatable DNA sequence, which when introduced into a cell, desirably a plant cell, is not expressed, i.e. is silent, unless a complete hybrid transcription factor capable of binding to and activating the synthetic promoter is present.
  • the activatable DNA construct is introduced into cells, tissues, or plants to form stable transgenic lines capable of expressing the activatable DNA sequence.
  • Co-factor natural reactant, such as an organic molecule or a metal ion, required in an enzyme-catalyzed reaction.
  • a co-factor is e.g. NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.
  • Coupled synthesis a enzymatic biosynthesis, in which a final product is synthesized by two sequential enzymatic steps, wherein the substrate for the first enzymatic step is converted by the first enzyme to an intermediate product, which serves as a substrate for the second enzymatic step and is converted by the second enzyme to the final product, without external addition of the intermediate product.
  • DNA shuffling is a method to introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly.
  • the DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturaHy occurring DNA molecule derived from at least one template DNA molecule.
  • the shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.
  • Enzyme activity means herein the ability of an enzyme to catalyze the conversion of a substrate into a product.
  • a substrate for the enzyme comprises the natural substrate of the enzyme but also comprises analogues of the natural substrate which can also be converted by the enzyme into a product or into an analogue of a product.
  • the activity of the enzyme is measured for example by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time.
  • the activity of the enzyme is also measured by determining the amount of an unused co-factor of the reaction remaining in the reaction mixture after a certain period of time or by determining the amount of used co-factor in the reaction mixture after a certain period of time.
  • the activity of the enzyme is also measured by determining the amount of a donor of free energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine) remaining in the reaction mixture after a certain period of time or by determining the amount of a used donor of free energy or energy-rich molecule (e.g. ADP, pyruvate, acetate or creatine) in the reaction mixture after a certain period of time.
  • a donor of free energy or energy-rich molecule e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine
  • Herbicide a chemical substance used to kill or suppress the growth of plants, plant cells, plant seeds, or plant tissues.
  • Heterologous DNA Sequence a DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • Homologous DNA Sequence a DNA sequence naturally associated with a host cell into which it is introduced.
  • Inhibitor a chemical substance that inactivates the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the plant.
  • an inhibitor is a chemical substance that inactivates the enzymatic activity of AIR synthetase from a plant.
  • the term "herbicide” is used herein to define an inhibitor when applied to plants, plant cells, plant seeds, or plant tissues.
  • Isogenic plants which are genetically identical, except that they may differ by the presence or absence of a transgene.
  • an isolated DNA molecule or an isolated enzyme in the context of the present invention, is a DNA molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • Mature protein protein which is normally targeted to a cellular organelle, such as a chloroplast, and from which the transit peptide has been removed.
  • Minimal Promoter promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
  • Modified Enzyme Activity enzyme activity different from that which naturally occurs in a plant (i.e. enzyme activity that occurs naturally in the absence of direct or indirect manipulation of such activity by man), which is tolerant to inhibitors that inhibit the naturally occurring enzyme activity.
  • Pre-protein protein which is normally targeted to a cellular organelle, such as a chloroplast, and still comprising its transit peptide.
  • an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
  • the amount of a product of an enzymatic reaction is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2-fold or greater of the activity of the wild-type enzyme in the absence of the inhibitor, more preferably an decrease by about 5-fold or greater, and most preferably an decrease by about 10-fold or greater.
  • the term "substantially similar”, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur.
  • the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence.
  • the percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 65%, more desirably at least 75%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, yet still more preferably at least 99%. Sequence comparisons are carried out using a Smith-Waterman sequence alignment algorithm (see e.g. Waterman, M.S. Introduction to Computational Biology: Maps, sequences and genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391 -0, or at http://www.hto.usc.edu/software/seqaln/index.html).
  • the locals program version 1 .16, is used with following parameters: match: 1 , mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.
  • a nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1 % SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1 %
  • substantially similar when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. where only changes in amino acids sequence not affecting the polypeptide function occur.
  • the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is at least 65%, more desirably at least 75%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, yet still more preferably at least 99%.
  • a substrate is the molecule that the enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function, or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.
  • Tolerance the ability to continue normal growth or function when exposed to an inhibitor or herbicide.
  • Transformation a process for introducing heterologous DNA into a cell, tissue, or plant.
  • Transformed cells, tissues, or plants are understood to encompass-not only the end product of a transformation process, but also transgenic progeny thereof.
  • Transgenic stably transformed with a recombinant DNA molecule that preferably comprises a suitable promoter operatively linked to a DNA sequence of interest.
  • SEQ ID NO:1 DNA sequence encoding the Arabidopsis AIR synthetase pre-protein
  • SEQ ID NO:3 DNA sequence encoding the putative mature Arabidopsis AIR synthetase
  • the Arabidopsis AIR synthetase gene was re-sequenced by the inventors of the present invention and compared to a published DNA sequence for the Arabidopsis AIR synthetase gene (Genbank accession L12457, Senecoff and Meagher (1993) Plant Physiol. 102: 387-399). Sequencing results revealed a substantial error in the published DNA sequence, resulting in the insertion of a cytosine base at the position corresponding to position 1 ,027 in SEQ ID NO:1 . This insertion leads to a frame-shift mutation in the amino acid sequence and therefore teaches away from the correct deduced amino acid sequence for the Arabidopsis AIR synthetase.
  • the present invention discloses fo he first time the correct nucleotide sequence of the Arabidopsis AIR synthetase gene as well as the correct amino acid sequence of the Arabidopsis AIR synthetase.
  • the nucleotide sequence encoding the pre-protein is set forth in SEQ ID NO:1 and the nucleotide sequence encoding the mature protein is set forth in SEQ ID NO:3.
  • the correct amino acid sequence of the Arabidopsis AIR synthetase pre-protein encoded by the nucleotide sequence set forth in SEQ ID NO:1 is set forth in SEQ ID NO:2 and the correct amino acid sequence of the putative mature Arabidopsis AIR synthetase encoded by the nucleotide sequence set forth in SEQ ID NO:2 is set forth in SEQ ID NO:4.
  • the nucleotide sequence encoding the Arabidopsis AIR synthetase pre-protein was deposited in E. coli strain DH5apASM and designated as NRRL accession number B-21976.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, wherein said amino acid sequence has 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase activity.
  • the present invention also further encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4, wherein said amino acid sequence has 5'- phosphoribosyl-5-aminoimidazole (AIR) synthetase activity.
  • an activatable DNA construct is made that comprises a synthetic promoter operatively linked to an activatable DNA sequence.
  • the hybrid transcription factor gene and synthetic promoter are selected or designed such that the DNA binding domain of the hybrid transcription factor is capable of binding specifically to the synthetic promoter, whi €h then activates expression of the activatable DNA sequence.
  • a first plant is transformed with the hybrid transcription factor gene, and a second plant is transformed with the activatable DNA construct.
