EP1276883A2 - Genes d'acide amine:glyoxylate aminotransferase d'origine vegetale et utilisations de ceux-ci - Google Patents

Genes d'acide amine:glyoxylate aminotransferase d'origine vegetale et utilisations de ceux-ci

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Publication number
EP1276883A2
EP1276883A2 EP01921374A EP01921374A EP1276883A2 EP 1276883 A2 EP1276883 A2 EP 1276883A2 EP 01921374 A EP01921374 A EP 01921374A EP 01921374 A EP01921374 A EP 01921374A EP 1276883 A2 EP1276883 A2 EP 1276883A2
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EP
European Patent Office
Prior art keywords
amino acid
plant
glyoxylate aminotransferase
nucleotide sequence
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01921374A
Other languages
German (de)
English (en)
Inventor
Hong Gil Nam
Ji H. Stud. Dorm. 1-104 JUN
Chul Soo Kim
Joshua Zvi Levin
Michael William Bauer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenta Participations AG
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Syngenta Participations AG
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Publication date
Application filed by Syngenta Participations AG filed Critical Syngenta Participations AG
Publication of EP1276883A2 publication Critical patent/EP1276883A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)

Definitions

  • the invention relates generally to enzymatic activity involved in amino acid: glyoxylate aminotransferase in plants.
  • the invention relates to plant genes that encode a polypeptide having amino acid:glyoxylate aminotransferase activity.
  • the invention has various utilities, including the recombinant production of polypeptides having amino acid:glyoxylate aminotransferase activity in heterologous hosts, the screening of chemicals for herbicidal activity, and the use of thereby identified herbicidal chemicals to control the growth of undesired vegetation.
  • the invention may also be applied to the development of herbicide tolerance in plants, plant tissues, plant seeds, and plant cells.
  • Novel herbicides can now be discovered using high-throughput screens that implement recombinant DNA technology. Once identified, metabolic enzymes essential to plant growth and development can be recombinantly produced through standard molecular biological techniques and utilized as herbicide targets in screens for novel inhibitors of the enzyme's activity. The novel inhibitors discovered through such screens may then be used as herbicides to control undesirable vegetation. Such herbicides are also useful for selecting herbicide tolerant plants, and seed plants tolerant to the herbicide can be produced, for example by genetic engineering techniques. Thus, herbicides that exhibit greater potency, broader weed spectrum, and more rapid degradation in soil can be applied to crops that are resistant or tolerant to herbicides in order to kill weeds without attendant risk of damage to the crop.
  • one object of the invention is to provide a method for identifying new or improved herbicides. Another object of the invention is to provide a method 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, and seed thereof, that are tolerant to such new or improved herbicides.
  • the present invention provides a DNA molecule comprising a nucleotide sequence, preferably isolated from a plant, that encodes a polypeptide having amino acid:glyoxylate aminotransferase activity.
  • the present invention provides a DNA molecule isolated from Arabidopsis thaliana that encodes amino acidrglyoxylate aminotransferase, preferably a serine:glyoxylate aminotransferase or an alanine:glyoxylate aminotransferase, more preferably an alanine:glyoxylate aminotransferase, and DNA molecules substantially similar thereto that encode enzymes having amino acid:glyoxylate aminotransferase activity, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably alanine:glyoxylate activity.
  • the present invention provides a DNA molecule comprising a nucleotide sequence isolated from a plant that encodes the polypeptide set forth in SEQ ID NO:2.
  • the DNA molecule of the invention may comprise a nucleotide sequence set forth in SEQ ID NO:1.
  • the DNA molecule of the invention comprises a nucleotide sequence that is substantially similar to the coding sequence set forth in SEQ ID NO:1 and that encodes a polypeptide having amino acid:glyoxylate aminotransferase activity, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably alanine:glyoxylate activity.
  • SEQ ID NO:1 is isolated from Arabidopsis thaliana, using the information provided by the present invention, other nucleotide sequences that encode a polypeptide having amino acid:glyoxylate aminotransferase activity are obtained from other sources, e.g. from other plants, using standard methods known in the art.
  • the present invention also provides a nucleotide sequence construct comprising a promoter operatively linked to a DNA molecule of the invention. Further, the present invention provides methods to stably transform such a nucleotide sequence construct into a host cell, and host cells comprising such a nucleotide sequence construct, wherein the host cell is capable of expressing the DNA molecule encoding a polypeptide having amino acid:glyoxylate aminotransferase activity. Any suitable cell may be used as a host cell, e.g. a bacterial cell, a yeast cell, or a plant cell.
  • the present invention also relates to the recombinant production of a amino acid:glyoxylate aminotransferase polypeptide and methods of use of amino acid:glyoxylate aminotransferase in assays for identifying compounds that interact with amino acid:glyoxylate aminotransferase polypeptide.
  • the present invention provides a plant polypeptide having amino acid:glyoxylate aminotransferase activity useful for identifying inhibitors of amino acid:glyoxylate aminotransferase activity in in vivo and in vitro assays.
  • the isolated polypeptide of the present invention comprises an amino acid sequence substantially similar to the amino acid sequence set forth in SEQ ID NO:2.
  • this enzyme comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the present invention further provides methods of using purified polypeptides having amino acid:glyoxylate aminotransferase activity, preferably polypeptides derived from plant sources, in assays to screen for and identify compounds that interact with a amino acid:glyoxylate aminotransferase polypeptide.
  • Such compounds are preferably inhibitors of amino acid:glyoxylate aminotransferase activity, and are potentially herbicides of future commercial interest.
  • the inhibitors are used as herbicides to suppress the growth of undesirable vegetation in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • an assay useful for identifying inhibitors of essential plant genes comprises the steps of: a) reacting a plant amino acid:glyoxylate aminotransferase enzyme and a substrate thereof in the presence of a suspected inhibitor of the enzyme's function; b) comparing the rate of enzymatic activity in the presence of the suspected inhibitor to the rate of enzymatic activity under the same conditions in the absence of the suspected inhibitor; and c) determining whether the suspected inhibitor inhibits the amino acid:glyoxylate aminotransferase enzyme.
