EP1093520A1

EP1093520A1 - Hmp-p kinase and tmp-ppase from arabidopsis thaliana and their use in herbicide screening

Info

Publication number: EP1093520A1
Application number: EP99931202A
Authority: EP
Inventors: Joshua Zvi Levin; Sharon Lee Potter; Michael William Bauer
Original assignee: Syngenta Participations AG
Current assignee: Syngenta Participations AG
Priority date: 1998-06-30
Filing date: 1999-06-28
Publication date: 2001-04-25
Also published as: CA2331881A1; AU4778999A; CN1317051A; WO2000000623A1; JP2002519035A

Abstract

The present invention discloses methods to screen chemicals for herbicidal activity using recombinantly produced enzymes having HMP-P kinase activity or TMP-PPase activity, and the use of thereby to identify herbicidal chemicals to suppress the growth of undesired vegetation. Furthermore, the present invention provides methods for the development of herbicide tolerance in plants, plant tissues, plant seeds, and plant cells using genes encoding enzymes having HMP-P kinase activity or TMP-PPase activity, and methods of using such transgenic plants to selectively suppress weed growth in crop fields.

Description

HMP-P KINASE AND TMP-PPASE FROM ARABIDOPSIS THALIANA AND THEIR USE IN HERBICIDE SCREENING

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.

The use of herbicides to control undesirable vegetation such as weeds in crop fields has become almost a universal practice. The herbicide market exceeds 15 billion dollars annually. Despite this extensive use, weed control remains a significant and costly problem for farmers.

Effective use of herbicides requires sound management. For instance, the time and method of application and stage of weed plant development are critical to getting good weed control with herbicides. Since various weed species are resistant to herbicides, the production of effective new herbicides becomes increasingly important. Novel herbicides can now be discovered using high-throughput screens that implement recombinant DNA technology. Metabolic enzymes found to be essential to plant growth and development can be recombinantly produced and utilized as herbicide targets in screens for novel inhibitors of the enzymes' activities. The novel inhibitors discovered through such screens may then be used as herbicides to control undesirable vegetation.

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. For example, U.S. Patent No. 4,761 ,373 to Anderson et al. is directed to plants resistant to various imidazolinone or sulfonamide herbicides. The resistance is conferred by an altered acetohydroxyacid synthase (AHAS) enzyme. 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. 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. U.S. Patent No. 5,162,602 to Somers et al. discloses plants tolerant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The tolerance is conferred by an altered acetyl coenzyme A carboxylase (ACCase).

DEFINITIONS

For clarity, certain terms used in the specification are defined and presented as follows:

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. In an especially preferred embodiment, the target gene encodes an enzyme having HMP-P kinase activity or TMP-PPase activity. The interaction of the antisense sequence and the target gene results in substantial inhibition of the expression of the target gene so as to kill the plant, or at least inhibit normal plant growth or development.

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. Optionally, 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: 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.

Expression refers to the transcription and/or translation of an endogenous gene or a transgene in plants. In the case of antisense constructs, for example, 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. Examples of 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. In the context of the instant invention, 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.

Isolated: in the context of the present invention, an isolated DNA molecule or an isolated enzyme 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.

Significant Increase: 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. Significantly less: means that 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.

In its broadest sense, 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. Desirably 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 NaPO₄, 1 iτiM 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 NaPO₄, 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 NaPO₄, 1 mM EDTA at 50°C with washing in 0.2X SSC, 0.1% SDS at 65°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 65°C.

The term "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. When used for a protein or an amino acid sequence 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%.

Substrate: 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.

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.

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.

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 discloses for the first time the correct nucleotide sequence of the Arabidopsis HMP-P kinase/TMP-PPase gene. The nucleotide sequence encoding the 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 HMP-P kinase and TMP-PPase pre-protein is set forth in SEQ ID NO:2 and of the correct amino acid sequence of the putative mature Arabidopsis HMP-P kinase and TMP-PPase is set forth in SEQ ID NO:4. 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. In a preferred embodiment, such 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

The 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). The HMP-P kinase enzymatic activity catalyzes the pyrophophoshorylation of 2-methyl-4-amino-5- hydroxymethylpyrimidine monophosphate (HMP-P) to form 2-methyl-4-amino-5- hydroxymethylpyrimidine pyrophosphate (HMP-PP). In E. coli, this step is carried out by a protein encoded by the thiD gene. The TMP-PPase enzymatic activity corresponds to the next to last step in the de novo biosynthesis of thiamine pyrophosphate and catalyzes the coupling of 4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate (THZ-P) and HMP-PP to form thiamine monophosphate (TMP). In E. coli, this step is carried out by a protein encoded by the thiE gene.

With the knowledge of the correct nucleotide sequence of the HMP-P kinase/TMP- PPase gene, the inventors of the present invention have shown that thiamine auxotroph mutants, which require to be supplemented with exogenous thiamine to be able to grow, have a mutation in the coding sequence of their HMP-P kinase/TMP-PPase gene. This unequivocally demonstrates the essentiality of this gene in plants at the molecular level. With such knowledge it is possible to develop screens for chemicals which are able to inhibit HMP-P kinase activity or TMP-PPase activity. Due to the essentiality of the enzyme activity encoded by the HMP-P kinase/TMP-PPase gene, such chemicals are also expected to inhibit growth or viability of a plant and therefore be potentially good herbicides. It is therefore an object of the present invention to provide methods of using a purified HMP-P kinase or TMP-PPase to identify inhibitors thereof, which can then be used as herbicides to suppress the growth of undesirable vegetation, e.g. 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.

Encompassed by the present invention is a chimeric gene comprising a promoter operatively linked to a nucleotide sequence which encodes for an enzyme having HMP-P kinase activity or TMP-PPase activity according to the invention.

Further encompassed is a recombinant vector comprising said chimeric gene according to the invention, wherein said vector is capable of being stably transformed into a host cell. Further encompasse d is 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.

Further preferred is 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.

