Classifications

• • 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
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
• • 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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • 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
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

• the invention relates to methods for screening herbicidal compounds which inhibit the enzymatic activities of 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate kinase (HMP-P kinase) or thiamine monophosphate pyrophosphorylase (2-methyl-4-amino-5- (hydroxymethyl)pyrimidine diphosphate:4-methyl-5-(2-phosphoethyl)thiazole 2-methyl-4- aminopyrimidine-5-methenyl transferase) or TMP-PPase, both of which are enzymatic activities involved in de novo thiamine biosynthesis.
• the invention relates to the use of thereby identified herbicidal compounds to control the growth of undesired vegetation.
• the invention may also be applied to the development of herbicide tolerant plants, plant tissues, plant seeds, and plant cells.
• 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 for 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 et al. is directed to plants resistant to various imidazolinone or sulfonamide herbicides.
• U.S. Patent No. 4,975,374 to Goodman er a/ 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.
• 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 HMP-P kinase activity or TMP-PPase 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 r S- adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.
• a co- factor can be regenerated and reused.
• Coupled synthesis an enzymatic biosynthesis, in which the final product is synthesized by two or more sequential enzymatic steps, wherein the substrate for the first enzymatic step in one or more of the branches of a biochemical pathway is supplemented to the reaction mixture and is converted by the first enzyme to an intermediate product, which is converted by a second enzyme or enzymes to the final product without addition of the intermediate product.
• chimeric is used to indicate that a DNA sequence, such as a vector or a gene, is comprised of more than one DNA sequences of distinct origin which are fused together by recombinant DNA techniques resulting in a DNA sequence, which does not occur naturally, and which particularly does not occur in the plant to be transformed.
• 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-naturally 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
• Expression refers to the transcription and/or translation of an endogenous gene or a transgene in plants.
• expression may refer to the transcription of the antisense DNA only.
• Gene refers to a coding sequence and associated regulatory sequences wherein the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
• regulatory sequences are promoter sequences, 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns
• Herbicide a chemical substance ⁇ used to kill or suppress the growth of plants, plant cells, plant seeds, or plant tissues.
• Heteroloqous 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 HMP-P kinase/TMP-PPase 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.
• Operativelv linked refers to a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence.
• Plant refers to any plant, particularly to seed plants.
• Plant cell refers to structural and physiological unit of the plant, comprising a protoplast and a cell wall.
• the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
• Plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
• Pre-protein protein which is normally targeted to a cellular organelle, such as a chloroplast, and still comprising its transit peptide.
• Recombinant DNA refers to a combination of DNA sequences that are joined together using recombinant DNA technology.
• Recombinant DNA technology refers to procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
• 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 85%, more desirably at least 90%, preferably at least 92%, more preferably at least 95%, still more preferably at least 97%, 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 i ⁇ iM EDTA at 50°C with washing in 1X SSC, 0.1 % SDS at 65°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 65°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.2X SSC, 0.1% SDS at 65°C
• 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%.
• Synthetic refers to a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and/or monocot genes is said to be synthetic.
• Tolerance the ability to continue normal growth or function when exposed to an inhibitor or herbicide.
• 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 present invention therefore encompasses nucleotide sequences derived from a plant which encode for an enzyme having HMP-P kinase activity or TMP-PPase activity.
• nucleotide sequences are derived from Arabidopsis thaliana and in a further preferred embodiment, such nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 and encodes for an enzyme having HMP-P kinase activity or TMP-PPase 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
• HMP-P kinase and TMP-PPase are enzymatic steps in the de novo thiamine biosynthesis pathway (see below).
• De novo thiamine biosynthesis leads ultimately to the formation of thiamine pyrophosphate (also known as thiamine diphosphate or vitamin B1 ), the form of this cofactor found in several enzymes (Schellenberger et al. (1997) Meth. Enz. 279, 131 -146).
• This de novo biosynthetic pathway occurs in bacteria, yeast, and plants but is absent from humans (Begley, T. P. (1996) Natl. Prod. Rep. 13, 177-186).
• 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.
• a recombinant vector comprising said chimeric gene according to the invention, wherein said vector is capable of being stably transformed into a host cell.
• a host cell comprising a vector according to the invention, wherein said nucleotide sequence is expressible in said cell.
• Preferred is a host ceil according to the invention, wherein said host cell is an eukaryotic cell. More preferred is a host cell according to the invention, wherein said host cell is selected from the group consisting of an insect cell, a yeast cell, and a plant cell.
• a host cell wherein said host cell is a procaryotic cell. More preferred is a host cell according to the invention, wherein said host cell is a bacterial cell. Further encompassed is an isolated plant protein involved in thiamine biosynthesis, wherein said protein has HMP-P kinase activity or TMP-PPase activity. Preferred is an isolated protein, wherein said plant is Arabidopsis thaliana. More preferred is an protein according to the invention, wherein said protein has HMP-P kinase activity. In particularly preferred is an protein according to the invention, wherein said protein comprises an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2. In particularly preferred is an protein, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:2.