  • the inhibitory effect on plant amino acid:glyoxylate aminotransferase may be determined by a reduction or complete inhibition of amino acid:glyoxylate aminotransferase activity in the assay. Such a determination may be made by comparing, in the presence and absence of the candidate inhibitor, the amount of substrate used or intermediate or product made during the reaction.
  • the present invention further embodies plants, plant tissues, plant seeds, and plant cells that have modified amino acid:glyoxylate aminotransferase activity and that are therefore tolerant to inhibition by a chemical at levels normally inhibitory to naturally occurring amino acid'.glyoxylate aminotransferase enzyme activity.
  • Herbicide tolerant plants encompassed by the invention include those that would otherwise be potential targets for normally inhibiting herbicides, particularly the agronomically important crops mentioned above.
  • plants, plant tissue, plant seeds, or plant cells are stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operatively linked to a nucleotide sequence that encodes an enzyme having modified amino acid:g!yoxy!ate aminotransferase activity that is tolerant to a concentration of a amino acid:glyoxylate aminotransferase inhibitor that would normally inhibit the activity of wild-type, unmodified amino acid:glyoxylate aminotransferase in the plant.
  • Modified amino acid:glyoxylate aminotransferase activity may also be conferred upon a plant by increasing expression of wild-type (i.e.
  • transgenic plants, plant tissue, plant seeds, or plant ceils thus created are then selected by conventional selection techniques, whereby inhibitor tolerant descendants (lines) are isolated, characterized, and developed. Alternately, random or site-specific mutagenesis may be used to generate amino acid:glyoxylate aminotransferase inhibitor tolerant lines. Still further, inhibitor tolerant lines can be developed via selection of natural variants.
  • the present invention provides a plant, plant cell, plant seed, or plant tissue comprising a DNA molecule comprising a nucleotide sequence, preferably isolated from a plant, that encodes an enzyme having amino acid:glyoxylate aminotransferase and wherein the DNA molecule confers upon the plant, plant cell, plant seed, or plant tissue tolerance to a amino acid:glyoxylate aminotransferase inhibitor in amounts that normally naturally occurring amino acid:glyoxylate aminotransferase activity.
  • the enzyme comprises an amino acid sequence substantially similar to the amino acid sequence set forth in SEQ ID NO:2.
  • the DNA molecule is substantially similar to the coding sequence set forth in SEQ ID NO:1.
  • 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 applying to crops or crop seeds that are tolerant to an inhibitor that inhibits naturally occurring amino acid:glyoxylate aminotransferase activity and the weeds in the field an amino acid:glyoxylate aminotransferase inhibitor in amounts that inhibit naturally occurring amino acid:glyoxylate aminotransferase activity, wherein the inhibitor suppresses the growth of the weeds without significantly suppressing the growth of the crops.
  • the invention thus provides: An isolated DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO:1.
  • the nucleotide sequence encodes an amino acid sequence substantially similar to SEQ ID NO:2.
  • the nucleotide sequence is SEQ ID NO:1.
  • the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:2.
  • the nucleotide sequence is a plant nucleotide sequence, which preferably encodes a polypeptide having amino acid:glyoxylate aminotransferase activity, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably alanine.-glyoxylate activit .
  • the invention further provides: A polypeptide comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO:1.
  • the amino acid sequence is encoded by SEQ ID NO:1.
  • the polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO:2.
  • the amino acid sequence is SEQ ID NO:2.
  • the amino acid sequence preferably has amino acid:glyoxylate aminotransferase activity, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably alanine:glyoxylate activity .
  • the amino acid sequence comprises at least 20 consecutive amino acid residues of the amino acid sequence encoded by SEQ ID NO:1. Or, alternatively, the amino acid sequence comprises at least 20 consecutive amino acid residues of the amino acid sequence of SEQ ID NO:2.
  • the invention further provides: An expression cassette comprising a promoter operatively linked to a DNA molecule according to the present invention, wherein the promoter is preferably functional in a eukaryote, wherein the promoter is preferably heterologous to the DNA molecule.
  • the present invention further provides recombinant vector comprising an expression cassette according to the present invention, wherein said vector is preferably capable of being stably transformed into a host cell, a host cell comprising a DNA molecule according to the present invention, wherein said DNA molecule is preferably expressible in the cell.
  • the host cell is preferably selected from the group consisting of an insect cell, a yeast cell, a prokaryotic cell and a plant cell.
  • the invention further provides a plant or seed comprising a plant cell of the present invention, wherein the plant or seed is preferably tolerant to an inhibitor of amino acid:glyoxylate aminotransferase activity, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably alanine:glyoxylate activity .
  • the invention further provides: A process for making nucleotides sequences encoding gene products having altered amino acid:glyoxylate aminotransferase activity comprising: a) shuffling an unmodified nucleotide sequence of the present invention, b) expressing the resulting shuffled nucleotide sequences, and c) selecting for altered amino acid:glyoxylate aminotransferase activity as compared to the amino acid:glyoxylate aminotransferase activity of the gene product of said unmodified nucleotide sequence.
  • the gene product has preferably altered serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably altered alanine:glyoxylate activity.
  • the unmodified nucleotide sequence is identical or substantially similar to SEQ ID NO:1 , or a homolog thereof.
  • the present invention further provides a DNA molecule comprising a shuffled nucleotide sequence obtainable by the process described above, a DNA molecule comprising a shuffled nucleotide sequence produced by the process described above.
  • a shuffled nucleotide sequence obtained by the process described above has enhanced tolerance to an inhibitor of amino acid: glyoxylate aminotransferase activity.
  • the invention further provides an expression cassette comprising a promoter operatively linked to a DNA molecule comprising a shuffled nucleotide sequence a recombinant vector comprising such an expression cassette, wherein said vector is preferably capable of being stably transformed into a host cell, a host cell comprising such an expression cassette, wherein said nucleotide sequence is preferably expressible in said cell.
  • a preferred host cell is selected from the group consisting of an insect cell, a yeast cell, a prokaryotic cell and a plant cell.
  • the invention further provides a plant or seed comprising such plant cell, wherein the plant is preferably tolerant to an inhibitor of amino acid:glyoxylate aminotransferase activity.