  • the first plant and second plants are crossed to produce a progeny plant containing both the sequence encoding the hybrid transcription factor and the synthetic promoter, wherein the activatable DNA sequence is expressed in the progeny plant.
  • the activatable DNA sequence is an antisense sequence capable of inactivating expression of an endogenous gene such as the AIR synthetase gene. Hence, the progeny plant will be unable to normally express the endogenous gene.
  • This antisense validation system is especially useful for allowing expression of traits that might otherwise be unrecoverable as constitutively driven transgenes. For instance, foreign genes with potentially lethal effect or antisense genes or dominant-negative mutations designed to abolish function of essential genes, while of great interest in basic studies of plant biology, present inherent experimental problems. Decreased transformation frequencies are often cited as evidence of lethality associated with a particular constitutively driven transgene, but negative results of this type are laden with alternative trivial explanations.
  • the present invention is an important advancement in the field of agriculture because it allows stable maintenance and propagation of a test transgene separate from its expression. This ability to separate transgene insertion from expression is especially useful for firm conclusions about essentiality of gene function to be drawn. A substantial benefit of the present invention is that plant genes essential for normal growth or development can thus be identified in this manner. The identification of such genes provide useful targets for screening compound libraries for identification of effective herbicides. Below, the antisense validation system is described in greater detail:
  • a hybrid transcription factor gene for use in the antisense validation system described herein comprises DNA sequences encoding (1 ) a DNA-binding domain and (2) an activation domain that interacts with components of transcriptional machinery assembling at a promoter. Gene fragments are joined, typically such that the DNA binding domain is toward the 5' terminus and the activator domain is toward the 3' terminus, to form a hybrid gene whose expression produces a hybrid transcription factor.
  • One skilled in the art is capable of routinely combining various DNA sequences encoding DNA binding domains with various DNA sequences encoding activation domains to produce a wide array of hybrid transcription factor genes.
  • DNA sequences encoding DNA-binding domains include, but are not limited to, those encoding the DNA binding domains of GAL4, bacteriophage 434, lexA, lacl, and phage lambda repressor.
  • DNA sequences encoding the activation domain include, but are not limited to, those encoding the acidic activation domains of herpes simplex VP16, maize C1 , and P1 .
  • suitable activation domains can be isolated by fusing DNA pieces from an organism of choice to a suitable DNA binding domain and selecting directly for function (Estruch et al., (1994) Nucleic Acids Res. 22: 3983-3989).
  • a preferable hybrid transcription factor gene comprises DNA sequences encoding the GAL4 DNA binding domain fused to the maize C1 activation domain.
  • An activatable DNA construct for use in the antisense validation system described herein comprises (1 ) a synthetic promoter operatively linked to (2) an activatable DNA sequence.
  • the synthetic promoter comprises at least one DNA binding site recognized by the DNA binding domain of the hybrid transcription factor, and a minimal promoter, preferably a TATA element derived from a promoter recognized by plant cells. More particularly the TATA element is derived from a promoter recognized by the plant cell type into which the synthetic promoter will be incorporated.
  • the DNA binding site is repeated multiple times in the synthetic promoter so that the minimal promoter may be more effectively activated, such that the activatable DNA sequence associated with the synthetic promoter is more effectively expressed.
  • DNA binding sites that can be used to make synthetic promoters useful in the invention include, but are not limited to, the upstream activating sequence (UAS G ) recognized by the GAL4 DNA binding protein, the lac operator, and the lexA binding site.
  • UAS G upstream activating sequence
  • promoter TATA elements recognized by plant cells include those derived from CaMV 35S, the maize Bz1 promoter, and the UBQ3 promoter.
  • An especially preferable synthetic promoter comprises a truncated CaMV 35S sequence containing the TATA element (nucleotides -59 to +48 relative to the start of transcription), fused at its 5' end to approximately 10 concatemeric direct repeats of the upstream activating sequence (UAS G ) recognized by the GAL4 DNA binding domain.
  • the activatable DNA sequence encompasses any DNA sequence for which stable introduction and expression in a plant cell is desired. Particularly desirable- activatable DNA sequences are sense or antisense sequences, whose expression results in decreased expression of their endogenous counterpart genes, thereby inhibiting normal plant growth or development.
  • the activatable DNA sequence is operatively linked to the synthetic promoter to form the activatable DNA construct.
  • the activatable DNA sequence in the activatable DNA construct is not expressed, i.e. is silent, in transgenic lines, unless a hybrid transcription factor capable of binding to and activating the synthetic promoter, is also present.
  • the activatable DNA construct subsequently is introduced into cells, tissues or plants to form stable transgenic lines expressing the activatable DNA sequence, as described more fully below.
  • the activatable DNA sequence preferably comprises an antisense AIR synthetase sequence.
  • the antisense validation system described herein utilizes a first plant containing the hybrid transcription factor gene and a second plant containing the activatable DNA construct.
  • the hybrid transcription factor genes and activatable DNA constructs described above are introduced into the plants by methods well known and routinely used in the art, including but not limited to crossing, >4 ⁇ ;ro-.ac.er/ ' -.m-mediated transformation, Ti plasmid vectors, direct DNA uptake such as microprojectile bombardment, liposome mediated uptake, micro-injection, etc. Transformants are screened for the presence and functionality of the transgenes according to standard methods known to those skilled in the art.
  • F1 plants containing both the hybrid transcription factor gene and the activatable DNA construct are generated by cross-pollination and selected for the presence of an appropriate marker.
  • th e F1 plants generate high levels of activatable DNA sequence expression product, comparable to those obtained with strong constitutive promoters such as CaMV 35S.
  • a useful assay in the system described herein comprises the following steps: providing a first transgenic plant stably transformed with a hybrid transcription factor gene encoding a hybrid transcription factor capable of activating a synthetic promoter when said synthetic promoter is present in the plant, wherein the first transgenic plant is homozygous for the hybrid transcription factor; b) providing a second transgenic plant stably transformed with an activatable DNA construct comprising a synthetic promoter activatable by the hybrid transcription factor of step a) operatively linked to an activatable DNA sequence, such as an antisense AIR synthetase sequence; c) crossing the first transgenic plant with the second transgenic plant to yield F1 plants expressing the activatable DNA sequence in the presence of the hybrid transcription factor; and d) determining the effect of expression of the activatable DNA sequence on the F1 plants.
  • a nucleotide sequence encoding an enzyme having AIR synthetase is inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced.
  • the choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the routineer in the art.
  • the resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E.
  • coli coli, yeast, and insect cells (see, e.g., Luckow and Summers, Bio/Technol. 6: 47 (1988)).
  • plasmids such as pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, La Jolla, CA), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV).
  • a preferred baculovirus/insect system is pVI1 1392/Sf21 cells (Invitrogen, La Jolla, CA).