• the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant, preferably Arabidopsis thaliana.
• the enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 , or has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2.
• the substrate of HMP-P kinase is 2-methyl-4-amino-5- hydroxymethylpyrimidine phosphate (HMP-P) and in another preferred embodiment, the substrate of TMP-PPase is 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate (THZ-P). In yet another embodiment, the substrate of TMP-PPase is 2-methyl-4-amino-5- hydroxymethylpyrimidine pyrophosphate (HMP-PP). In yet another preferred embodiment, the activity of the enzyme is determined by measuring the TMP produced in the reaction mixture. In another preferred embodiment, the activity of the enzyme is determined by measuring the ADP derived from ATP in the reaction mixture.
• the present invention further describes an assay comprising the steps of: (a) combining an enzyme having HMP-P kinase activity or TMP-PPase activity in a first reaction mixture with a substrate of HMP-P kinase or a substrate of TMP-PPase under conditions in which the enzyme is capable of catalyzing the synthesis of its product; (b) combining the chemical and the enzyme in a second reaction mixture with the substrate under the same conditions and for the same period of time as in the first reaction mixture; (c) determining the activity of the enzyme in the first and second reaction mixtures; wherein the chemical is capable of inhibiting the activity of the enzyme if the activity of the coupled enzymes in the second reaction mixture is significantly less than the activity of the enzyme in the first reaction mixture.
• the present invention further describes a method for identifying a chemical to be tested for the ability to inhibit plant growth and viability, comprising the steps of: (a) combining an enzyme having HMP kinase activity and an enzyme having HMP-P kinase activity or TMP-PPase activity in a first reaction mixture with a substrate of HMP kinase and a substrate of TMP-PPase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of TMP; (b) combining the chemical and the enzymes in a second reaction mixture with the substrates under the same conditions and for the same period of time as in the first reaction mixture; (b) determining the activity of the enzyme having HMP- P kinase activity or TMP-PPase activity in the first and second reaction mixtures; and (c) selecting the chemical to be tested for the ability to inhibit plant growth or viability when the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the second reaction mixture
• the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant, preferably Arabidopsis thaliana.
• the enzyme having HMP- P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 , or has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2.
• the substrate of HMP kinase is HMP and in yet another preferred embodiment, the substrate of TMP-PPase is THZ-P.
• the activity of the enzyme is measured by determining the TMP produced in the reaction mixture.
• the invention also further describes an assay comprising the steps of: (a) combining an enzyme having HMP kinase activity and an enzyme having HMP-P kinase activity or TMP-PPase activity in a first reaction mixture with a substrate of HMP kinase and a substrate of TMP-PPase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of TMP; (b) combining the chemical and the enzymes in a second reaction mixture with the substrates 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 HMP-P kinase activity or TMP-PPase activity in the first and second reaction mixtures; wherein the chemical is capable of inhibiting the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity if the activity of the enzyme having HMP-P
• the present invention further describes a method for identifying a chemical to tested for the ability to inhibit plant growth or viability, comprising the steps of: (a) combining an enzyme having THZ kinase activity and an enzyme having HMP-P kinase activity or TMP- PPase activity in a first reaction mixture with a substrate of HMP-P kinase and a substrate of THZ kinase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of TMP; (b) combining the chemical and the enzymes in a second reaction mixture with the substrates 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 HMP-P kinase activity or TMP-PPase activity in the first and second reaction mixtures; and (d) selecting the chemical to be tested for the ability to inhibit plant growth or viability when the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in
• the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant, preferably Arabidopsis thaliana.
• the enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 , or has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2.
• the substrate of HMP-P kinase is HMP-P and in yet another embodiment, the substrate of THZ kinase is THZ.
• the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity is measured by determining the TMP produced in the reaction mixture.
• the invention further describes an assay comprising the steps of: (a) combining an enzyme having THZ kinase activity and an enzyme having HMP-P kinase activity or TMP-PPase activity in a first reaction mixture with a substrate of THZ kinase and a substrate of HMP-P kinase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of TMP; (b) combining the chemical and the enzymes in a second reaction mixture with the substrates 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 HMP-P kinase activity or TMP- PPase activity in the first and second reaction mixtures; wherein the chemical is capable of inhibiting the activity of the enzyme having HMP-P kinase
• the present invention describes a method for identifying chemicals having the ability to inhibit HMP-P kinase or TMP-PPase activity in plants preferably comprising the steps of: a) obtaining transgenic plants, plant tissue, plant seeds or plant cells, preferably stably transformed, comprising a non-native nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity and capable of overexpressing an enzymatically active HMP-P kinase or TMP-PPase; b) applying the chemical 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; d) comparing the growth or viability of the transgenic and non-transf ⁇ rmed plants, plant cells, tissues after application of 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 HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence derived from a plant, preferably Arabidopsis thaliana, desirably identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.