  • the invention further provides: A method for selecting compounds that interact with the protein encoded by SEQ ID NO:1 , comprising: a) expressing a DNA molecule comprising SEQ ID NO:1 or a sequence substantially similar to SEQ ID NO:1 , or a homolog thereof, to generate the corresponding protein, b) testing a compound suspected of having the ability to interact with the protein expressed in step (a), and c) selecting compounds that interact with the protein in step (b).
  • the invention further provides: A process of identifying an inhibitor of amino acid:glyoxylate aminotransferase activity, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably alanine:glyoxylate activity, comprising: a) introducing a DNA molecule comprising a nucleotide sequence of SEQ ID NO:1 and having amino acid:glyoxylate aminotransferase activity, or nucleotide sequences substantially similar thereto, or a homolog thereof, into a plant cell, such that said sequence is functionally expressible at levels that are higher than wild-type expression levels, b) combining said plant cell with a compound to be tested for the ability to inhibit the amino acid:glyoxylate aminotransferase activity under conditions conducive to such inhibition, c) measuring plant cell growth under the conditions of step (b), d) comparing the growth of said plant cell with the growth of a plant cell having unaltered amino acid:g
  • the invention further comprises a compound having herbicidal activity identifiable according to the process described immediately above.
  • the invention further comprises: A process of identifying compounds having herbicidal activity comprising: a) combining a protein of the present invention and a compound to be tested for the ability to interact with said protein, under conditions conducive to interaction, b) selecting a compound identified in step (a) that is capable of interacting with said protein, c) applying identified compound in step (b) to a plant to test for herbicidal activity, and d) selecting compounds having herbicidal activity.
  • the invention further comprises a compound having herbicidal activity identifiable according to the process described immediately above.
  • the invention further comprises: A method for suppressing the growth of a plant comprising, applying to said plant a compound that inhibits the activity of a polypeptide of the present invention in an amount sufficient to suppress the growth of said plant.
  • the invention further comprises: A method for recombinantly expressing a protein having amino acid:glyoxylate aminotransferase activity comprising introducing a nucleotide sequence encoding a protein having one of the above activities into a host cell and expressing the nucleotide sequence in the host cell.
  • a preferred host cell is selected from the group consisting of an insect cell, a yeast cell, a prokaryotic cell and a plant cell.
  • a preferred prokaryotic cell is a bacterial cell, e.g. E. coli.
  • SEQ ID NO:1 is an mRNA sequence encoding amino acid:glyoxylate aminotransferase from Arabidopsis thaliana.
  • SEQ ID NO:2 is the predicted amino acid sequence of Arabidopsis thaliana amino acid:glyoxylate aminotransferase encoded by SEQ ID NO:1.
  • SEQ ID NO:3 is the 5' portion of the partial cDNA sequence of the amino acid:glyoxylate aminotransferase gene isolated from the 35-191 line of Arabidopsis thaliana.
  • SEQ ID NO:4 is the 3' portion of the partial cDNA sequence of the amino acid:glyoxylate aminotransferase gene isolated from the 35-191 line of Arabidopsis thaliana.
  • SEQ ID NO:5 is the oligonucleotide
  • SEQ ID NO:6 is the oligonucleotide P1
  • SEQ ID NO:7 is the oligonucleotide P4
  • 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, in this case the gene encoding amino acid:glyoxylate aminotransferase.
  • 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 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.
  • Antiparallel refers herein to two nucleotide sequences paired through hydrogen bonds between complementary base residues with phosphodiester bonds running in the 5'-3' direction in one nucleotide sequence and in the 3'-5' direction in the other nucleotide sequence.
  • Co-factor natural reactant, such as an organic molecule or a metal ion, required in an enzyme-catalyzed reaction.
  • a co-factor is e.g. NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.
  • a co- factor can be regenerated and reused.
  • DNA shuffling is a method to rapidly, easily and efficiently introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly.
  • the DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non- naturally occurring DNA molecule derived from at least one template DNA molecule.
  • the shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.
  • Enzyme activity means herein the ability of an enzyme to catalyze the conversion of a substrate into a product.
  • a substrate for the enzyme comprises the natural substrate of the enzyme but also comprises analogues of the natural substrate, which can also be converted, by the enzyme into a product or into an analogue of a product.
  • the activity of the enzyme is measured for example by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time.
  • the activity of the enzyme is also measured by determining the amount of an unused co-factor of the reaction remaining in the reaction mixture after a certain period of time or by determining the amount of used co- factor in the reaction mixture after a certain period of time.
  • the activity of the enzyme is also measured by determining the amount of a donor of free energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine) remaining in the reaction mixture after a certain period of time or by determining the amount of a used donor of free energy or energy-rich molecule (e.g. ADP, pyruvate, acetate or creatine) in the reaction mixture after a certain period of time.
  • a donor of free energy or energy-rich molecule e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine
  • essential gene is a gene encoding a protein such as e.g. a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the plant.
  • Expression cassette means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue or organ or stage of development.
  • additional elements i.e. ribosome binding sites, may be required.
  • Herbicide a chemical substance used to kill or suppress the growth of plants, plant cells, plant seeds, or plant tissues.
  • Heterologous DNA Sequence a DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • Homologous DNA Sequence a DNA sequence naturally associated with a host cell.
  • Inhibitor a chemical substance that inactivates the enzymatic activity of amino acid:glyoxylate aminotransferase.
  • 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 heterologous DNA sequence.
  • an isolated DNA molecule or an isolated enzyme in the context of the present invention, is a DNA molecule or enzyme which, 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, in 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.
  • a “native” refers to a gene which is present in the genome of the untransformed plant cell.
  • Plant refers to any plant or part of a plant at any stage of development. Therein are also included cuttings, cell or tissue cultures and seeds. As used in conjunction with the present invention, the term “plant tissue” includes, but is not limited to, whole plants, plant cells, plant organs, plant seeds, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and/or functional units.
  • an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
  • the 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.
  • the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence.
  • the term “substantially similar” is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells.
  • substantially similar refers to nucleotide sequences that encode a protein having at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, yet still more preferably at least 99% identity, to SEQ ID NO:2, wherein said protein sequence comparisons are conducted using GAP analysis as described below.
  • substantially similar preferably also refers to nucleotide sequences having at least 73% identity, more preferably 85% identity, more preferably at least 90% identity, still more preferably 95% identity, yet still more preferably at least 99% identity, to SEQ ID NO:1 , wherein said nucleotide sequence comparisons are conducted using GAP analysis as described below.