  • the nucleotide sequence encoding an enzyme having a AIR synthetase activity is derived from an eukaryote, such as a mammal, a fly or a yeast, but is preferably derived from a plant.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, or encodes an enzyme having AIR synthetase activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the nucleotide sequence set forth in SEQ ID NO:1 encodes the Arabidopsis AIR synthetase pre-protein, whose amino acid sequence is set forth in SEQ ID NO:2, and the nucleotide sequence set forth in SEQ ID NO:3 encodes the Arabidopsis putative mature AIR synthetase, whose amino acid sequence is set forth in SEQ ID NO:4.
  • the nucleotide sequence is derived from a prokaryote, preferably a bacteria, e.g. E. coli.
  • the enzyme having AIR synthetase activity is encoded by the purM gene.
  • Recombinantly produced AIR synthetases are isolated and purified using a variety of standard techniques. The actual techniques that may be used will vary depending upon the host organism used, whether the enzyme is designed for secretion, and other such factors familiar to the skilled artisan (see, e.g. chapter 16 of Ausubel, F. et al., "Current Protocols in Molecular Biology", pub. by John Wiley & Sons, Inc. (1994).
  • Recombinantly produced AIR synthetases are useful for a variety of purposes. For example, they can be used in in vitro assays to screen known herbicidal chemicals whose target has not been identified to determine if they inhibit AIR synthetases. Such in vitro assays may also be used as more general screens to identify chemicals that inhibit such enzymatic activity and that are therefore novel herbicide candidates. Alternatively, recombinantly produced AIR synthetases may be used to elucidate the complex structure of these molecules and to further characterize their association with known inhibitors in order to rationally design new inhibitory herbicides as well as herbicide tolerant forms of the enzymes.
  • An in vitro assay useful for identifying inhibitors of enzymes encoded by essential plant genes, such as the AIR synthetase preferably comprises the steps of: a) reacting an enzyme having AIR synthetase activity and a substrate thereof in the presence of a suspected inhibitor of the enzyme's function; b) comparing the rate of enzymatic activity in the presence of the suspected inhibitor to the rate of enzymatic activity under the same conditions in the absence of the suspected inhibitor; and c) determining whether the suspected inhibitor inhibits the AIR synthetase enzymatic activity.
  • such a determination is made by comparing, in the presence and absence of the candidate inhibitor, the amount of AIR synthesized in the in vitro assay using fluorescence or absorbance detection. In another preferred embodiment, such a determination is made by comparing, in the presence and absence of the candidate inhibitor, the amount of ADP formed in the in vitro assay using fluorescence or absorbance detection.
  • a preferred substrate for AIR synthetase is 5'-phosphoribosyl-N- formylglycinamidine (FGAM), in particular the b isomer, b-FGAM.
  • a coupled FGAM synthetase/AIR synthetase assay is used, thereby increasing the detection limit of the assay and resulting in an improved screening procedure for a chemical inhibiting AIR synthetase activity.
  • Such a coupling assay preferably comprises the steps of: a) reacting an enzyme having 5'- phosphoribosyl-N-formylglycinamidine (FGAM) synthetase activity, an enzyme having AIR synthetase activity and a substrate of FGAM synthetase in the presence of a suspected inhibitor of the enzyme's function; b) comparing the rate of enzymatic activity in the presence of the suspected inhibitor to the rate of enzymatic activity under the same conditions in the absence of the suspected inhibitor; and c) determining whether the suspected inhibitor inhibits the AIR synthetase enzymatic activity.
  • FGAM 5'- phosphoribosyl-N-formylglycinamidine
  • such a determination is made by comparing, in the presence and absence of the candidate inhibitor, the amount of AIR synthesized in the in vitro assay using fluorescence or absorbance detection. In another preferred embodiment, such a determination is made by comparing, in the presence and absence of the candidate inhibitor, the amount of ADP formed in the in vitro assay using fluorescence or absorbance detection.
  • a preferred substrate for FGAM synthetase is 5'-phosphoribosyl-N- formylglycinamide (FGAR), in particular the b isomer, b-FGAR.
  • the enzyme having FGAM synthetase activity is derived from a bacteria, and is preferably the E.
  • the purL gene is preferably recombinantly produced in E. coli. While any suitable AIR synthetase may be used, preferably the AIR synthetase used in such in vitro assays is derived from a plant. In another preferred embodiment, an assay coupling more than one enzymatic activity preceding AIR synthetase in the purine biosynthesis pathway is used.
  • an enzyme used in an in vitro assay is derived from cells comprising the enzyme, preferably, from a crude extract of the cells.
  • the enzyme is preferably isolated and purified from the cells or from the crude extract.
  • the enzyme is preferably produced recombinantly and is preferably isolated and purified prior to be used in the assay. Chemicals identified in an in vitro assay are then tested for their ability to inhibit plant growth or viability.
  • a suspected herbicide for example identified by in vitro screening, is applied to plants at various concentrations.
  • the suspected herbicide is preferably sprayed on the plants. After application of the suspected herbicide, its effect on the plants, for example death or suppression of growth is recorded.
  • an in vivo screening assay for inhibitors of the AIR synthetase activity uses transgenic plants, plant tissue, plant seeds or plant cells capable of overexpressing a nucleotide sequence having AIR synthetase activity, wherein the AIR synthetase is enzymatically active in the transgenic plants, plant tissue, plant seeds or plant cells.
  • the nucleotide sequence is preferably derived from an eukaryote, such as a mammal, a fly or a yeast, but is preferably derived from a plant.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, or encodes an enzyme having AIR synthetase activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
  • the nucleotide sequence is derived from a prokaryote, preferably a bacteria, e.g. E. coli. In this case, the enzyme having AIR synthetase activity is encoded by the purM gene.
  • a chemical is then applied to the transgenic plants, plant tissue, plant seeds or plant cells and to the isogenic non-transformed plants, plant tissue, plant seeds or plant cells, and the growth or viability of the transgenic and non-transformed plants, plant tissue, plant seeds or plant cells are determined after application of the chemical and compared.
  • the present invention is further directed to plants, plant tissue, plant seeds, and plant cells tolerant to herbicides that inhibit the naturally occurring AIR synthetase activity in these plants, wherein the tolerance is conferred by an altered AIR synthetase activity.
  • Altered AIR synthetase activity may be conferred upon a plant according to the invention by increasing expression of wild-type herbicide-sensitive AIR synthetase by providing additional wild-type AIR synthetase genes to the plant, by expressing modified herbicide-tolerant AIR synthetases in the plant, or by a combination of these techniques.
  • Representative plants include any plants to which these herbicides are applied for their normally intended purpose.
  • Achieving altered AIR synthetase activity through increased expression results in a level of a AIR synthetase in the plant cell at least sufficient to overcome growth inhibition caused by the herbicide.
  • the level of expressed enzyme generally is at least two times, preferably at least five times, and more preferably at least ten times the natively expressed amount.