• the enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
• the enzyme having HMP-P kinase activity or TMP-PPase activity 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 present invention further embodies plants, plant tissues, plant seeds, and plant cells that have modified HMP-P kinase activity or TMP-PPase activity and that are therefore tolerant to inhibition by a herbicide at levels normally inhibitory to naturally occurring HMP-P kinase activity or TMP-PPase 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 HMP-P kinase or TMP-PPase that is tolerant to inhibition by a herbicide at a concentration that would normally inhibit the activity of wild-type, unmodified HMP-P kinase or TMP-PPase.
• Modified HMP-P kinase activity or TMP-PPase activity may also be conferred upon a plant by increasing expression of wild-type herbicide-sensitive HMP-P kinase or TMP-PPase by providing multiple copies of wild-type HMP-P kinase or TMP- PPase genes to the plant or by overexpression of wild-type HMP-P kinase or TMP-PPase 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 HMP-P kinase activity or TMP-PPase activity, wherein the enzyme has HMP-P kinase activity or TMP-PPase 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 HMP-P kinase activity or TMP-PPase activity.
• the enzyme having HMP-P kinase activity or TMP-PPase activity 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, 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 HMP-P kinase activity or TMP-PPase 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 HMP-P kinase activity or TMP-PPase activity; and (b) applying to the crops or crop seeds and the weeds in the field a herbicide in amounts that inhibit naturally occurring HMP-P kinase activity or TMP-PPase activity, wherein the herbicide suppresses the growth of the weeds without significantly suppressing the growth of the crops.
• the th1 mutant was later shown to lack the TMP-PPase enzymatic activity using biochemical methods ( Komeda et al. (1988) Plant Physiol. 88, 248-250).
• BAC F19G10 BAC F19G10
• GenBank GenBank that covered this region of chromosome 1.
• one gene had significant amino acid sequence similarities to both thiD and thiE irom E. coli (nucleotide positions 46133 to 48657 on the BAC sequence).
• the inventors have isolated an Arabidopsis c- DNA encoding a novel protein with two functional domains: the amino acid sequence of the N-terminal domain (up to approximately nucleotide 906 in SEQ ID NO:1 or amino acid 302 in SEQ ID NO:2) has homology to HMP-P kinases (thiD) and the amino acid sequence of the C-terminal domain (from approximately nucleotide 925 in SEQ ID NO:1 or amino acid 309 in SEQ ID NO:2) has homology to TMP-PPases (thiE).
• a putative chloroplast transit peptide is also predicted in the first 99 nucleotides in SEQ ID NO:1 (first 33 amino acids in SEQ ID NO:2).
• the inventors have also obtained the sequence of the HMP-P kinase/TMP-PPase gene derived from two different th1 mutants, CS79 and CS3530 (Arabidopsis Stock Center, Nottingham, UK). They found that in both mutants a mutation is present in the HMP-P kinase/TMP-PPase gene.
• CS79 has a single nucleotide change from C to T at position 188 in SEQ ID NO:1 that changes amino acid 63 in SEQ ID NO:2 from a serine to a phenylalanine.
• CS3530 has a seven nucleotide deletion from either positions 259-265 or positions 260-266 in SEQ ID NO:1 that changes amino acid 87 in SEQ ID NO:2 from an isoleucine to an asparagine.
• the codon for asparagine 87 is followed by codons for 13 amino acids and then a TGA stop codon in the reading frame.
• the mutation should result in the translation of a protein of only 100 amino acids comprising only a small portion of the domain homologous to thiD and lacking the domain homologous to thiE.
• the HMP-P kinase/TMP-PPase gene is further referred as encoding an enzyme having HMP-P kinase activity or TMP-PPase activity.
• the HMP-P kinase/TMP-PPase gene encodes a single polypeptide having HMP-P kinase activity or TMP-PPase activity.
• the single polypeptide encoded by the HMP-P kinase/TMP-PPase gene has HMP-P kinase activity and TMP- PPase activity.
• the nucleotide sequence encoding the mature HMP-P kinase/TMP-PPase gene as set forth in SEQ ID NO:3 has been deposited in the Agricultural Research Culture Collection (NRRL), 1815 N, University Street, Peoria, Illinois 61604, USA, as International Depositary Authority as established under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure under the clone name aththiDE-ctp, Accession number NRRL B-30040, on July 8, 1998.
• NRRL Agricultural Research Culture Collection
• Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli, yeast, and insect cells (see, e.g., Luckow and Summers, Bio/Technol. 6: 47 (1988)).
• host organisms such as E. coli, yeast, and insect cells
• 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 the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from an eukaryote, such as a yeast, but is preferably derived from a plant.