  • a nucleotide sequence "substantially similar" to the reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1 % SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA
  • amino acid:glyoxylate aminotransferase gene refers to a DNA molecule comprising SEQ ID N0:1 or comprising a nucleotide sequence substantially similar to SEQ ID NO:1.
  • homologs of the amino acid:glyoxylate aminotransferase gene include nucleotide sequences that encode an amino acid sequence that is at least 28% identical to SEQ ID NO:2, more preferably at least 30%, still more preferably at least 35%, yet still more preferably at least 50%, still more preferably at least 85%, yet still more preferably at least 90%, as measured, using the parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the amino acid:glyoxylate aminotransferase protein.
  • substantially similar when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. where only changes in amino acids sequence not affecting the polypeptide function occur.
  • the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is preferably at least 85%, more preferably 90%, more preferably at least 95%, still more preferably at least 99% using default GAP analysis parameters with the University of Wisconsin GCG (version 10), SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453).
  • amino acid:glyoxylate aminotransferase protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO:1.
  • homologs of the amino acid:glyoxy!ate aminotransferase protein are amino acid sequences that are at least 28% identical to SEQ ID NO:2, more preferably at least 30%, still more preferably at least 35%, yet still more preferably at least 50%, still more preferably at least 85%, yet still more preferably at least 90%, as measured using the parameters described above, wherein the amino acid sequence encoded by the homolog has the biological activity of the amino acid:glyoxylate aminotransferase protein.
  • a substrate is the molecule that an 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.
  • Target gene is any gene in a plant cell.
  • a target gene is a gene of known function or is a gene whose function is unknown, but whose total or partial nucleotide sequence is known.
  • the function of a target gene and its nucleotide sequence are both unknown.
  • a target gene is a native gene of the plant cell or is a heterologous gene which had previously been introduced into the plant cell or a parent cell of said plant cell, for example by genetic transformation.
  • a heterologous target gene is stably integrated in the genome of the plant cell or is present in the plant cell as an extrachromosomal molecule, e.g. as an autonomously replicating extrachromosomal molecule.
  • Tolerance the ability to continue essentially normal growth or function (i.e. no more than 5% of herbicide tolerant plants show phytotoxicity) when exposed to an inhibitor or herbicide in an amount sufficient to suppress the normal growth or function of native, unmodified plants.
  • 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.
  • the present invention is directed to a DNA molecule comprising a nucleotide sequence isolated from a plant source that encodes amino acid:glyoxylate aminotransferase, preferably the nucleotide sequence encodes an alanine:glyoxylate aminotransferase or a serine:glyoxylate aminotransferase, more preferably the nucleotide sequence encodes an alanine:glyoxylate aminotransferase.
  • the present invention provides a DNA molecule isolated from Arabidopsis thaliana that encodes amino acid: glyoxylate aminotransferase, preferably a serine:glyoxylate aminotransferase or an alanine:glyoxylate aminotransferase, more preferably an alanine:glyoxylate aminotransferase, and DNA molecules substantially similar thereto that encode enzymes having amino acid:glyoxylate aminotransferase activity, preferably serin e -.glyoxylate aminotransferase or alanine.-glyoxylate aminotransferase activity, more preferably alanine:glyoxylate activity.
  • the DNA coding sequence for amino acid:glyoxylate aminotransferase from Arabidopsis thaliana is provided in SEQ ID NO:1.
  • the DNA sequence corresponding to the genomic sequence of the amino acid:glyoxylate aminotransferase gene from Arabidopsis thaliana is found in Genbank accession # AC007209. .
  • a nucleotide sequence of the present invention preferably encodes a protein having amino acid: glyoxylate aminotransferase activity, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase activity, more preferably alanine:glyoxylate activity.
  • Such protein is referred to as amino acid: glyoxylate aminotransferase, preferably serine:glyoxylate aminotransferase or alanine:glyoxylate aminotransferase, more preferably alanine:glyoxylate.
  • Alanine:glyoxylate aminotransferase also known as alanine-glyoxylate transaminase, (EC 2.6.1.44) is an enzyme catalyzing a biochemical reaction involved in amino acid metabolism and the glycolate pathway. This enzyme catalyzes the conversion of L-alanine and glyoxylate to pyruvate and glycine.
  • Arabidopsis thaliana protein from line 35- 191 encoded by SEQ ID NO:2
  • this protein has significant similarity to serine-glyoxylate transaminases, also known as serine-glyoxylate aminotransferases (E.C. 2.6.1.45).
  • This enzyme catalyzes the conversion of L-serine and glyoxylate to 3-hydroxypyruvate and glycine as part of amino acid metabolism and the glycolate pathway.
  • serine-glyoxylate aminotransferase is required in the photorespiratory pathway (Somerville (1984) Oxford Surv. of Plant. Mol. & Cell Bio!., 1 :103-131 ) and Arabidopsis thaliana mutants lacking this enzymatic activity are shown to have a conditional lethal phenotype (Somerville and Ogren, (1980) Proc. Natl. Acad. Sci. USA, 77:2684-2687).
  • Proteins similar to that encoded by the nucleotide sequence isolated from the 35-191 line are identified based on DNA sequences from many organisms, including Arabidopsis thaliana (GenPept accession # AAC26854), Fritillaria agrestis (GenPept accession # AAB95218), Anabaena cylindrica (GenPept accession # CAA35518), Synechocystis sp. (GenPept accession # BAA18375), human (GenPept accession # AAA51680), and Saccharomyces cerevisiae (GenPept accession # BAA09208).
  • DNA sequences encoding amino acid:glyoxylate aminotransferase enzymes are isolated from the genome of any desired plant.
  • amino acid:glyoxylate aminotransferase gene sequences can be isolated from any plant according to well known techniques based on their sequence similarity to the Arabidopsis thaliana coding sequences (SEQ ID NO:1) taught by the present invention. In these techniques, all or part of a known amino acid:glyoxylate aminotransferase gene's coding sequence is used as a probe that selectively hybridizes to other amino acid:glyoxylate aminotransferase gene sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e.
  • genomic or cDNA libraries from a chosen source organism.
  • techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g. Sambrook et al., "Molecular Cloning", eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers corresponding to sequence domains conserved among known amino acid:glyoxylate aminotransferase enzyme's amino acid sequences (see, e.g. Innis et al., "PCR Protocols, a Guide to Methods and Applications", Academic Press (1990)).
  • an entire plant amino acid: glyoxylate aminotransferase gene sequence or portions thereof may be used as a probe capable of specifically hybridizing to coding sequences and messenger RNAs.
  • probes include, e.g. sequences that are unique among plant amino acid:glyoxylate aminotransferase gene sequences and are at least 10 nucleotides in length, preferably at least 20 nucleotides in length, and most preferably at least 50 nucleotides in length.
  • Such probes are .used to amplify and analyze amino acid:glyoxylate aminotransferase gene sequences from a chosen organism via PCR.
  • This technique is useful to isolate additional plant amino acid'.glyoxylate aminotransferase gene sequences from a desired organism or as a diagnostic assay to determine the presence of amino acid:glyoxylate aminotransferase gene sequences in an organism. This technique also is used to detect the presence of altered amino acid:glyoxylate aminotransferase gene sequences associated with a particular condition of interest such as herbicide tolerance, poor health, etc.
  • Amino acid:glyoxylate aminotransferase specific hybridization probes also are used to map the location of these native genes in the genome of a chosen plant using standard techniques based on the selective hybridization of the probe to genomic sequences. These techniques include, but are not limited to, identification of DNA polymorphisms identified or contained within the probe sequence, and use of such polymorphisms to follow segregation of the gene relative to other markers of known map position in a mapping population derived from self fertilization of a hybrid of two polymorphic parental lines (see e.g. Helentjaris et al., Plant Mol. Biol. 5: 109 (1985); Sommer et al. Biotechniques 12:82 (1992); D'Ovidio et al., Plant Mol.
  • Amino acid:glyoxylate aminotransferase specific hybridization probes also are used to quantify levels of amino acid:glyoxylate aminotransferase gene mRNA in a plant using standard techniques such as Northern blot analysis. This technique is useful as a diagnostic assay to detect altered levels of amino acid:glyoxylate aminotransferase gene expression that are associated with particular conditions such as enhanced tolerance to herbicides that target amino acid:glyoxylate aminotransferase genes.
  • a amino acid:glyoxylate aminotransferase coding sequence preferably a plant coding sequence
  • an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced.
  • 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 operably linked in proper reading frame, is 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)).
  • host organisms such as E. coli, yeast, and insect cells
  • 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 pVI11392/Sf21 cells (Invitrogen, La Jolla, CA).
  • Recombinantly produced amino acid:glyoxylate aminotransferase enzymes is isolated and purified using a variety of standard techniques. The actual techniques used varies depending upon the host organism used, whether the enzyme is designed for secretion, and other such factors. Such techniques are well known 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 amino acid:glyoxylate aminotransferase enzymes are useful for a variety of purposes. For example, they are used in in vitro assays to screen known herbicidal chemicals, whose target has not been identified, to determine if they inhibit amino acid:glyoxylate aminotransferase enzymes. Such in vitro assays also are useful as screens to identify new chemicals that inhibit such enzymatic activity and that are therefore novel herbicide candidates. Alternatively, recombinantly produced amino acid:glyoxylate aminotransferase enzymes are used 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 amino acid:glyoxylate aminotransferase comprises the steps of: a) reacting an enzyme having amino acid:glyoxylate aminotransferase activity and the substrate 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 amino acid:glyoxylate aminotransferase enzymatic activity.
  • the inhibitory effect on amino acid:glyoxylate aminotransferase activity is determined by a reduction or complete inhibition of product formation 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 product formed in the in vitro assay using fluorescence or absorbance detection.
  • the preferred substrates are L(+) aianine and glyoxylate.
  • FCS Fluorescence Correlation Spectroscopy
  • FCS measures the average diffusion rate of a fluorescent molecule within a small sample volume.
  • the sample size can be as low as 10 3 fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium.
  • the diffusion rate is a function of the mass of the molecule and decreases as the mass increases. FCS can therefore be applied to protein-ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding.
  • the target to be analyzed is expressed as a recombinant protein with a sequence tag, such as a poly-histidine sequence, inserted at the N or C-terminus.
  • a sequence tag such as a poly-histidine sequence
  • the expression takes place in E. coli, yeast or insect cells.
  • the protein is purified by chromatography.
  • the poly-histidine tag can be used to bind the expressed protein to a metal chelate column such as Ni2+ chelated on iminodiacetic acid agarose.
  • the protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPY® (Molecular Probes, Eugene, OR).
  • the protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thomwood, NY). Ligand binding is determined by changes in the diffusion rate of the protein.
  • SMDI Surface-Enhanced Laser Desorption/lonization
  • Hutchens and Yip during the late 1980's (Hutchens and Yip (1993) Rapid Commun. Mass Spectrom. 7: 576- 580).
  • TOF time-of-flight mass spectrometer
  • SELDI provides a mean to rapidly analyze molecules retained on a chip.
  • the chip is then submitted to washes of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI-TOF. Ligands that specifically bind the target will be identified by the stringency of the wash needed to elute them.
  • Biacore relies on changes in the refractive index at the surface layer upon binding of a ligand to a protein immobilized on the layer.
  • a collection of small ligands is injected sequentially in a 2-5 microlitre cell with the immobilized protein. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface.
  • SPR surface plasmon resonance
  • the refractive index change for a given change of mass concentration at the surface layer is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein (Liedberg et al. (1983) Sensors Actuators 4: 299-304; Malmquist (1993) Nature, 361 : 186-187).
  • the target to be analyzed is expressed as described for FCS.
  • the purified protein is then used in the assay without further preparation. It is bound to the Biacore chip either by utilizing the poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction.
  • the chip thus prepared is then exposed to the potential ligand via the delivery system incorporated in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler).
  • the SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics on rate and off rate allows the discrimination between non-specific and specific interaction.
  • an assay for small molecule ligands that interact with a polypeptide is an inhibitor assay.