  • Increased expression may be due to multiple copies of a wild-type AIR synthetase gene; multiple occurrences of the coding sequence within the gene (i.e. gene amplification) or a mutation in the non-coding, regulatory sequence of the endogenous gene in the plant cell. Plants having such altered gene activity can be obtained by direct selection in plants by methods known in the art (see, e.g. U.S. Patent No.
  • Increased expression of a herbicide-sensitive AIR synthetase gene can also be accomplished by transforming a plant cell with a recombinant or chimeric DNA molecule comprising a promoter capable of driving expression of an associated structural gene in a plant cell operatively linked to a homologous or heterologous structural gene encoding the AIR synthetase.
  • the transformation is stable, thereby providing a heritable transgenic trait.
  • plants, plant tissue, plant seeds, or plant cells are stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operatively linked to a coding sequence encoding a herbicide tolerant form of an AIR synthetase.
  • a herbicide tolerant form of the enzyme has at least one amino acid substitution, addition or deletion that confers tolerance to a herbicide that inhibits the unmodified, naturally occurring form of the enzyme.
  • the transgenic plants, plant tissue, plant seeds, or plant cells thus created are then selected by conventional selection techniques, whereby herbicide tolerant lines are isolated, characterized, and developed. Below are described methods for obtaining genes that encode herbicide tolerant forms of AIR synthetases:
  • a genetically manipulatable microbe such as E. coli or S. cerevisiae may be subjected to random mutagenesis in vivo with mutagens such as UV light or ethyl or methyl methane sulfonate.
  • mutagens such as UV light or ethyl or methyl methane sulfonate.
  • Mutagenesis procedures are described, for example, in Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972); Davis et al., Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1980); Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983); and U.S. Patent No. 4,975,374.
  • the microbe selected for mutagenesis contains a normal, inhibitor-sensitive AIR synthetase gene and is dependent upon the activity conferred by this gene.
  • the mutagenized cells are grown in the presence of the inhibitor at concentrations that inhibit the unmodified gene. Colonies of the mutagenized microbe that grow better than the unmutagenized microbe in the presence of the inhibitor (i.e. exhibit resistance to the inhibitor) are selected for further analysis.
  • AIR synthetase genes from these colonies are isolated, either by cloning or by PCR amplification, and their sequences are elucidated. Sequences encoding altered gene products are then cloned back into the microbe to confirm their ability to confer inhibitor tolerance.
  • a method of obtaining mutant herbicide-tolerant alleles of a plant AIR synthetase gene involves direct selection in plants.
  • the effect of a mutagenized AIR synthetase gene on the growth inhibition of plants is determined by plating seeds sterilized by art-recognized methods on plates on a simple minimal salts medium containing increasing concentrations of the inhibitor.
  • concentrations are in the range of 0.001 , 0.003, 0.01 , 0.03, 0.1 , 0.3, 1 , 3, 10, 30, 1 10, 300, 1000 and 3000 parts per million (ppm).
  • the lowest dose at which significant growth inhibition can be reproducibly detected is used for subsequent experiments. Determination of the lowest dose is routine in the art.
  • Mutagenesis of plant material is utilized to increase the frequency at which resistant alleles occur in the selected population.
  • Mutagenized seed material is derived from a variety of sources, including chemical or physical mutagenesis or seeds, or chemical or physical mutagenesis or pollen (Neuffer, In Maize for Biological Research Sheridan, ed. Univ. Press, Grand Forks, ND., pp. 61 -64 (1982)), which is then used to fertilize plants and the resulting Mi mutant seeds collected.
  • M2 seeds (Lehle Seeds, Arlington, AZ), which are progeny seeds of plants grown from seeds mutagenized with chemicals, such as ethyl methane sulfonate, or with physical agents, such as gamma rays or fast neutrons, are plated at densities of up to 10,000 seeds/plate (10 cm diameter) on minimal salts medium containing an appropriate concentration of inhibitor to select for tolerance. Seedlings that continue to grow and remain green 7-21 day& after plating are transplanted to soil and grown to maturity and seed set. Progeny of these seeds are tested for tolerance to the herbicide.
  • AIR synthetase inhibitors can be either Arabidopsis plants or any other plant whose growth is susceptible to the AIR synthetase inhibitors.
  • the inserted AIR synthetase genes are mapped relative to known restriction fragment length polymorphisms (RFLPs) (See, for example, Chang et al. Proc. Natl. Acad, Sci, USA 85: 6856-6860 (1988); Nam et al., Plant Cell 1 : 699-705 (1989).
  • RFLPs restriction fragment length polymorphisms
  • the AIR synthetase inhibitor tolerance trait is independently mapped using the same markers. When tolerance is due to a mutation in that AIR synthetase gene, the tolerance trait maps to a position indistinguishable from the position of the AIR synthetase gene.
  • Another method of obtaining herbicide-tolerant alleles of a AIR synthetase gene is by selection in plant cell cultures. Explants of plant tissue, e.g. embryos, leaf disks, etc. or actively growing callus or suspension cultures of a plant of interest are grown on medium in the presence of increasing concentrations of the inhibitory herbicide or an analogous inhibitor suitable for use in a laboratory environment. Varying degrees of growth are recorded in different cultures. In certain cultures, fast-growing variant colonies arise that continue to grow even in the presence of normally inhibitory concentrations of inhibitor. The frequency with which such faster-growing variants occur can be increased-by treatment with a chemical or physical mutagen before exposing the tissues or cells to the inhibitor.
  • Explants of plant tissue e.g. embryos, leaf disks, etc. or actively growing callus or suspension cultures of a plant of interest are grown on medium in the presence of increasing concentrations of the inhibitory herbicide or an analogous inhibitor suitable for use in a laboratory environment. Varying degrees of growth are recorded in different cultures. In certain cultures,
  • Putative tolerance-conferring alleles of the AIR synthetase gene are isolated and tested as described in the foregoing paragraphs. Those alleles identified as conferring herbicide tolerance may then be engineered for optimal expression and transformed into the plant. Alternatively, plants can be regenerated from the tissue or cell cultures containing these alleles.
  • Still another method involves mutagenesis of wild-type, herbicide sensitive plant AIR synthetase genes in bacteria or yeast, followed by culturing the microbe on medium that contains inhibitory concentrations of the inhibitor and then selecting those colonies that grow in the presence of the inhibitor. More specifically, a plant cDNA, such as the Arabidopsis cDNA encoding the AIR synthetase is cloned into a microbe that otherwise lacks the selected gene's activity. The transformed microbe is then subjected to in vivo mutagenesis or to in vitro mutagenesis by any of several chemical or enzymatic methods known in the art, e.g. sodium bisulfite (Shortle et al., Methods Enzymol.
  • Colonies that grow in the presence of normally inhibitory concentrations of inhibitor are picked and purified by repeated restreaking. Their plasmids are purified and tested for the ability to confer tolerance to the inhibitor by retransforming them into the microbe lacking AIR synthetase gene activity. The DNA sequences of cDNA inserts from plasmids that pass this test are then determined.