• the nucleotide sequence set forth in SEQ ID NO:1 encodes the Arabidopsis HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP-PPase, 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 HMP-P kinase activity and the enzyme having TMP-PPase activity are encoded by the thiD and thiE genes, respectively.
• HMP-P kinase/TMP-PPase is 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).
• HMP-P kinase/TMP-PPase is useful for a variety of purposes.
• it can be used in in vitro assays to screen known herbicidal chemicals whose target has not been identified to determine if they inhibit HMP-P kinase activity or TMP-PPase activity.
• 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.
• HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP-PPase comprises the steps of: a) reacting an enzyme having HMP-P kinase activity or TMP-PPase activity and the substrates thereof in the presence of a suspected inhibitor of the enzyme's function; b) comparing the rate of enzymatic activities in the presence of the suspected inhibitor to the rate of enzymatic activities under the same conditions in the absence of the suspected inhibitor; and c) determining whether the suspected inhibitor inhibits the HMP-P kinase enzymatic activity or TMP-PPase enzymatic activity.
• the inhibitory effect on HMP-P kinase activity or TMP- PPase activity is determined by a reduction or complete inhibition of TMP synthesis in the assay. In a preferred embodiment, such a determination is made by comparing, in the presence and absence of the candidate inhibitor, the amount of TMP synthesized in the in vitro assay using fluorescence 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 absorbance detection.
• a preferred substrate for HMP-P kinase is 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P).
• Preferred substrates for TMP-PPase are 2-methyl-4-amino-5- hydroxymethylpyrimidine pyrophosphate (HMP-PP) and 4-methyl-5-(beta- hydroxyethyl)thiazole phosphate (THZ-P).
• the amount of HMP-P available for the HMP-P kinase is increased by using a coupled HMP kinase/HMP-P kinase/TMP-PPase assay, thereby increasing the detection limit of the assay and resulting in an improved screening procedure for chemical inhibiting HMP-P kinase activity or TMP-PPase activity.
• Such a coupling assay comprises the steps of: (a) combining an enzyme having HMP kinase activity and an enzyme having HMP-P kinase activity or TMP-PPase activity in a first reaction mixture with a substrate of HMP kinase and a substrate of TMP-PPase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of TMP; (b) combining the chemical and the enzymes in a second reaction mixture with the substrates under the same conditions and for the same period of time as in the first reaction mixture; (b) determining the activity of the enzyme having HMP- P kinase activity or TMP-PPase activity in the first and second reaction mixtures; and (c) selecting the chemical to be tested for the ability to inhibit plant growth or viability when the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the second reaction mixture is significantly less than the activity of the enzyme having HMP-P kinase activity or T
• the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant.
• the enzyme having HMP-P kinase activity or TMP-PPase 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, 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 substrate of HMP kinase is HMP and in yet another preferred embodiment, the substrate of TMP-PPase is THZ-P.
• the activity of the enzyme is measured by determining the TMP produced in the reaction mixture.
• Such a coupling assay comprises the steps of: (a) combining an enzyme having THZ kinase activity and an enzyme having HMP-P kinase activity or TMP-PPase activity in a first reaction mixture with a substrate of HMP-P kinase and a substrate of THZ kinase under conditions in which the enzymes are capable of catalyzing the coupled synthesis of TMP; (b) combining the chemical and the enzymes in a second reaction mixture with the substrates 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 HMP-P kinase activity or TMP-PPase activity in the first and second reaction mixtures; and (d) selecting the chemical to be tested for the ability to inhibit plant growth or viability when the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the second reaction mixture is significantly less than the activity of the enzyme having HMP-P kinas
• the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant.
• the enzyme having HMP-P kinase activity or TMP-PPase 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, 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 substrate of HMP-P kinase is HMP-P and in yet another embodiment, the substrate of THZ kinase is THZ.
• the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity is measured by determining the TMP produced in the reaction mixture.
• 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 HMP-P kinase activity or TMP-PPase activity uses transgenic plants, plant tissue, plant seeds or plant cells capable of overexpressing a nucleotide sequence having HMP-P kinase activity or TMP- PPase activity, wherein the HMP-P kinase and TMP-PPase are 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 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 HMP-P kinase or TMP-PPase 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.
• the enzymes having HMP-P kinase and TMP-PPase activities are encoded by the thiD and thiE genes, respectively.
• 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 HMP-P kinase activity or TMP- PPase activity in these plants, wherein the tolerance is conferred by an altered HMP-P kinase activity or TMP-PPase activity.
• Altered HMP-P kinase activity or TMP-PPase activity may be conferred upon a plant according to the invention by increasing expression of wild-type herbicide-sensitive HMP-P kinase/TMP-PPase by providing additional wild-type HMP-P kinase/TMP-PPase genes to the plant, by expressing a modified herbicide-tolerant HMP-P kinase/TMP-PPase 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.