  • an inhibitor assay useful for identifying inhibitors of essential plant genes, such as plant amino acid:glyoxylate aminotransferase genes comprises the steps of: a) reacting a plant amino acid:glyoxylate aminotransferase enzyme and a substrate thereof in the presence of a suspected inhibitor of the enzyme's function; b) comparing the rate of enzymatic activity in the presence of the suspected inhibitor to the rate of enzymatic activity under the same conditions in the absence of the suspected inhibitor; and c) determining whether the suspected inhibitor inhibits the amino acid:glyoxylate aminotransferase enzyme.
  • the inhibitory effect on plant amino acid:glyoxylate aminotransferase may be determined by a reduction or complete inhibition of amino acid:glyoxylate aminotransferase activity in the assay. Such a determination may be made by comparing, in the presence and absence of the candidate inhibitor, the amount of substrate used or intermediate or product made during the reaction.
  • a suspected herbicide for example identified by in vitro screening, is applied to plants at various concentrations.
  • the suspected herbicide is preferably sprayed on the plants. After application of the suspected herbicide, its effect on the plants, for example death or suppression of growth is recorded.
  • an in vivo screening assay for inhibitors of the amino acid:glyoxylate aminotransferase activity uses transgenic plants, plant tissue, plant seeds or plant cells capable of overexpressing a nucleotide sequence having amino acid:glyoxylate aminotransferase activity, wherein the amino acid:glyoxylate aminotransferase gene product is enzymatically active in the transgenic plants, plant tissue, plant seeds or plant cells.
  • the nucleotide sequence is preferably derived from an eukaryote, such as a yeast, but is preferably derived from a plant.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1, or encodes an enzyme having amino acid:glyoxylate aminotransferase activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2.
  • the nucleotide sequence is derived from a prokaryote.
  • a chemical is then applied to the transgenic plants, plant tissue, plant seeds or plant cells and to the isogenic non-transgenic 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.
  • Compounds capable of inhibiting the growth of the non-transgenic plants, but not affecting the growth of the transgenic plants are selected as specific inhibitors of amino acid:glyoxylate aminotransferase activity.
  • U.S. Patent No. 4,761 ,373 to Anderson ef al. is directed to plants resistant to various imidazolinone or sulfonamide herbicides. The resistance is conferred by an altered acetohydroxyacid synthase (AHAS) enzyme.
  • AHAS acetohydroxyacid synthase
  • U.S. Patent No. 4,975,374 to Goodman et al. relates to plant cells and plants containing a gene encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that were known to inhibit GS, e.g.
  • 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 ef al. discloses plants tolerant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The tolerance is conferred by an altered acetyl coenzyme A carboxylase (ACCase).
  • the present invention is further directed to plants, plant tissue, plant seeds, and plant cells tolerant to herbicides that inhibit the naturally occurring amino acid:glyoxylate aminotransferase in these plants, wherein the tolerance is conferred by altered amino acid:glyoxylate aminotransferase enzyme activity.
  • Altered amino acid:glyoxylate aminotransferase enzyme activity is conferred upon a plant according to the invention by increasing expression of wild-type herbicide-sensitive amino acid:glyoxylate aminotransferase enzyme by providing additional wild-type amino acid: glyoxylate aminotransferase genes to the plant, by expressing modified herbicide-tolerant amino acid:glyoxylate aminotransferase enzymes 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 amino acid:glyoxylate aminotransferase enzyme activity through increased expression results in a level of a amino acid:glyoxylate aminotransferase enzyme 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 is conferred in a number of ways, e.g., providing multiple copies of a wild-type amino acid:glyoxylate aminotransferase 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 are 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 amino acid:glyoxylate aminotransferase gene also is accomplished by stably 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 amino acid:glyoxylate aminotransferase enzyme.
  • 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 amino acid:glyoxylate aminotransferase enzyme.
  • a herbicide tolerant form of the enzyme has at least one amino acid substitution, addition or deletion that confers tolerance to an amount of a herbicide effective to inhibit the unmodified, naturally occurring form of the amino acid:glyoxylate aminotransferase enzyme.
  • the transgenic plants, plant tissue, plant seeds, or plant cells thus created are 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 amino acid:g!yoxylate aminotransferase enzymes:
  • a genetically manipulatable microbe such as E. coli or S. cerevisiae may be subjected to random mutagenesis in vivo with mutagens such as UV light or ethyl or methyl methane sulfonate.
  • mutagens such as UV light or ethyl or methyl methane sulfonate.
  • Mutagenesis procedures are described, for example, in Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972); Davis ef al., Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1980); Sherman ef al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983); and U.S. Patent No. 4,975,374.
  • the microbe selected for mutagenesis contains a normal, inhibitor-sensitive amino acid:glyoxylate aminotransferase 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. Amino acid:glyoxylate aminotransferase 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 amino acid:glyoxylate aminotransferase gene involves direct selection in plants.
  • the effect of a mutagenized amino acid:glyoxylate aminotransferase 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.
  • concentrations are in the range of 0.001 , 0.003, 0.01 , 0.03, 0.1 , 0.3, 1 , 3, 10, 30, 110, 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.
  • 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 of seeds, or chemical or physical mutagenesis of pollen (Neuffer, In Maize for Biological Research Sheridan, ed. Univ. Press, Grand Forks, ND., pp. 61-64 (1982)), which is then used to fertilize plants and the resulting Mi mutant seeds collected.
  • Arabidopsis M2 seeds 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 a amino acid:glyoxylate aminotransferase inhibitor.
  • 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).
  • mutant seeds to be screened for herbicide tolerance are obtained as a result of fertilization with pollen mutagenized by chemical or physical means.
  • 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 are Arabidopsis plants or any other plant whose growth is susceptible to the inhibitors.
  • the amino acid:glyoxylate aminotransferase 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 ef al., Plant Cell 1 : 699-705 (1989).
  • the tolerance trait is independently mapped using the same markers. When tolerance is due to a mutation in that amino acid:glyoxylate aminotransferase gene, the tolerance trait maps to a position indistinguishable from the position of the amino acid:glyoxylate aminotransferase gene.
  • Another method of obtaining herbicide-tolerant alleles of a amino acid:glyoxylate aminotransferase 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 a amino acid:glyoxylate aminotransferase inhibitor. 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 amino acid:glyoxylate aminotransferase 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 amino acid:glyoxylate aminotransferase 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.