  • Herbicide resistant AIR synthetase enzymes are also obtained using methods involving in vitro recombination, also called DNA shuffling.
  • DNA shuffling mutations, preferably random mutations, are introduced in AIR synthetase genes.
  • DNA shuffling also leads to the recombination and rearrangement of sequences within an AIR synthetase genes or to recombination and exchange of sequences between two or more different of AIR synthetase genes. These methods allows for the production of millions of mutated AIR synthetase genes.
  • the mutated genes, or shuffled genes are screened for desirable properties, e.g. improved tolerance to herbicides and for mutations that provide broad spectrum tolerance to the different classes of inhibitor chemistry. Such-screens are well within the skills of a routineer in the art.
  • a mutagenized AIR synthetase gene is formed from at least one template AIR synthetase gene, wherein the template AIR synthetase gene has been cleaved into double-stranded random fragments of a desired size, and comprising the steps of adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded random fragments; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single- stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other
  • the concentration of a single species of double- stranded random fragment in the population of double-stranded random fragments is less than 1 % by weight of the total DNA.
  • the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides.
  • the size of the double-stranded random fragments is from about 5 bp to 5 kb.
  • the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles. Such method is described e.g. in Stemmer et al. (1994) Nature 370: 389-391 , in US Patent 5,605,793 and in Crameri et al.
  • any combination of two or more different AIR synthetase genes are mutagenized in vitro by a staggered extension process (StEP), as described e.g. in Zhao et al. (1998) Nature Biotechnology 16: 258-261.
  • StEP staggered extension process
  • AIR synthetase genes are used as template for PCR amplification with the extension cycles of the PCR reaction preferably carried out at a lower temperature than the optimal polymerization temperature of the polymerase.
  • the temperature for the extension reaction is desirably below 72°C, more desirably below 65°C, preferably below 60°C, more preferably the temperature for the extension reaction is 55°C.
  • the duration of the extension reaction of the PCR cycles is desirably shorter than usually carried out in the art, more desirably it is less than 30 seconds, preferably it is less than 15 seconds, more preferably the duration of the extension reaction is 5 seconds.
  • extension reaction Only a short DNA fragment is polymerized in each extension reaction, allowing template switch of the extension products between the starting DNA molecules after each cycle of denaturation and annealing, thereby generating diversity among the extension products.
  • the optimal number of cycles in the PCR reaction depends on the length of the AIR synthetase coding regions to be mutagenized but desirably over 40 cycles, more desirably over 60 cycles, preferably over 80 cycles are used.
  • Optimal extension conditions and the optimal number of PCR cycles for every combination of AIR synthetase genes are determined as described in using procedures well-known in the art.
  • the other parameters for the PCR reaction are essentially the same as commonly used in the art.
  • the primers for the amplification reaction are preferably designed to anneal to DNA sequences located outside of the coding sequence of the AIR synthetase genes, e.g.
  • the primers desirably anneal to sequences located less than 500 bp away from the AIR synthetase coding sequences, preferably less than 200 bp away from the AIR synthetase coding sequences, more preferably less than 120 bp away from the AIR synthetase coding sequences.
  • the AIR synthetase coding sequences are surrounded by restriction sites, which are included in the DNA sequence amplified during the PCR reaction, thereby facilitating the cloning of the amplified products into a suitable vector.
  • fragments of AIR synthetase genes having cohesive ends are produced as described in WO 98/05765.
  • the cohesive ends are produced by ligating a first oligonucleotide corresponding to a part of a AIR synthetase gene to a second oligonucleotide not present in the gene or corresponding to a part of the gene not adjoining to the part of the gene corresponding to the first oligonucleotide, wherein the second oligonucleotide contains at least one ribonucleotide.
  • a double-stranded DNA is produced using the first oligonucleotide as template and the second oligonucleotide as primer.
  • the ribonucleotide is cleaved and removed.
  • the nucleotide(s) located 5' to the ribonucleotide is also removed, resulting in double-stranded fragments-having cohesive ends. Such fragments are randomly reassembled by ligation to obtain novel combinations of gene sequences.
  • any AIR synthetase gene or any combination of AIR synthetase genes is used for in vitro recombination in the context of the present invention, for example, an AIR synthetase gene derived from a plant, such as, e.g. Arabidopsis thaliana, e.g. an AIR synthetase gene set forth in SEQ ID NO:1 or SEQ ID NO:3, an AIR synthetase gene from a bacteria, such as Bacillus subtilis (Ebbole and Zalkin (1987) J. Biol. Chem. 262: 8274-8287) or E. coli (Smith and Daum (1986) J. Biol. Chem.
  • AIR synthetase genes or portions thereof are used in the context of the present invention.
  • the library of mutated AIR synthetase genes obtained by the methods described above are cloned into appropriate expression vectors and the resulting vectors are transformed into an appropriate host, for example an algae like Chlamydomonas, a yeast or a bacteria.
  • An appropriate host is preferably a host that otherwise lacks AIR synthetase gene activity, for example E. coli strain S066O9/IKC (Schnorr et al. (1994) Plant Journal 6: 113-121 ).
  • Host cells transformed with the vectors comprising the library of mutated AIR synthetase genes are cultured on medium that contains inhibitory concentrations of the inhibitor and those colonies that grow in the presence of the inhibitor are selected. Colonies that grow in the presence of normally inhibitory concentrations of inhibitor are picked and purified by repeated restreaking. Their plasmids are purified and the DNA sequences of cDNA inserts from plasmids that pass this test are then determined.
  • An assay for identifying a modified AIR synthetase gene that is tolerant to an inhibitor may be performed in the same manner as the assay to identify inhibitors of the AIR synthetase activity (Inhibitor Assay, above) with the following modifications: First, a mutant AIR synthetase is substituted in one of the reaction mixtures for the wild-type AIR synthetase of the inhibitor assay. Second, an inhibitor of wild-type enzyme is present in both reaction mixtures.
  • mutated activity activity in the presence of inhibitor and mutated enzyme
  • unmutated activity activity in the presence of inhibitor and wild-type enzyme
  • a significant increase is defined as an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, most preferably an increase by about 10-fold or greater.
  • genes encoding herbicide tolerant AIR synthetases can also be used as selectable markers in plant cell transformation methods.
  • plants, plant tissue, plant seeds, or plant cells transformed with a transgene can also be transformed with a gene encoding an altered AIR synthetase capable of being expressed by the plant.
  • the transformed cells are transferred to medium containing an inhibitor of the enzyme in an amount sufficient to inhibit the survivability of plant cells not expressing the modified gene, wherein only the transformed cells will survive.
  • the method is applicable to any plant cell capable of being transformed with a modified AIR synthetase-encoding gene, and can be used with any transgene of interest. Expression of the transgene and the modified gene can be driven by the same promoter functional in plant cells, or by separate promoters.