Preferred is a protein according to the invenion, wherein said protein has TMP-PPase activity. More preferred is a protein according to the invention, wherein said protein comprises an amino acid sequence substantially similar to the amino acid sequence set forth in SEQ ID NO:2. More preferred is an protein according to the invention wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:2.

In a preferred embodiment, the present invention describes a method for identifying a chemical to be tested for the ability to inhibit plant growth or viability, 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 to be tested and the enzyme in a second reaction mixture with the substrate used in the first reaction mixture 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; and (d) selecting the chemical to be tested for the ability to inhibit plant growth or viability when the activity of the enzyme in the second reaction mixture is less, desirably significantly less, than the activity of the enzyme in the first reaction mixture. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant, preferably Arabidopsis thaliana. In another preferred embodiment, 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. In another preferred embodiment, 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 is significantly less than the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the first reaction mixture. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant, preferably Arabidopsis thaliana. In another preferred embodiment, 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. In another preferred embodiment, the substrate of HMP kinase is HMP and in yet another preferred embodiment, the substrate of TMP-PPase is THZ-P. In a further preferred embodiment, 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 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 TMP-PPase activity in the first reaction mixture.

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 second reaction mixture is significantly less than the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the first reaction mixture. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant, preferably Arabidopsis thaliana. In another preferred embodiment, 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. In another preferred embodiment, the substrate of HMP-P kinase is HMP-P and in yet another embodiment, the substrate of THZ kinase is THZ. In another preferred embodiment, 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 activity or TMP-PPase activity if 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 TMP-PPase activity in the first reaction mixture.

In another preferred embodiment, 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-transfαrmed plants, plant cells, tissues after application of the chemical. Desirably, 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. In a preferred embodiment, 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. In another embodiment, 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. In yet another embodiment, 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. According to this embodiment, 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.

Therefore, 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. According to one example of this embodiment, 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. In a related aspect, 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.

Other objects and advantages of the present invention will become apparent to those skilled in the art from a study of the following description of the invention and non-limiting examples.

Several obligate organoauxotrophic mutants requiring exogenous thiamine or thiamine precursors {i.e., 4-methyl-5-(beta-hydroxyethyl)thiazole (THZ) and/or 2-methyl-4-amino-5- hydroxymethylpyrimidine (HMP)) have been identified in Arabidopsis thaliana and mapped to four loci (Li and Redei (1969) Biochemical Genetics 3, 163-170). One locus, f/?1 , was mapped to chromosome 1 (Koornneef and Hanhart (1981 ) Arab. Info. Service 18, 52-58). Although the exact position of the block between the pyrimidine and thiazole precursors and thiamine in the th1 mutant could not be located by Koomeef and Hanhart, 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).

Recently, a BAC sequence (BAC F19G10) was submitted to GenBank that covered this region of chromosome 1. On this BAC, 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). Based on this information, 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. In this mutant, 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. These results show that the thiamine auxotrophic phenotype is due to a mutation in the HMP-P kinase/TMP-PPase gene and unequivocally demonstrate that the HMP-P kinase/TMP-PPase gene is essential in plants and potentially represents a good target for herbicides.

Based on the c-DNA sequence, the inventors further discovered that two inaccurate splicing junctions had been predicted in the BAC clone. A first predicted splicing junction had inaccurately resulted in the addition of 24 bases of intron sequence at position 383 in SEQ ID NO:1 (figure 2) and a second predicted splicing junction had inaccurately resulted in the deletion of 24 bases and addition of 9 wrong bases between positions 1066 and 1089 in SEQ ID NO:1 (figure 2). Based on this predictions, a HMP-P kinase/TMP-PPase gene would have been inoperative and could not have been used for the purposes of the present invention.

The HMP-P kinase/TMP-PPase gene is further referred as encoding an enzyme having HMP-P kinase activity or TMP-PPase activity. Hereby is preferably meant that the HMP-P kinase/TMP-PPase gene encodes a single polypeptide having HMP-P kinase activity or TMP-PPase activity. In another preferred embodiment, 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.

For recombinant production of HMP-P kinase/TMP-PPase in a host organism, a nucleotide sequence encoding the enzyme having HMP-P kinase actuvity or TMP-PPase activity 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, yeast, and insect cells (see, e.g., Luckow and Summers, Bio/Technol. 6: 47 (1988)). Specific examples include 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). ln a preferred embodiment, 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. In a further preferred embodiment, 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 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. 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. In another preferred embodiment, the nucleotide sequence is derived from a prokaryote, preferably a bacteria, e.g. E. coli. In this case, the enzyme having HMP-P kinase activity and the enzyme having TMP-PPase activity are encoded by the thiD and thiE genes, respectively.

Recombinantly produced 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).

Recombinantly produced HMP-P kinase/TMP-PPase is useful for a variety of purposes. For example, 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. Alternatively, recombinantly produced 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 TMP-PPase activity in the first reaction mixture. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant. In another preferred embodiment, 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. In another preferred embodiment, the substrate of HMP kinase is HMP and in yet another preferred embodiment, the substrate of TMP-PPase is THZ-P. In a further preferred embodiment, the activity of the enzyme is measured by determining the TMP produced in the reaction mixture.

Alternatively, the amount of THZ-P available for the TMP-PPase is increased by using a coupled THZ 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 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 kinase activity or TMP-PPase activity in the first reaction mixture. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant. In another preferred embodiment, 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. In another preferred embodiment, the substrate of HMP-P kinase is HMP-P and in yet another embodiment, the substrate of THZ kinase is THZ. In another preferred embodiment, 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.

In one embodiment, 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. ln another embodiment, 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. In a further preferred embodiment, 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. In another preferred embodiment, the nucleotide sequence is derived from a prokaryote, preferably a bacteria, e.g. E. coli. In this case, 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. Preferred are agronomically important crops such as cotton, soybean, oilseed rape, sugar beet, maize, rice, wheat, barley, oats, rye, sorghum, millet, turf, forage, turf grasses, and the like.

Achieving altered HMP-P kinase activity or TMP-PPase activity through increased expression 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. Preferably, the transformation is stable, thereby providing a heritable transgenic trait.