  • a plant cDNA such as the Arabidopsis cDNA encoding amino acid:glyoxylate aminotransferase (SEQ ID NO:1 ) 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.
  • Herbicide resistant amino acid:glyoxylate aminotransferase proteins are also obtained using methods involving in vitro recombination, also called DNA shuffling.
  • DNA shuffling mutations, preferably random mutations, are introduced into nucleotide sequences encoding amino acid:glyoxylate aminotransferase activity.
  • DNA shuffling also leads to the recombination and rearrangement of sequences within a amino acid:glyoxylate aminotransferase gene or to recombination and exchange of sequences between two or more different of amino acid:glyoxylate aminotransferase genes. These methods allow for the production of millions of mutated amino acid:glyoxylate aminotransferase coding sequences.
  • mutated genes are screened for desirable properties, e.g. improved tolerance to herbicides and for mutations that provide broad spectrum tolerance to the different classes of inhibitor chemistry. Such screens are well within the skills of a routineer in the art.
  • a mutagenized amino acid:glyoxylate aminotransferase gene is formed from at least one template amino acid:glyoxylate aminotransferase gene, wherein the template amino acid:glyoxylate aminotransferase 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
  • the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1 % by weight of the total DNA.
  • the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides.
  • the size of the double-stranded random fragments is from about 5 bp to 5 kb.
  • the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles. Such method is described e.g. in Stemmer et al.
  • any combination of two or more different amino acid:glyoxylate aminotransferase 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 amino acid:glyoxylate aminotransferase genes are used as template for PCR amplification with the extension cycles of the PCR reaction preferably carried out at a lower temperature than the optimal polymerization temperature of the polymerase.
  • the temperature for the extension reaction is desirably below 72°C, more desirably below 65°C, preferably below 60°C, more preferably the temperature for the extension reaction is 55°C.
  • the duration of the extension reaction of the PCR cycles is desirably shorter than usually carried out in the art, more desirably it is less than 30 seconds, preferably it is less than 15 seconds, more preferably the duration of the extension reaction is 5 seconds. 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 amino acid:glyoxylate aminotransferase genes 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 amino acid:glyoxylate aminotransferase 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 amino acid:glyoxylate aminotransferase genes, e.g.
  • the primers desirably anneal to sequences located less than 500 bp away from amino acid:glyoxylate aminotransferase sequences, preferably less than 200 bp away from the amino acid:glyoxylate aminotransferase sequences, more preferably less than 120 bp away from the amino acid:glyoxylate aminotransferase sequences.
  • the amino acid: glyoxylate aminotransferase sequences are surrounded by restriction sites, which are included in the DNA sequence amplified during the PCR reaction, thereby facilitating the cloning of the amplified products into a suitable vector.
  • fragments of amino acid:glyoxylate aminotransferase 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 amino acid:glyoxylate aminotransferase 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 amino acid:glyoxylate aminotransferase gene or any combination of amino acid:glyoxylate aminotransferase genes, or homologs thereof, is used for in vitro recombination in the context of the present invention, for example, a amino acid:glyoxylate aminotransferase gene derived from a plant, such as, e.g. Arabidopsis thaliana, e.g. a amino acid:glyoxylate aminotransferase gene set forth in SEQ ID NO:1.
  • Whole amino acid.-glyoxylate aminotransferase genes or portions thereof are used in the context of the present invention.
  • the library of mutated amino acid:glyoxylate aminotransferase 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 a plant cell, an algae like Chlamydomonas, a yeast or a bacteria.
  • An appropriate host requires amino acid:glyoxylate aminotransferase gene product activity for growth.
  • Host cells transformed with the vectors comprising the library of mutated amino acid:glyoxylate aminotransferase 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 amino acid:glyoxylate aminotransferase gene that is tolerant to an inhibitor may be performed in the same manner as the assay to identify inhibitors of the amino acid:glyoxylate aminotransferase enzyme (Inhibitor Assay, above) with the following modifications: First, a mutant amino acid:glyoxylate aminotransferase enzyme is substituted in one of the reaction mixtures for the wild-type amino acid:glyoxylate aminotransferase enzyme of the inhibitor assay. Second, an inhibitor of wild-type enzyme is present in both reaction mixtures.
  • mutated activity activity in the presence of inhibitor and mutated enzyme
  • unmutated activity activity in the presence of inhibitor and wild-type enzyme
  • a significant increase is defined as an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, most preferably an increase by about 10-fold or greater.
  • genes encoding herbicide tolerant amino acid:glyoxylate aminotransferase enzymes also are used as selectable markers in plant cell transformation methods. For example, plants, plant tissue, plant seeds, or plant cells transformed with a transgene are transformed with a gene encoding an altered amino acid:glyoxylate aminotransferase enzyme capable of being expressed by the plant. The transformed cells are transferred to medium containing an amino acid:glyoxylate aminotransferase inhibitor 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 amino acid:glyoxylate aminotransferase enzyme-encoding gene, and can be used with any transgene of interest.
  • Expression of the transgene and the inhibitor-tolerant amino acid:glyoxylate aminotransferase gene can be driven by the same promoter functional in plant cells, or by separate promoters.
  • herbicide-resistant amino acid:glyoxylate aminotransferase proteins are produced using the incremental truncation for the creation of hybrid enzymes (ITCHY), as described in Ostermeier et al. (1999) Nature Biotechnology 17:1205-1209), and this reference is incorporated herein by reference.
  • a wild-type or herbicide-tolerant form of the amino acid:glyoxylate aminotransferase gene can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting a DNA molecule encoding the amino acid:glyoxylate aminotransferase enzyme 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 optionally are modified to increase expression.
  • truncated sequences, nucleotide substitutions or other modifications optionally are employed.
  • Expression systems known in the art are used to transform virtually any crop plant cell under suitable conditions. Transformed cells are regenerated into whole plants such that the chosen form of the amino acid:glyoxylate aminotransferase gene confers herbicide tolerance in the transgenic plants.
  • Gene sequences intended for expression in transgenic plants are first operably linked to a suitable promoter expressible in plants.
  • Such expression cassettes optionally comprise further sequences required or selected for the expression of the transgene.
  • 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.
  • Promoters The selection of the promoter used determines the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters known in the art can be used.