  • a wild-type or herbicide-tolerant form of the AIR synthetase gene can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting a DNA molecule encoding the AIR synthetase into an expression system to which the DNA molecule is heterologous (i.e., not normally present) using standard cloning procedures known in the art.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences in a host cell containing the vector.
  • a large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.
  • the components of the expression system may also be modified to increase expression.
  • a transgene comprising a wild-type or herbicide- tolerant form of the AIR synthetase gene is preferably stably transformed and integrated into the genome of the host cells.
  • the transgene comprising a wild-type or herbicide-tolerant form of the AIR synthetase gene located on a self-replicating vector are viruses, in particular gemini viruses.
  • Transformed cells can be regenerated into whole plants such that the chosen form of the AIR synthetase gene confers herbicide tolerance in the transgenic plants.
  • A. Requirements for Construction of Plant Expression Cassettes Gene sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants.
  • the expression cassettes may also comprise any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
  • These expression cassettes can then be easily transferred to the plant transformation vectors described infra. The following is a description of various components of typical expression cassettes.
  • the selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters known in the art can be used.
  • the CaMV 35S promoter for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter may be used.
  • the chemically inducible PR-1 promoter from tobacco or Arabidopsis may be used (see, e.g., U.S. Patent No. 5,689,044).
  • transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledonous and dicotyledonous plants.
  • intron sequences such as introns of the maize Adhl gene have been shown to enhance expression, particularly in monocotyledonous cells.
  • non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
  • the coding sequence of the selected gene may be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest. Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (see, e.g. Perlak er a/., Proc. Natl. Acad. Sci. USA 88: 3324 (1991 ); and Koziel er a/., Bio/technol. 11: 194 (1993)).
  • the cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles.
  • sequences have been characterized which cause the targeting of gene products to other cell compartments.
  • Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)).
  • amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
  • transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors.
  • the selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al.
  • vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001 , as well as the binary vector pCIBI O and hygromycin selection derivatives thereof. (See, for example, U.S. Patent No. 5,639,949).
  • Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector -and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non- Agrobacterium transformation include pCIB3064, pSOG19, and pSOG35. (See, for example, U.S. Patent No. 5,639,949).
  • the coding sequence of interest Once the coding sequence of interest has been cloned into an expression system, it is transformed into a plant cell.
  • Methods for transformation and regeneration of plants are well known in the art.
  • Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles.
  • bacteria from the genus Agrobacterium can be utilized to transform plant cells.
  • Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium.
  • Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
  • Transformation of most monocotyledon species has now also become routine.
  • Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, particle bombardment into callus tissue, as well as /4 ⁇ o/.ac.e/7-.m-mediated transformation. VIII. Breeding
  • the wild-type or altered form of a AIR synthetase gene of the present invention can be utilized to confer herbicide tolerance to a wide variety of plant cells, including those of gymnosperms, monocots, and dicots.
  • the gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • the high-level expression of a wild-type AIR synthetase gene and/or the expression of herbicide-tolerant forms of a AIR synthetase gene conferring herbicide tolerance in plants, in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding approaches and techniques known in the art.
  • a herbicide tolerant AIR synthetase gene allele is obtained by direct selection in a crop plant or plant cell culture from which a crop plant can be regenerated, it is moved into commercial varieties using traditional breeding techniques to develop a herbicide tolerant crop without the need for genetically engineering the allele and transforming it into the plant.
  • Example 1 Construction of a Vector Containing a GAL4 Binding Site/Minimal 35S CaMV Promoter Fused to Antisense AIR Synthetase
  • GAL4 binding sites and the minimal 35S promoter (-59 to +1 ) are excised from pGALLuc2 (Goff, et al., (1991 ) Genes & Development 5: 298-309) as an EcoRI-Pstl fragment and inserted into the respective sites of pBluescript, yielding pAT52.
  • pAT66 is constructed with a three-way ligation between the Hindlll-Pstl fragment of pAT52, a Pstl- EcoRI fragment of pCIB1716 (contains a 35S untranslated leader, GUS gene, 35S terminator) and Hindlll-EcoRI cut pUC18.
  • the 35S leader of pAT66 is excised with Pstl- Ncol and replaced with a PCR-generated 35S leader extending from +1 to +48 to yield pAT71 .
  • Plasmid pBS SK+ (Stratagene, LaJolla, CA) is linearized with Sacl, treated with mung bean nuclease to remove the Sacl site, and re-ligated with T4 ligase to make pJG201.
  • the 10XGAL4 consensus binding site/CaMV 35S minimal promoter/GUS gene/CaMV terminator cassette is removed from pAT71 with Kpnl and cloned into the Kpnl site of pJG201 to make pJG304.
  • pJG304 is partially digested with restriction endonuclease Asp718 to isolate a full- length linear fragment.
  • a fragment of the 5' phosphoribosyl-5-aminoimidazole (AIR) synthetase cDNA clone (Senecoff and Meagher (1993) Plant Physiology, 102: 387-399) is PCR-amplified from the Arabidopsis thaliana cDNA plasmid library pFL61 (Minet et al, (1992) Plant Journal, 2: 417- 422) using the oligonucleotides AS-1 (5' GAT CGA GCT CGT TCT CTTOTG TGT CAT C 3', SEQ ID NO:6) and AS-2 (5' GAT CCC ATG GTC CCC AGG TAA AGA CGT C 3', SEQ ID NO:7).
  • AS-1 5' GAT CGA GCT CGT TCT CTTOTG TGT CAT C 3', SEQ ID NO:6
  • AS-2 5' GAT CCC ATG GTC CCC AGG TAA AGA CGT C 3', SEQ ID NO:
  • the vector pJG304 ⁇ Xhol is digested with Sacl and Ncol to excise the GUS gene coding sequence.
  • the AIR synthetase PCR fragment is digested with Sacl and Ncol and ligated into pJG304 ⁇ Xhol to make pDG3.
  • Vector pGPTV (Becker, et al., (1992) Plant Molecular Biology 20: 1 195-1197) is digested with EcoRI and Hindlll to remove the nopaline synthase promoter/GUS cassette. Concurrently, the superlinker is excised from pSE380 (Invitrogen, San Diego, CA) with EcoRI and Hindlll and cloned into the EcoRVHindlll linearized pGPTV, to make pJG261.
  • PDG4 pDG3 is cut with Xhol to excise the cassette containing the GAL4 DNA binding site/35S minimal promoter/antisense AIR synthetase/CaMV terminator fusion. This cassette is ligated into Xhol digested pJG261 , such that transcription is divergent from that of the BAR selectable marker, producing pDG4.
  • pDG4 is electro-transformed (Bio-Rad Laboratories, Hercules, CA) into Agrobacterium tumefaciens strain C58C1 (pMP90), and Arabidopsis plants (Ecotype Columbia) are transformed by infiltration (Bechtold et al, C. R. Acad. Sci. Paris, 316: 1 188- 93 (1993).