B. Expression of Modified Herbicide-Tolerant HMP-P Kinases/TMP-PPases According to this embodiment, 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 a HMP- P kinase/TMP-PPase. 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 HMP-P kinase/TMP-PPases:

One general strategy involves direct or indirect mutagenesis procedures on microbes. For instance, 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. 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. 4,975,374. The microbe selected for mutagenesis contains a normal, inhibitor-sensitive HMP-P kinase/TMP-PPase 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. HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP- PPase gene involves direct selection in plants. For example, the effect of a mutagenized HMP-P kinase/TMP-PPase gene on the growth inhibition of plants such as Arabidopsis, soybean, or maize is determined by plating seeds sterilized by art-recognized methods on plates on a simple minimal salts medium containing increasing concentrations of the inhibitor. Such 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 M-\ mutant seeds collected. Typically for Arabidopsis, M2 seeds (Lehle Seeds, Tucson, 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 days after plating are transplanted to soil and grown to maturity and seed set. Progeny of these seeds are tested for tolerance to the herbicide. If the tolerance trait is dominant, plants whose seed segregate 3:1 / resistant:sensitive are presumed to have been heterozygous for the resistance at the M2 generation. Plants that give rise to all resistant seed are presumed to have been homozygous for the resistance at the M2 generation. Such mutagenesis on intact seeds and screening of their M2 progeny seed can also be carried out on other species, for instance soybean (see, e.g. U.S. Pat. No. 5,084,082). Alternatively, mutant seeds to be screened for herbicide tolerance are obtained as a result of fertilization with pollen mutagenized by chemical or physical means.

Confirmation that the genetic basis of the herbicide tolerance is a modified HMP-P kinase/TMP-PPase gene is ascertained as exemplified below. First, 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. After sequencing the alleles to determine the presence of mutations in the coding sequence, the alleles are tested for their ability to confer tolerance to the inhibitor on plants into which the putative tolerance-conferring alleles have been transformed. These plants can be either Arabidopsis plants or any other plant whose growth is susceptible to the HMP-P kinase/TMP-PPase inhibitors. Second, the inserted HMP-P kinase/TMP-PPase 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 er a/., Plant Cell 1 : 699-705 (1989). The HMP-P kinase/TMP-PPase inhibitor tolerance trait is independently mapped using the same markers. When tolerance is due to a mutation in that HMP-P kinase/TMP-PPase gene, the tolerance trait maps to a position indistinguishable from the position of the HMP-P kinase/TMP-PPase gene.

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. Putative tolerance-conferring alleles of the HMP-P kinase/TMP-PPase 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 HMP- P kinase/TMP-PPase 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 HMP-P kinase/TMP-PPase 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 er a/., Methods Enzymol. 700:457-468 (1983); methoxylamine (Kadonaga et ai, Nucleic Acids Res. 73:1733-1745 (1985); oligonucleotide-directed saturation mutagenesis (Hutchinson er a/., Proc. Natl. Acad. Sci. USA, 83:710-714 (1986); or various polymerase misincorporation strategies (see, e.g. Shortle er a/., Proc. Natl. Acad. Sci. USA, 79:1588-1592 (1982); Shiraishi er a/., Gene 64:313-319 (1988); and Leung er a/., Technique 7:1 1 -15 (1989). 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 HMP-P kinase/TMP-PPase gene activity. The DNA sequences of cDNA inserts from plasmids that pass this test are then determined.

Herbicide resistant HMP-P kinase/TMP-PPase enzymes are also obtained using methods involving in vitro recombination, also called DNA shuffling. By DNA shuffling, mutations, preferably random mutations, are introduced in HMP-P kinase/TMP-PPase genes. DNA shuffling also leads to the recombination and rearrangement of sequences within HMP-P kinase/TMP-PPase genes or to recombination and exchange of sequences between two or more different of HMP-P kinase/TMP-PPase genes. These methods allows for the production of millions of mutated HMP-P kinase/TMP-PPase 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. Encompassed by the present invention is a shuffled DNA molecule, wherein said shuffled DNA molecule encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP-PPase activity encoded by a template DNA molecule from which said shuffled DNA molecule is derived. Further encompassed is a mutagenized DNA molecule obtained by shuffling a template DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity, wherein said mutagenized DNA molecule encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits HMP-P kinase activity or TMP- PPase activity encoded by said template DNA molecule.

In a preferred embodiment, 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 of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide, wherein the mutagenized polynucleotide is a mutated HMP-P kinase/TMP-PPase gene having enhanced tolerance to a herbicide which inhibits naturally occurring HMP-P kinase activity or TMP-PPase activity.

In a further preferred embodiment 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 forming a mutagenized double-stranded polynucleotide comprising sequences derived from different template DNA molecules, wherein the mutagenized double-stranded polynucleotide encodes an HMP-P kinase activity or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP- PPase activity encoded by the template DNA molecules.

In a preferred embodiment, 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. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides. In another preferred embodiment, the size of the double-stranded random fragments is from about 5 bp to 5 kb. In a further preferred embodiment, 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.

In another preferred embodiment, any combination of two or more different HMP-P kinase/TMP-PPase 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. The two or more HMP-P kinase/TMP-PPase 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. For example, when a thermostable polymerase with an optimal temperature of approximately 72°C is used, 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. Additionally, 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. 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 HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP-PPase 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 HMP-P kinase/TMP-PPase genes, e.g. to DNA sequences of a vector comprising the HMP-P kinase/TMP-PPase genes, whereby the different HMP-P kinase/TMP-PPase genes used in the PCR reaction are preferably comprised in separate vectors. The primers desirably anneal to sequences located less than 500 bp away from the HMP-P kinase/TMP-PPase coding sequences, preferably less than 200 bp away from the HMP-P kinase/TMP-PPase coding sequences, more preferably less than 120 bp away from the HMP-P kinase/TMP-PPase coding sequences. Preferably, the HMP-P kinase/TMP-PPase 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.

In another preferred embodiment, 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.