• HMP-P kinase activity or TMP-PPase activity results in levels of HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP-PPase 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. 5,162,602, and U.S. Patent No. 4,761 ,373, and references cited therein). These plants also may be obtained by genetic engineering techniques known in the art.
• Increased expression of a herbicide-sensitive HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP-PPase.
• the transformation is stable, thereby providing a heritable transgenic trait.
• a genetically manipulatable microbe such as E. co// ' 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 er a/., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983); and U.S. Patent No.
• a method of obtaining mutant herbicide-tolerant alleles of a plant HMP-P kinase/TMP- PPase gene involves direct selection in plants.
• the effect of a mutagenized HMP-P kinase/TMP-PPase 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.
• HMP-P kinase/TMP-PPase gene Confirmation that the genetic basis of the herbicide tolerance is a modified HMP-P kinase/TMP-PPase gene is ascertained as exemplified below.
• alleles of the HMP-P kinase/TMP-PPase gene from plants exhibiting resistance to the inhibitor are isolated using PCR with primers based either upon the Arabidopsis cDNA coding sequences shown in SEQ ID NO:? or, more preferably, based upon the unaltered HMP-P kinase/TMP-PPase gene sequence from the plant used to generate tolerant alleles.
• Another method of obtaining herbicide-tolerant alleles of a HMP-P kinase/TMP-PPase 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.
• a mutagenized HMP-P kinase/TMP-PPase gene is formed from at least one template HMP-P kinase/TMP-PPase gene, wherein the template HMP-P kinase/TMP-PPase 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
• a mutagenized DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity is formed from at least two non- identical template DNA molecules encoding enzymes having HMP-P kinase activity or TMP- PPase activity, comprising the steps of adding to the template DNA molecules at least one oligonucleotide comprising an area of identity to each of the template DNA molecule, denaturing the resultant mixture into single-stranded molecules, 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, 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
• 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. (1998) Nature 391 : 288-291 , as well as in WO 97/20078, and these references are incorporated herein by reference.
• fragments of HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP- PPase 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.
• a bacteria such as a E. coli thiD or thiE gene (Vander Horn et al. (1993) J. Bacteriol. 175, 982-992; Backstrom er a/. (1995) J. Am. Chem. Soc. 1.17, 2351 - 2352), a Salmonella typhimurium thiD gene (Petersen and Downs (1997) J. Bacteriol. 179, 4894-4900), a Neurospora crassa thiD gene (Akiyama and Nakashima (1996) Curr. Genet. 30, 62-67; Ouzonis and Kyrpides (1997) J. Mol. Evol.
• HMP-P kinase/TMP-PPase genes or portions thereof are used in the context of the present invention.
• the library of mutated HMP-P kinase/TMP-PPase 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 assay for identifying a modified HMP-P kinase/TMP-PPase gene that is tolerant to an inhibitor may be performed in the same manner as the assay to identify inhibitors of the HMP-P kinase activity or TMP-PPase activity (Inhibitor Assay, above) with the following modifications: First, a mutant HMP-P kinase/TMP-PPase is substituted in one of the reaction mixtures for the wild-type HMP-P kinase/TMP-PPase of the inhibitor assay.
• the method is applicable to any plant cell capable of being transformed with a modified HMP-P kinase/TMP-PPase-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 method for forming a mutagenized DNA molecule according to the invention wherein one template DNA molecule is derived from an eukaryote.
• Paricularly preferred is a method according to the invention, wherein said eukaryote is a plant.
• this involves inserting a DNA molecule encoding the HMP-P kinase/TMP-PPase 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. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. Expression systems known in the art can be used to transform virtually any crop plant cell under suitable conditions.
• 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.
• 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.
• 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. 77: 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.
• Transformation without the use of Agrobactenum 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 Agrobactenum include transformation via particle bombardment, protoplast uptake (e.gr. 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 Agrobactenum 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 Agrobactenum.
• 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 /4grojbacferu/77-mediated transformation.
• HMP-P kinase/TMP-PPase 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.
• 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
• the high-level expression of a wild-type HMP-P kinase/TMP-PPase gene and/or the expression of herbicide-tolerant forms of a HMP-P kinase/TMP-PPase 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.
• HMP-P kinase/TMP-PPase 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.
• SEQ ID NO:1 DNA sequence of the Arabidopsis HMP-P kinase/TMP-PPase pre-protein
• Example 1 Expression of Recombinant Plant HMP-P Kinase and TMP-PPase in E. coli
• An Arabidopsis thaliana ecotype Landsberg cDNA library in the plasmid vector pFL61 (Minet et al., (1992) Plant J. 2, 417-422) is obtained and amplified.
• PCR primers to amplify protein coding sequence to Arabidopsis HMP-P kinase and TMP-PPase are designed from the SEQ ID NO:? and used to amplify the HMP-P kinase and TMP-PPase coding sequence from the plasmid library with Pfu DNA Polymerase (Stratagene).