  • Seeds from the infiltrated plants are selected on germination medium (Murashige-Skoog salts at 4.3 g/liter, Mes at 0.5 g/liter, 1 % sucrose, thiamine at 10 ug/liter, pyridoxine at 5 ug/liter, nicotinic acid at 5 ug/liter, myo-inositol at 1 mg/liter, pH 5.8) containing Basta at 15 mg/liter.
  • germination medium Merashige-Skoog salts at 4.3 g/liter, Mes at 0.5 g/liter, 1 % sucrose, thiamine at 10 ug/liter, pyridoxine at 5 ug/liter, nicotinic acid at 5 ug/liter, myo-inositol at 1 mg/liter, pH 5.8
  • pSGZLI is constructed by ligating the GAL4-C1 EcoRI fragment from pGALCI (Goff, er a/., (1991 ) Genes & Development, 5: 298-309) into the EcoRI site of plC20H.
  • the GAL4-C1 fragment of pSGZLI is excised with BamHI-Bglll and inserted into the BamHI site of pCIB770 (Rothstein, er a/., (1987) Gene 53: 153-161) yielding pAT53.
  • Arabidopsis root explants are transformed with pAT53 as described in Valvekens, et al., (1985) PNAS USA 85: 5536-5540.
  • Transgenic plants with single site insertion and positive for GAL4/C1 expression are taken to homozygosity.
  • Example 5 Antisense Inhibition of AIR Synthetase Using a GAL4/C1 Transactivator and a GAL4 Binding Site/Minimal CaMV 35S Promoter
  • Fifteen transgenic plants containing the GAL4 binding site/minimal CaMV 35S promoter/antisense AIR synthetase construct are transplanted to soil and grown to maturity in the greenhouse. Flowers borne on the primary transformants are crossed to pollen from the homozygous GAL4/C1 transactivator line pAT53-103. F1 seeds are plated on germination medium and germination medium containing 15 mg/liter Basta. Seedlings from five F1 lines are transplanted to soil and grown to maturity in the greenhouse. Half of the seedlings from two F1 lines die while in soil. Half of the seedlings from three F1 lies are bleached and severely retarded in growth. These results show that the AIR synthetase gene is essential in plants.
  • Arabidopsis thaliana (Landsberg) cDNA library in the plasmid vector pFL61 is obtained and amplified.
  • PCR primers to amplify protein coding sequence of Arabidopsis AIR synthetase are designed from a published DNA sequence (Genbank accession L12457, Senecoff and Meagher, Plant Physiol., 102: 387-399 (1993)) and used to amplify the AIR synthetase coding sequence from the plasmid library with Pfu DNA polymerase (Stratagene).
  • Sequencing of the PCR product reveals an error in the published DNA sequence resulting in the insertion of a cytosine base at the position corresponding to position 1027 in SEQ ID NO:1 , resulting in an incorrect predicted protein.
  • primers slp242 (5' CGC GGA TCC TCA CTA CTG ATA GCT TAC GCC TTC ACC 3', SEQ ID NO:8) and slp244 (5' TTG AAG CCA TGG AAG CTC GGA TTT TG 3', SEQ ID NO:9) are used, and for the construct including the coding region of the putative mature AIR synthetase, primers slp242 and slp243 (5' CGC ATG CCA TGG ATA AAG ATG ATG ACA CTG ATA GTC T 3', SEQ ID NO:10) are used.
  • the coding regions of the pre-protein and of the putative mature protein are subcloned into the expression vector pET32a (Novagen) and both are transformed into E.coli BL21 DE3 pLysS (Novagen) by electroporation using the Biorad Gene Pulser and the manufacturer's conditions.
  • E. coli strain TX635/pJS1 13 (Schendel et al. (1989) Biochemistry 28, 2459-2471 ) is grown in Luria broth (LB) containing 50 ⁇ g/mL carbenicillin at 30°C in an incubator/shaker. When the cells reach an optical density of approximately 1 OD at 600 nm, an equal volume of LB carbenicillin at 56°C is added to heat-shock the cells. Subsequently, the cells are placed in an incubator-shaker and grown at 42°C. The cells are harvested at the end of log phase using low speed centrifugation. The centrifuge bottle is inverted and the media is allowed to drain.
  • LB Luria broth
  • the cell pellet is resuspended with a small paintbrush in buffer A (50 mM EPPS, pH 7.5, 1 mM EDTA, 2 mM DTT, 150 mM KCI, 10% glycerol) and then disrupted in a trench pressure cell at 18,000 PSI. Following a high speed centrifugation to remove cell debris, the enzyme is precipitated with ammonium sulfate (40-60%) and the pellets stored at -80°C. The enzyme is resuspended in a small volume of Buffer A and applied to a Sephadex G-25 column for desalting into Buffer A. The activity is assayed as described below.
  • Example 8 Growth and Extraction of AIR Synthetase
  • E.coli strain pJS24/Tx393 (Schrimsher et al. (1986) Biochemistry 25, 4366-4371) containing multiple gene copies of the native AIR synthetase is grown in LB broth containing 50 ⁇ g/mL of carbenicillin at 37°C in an incubator-shaker.
  • the cells are harvested at the end of the log phase of growth and pelleted in a centrifuge at low speed, the growth media is discarded and the centrifuge bottle is inverted and allowed to drain.
  • the cells are resuspended in buffer A with a small paintbrush and disrupted in a French Pressure Cell at approximately 18,000 PSI. Following a high speed centrifugation to pellet cell debris, the supernatant is precipitated with ammonium sulfate and stored at -80°C.
  • the AIR synthetase activity assay is essentially derived from Schrimsher et al. (1986) Biochemistry 25, 4356-4365.
  • the reaction volumes are preferably the ones described below, but can be varied depending on the experimental requirements.
  • 0.2-1.0 x 10 "4 unit of an enzyme having AIR synthetase activity one unit of activity is defined as the amount of enzyme required to produce 1 mmol/min of product
  • 0.1 mM 5'- phosphoribosyl-N-formylglycinamidine (FGAM) are mixed in a final volume of 96 ml 50 mM HEPES (pH 7.4-8.1 , but preferably 7.7), 20 mM MgCI 2 , 150 mM KCI and 0.01 -10 mM, but preferably 2.0 mM ATP.
  • AIR is determined preferably according to Bratton and Marshall (J. Biol. Chem. (1939) 128, 537-550) by adding 32 ml of 1 .33 M potassium phosphate in 20% (w/v) trichloroacetic acid (pH 1.4). The mixture is centrifuged to remove precipitated protein and 32 ml of 0.1 % (w/v) sodium nitrite is added. After 3 min., 32 ml of 0.5% (w/v) ammonium sulfamate is added and, after an additional minute, 8 ml of 25% N-(1 -naphthyl)ethylenediamine dihydrochloride is added. The absorbance is measured at 530 nm after 10 min.