Any HMP-P kinase/TMP-PPase gene or any combination of HMP-P kinase/TMP- PPase genes is used for in vitro recombination in the context of the present invention. For example, the HMP-P kinase/TMP-PPase gene is derived from a plant, such as, e.g. Arabidopsis thaliana, e.g. the HMP-P kinase/TMP-PPase gene as set forth in SEQ ID NO:1 or SEQ ID NO:3. Other genes suitable for in vitro recombination are HMP-P kinase/TMP- PPase genes derived from e.g. a bacteria, such 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. 45, 708-71 1 ) or a Saccharomyces cerevisiae thiE gene (Nosaka et al. (1994) J. Biol. Chem. 269, 30510-30516), all incorporated herein by reference. Whole 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 appropriate host is preferably a host that otherwise lacks HMP-P kinase acitivity, such as E. coli strain NI500 (Genetic Stock Center, New Haven, USA) or a host that lacks TMP-PPase activity, for example E. coli strain NI400 (Nakayama and Hayashi (1972) J. Bacteriology 112: 1118-1126). Host cells transformed with the vectors comprising the library of mutated HMP-P kinase/TMP-PPase 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 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. Second, an inhibitor of wild-type enzyme is present in both reaction mixtures. Third, mutated activity (activity in the presence of inhibitor and mutated enzyme) and unmutated activity (activity in the presence of inhibitor and wild-type enzyme) are compared to determine whether a significant increase in enzymatic activity is observed in the mutated activity when compared to the unmutated activity. Mutated activity is any measure of activity of the mutated enzyme while in the presence of a suitable substrate and the inhibitor. Unmutated activity is any measure of activity of the wild-type enzyme while in the presence of a suitable substrate and the inhibitor. 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.

In addition to being used to create herbicide-tolerant plants, genes encoding herbicide tolerant HMP-P kinase/TMP-PPase can also be used as selectable markers in plant cell transformation methods. For example, plants, plant tissue, plant seeds, or plant cells transformed with a transgene can also be transformed with a gene encoding an altered HMP-P kinase/TMP-PPase 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 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.

Preferred is a method for forming a mutagenized HMP-P kinase/TMP-PPase gene according to the invention.

Further preferred is a method for forming said mutagenized HMP-P kinase/TMP- PPase gene.

More preferred is 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.

More preferred is method according to the invention, wherein said species of template DNA molecule is identical of substantially similar to the nucleotide sequences set forth in SEQ ID NO:1.

More preferred is a mutagenized DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity obtained by the method of the invention , wherein said mutagenized DNA molecule encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP-PPase activity encoded by said template DNA molecule. A wild-type or herbicide-tolerant form of the HMP-P kinase/TMP-PPase gene can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, 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. A transgene comprising a wild-type or herbicide- tolerant form of the HMP-P kinase/TMP-PPase gene is preferably stably transformed and integrated into the genome of the host cells. In another preferred embodiment, the transgene comprising a wild-type or herbicide-tolerant form of the HMP-P kinase/TMP- PPase gene located on a self-replicating vector. Examples of self-replicating vectors are viruses, in particular gemini viruses. Transformed cells can be regenerated into whole plants such that the chosen form of the HMP-P kinase/TMP-PPase 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. 1. Promoters

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. For example, for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter may be used. For regulatable expression, the chemically inducible PR-1 promoter from tobacco or Arabidopsis may be used (see, e.g., U.S. Patent No. 5,689,044).

2. Transcriptional Terminators

A variety of 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.

3. Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants. For example, various intron sequences such as introns of the maize Adhl gene have been shown to enhance expression, particularly in monocotyledonous cells. In addition, a number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. 4. Coding Sequence Optimization

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)).

5. Targeting of the Gene Product Within the Cell

Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411 -418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. In addition, 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)). Additionally, 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)). By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment.

B. Construction of Plant Transformation Vectors

Numerous 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. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929- 2931 ), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642).

1. Vectors Suitable for Agrobactenum Transformation

Many vectors are available for transformation using Agrobactenum 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 Agrobactenum transformation include the binary vectors pCIB200 and pCIB2001 , as well as the binary vector pCIB10 and hygromycin selection derivatives thereof. (See, for example, U.S. Patent No. 5,639,949).

2. Vectors Suitable for non- Agrobactenum Transformation

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).

C. Transformation Techniques

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. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. In addition, 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.

The wild-type or altered form of a 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. Although 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 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.

Where a herbicide tolerant 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. BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO:1 DNA sequence of the Arabidopsis HMP-P kinase/TMP-PPase pre-protein

SEQ ID NO:2 amino acid sequence of the Arabidopsis HMP-P kinase/TMP-PPase pre- protein

SEQ ID NO:3 DNA sequence of the putative mature Arabidopsis HMP-P kinase/TMP- PPase

SEQ ID NO:4 amino acid sequence of the putative mature Arabidopsis HMP-P kinase/TMP-PPase

SEQ ID NO:5 oligonucleotide aththiDEfor SEQ ID NO:6 oligonucleotide aththiDErev

SEQ ID NO:7 oligonucleotide aththiDEfor-ctp

DEPOSIT

Clone Accession number Date of Deposit aththiDE-ctp NRRL B-30040 July 8, 1998

The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, er a/., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and by T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F.M. etal., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-lnterscience (1987).

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). For the construct including the coding region of the HMP-P kinase and TMP-PPase pre-protein, 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 coding regions of the pre-protein and the putative mature protein are subcloned into the expression vector pET35b(+) Xa/LIC (Novagen) and both are transformed into E. coli BL21 (DE3) (Novagen) by chemical transformation using the manufacturer's protocols. Exampie 2: HMP-P Kinase and TMP-PPase Activity Assay

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. 0.01-1.0 x 10^"3 unit of an enzyme having HMP-P kinase activity or TMP-PPase activity (one unit of activity is defined as the amount of enzyme required to produce 1 mmol/min of product) and 10^"3-10^"5 M, but preferably 10^"4 M 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P) and 10^"3-10^"5 M, but preferably 10^~4 M 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate (THZ-P) are mixed in a final volume of 100 mL 50 mM Tris-HCI (pH 7.0-8.0, but preferably 7.4), 1 -20mM, but preferably 10 mM MgCI₂, and 0.1-10 mM, but preferably 1 mM ATP. The production of thiamine monophosphate (TMP) is determined preferably according to Kawasaki et al. (1990) J. Bacteriol. 172, 6145-6147 by adding 50 mL of 10% (w/v) metaphosphoric acid or 20% (w/v) trichloroacetic acid (pH 1.4). The mixture is then centrifuged to remove precipitated proteins. A 100 mL aliquot of the supernatant is removed and 50 mL of 0.3 M cyanogen bromide is added and mixed, followed by addition of 100 mL of 1 M NaOH. Fluorescence intensity is measured for the solution with an excitation wavelength of 360 ± 10nm and an emission wavelength of 430 ± 10nm.