• primers aththiDEfor (5' ggt att gag ggt cgc atg aat age tta gga gga at 3', SEQ ID NO:5)and aththiDErev (5' aga gga gag tta gag cct caa att ccc ctt ttg etc tct tta a 3', SEQ ID NO:6) are used, and for the construct of the putative mature HMP-P kinase and TMP-PPase, primers aththiDErev and aththiDEfor-ctp (5' ggt att gag ggt cgc tta aca gtg gcg gga tea gat 3', SEQ ID NO:7) are used.
• the HMP-P kinase and TMP-PPase activity assay is essentially derived from Komeda et al. (1988) Plant Physiol. 88, 248-250.
• the reaction volumes are preferably the ones described below, but can be varied depending on the experimental requirements.
• TMP thiamine monophosphate
• 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.
• the conversion of HMP to HMP-P 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 conversion of NADH to NAD + in the presence of phosphoenolpyruvate (PEP). This is monitored at 340nm. Pyruvate kinase and PEP facilitate the regeneration of ATP from ADP. ATP is a required substrate for both HMP kinase and HMP-P kinase.
• the assay buffer is 50 mM Tris-HCI, pH 7.5 (Buffer A) with the addition of 10 mM MgCI 2 .
• HMP-P kinase and TMP-PPase it is necessary to provide the substrates HMP-P and THZ-P.
• the HMP-P is provided by the conversion of HMP to HMP-P in the same reaction mixture. If NADH is added, the conversion can be followed utilizing the HMP kinase assay. When the HMP to HMP-P conversion proceeds sufficiently (approximately 50 mM), then HMP-P kinase, TMP-PPase, and THZ-P are added.
• the assays are carried out in the same way independent of the original source of the enzymes.
• the assays are performed in 300 mL 96 well microtiter plates.
• the total assay reaction volume is 100 mL.
• Substrates are mixed in a ratio such that the final concentration (in the microtiter plate) are as follows: HMP (100 mM), ATP (1000 mM), PEP (1000 mM), and NADH (200 mM).
• a mixture of the substrates at 10X can be pipetted at 10 mL/well.
• the reagent enzymes and HMP kinase can also be mixed to be added simultaneously.
• the suggested amount of the ADP detecting/regeneration mix is 1.0 units pyruvate kinase and 1.0 units lactate dehydrogenase per reaction. This should be used as a guideline and the amounts of enzyme adjusted empirically.
• the HMP kinase reaction proceeds to completion at a rate of approximately 5 mM/min (this is within 10-15 minutes)
• the THZ-P is added to a final concentration of 50 mM and the HMP-P kinase and TMP-PPase are added.
• the reaction is stopped by the addition of 50 mL of 10% (w/v) metaphosphoric acid or 20% (w/v) trichloroacetic acid (pH 1.4).
• the plate is spun in a centrifuge to pellet the precipitated protein, then the supernatant is transferred to a separate microtiter plate.
• 50 mL of 0.3 M cyanogen bromide is added and mixed, followed by addition of 100 mL of 1 M NaOH.
• the plate is read by a fluorimetric microtiter plate reader with an excitation wavelength of 360 ⁇ 10nm and an emission wavelength of 430 ⁇ 10nm.
• Thiamine monophosphate is used as a standard.
• the conversion of THZ to THZ-P is followed by detecting the concomitant formation of ADP.
• the ADP formation is followed utilizing the enzymes pyruvate kinase and lactate dehydrogenase (reagent enzymes) and detecting conversion of NADH to NAD + in the presence of phosphoenolpyruvate (PEP). This is monitored at 340nm. Pyruvate kinase and PEP facilitate the regeneration of ATP from ADP. ATP is a required substrate for both THZ kinase and HMP-P kinase.
• the assay buffer is Buffer A with the addition of 10 mM MgCI 2 .
• HMP-P kinase and TMP-PPase it is necessary to provide the substrates HMP-P and THZ-P.
• the THZ-P is provided by the conversion of THZ to THZ-P in the same reaction mixture. If NADH is added, the conversion can be followed utilizing the THZ kinase assay. When the THZ to THZ-P conversion proceeds sufficiently (approximately 20 mM), then HMP-P kinase, TMP-PPase, and HMP-P are added.
• Adding the HMP-P kinase, TMP- PPase, and HMP-P after the production of THZ-P insures that the initial concentration of THZ-P is constant in all reaction wells.
• the amount of TMP formed is assayed by the method of Kawasaki et al. (1990) J. Bacteriol. 172, 6145-6147. After a sufficient time for TMP production (typically 15 minutes), the enzyme reaction is stopped with metaphosphoric acid or TCA. The TMP is oxidized under alkaline conditions. The fluorescence of the resultant thiachrome derivative is measured with an excitation wavelength of 360 ⁇ 10 nm and an emission wavelength of 430 ⁇ 10 nm.