  • ADP formation is quantitated by a coupled reaction procedure.
  • 3.5 units of pyruvate kinase, 4,7 units of lactate dehydrogenase, 1 .0 mM phosphenolpyruvate and 0.2 mM NADH are added and absorbance is measured at 340 nm.
  • Example 10 Coupled FGAM Synthetase and AIR Synthetase Enzyme Assays
  • the conversion of FGAR to FGAM is followed by detecting the concommittant formation of ADP.
  • the ADP formation is followed utilizing the enzymes pyruvate kinase, and lactate dehydrogenase (reagent enzymes) and detecting the conversion of NADH to NAD + in the presence of phosphoenolpyruvate (PEP). This is monitored at 340 nm. Pyruvate kinase and PEP facilitate the regeneration of ATP from ADP. ATP is a required substrate for both FGAM synthetase and AIR synthetase.
  • the assay buffer is buffer A with the addition of 20 mM MgCI 2 .
  • AIR synthase it is necessary to provide the substrate FGAM.
  • the FGAM is provided by the conversion of FGAR to FGAM in the same reaction mixture. If NADH is added the conversion can be followed utilizing the FGAM synthetase assay. When the FGAR-FGAM conversion proceeds sufficiently (approximately 50 ⁇ M) then AIR synthetase is added. Adding the AIR synthetase after the production of FGAM insures that the initial concentration of FGAM is constant in all reaction wells. The AIR synthetase is assayed by the method of Bratton and Marshall (J. Biol. Chem. (1939) 128, 537-550).
  • AIR N-(1 -napthyl)ethylene- diamine dihydrochloride
  • the assays are carried out in the same way independent of the original source of the enzymes.
  • the assays are performed in 300 ⁇ L 96 well microtiter plates.
  • the total assay reaction volume is 200 ⁇ L.
  • Substrates (except FGAR) are mixed in a ratio such that the final concentrations (in the microtiterplate) are as follows: L-glutamine (600 ⁇ M), ATP (600 ⁇ M), PEP (1 mM), and NADH (200 ⁇ M).
  • a mixture of substrates at 10X concentration can be pipetted at 20 ⁇ L/well.
  • the reagent enymes and FGAM synthetase can also be mixed to be added simultaneously.
  • the suggested amounts of the ADP detecting/regeneration mix is 0.7 units pyruvate kinase and 0.97 units lactate dehydrogenase per reaction. This should be used as a guideline and the amounts of enzyme adjusted empirically.
  • the FGAR (200 ⁇ M) should be added after a two minute incubation period. After the FGAM synthase reaction proceedes to completion at a rate of approximately 10 ⁇ M/minute (this is within 10- 15 minutes), the AIR synthetase is added.
  • the reaction is stopped with 66 ⁇ L of 20% TCA in 1 M K 3 PO 4
  • the plate in spun in a centrifuge to pellet the precipitated protein, then the supernatant is transferred to a separate microtiterplate for color development and reading.
  • 1.2 ⁇ L of 10% sodium nitrite is added and after 3 minutes 1 .2 ⁇ L of 50% ammonium sulfamate is added (neutralizes excess nitrite).
  • 8.3 ⁇ L of 1 % NEDD are added and after 5 minutes, the plate is read at 530 nm using a microtiter plate reading UV/VIS spectrophotometer.
  • AICAR is used as a standard since AIR is not available for that purpose. Based on AICAR a reasonable detection limit (3-fold OD over background) of 10 ⁇ M is easily attainable.
  • L-Glutamine, ATP, sodium nitrite, ammonium sulfamate, and NEDD are available from Sigma Chemicals.
  • FGAR is synthesized by the methods of Chen and Henderson (Can. J. Chemistry (1970) 48: 2306-2309) or Carrington et al. (J.Chem. Soc. (1968) 6864).
  • Example 1 1 In vitro Recombination of AIR Synthetase Genes by DNA Shuffling
  • the A. thaliana AIR synthetase gene encoding the pre-protein is amplified by PCR as described in example 6.
  • the resulting DNA fragment is digested by DNasel treatment essentially as described (Stemmer et al. (1994) PNAS 91 : 10747-10751) and the PCR primers are removed from the reaction mixture.
  • a PCR reaction is carried out without primers and is followed by a PCR reaction with the primers, both as described (Stemmer et al. (1994) PNAS 91 : 10747-10751 ).
  • the resulting DNA fragments are cloned into pTRC99a (Pharmacia, Cat no: 27-5007-01 ) and transformed into E.coli strain S066O9/IKC (Schnorr et al. (1994) Plant Journal 6: 1 13-121 ) by electroporation using the Biorad Gene Pulser and the manufacturer's conditions.
  • the transformed bacteria are grown on medium that contains inhibitory concentrations of the inhibitor and those colonies that grow in the presence of the inhibitor are selected. Colonies that grow in the presence of normally inhibitory concentrations of inhibitor are picked and purified by repeated restreaking. Their plasmids are purified and the DNA sequences of cDNA inserts from plasmids that pass this test are then determined.
  • PCR-amplified DNA fragments comprising the A. thaliana AIR synthetase gene encoding the pre-protein and PCR-amplified DNA fragments comprising the E.coli purM gene are recombined in vitro and resulting variants with improved tolerance to the inhibitor are recovered as described above.
  • the A. thaliana AIR synthetase gene encoding the mature protein and the E.coli purM gene are each cloned into the polylinker of a pBluescript vector.
  • a PCR reaction is carried out essentially as described (Zhao et al. (1998) Nature Biotechnology 16: 258-261 ) using the "reverse primer” and the "M13 20 primer” (Stratagene Catalog). Amplified PCR fragments are digested with appropriate restriction enzymes and cloned into pTRC99a and mutated AIR synthetase genes are screened as described in example 1 1 .

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Abstract

L'invention concerne des procédés de criblage de composés chimiques, destinés à rechercher l'activité herbicide de ceux-ci, au moyen d'enzymes produits de manière recombinée et possédant une activité de 5'-phosphoribosyl-5-amino-imidazole synthétase (AIR synthétase). Elle concerne également l'utilisation de ces procédés pour identifier des composés chimiques herbicides destinés à supprimer la croissance de la végétation indésirable. En outre, l'invention concerne des procédés visant à rendre tolérants aux herbicides, des plantes, tissus végétaux, graines et cellules végétales, et mettant en oeuvre des gènes codant pour des enzymes possédant une activité AIR synthétase. Elle concerne enfin des procédés d'utilisation de telles plantes transgéniques, destinés à supprimer de manière sélective la croissance des mauvaises herbes dans les champs de culture.
EP99936458A 1998-06-24 1999-06-22 Procedes de criblage de composes herbicides utilisant air synthetase d'arabidopsis thaliana Withdrawn EP1090135A2 (fr)

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