Alternatively, ADP formation is quantitated by a coupled reaction procedure. In this case, 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 3: Coupled HMP Kinase and HMP-P Kinase and TMP-PPase Activity Assay

HMP kinase assay

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 MgCI₂. 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 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. Adding the HMP-P kinase, TMP-PPase, and THZ-P after the production of HMP-P insures that the initial concentration of HMP-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 15minutes), 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.

Assay Protocols

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. After 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. After an interval (determined by the activities of HMP-P kinase and TMP-PPase, 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. 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 4: Coupled THZ Kinase and HMP-P Kinase and TMP-PPase Activity Assay

A. THZ kinase assay

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 MgCI₂.

B. 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 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.

C. Assay Protocols

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. After the THZ kinase reaction proceeds to completion at a rate of approximately 5 mM/min (this is within 5-10 minutes), the HMP-P is added to a final concentration of 100 mM and the HMP-P kinase and TMP-PPase are added. After an interval (determined by the activities of HMP-P kinase and TMP-PPase), 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.

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 MgCI₂. 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. When the HMP to HMP-P and THZ to THZ-P conversions proceed sufficiently (approximately 50 and 20 mM, respectively), then 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.

Assay Protocols 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. After an interval (determined by the activities of HMP-P kinase and TMP-PPase), 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. 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 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). The resulting DNA fragments are cloned into pTRC99a (Pharmacia, Cat no: 27-5007-01 ) and transformed into E.coli strain NI500 (Genetic Stock Center, New Haven, USA) or into E. coli strain NI400 (Nakayama and Hayashi (1972) J. Bacteriology 112: 1118-1126) 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.

Example 7: In vitro Recombination of HMP-P kinase/TMP-PPase Genes by Staggered Extension Process

The A. thaliana HMP-P kinase/TMP-PPase gene encoding the mature protein and the E.coli thiD 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. B. The A. 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.

C. The A. thaliana HMP-P kinase/TMP-PPase gene encoding the mature protein and the E.coli thiD and 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

Immature spikelets with milky endosperm of the Japonica rice variety "Taipei 309" are dehulled and surface sterilized with 70% (v/v) ethanol for 1 min and 6% calcium hypochlorite for 20 min, followed by three washes with sterile distilled water. The isolated immature embryos are cultured at 28°C on 0.35% agarose-solidified MS- medium (Murashige and Skoog, 1962) containing 3% sucrose, 2 mg/l 2,4- dichlorophenoxyacetic acid (2,4-D), pH 5.8. After one week, callus material produced from the scutella is divided and cultured by weekly transfers onto fresh medium. Four weeks after the initiation, three to four calli are transferred into a 50-ml-culture vessel containing 20 ml of R2-medium (R2 salts and vitamins [Ohira et al. 1973], 1 mg/l 2,4-D, 500 mg/l 2-morpholino ethanesulfonic acid [MES], 3% sucrose, pH 5.8). The cultures are maintained in dim light at 28°C on a rotary shaker at 220 rpm, and the medium is replaced weekly by an equal amount of fresh medium. Rapidly dividing, friable calli are selected and subcultured into a fresh container by transferring 2 ml of fine callus suspension into 20 ml of R2-medium. Example 9: Microprojectile bombardment

Two- to 3-month-old suspension cultures that have been subcultured 3 to 4 days in advance serve as target cells for the bombardments. Four hours before particle bombardment, approx. 500 mg of cells are spread as a single layer of 2 cm in diameter on 0.35% agarose-solidified plasmolysis medium (R2 salts and vitamins, 1 mg/l 2,4-D, 3% sucrose, 0.5 M sucrose, pH 5.8) contained in a 5.5-cm petri dish. A particle inflow gun (Finer et al., 1992) is used to deliver DNA-coated gold particles (Aldrich Cat. # 32,658-5, spherical gold powder 1.5-3.0 μm) into the embryogenic suspension cells. Particle coating is essentially performed as described by Vain et al. (1993): 5 μl aliquots of the plasmid solution are distributed into 0.5 ml-reaction tubes and placed on ice. Particles are suspended in 96% ethanol at 100 mg/ml and vortexed for 2 min. Ethanol is replaced by an equal volume of sterile ddH20 and the suspension vortexed for 1 min. This washing step has to be repeated once. The particles are finally resuspended in sterile ddH20 at 100 mg/ml. 25 μl of the particle suspension are added to each of the DNA aliquots and the tubes vortexed for 1 min, followed by immediate addition of 25 μl of sterile, ice-cold CaCI2 (2.5 M in ddH20) and further vortexing for 1 min. 10 μl of sterile spermidine (0.1 M in ddH20) are added, the suspension vortexed again and placed on ice for 5 min during which the particles sediment. 50 μl of the particle-free supernatant are removed and the remaining suspension (15 μl) used for 5 bombardments. Prior to each bombardment, the particles need to be resuspended by intense pipetting.

The cells are covered with a 500 μm mesh baffle and positioned at 14 cm below the filter unit containing the particles. Particles are released by a single 8-bar-pressure pulse of 50 msec in partial vacuum (2 x 104 Pa).

Example 10: Transformation of wheat

A preferred technique for wheat transformation involves particle bombardment of immature wheat embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashige and Skoog, 1962) and 3 mg/l 2,4-D for induction of somatic embryos which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. 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. After about one month, developed shoots are transferred to larger sterile containers known as GA7s which contained half-strength MS, 2% sucrose, and the same concentration of selection agent. The stable transformation of wheat is described in detail in patent application EP 0 674 715.