• the assays are carried out in the same way independent of the original source of the enzymes.
• the assays are performed in 300 mL 96 well microtiter plates.
• the total assay reaction volume is 100 mL.
• Substrates are mixed in a ratio such that the final concentration (in the microtiter plate) are as follows: THZ (50 mM), ATP (5 mM), PEP (1000 mM), and NADH (200 mM).
• a mixture of the substrates at 10X can be pipetted at 10 mL/well.
• the reagent enzymes and HMP kinase can also be mixed to be added simultaneously.
• the suggested amount of the ADP detecting/regeneration mix is 1.0 units pyruvate kinase and 1.0 units lactate dehydrogenase per reaction. This should be used as a guideline and the amounts of enzyme adjusted empirically.
• the HMP-P is added to a final concentration of 100 mM and the HMP-P kinase and TMP-PPase are added.
• Thiamine monophosphate, ATP, PEP, NADH, metaphosphoric acid, trichloroacetic acid, cyanogen bromide, and NaOH are available from Sigma Chemicals.
• HMP is synthesized by the methods of Schellenberger et al. (Hoppe-Seyler's Z. Physiol. Chem. (1967) 348, 501 -505) and THZ-P is synthesized according to the method of Leder et al. (1970) Meth. Enz. 18A, 166-167.
• Example 5 Coupled HMP Kinase, THZ Kinase, and HMP-P Kinase and TMP-PPase Activity Assay
• HMP kinase and THZ kinase assays The conversions of HMP to HMP-P and of THZ to THZ-P are followed by detecting the concomitant formation of ADP.
• the ADP formation is followed utilizing the enzymes pyruvate kinase and lactate dehydrogenase (reagent enzymes) and detecting conversion of NADH to NAD + in the presence of phosphoenolpyruvate (PEP). This is monitored at 340nm. Pyruvate kinase and PEP facilitate the regeneration of ATP from ADP. ATP is a required substrate for both HMP kinase and HMP-P kinase.
• the assay buffer is Buffer A with the addition of 10 mM MgCI 2 .
• HMP-P kinase and TMP-PPase To assay HMP-P kinase and TMP-PPase it is necessary to provide the substrates HMP-P and THZ-P.
• the HMP-P and THZ-P are provided by the conversion of HMP to HMP-P and of THZ to THZ-P in the same reaction mixture. If NADH is added, the conversions can be followed utilizing the HMP kinase and THZ kinase assays.
• HMP-P kinase and TMP-PPase are added. Adding the HMP-P kinase and TMP- PPase after the production of HMP-P and THZ-P insures that the initial concentration of these substrates is constant in all reaction wells.
• the amount of TMP formed is assayed by the method of Kawasaki et al. (1990) J. Bacteriol. 172, 6145-6147. After a sufficient time for TMP production (typically 15 minutes), the enzyme reaction is stopped with metaphosphoric acid or TCA. The TMP is oxidized under alkaline conditions. The fluorescence of the resultant thiachrome derivative is measured with an excitation wavelength of 360 ⁇ 10 nm and an emission wavelength of 430 ⁇ 10 nm.
• the assays are carried out in the same way independent of the original source of the enzymes.
• the assays are performed in 300 mL 96 well microtiter plates.
• the total assay reaction volume is 100 mL.
• Substrates are mixed in a ratio such that the final concentration (in the microtiter plate) are as follows: HMP (100 mM), THZ (50 mM), ATP (5 mM), PEP (1000 mM), and NADH (200 mM).
• a mixture of the substrates at 10X can be pipetted at 10 mL/well.
• the reagent enzymes and HMP kinase can also be mixed to be added simultaneously.
• the suggested amount of the ADP detecting/regeneration mix is 1.0 units pyruvate kinase and 1.0 units lactate dehydrogenase per reaction. This should be used as a guideline and the amounts of enzyme adjusted empirically. After the HMP kinase and THZ kinase reactions proceed to completion at a rate of approximately 5 mM/min (this is within 10-15 minutes), the HMP-P kinase and TMP-PPase are added.
• the reaction is stopped by the addition of 50 mL of 10% (w/v) metaphosphoric acid or 20% (w/v) trichloroacetic acid (pH 1.4).
• the plate is spun in a centrifuge to pellet the precipitated protein, then the supernatant is transferred to a separate microtiter plate.
• 50 mL of 0.3 M cyanogen bromide is added and mixed, followed by addition of 100 mL of 1 M NaOH.
• the plate is read by a fluorimetric microtiter plate reader with an excitation wavelength of 360 ⁇ 10nm and an emission wavelength of 430 ⁇ 10nm.
• Thiamine monophosphate is used as a standard.