Example 11 : Preparation of a special type of callus of Zea mays, elite inbred line Funk 2717

Zea mays plants of the inbred line Funk 2717 are grown to flowering in the greenhouse, and self pollinated. Immature ears containing embryos about 2 to 2.5 mm in length are removed from the plants and sterilized in 10% Chlorox solution for 20 minutes. Embryos are aseptically removed from the kernels and plated with the embryo axis downwards on OMS medium containing 0.1 mg/l 2,4-D, 6% sucrose and 25 mM L-proline solidified with 0.24% Gelrite (initiation medium). After two weeks culture in the dark at 27°C, 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. The callus is subcultured every two weeks to fresh medium. After a total of eight weeks after placing the embryos on the initiation medium, the special type of callus is identified by its characteristic morphology. This callus is subcultured further on the same medium. After a further period of two months, 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

The callus described above is subcultured for a total of at least six months. The type of callus chosen for subculture is relatively non-mucilaginous, granular and very friable, such that it separates into small individual cell aggregates upon placing into liquid medium. Cultures containing aggregates with large, expanded cells are not retained. Approximately 500 mg aliquots of the special callus of Zea mays elite inbred Funk 2717 are placed into 30 ml of N6 medium containing 2 mg/l 2,4-D in 125 ml Delong flasks. After one week of culture at 26°C in the dark on a gyratory shaker (130 rpm, 2.5 cm throw), the medium is replaced with fresh medium. The suspensions are again subcultured in this way after another week. At that time, the cultures are inspected, and those which do not show large numbers of expanded cells are retained. Suspension cultures containing aggregates with large, expanded cells are discarded. The preferred tissue consists of densely cytoplasmic dividing cell aggregates which have a characteristically smoother surface than the usual type of cell aggregates. The cultures retained have at least 50% of the cells represented in these small aggregates. This is the desired morphology. These suspensions also have a rapid growth rate, with a doubling time of less than one week. The suspension cultures are subcultured weekly by transferring 0.5 ml PCV into 25 ml of fresh medium. After four to six weeks of subculture in this fashion, the cultures increase two- to three-fold per weekly subculture. Cultures in which more than 75% of the cells are of the desired morphology are retained for further subculture. The lines are maintained by always choosing for subculture the flask whose contents exhibit the best morphology. Periodic filtration through 630 μm pore size stainless steel sieves every two weeks is used in some cases to increase the dispersion of the cultures, but is not necessary.

Example 13: Preparation of protoplasts from suspension cultures of Zea mays

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 μm mesh sieve, followed by a 50 μm 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 protoplast preparation is resuspended in 10 ml of fresh KMC salt solution, and centrifuged for five minutes at 60 to 100 x g. The supernatant is removed and discarded, and the protoplasts resuspended gently in the drop remaining, and then 10 ml of a 13/14 strength KMC solution gradually added. After centrifuging again for five minutes, the supernatant is again removed and the protoplasts resuspended in a 6/7 strength KMC solution. An aliquot is taken for counting, and the protoplasts again sedimented by centrifugation. The protoplasts are resuspended at 107 per ml in KM-8p medium or in 0.5 M mannitol containing 6 mM MgCI2 or other suitable medium for use in transformation as described in the following examples. This protoplast suspension is used for transformation and is cultured as described below.

Example 14: Transformation of Zea mays protoplasts by electroporation

A. All steps except the heat shock are carried out at room temperature (22 to 28°C). The protoplasts are resuspended in the last step of above in 0.5 M mannitol containing 0.1 % MES and 6 mM MgCI2. The resistance of this suspension is measured in the chamber of a Dialog Electroporator and adjusted to 1 to 1.2 k; using a 300 mM MgCI2 solution. The protoplasts are heat-shocked by immersing the tube containing the sample in a water bath at 45°C for five minutes, followed by cooling to room temperature on ice. 4 μg of linearized plasmid containing a plant-selectable hygromycin resistance gene such as described by Rothstein et al. (1987) or chimeric gene constructs as described and 20 μg of calf thymus carrier DNA are added to aliquots of 0.25 ml of this suspension. 0.125 ml of a 24% PEG solution (MW 8000) in 0.5 M mannitol containing 30 mM MgCI2 are added to the protoplasts. The mixture is mixed well but gently, and incubated for 10 minutes. The sample is transferred to the chamber of the electroporator and samples pulsed three times at 10 second intervals, at initial voltages of 1500, 1800, 2300 or 2800 Vcm-1 , and an exponential decay time of 10 msec.

The protoplasts are cultured as follows. The samples are plated in 6 cm petri dishes at room temperature. After a further 5 to 15 minutes, 3 ml of KM-8p medium containing 1.2% SeaPlaque agarose and 1 mg/l 2,4-D are added. The agarose and protoplasts are mixed well and the medium allowed to gel.

B. This is repeated with one or more of the following modifications:

(1 ) The resistance of the protoplast preparation is adjusted to 0.5 to 0.7 k;. (2) The PEG used is PEG with a MW of 4000.

(3) No PEG is added, or one-half volume of 12% PEG is added.

(4) The pulses are applied at intervals of three seconds.

(5) The protoplasts are plated after the electroporation in dishes placed on a plate cooled to a temperature of 16°C.

(6) 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.

(7) The calf thymus carrier DNA is not added.

Example 15: Transformation of Zea mays protoplasts by treatment with PEG

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. 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.

B. This is repeated and the protoplasts are washed after 30 minutes of incubation in the PEG solution of above, by adding 0.3 ml of W5 solution five times at two- to three-minute intervals. The protoplast suspension is centrifuged, the supernatant removed, and the protoplasts are cultured as described.

C. The above is repeated with the modification that the final concentration of PEG is between 13 and 25%.

Example 16: Regeneration of callus from protoplasts

The plates containing the protoplasts in agarose are placed in the dark at 26°C. After 14 days, colonies arise from the protoplasts. The agarose containing the colonies is transferred to the surface of a 9 cm diameter petri dish containing 30 ml of N6 medium containing 2 mg/l 2,4-D, solidified with 0.24% Gelrite. This medium is referred to as 2N6. The callus is cultured further in the dark at 26°C and callus pieces subcultured every two weeks onto fresh solid 2N6 medium.

Example 17: Selection of transformed callus of Zea mays

The above example is repeated with the modification that 100 mg/l or 200 mg/l hygromycin B is added to the 2N6 medium in order to select for transformed cells.

Example 18: Regeneration of corn plants

A. Callus is placed on 2N6 medium for maintenance and on ON6 (comprising N6 medium lacking 2,4-D) and N61 medium (comprising N6 medium containing 0.25 mg/l 2,4-D and 10 mg/l kinetin) to initiate regeneration. Callus growing on ON6 and N61 media is grown in the light (16 hours/day light of 840 to 8400 Ix from white fluorescent lamps). Callus growing on N61 medium is transferred to ON6 medium after two weeks, as prolonged time on N61 medium is detrimental. The callus is subcultured every two weeks even if the callus is to be transferred again on the same medium formulation. Plantlets appear in about four to eight weeks. Once the plantlets are at least 2 cm tall, they are transferred to ON6 medium in GA7 containers. Roots form in two to four weeks, and when the roots look well-formed enough to support growth, the plantlets are transferred to soil in peat pots, under a light shading for the first four to seven days. It is often helpful to invert a clear plastic cup over the transplants for two to three days to assist hardening off. Once the plants are established, they are treated as normal corn plants and grown to maturity in the greenhouse. In order to obtain progeny plants are self pollinated or crossed with wild type.

B. The above example is repeated with the modification that 100 mg/l or 200 mg/l hygromycin B is added to the medium used to maintain the callus.

Example 19: Introduction of DNA into protoplasts of Sorghum bicolor

Protoplasts of sorghum suspension FS 562 are prepared essentially as described for Zea mays above, and resuspended following the last wash at a density of 107 per ml in the following solution: 0.2 M mannitol, 0.1 % MES, 72 mM NaCI, 70 mM CaCI2, 2.5 mM KCI, 2.5 mM glucose, pH to 5.8 with KOH, at a density of 1.6 to 2 x 106 per ml. The protoplast suspension is distributed as 1 ml aliquots into plastic disposable cuvettes and 10 μg of DNA added as described. The resistance of the solution at this point when measured between the electrodes of the 471 electrode set of the electroporation apparatus described below is in the range of 6. For transformation, the DNA is added in 10 μl 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.

B. The above is repeated with one or more of the following modifications:

(1 ) The voltage used is 200 Vcm-1 , or between 100 Vcm-1 and 800 Vcm-1.

(2) The exponential decay constant is 5 ms, 15 ms or 20 ms.

(3) 50 μg of sheared calf thymus DNA in 25 μl sterile water is added together with the plasmid DNA.

(4) The plasmid DNA is linearized before use by treatment with an appropriate restriction enzyme (e.g. BamHI).

The protoplasts are cultured following transformation at a density of 2 x 106 per ml in KM-8p medium, with no solidifying agent added.

Example 20: Introduction of DNA into protoplasts of Glycine max

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.

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Claims

What Is Claimed Is:

1. A nucleotide sequence that encodes an enzyme involved in thiamine biosynthesis, wherein said enzyme has HMP-P kinase activity or TMP-PPase activity.

2. A nucleotide sequence according to claim 1 , wherein the enzyme comprises an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2.

3. A nucleotide sequence according to claim 1 , wherein the enzyme comprises the amino acid sequence set forth in SEQ ID NO:2.

4. A nucleotide sequence according to claim 1 , wherein the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1.

5. A nucleotide sequence according to claim 1 , wherein the nucleotide sequence is set forth in SEQ ID NO:1.

6. A chimeric gene comprising a promoter operatively linked to a nucleotide sequence according to claim 1.

7. A recombinant vector comprising a chimeric gene according to claim 7, wherein said vector is capable of being stably transformed into a host cell.

8. A host cell comprising a vector according to claim 14, wherein said nucleotide sequence is expressible in said cell.

9. A plant protein involved in thiamine biosynthesis, wherein said protein has HMP-P kinase activity or TMP-PPase activity.

10. A protein according to claim 9, wherein said enzyme comprises an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2.

11. A protein according to claim 9, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:2.

12. A method for identifying a chemical to be tested for the ability to inhibit plant growth and viability, comprising the steps of:

(a) combining the 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 to be tested and the enzyme in a second reaction mixture with the substrate used in the first reaction mixture 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; and

(d) selecting the chemical to be tested for the ability to inhibit plant growth or viability when the activity of the enzyme in the second reaction mixture is significantly less than the activity of the enzyme in the first reaction mixture.

13. A method according to claim 12, wherein the enzyme 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.

14. A method according to claim 12, wherein said substrate of HMP-P kinase is 2-methyl-4- amino-5-hydroxymethylpyrimidine phosphate (HMP-P).

15. A method according to claim 12, wherein said substrate of TMP-PPase is 4-methyl-5- (beta-hydroxyethyl) thiazole phosphate (THZ-P).

16. A method according to claim 12, wherein said substrate of TMP-PPase is 2-methyl-4- amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP).

17. A method according to claim 12, wherein the activity of the enzyme is measured by determining the TMP produced in the reaction mixture.

18. A method for suppressing the growth of undesired vegetation, comprising the step of applying to the undesired vegetation a chemical identified by the method of claim 12.

19. A plant, plant cell, plant seed, or plant tissue comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity, wherein the nucleotide sequence confers upon said plant, plant cell, plant seed, or plant tissue tolerance to a chemical identified by the method of claim 12 in amounts that normally inhibits the HMP-P. kinase activity in the wild-type plant.

20. A plant obtainable by a process comprising the step of transforming the plant or a parent of the plant or a parent of the plant with an isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity and capable of expressing the nucleotide sequence in the plant so as to render the plant tolerant to a chemical identified by the method of claim 12.