• Example 6 In vitro Recombination of HMP-P kinase/TMP-PPase Genes by DNA Shuffling
• the A. thaliana HMP-P kinase/TMP-PPase 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).
• thaliana HMP-P kinase/TMP-PPase gene encoding the mature protein and the E.coli thiE 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 HMP-P kinase/TMP-PPase genes are screened as described in example 6.
• Example 8 Initiation and maintenance of a rice embryogenic cell suspension
• Twenty embryos per target plate is typical, although not critical.
• An appropriate gene-carrying plasmid is precipitated onto micrometer size gold particles using standard procedures.
• Each plate of embryos is shot with the DuPont Biolistics helium device using a burst pressure of -1000 psi and using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent.
• the callus developing on the scutellum is removed from the embryo and plated on B5 medium (Gamborg et al., 1968) containing 0.5 mg/l 2,4-D and solidified with 0.24% Gelrite.
• B5 medium Gibborg et al., 1968
• the callus is subcultured every two weeks to fresh medium.
• the special type of callus is identified by its characteristic morphology. This callus is subcultured further on the same medium.
• the callus is transferred to, and serially subcultured on, N6 medium containing 2 mg/l 2,4-D and solidified with Gelrite.
• Example 12 Preparation of a suspension culture of Zea mays elite inbred line Funk 2717
• 1 to 1.5 ml PCV of the suspension culture cells from above are incubated in 10 to 15 ml of a filter-sterilized mixture consisting of 4% cellulase RS with 1 % Rhozyme in KMC (8.65 g/l KCI, 16.47 g/l MgCI2 x 6 H20 and 12.5 g/l CaCI2 x 2 H20, 5 g/l MES, pH 5.6) salt solution. Digestion is carried out at 30°C on a slow rocking table for a period of 3 to 4 hours. The preparation is monitored under an inverted microscope for protoplast release.
• the protoplasts which are released are collected as follows: The preparation is filtered through a 100 ⁇ m mesh sieve, followed by a 50 ⁇ m mesh sieve. The protoplasts are washed through the sieves with a volume of KMC salt solution equal to the original volume of enzyme solution. 10 ml of the protoplast preparation is placed in each of several disposable plastic centrifuge tubes, and 1.5 to 2 ml of 0.6 M sucrose solution (buffered to pH 5.6 with 0.1 % MES and KOH) layered underneath. The tube is centrifuged at 60 to 100 x g for 10 minutes, and the protoplasts banding at the interface collected using a pipette and placed in a fresh tube.
• the protoplasts are plated after the electroporation in dishes placed on a plate cooled to a temperature of 16°C.
• the protoplasts are placed in tubes after the electroporation step, washed with 10 ml of 6/7 strength KMC solution or with W5 solution (comprised of 380 mg/l KCI, 18.375 g/l CaCI2 x 2 H20, 9 g/l NaCI; 9 g/l glucose, pH 6.0), then collected by centrifugation at 60 x g for 10 minutes, resuspended in 0.3 ml of KM medium, and plated as in A.
• the protoplasts are resuspended at the last step of above in a 0.5 M mannitol solution containing 12 to 30 mM MgCI2. A heat shock of 45°C for five minutes is given as described.
• the protoplasts are distributed in aliquots for transformation in centrifuge tubes, 0.3 ml of suspended protoplasts per tube. During the next 10 minutes the following are added: DNA and PEG solution (MW 6000, 40% containing 0.1 M Ca(N03)2 and 0.4 M mannitol; pH 8 to 9 with KOH) to give a final concentration of 20% PEG.
• DNA and PEG solution MW 6000, 40% containing 0.1 M Ca(N03)2 and 0.4 M mannitol; pH 8 to 9 with KOH
• the aliquots are incubated for 30 minutes with occasional gentle shaking, and then the protoplasts are placed in petri dishes (0.3 ml original protoplast suspension per 6 cm diameter dish) and cultured as described.
• the DNA is added in 10 ⁇ l sterile distilled water, sterilized as described by Paszkowski et al. (1984).
• the solution is mixed gently and then subjected at room temperature (24 to 28°C) to a pulse of 400 Vcm-1 with an exponential decay constant of 10 ms from a BTX-Transfector 300 electroporation apparatus using the 471 electrode assembly.
• the exponential decay constant is 5 ms, 15 ms or 20 ms.
• the plasmid DNA is linearized before use by treatment with an appropriate restriction enzyme (e.g. BamHI).
• an appropriate restriction enzyme e.g. BamHI
• Protoplasts of Glycine max are prepared by the methods as described by Tricoli et al. (1986), or Chowhury and Widholm (1985), or Klein et al. (1981 ). DNA is introduced into these protoplasts essentially as described above. The protoplasts are cultured as described in Klein et al. (1981 ), Chowhury and Widholm (1986) or Tricoli et al. (1986) without the addition of alginate to solidify the medium.
• microorganism identified under I. above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) .

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