21. 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.

22. 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;

(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 kinase activity or TMP-PPase activity in the first reaction mixture.

23. A method according to claim 22, wherein the having HMP-P kinase activity or TMP- PPase activity enzyme is derived from a plant.

24. A method according to claim 22, wherein 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.

25. A method according to claim 22, wherein said substrate of HMP kinase is HMP.

26. A method according to claim 22, wherein said substrate of TMP-PPase is THZ-P.

27. A method according to claim 22, wherein 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.

28. A plant, plant cell, plant seed, or plant tissue transformed with an isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity, wherein the DNA molecule confers upon said plant, plant cell, plant seed, or plant tissue tolerance to a chemical identified by the method of claim 22 in amounts that normally inhibit the HMP-P kinase activity or TMP-PPase activity in the plant.

29. A plant obtainable by a process comprising the step of transforming the plant or a parent of the plant with an isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity and capable of expressing the nucleotide sequence in the plant so as to render the plant tolerant to a chemical identified by the method of claim 22.

30. 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 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 TMP-PPase activity in the first reaction mixture.

31. 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;

(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 kinase activity or TMP-PPase activity in the first reaction mixture.

32. A method according to claim 31 , wherein the enzyme having HMP-P kinase activity or TMP-PPase activity is derived from a plant.

33. A method according to claim 31 , wherein 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.

34. A method according to claim 31 , wherein the substrate of HMP-P kinase is HMP-P.

35. A method according to claim 31 , wherein the substrate of THZ kinase is THZ.

36. A method according to claim 31 , wherein 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.

37. A method for suppressing the growth of undesired vegetation, comprising the step of applying to the undesired vegetation a chemical identified by the method of claim 31.

38. A plant, plant cell, plant seed, or plant tissue transformed with an isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity and/or TMP-PPase activity, wherein the DNA molecule confers upon said plant, plant cell, plant seed, or plant tissue tolerance to a chemical identified by the method of claim 31 in amounts that normally inhibit the HMP-P kinase activity and/or TMP-PPase activity in the plant.

39. A plant obtainable by a process comprising the step of transforming the plant or a parent of the plant with an isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity and/or TMP-PPase activity and capable of expressing the nucleotide sequence in the plant so as to render the plant tolerant to a chemical identified by the method of claim 31.

40. 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;

41. A method for identifying a chemical with herbicidal activity, wherein the chemical inhibits the activity HMP-P kinase activity or TMP-PPase in a plant, comprising the steps of:

(a) obtaining transgenic plants, plant tissue, plant seeds or plant cells comprising an isolated nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP- PPase activity and capable of overexpressing an enzymatically active HMP-P kinase activity 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-transformed plants, plant cells, tissues after application of the chemical; wherein the chemical suppresses the viability or growth of the transgenic plants, plant cells, tissues or parts, without significantly suppressing the growth of the viability or growth of the isogenic non-transgenic plants, plant cells, tissues or parts.

42. A method according to claim 41 , wherein the enzyme having 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 of said enzyme identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2.

43. A method for suppressing the growth of undesired vegetation, comprising applying to the undesired vegetation a chemical identified by the method according to anyone of claims 22 or 41.

44. A plant, plant cell, plant seed, or plant tissue transformed with an isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity, wherein the DNA molecule confers upon said plant, plant cell, plant seed, or plant tissue tolerance to a chemical identified by the method of claim 41 in amounts that normally inhibit HMP-P kinase activity or TMP-PPase activity in the plant.

45. A plant obtainable by a process comprising the step of transforming the plant or a patent of the plant with an isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity and expressing the nucleotide sequence in the plant so as to render the plant tolerant to a chemical identified by the method of claim 41.

46. 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 transformed with an isolated DNA molecule comprising a nucleotide sequence having HMP-P kinase activity or TMP-PPase activity, wherein said nucleotide sequence is expressible in said plant or plant seed; 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.

47. A shuffled DNA molecule, wherein said shuffled DNA molecule encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP-PPase activity encoded by a template DNA molecule from which said shuffled DNA molecule is derived.

48. A mutagenized DNA molecule obtained by shuffling a template DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity, wherein said mutagenized DNA molecule encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits HMP-P kinase activity or TMP-PPase activity encoded by said template DNA molecule.

49. A method for forming a mutagenized DNA molecule encoding an enzyme having HMP- P kinase activity or TMP-PPase activity from a template DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase 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 or 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 fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide;

(d) repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide; wherein the mutagenized double-stranded polynucleotide encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP-PPase activity encoded by the template DNA molecule.

50. A method according to claim 49, wherein one template DNA molecule is derived from an eukaryote.

51. A method according to claim 49, wherein said eukaryote is a plant.

52. A method according to claim 49, wherein said species of template DNA molecule is identical of substantially similar to the nucleotide sequences set forth in SEQ ID NO:1.

53. A mutagenized DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity obtained by the method of claim 49, wherein said mutagenized DNA molecule encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP-PPase activity encoded by said template DNA molecule.

54. A method for forming a mutagenized DNA molecule encoding an enzyme having HMP- P kinase activity or TMP-PPase activity from at least two non-identical template DNA molecules encoding enzymes having HMP-P kinase activity or TMP-PPase 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 double-stranded polynucleotide comprising sequences derived from different template DNA molecules; wherein the mutagenized double-stranded polynucleotide encodes an HMP-P kinase activity or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP-PPase activity encoded by the template DNA molecules.

55. A method according to claim 54, wherein one template DNA molecule is derived from an eukaryote.

56. A method according to claim 54, wherein said eukaryote is a plant.

57. A method according to claim 54, wherein said species of template DNA molecule is identical of substantially similar to the nucleotide sequences set forth in SEQ ID NO:1.

58. A mutagenized DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity obtained by the method of claim 54, wherein said mutagenized DNA molecule encodes a HMP-P kinase or TMP-PPase enzyme having enhanced tolerance to a herbicide which inhibits the HMP-P kinase activity or TMP-PPase activity encoded by said template DNA molecule.