  • the CaMV 35S promoter for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter may be used.
  • the chemically inducible PR-1 promoter from tobacco or Arabidopsis may be used (see, e.g., U.S. Patent No. 5,689,044).
  • transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
  • sequences are known 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.
  • various intron sequences such as introns of the maize Adhl gene have been shown to enhance expression, particularly in monocotyledonous cells.
  • non-translated leader sequences derived from viruses also are known to enhance expression, and these are particularly effective in dicotyledonous cells.
  • the coding sequence of the selected gene optionally is 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 et al., Proc. Natl. Acad. Sci. USA 88: 3324 (1991); and Koziel et al., Bio/technol. 11: 194 (1993); Fennoy and Bailey-Serres. Nucl. Acids Res. 21 : 5294- 5300 (1993).
  • 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.
  • 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 peroxis ⁇ me (e.g. Unger et al. Plant Molec. Biol. 13: 411-418 (1989)).
  • the cDNAs encoding these products are manipulated to effect the targeting of heterologous gene products to these organelles.
  • sequences have been characterized which cause the targeting of gene products to other cell compartments.
  • Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)).
  • amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi ef al. Plant Molec. Biol. 14: 357-368 (1990)).
  • transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention are used in conjunction with any such vectors.
  • the selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al.
  • Vectors Suitable for Agrobacterium Transformation Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001 , as well as the binary vector pCIBIO and hygromycin selection derivatives thereof. (See, for example, U.S. Patent No. 5,639,949).
  • Vectors Suitable for non-Agrobacterium Transformation Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non- Agrobacterium transformation include pCIB3064, pSOG19, and pSOG35. (See, for example, U.S. Patent No. 5,639,949).
  • the coding sequence of interest Once the coding sequence of interest has been cloned into an expression system, it is transformed into a plant cell.
  • Methods for transformation and regeneration of plants are well known in the art.
  • Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles.
  • bacteria from the genus Agrobacterium can be utilized to transform plant cells.
  • Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-base ⁇ techniques and techniques that do not require Agrobacterium.
  • Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art. Transformation of most monocotyledon species has now also become routine.
  • Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, particle bombardment into callus tissue, as well as .4c;robac.er/ut7.-mediated transformation. D. Plastid Transformation
  • a nucleotide sequence encoding a polypeptide having amino acid:glyoxylate aminotransferase activity is directly transformed into the plastid genome.
  • Plastid expression in which genes are inserted by homologous recombination into the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein.
  • the nucleotide sequence is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplasmic for plastid genomes containing the nucleotide sequence are obtained, and are preferentially capable of high expression of the nucleotide sequence.
  • Plastid transformation technology is for example extensively described in U.S. Patent Nos. 5,451 ,513, 5,545,817, 5,545,818, and 5,877,462 in PCT application no. WO 95/16783 and WO 97/32977, and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 , 7301-7305, all incorporated herein by reference in their entirety.
  • the basic technique for plastid transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleotide sequence into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
  • the 1 to 1.5 kb flanking regions facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
  • point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45).
  • the wild-type or altered form of a amino acid:glyoxylate aminotransferase gene of the present invention is utilized to confer herbicide tolerance to a wide variety of plant cells, including those of gymnosperms, monocots, and dicots.
  • the gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • herbicide tolerant amino acid:glyoxylate aminotransferase gene allele is obtained by direct selection in a crop plant or plant cell culture from which a crop plant can be regenerated, it is moved into commercial varieties using traditional breeding techniques to develop a herbicide tolerant crop without the need for genetically engineering the allele and transforming it into the plant.
  • Example 1 Construction of a Plant Antisense Expression Vector pNB96
  • a plant antisense expression vector, pNB96 is constructed as follows. The EcoR USma I fragment of the TEV leader sequence in pTRL2 (Ling et al. (1991) Biotechnology, 9: 752- 758) is replaced by a synthetic oligonucleotide sequence containing Eco RI/ ⁇ /of I sites. The inserted sequence is 5'-AAT TGC GGC CGC AAA GAA TTC- 3' (oligonucleotide A: SEQ ID NO:5).
  • the Hind III fragment containing the 35S promoter-EcoR MNot I sites-35S terminator is, then, subcloned into the Hind Ill-digested pARK5 (Saito et al. (1992) Plant Cell Reports, 11 : 219-224), after filling the EcoR I and Not I sites in pARK5.
  • the resulting antisense expression vector is named pNB96.
  • the vector pNB96 contains the Not I and EcoR I sites downstream from the dual 35S promoter for cloning of the cDNA molecules in the antisense direction, and the 35S terminator is downstream of the EcoR I cloning site.
  • the npt II and bar gene encodes kanamycin and basta resistance, respectively. Expression of the npt II gene is driven by the nos promoter and the nos terminator is downstream of the npt II gene. Expression of the bar gene is driven by the 35S promoter and the nos terminator is downstream of the bar gene.
  • the positions of the primer sequences used for isolating inserted cDNAs by PCR are indicated by P1 P4, respectively.
  • primer P1 5'-TTC GCA AGA CCC TTC CTC TA -3': SEQ ID NO:6
  • primer P4 5'-CTT ATC TGG GAA CTA CTC-3': SEQ ID NO:7.
  • An antisense cDNA library of Arabidopsis thaliana in this vector is generated using RNA from leaf tissue of Arabidopsis. Total RNA isolated from leaf tissues of Arabidopsis thaliana is used for preparation of poly (A) + RNA. Double-stranded cDNA is constructed from 5 ⁇ g of poly (A) + RNA with the Time Saver cDNA synthesis Kit (Pharmacia) using Not l-dT ⁇ 8 as a primer. The cDNA is inserted into the antisense vector pNB96 after ligating an EcoR I adapter and digesting with Not I.
  • the antisense cDNA library is first established in E. coli.
  • the primary cDNA library established in the E. coli XL-1 Blue cells (Bullock et al. (1987) BioTechniques, 5: 376-378) contains 2 X 10 5 recombinant clones.
  • the sizes of the antisense cDNA inserts estimated from 50 random clones are between 150 and 3400 bp.
  • the plasmid DNA prepared from this library is then introduced into the Agrobacterium strain AGL1 (Lazo et al. (1991 ) Bio/Technology, 9: 963-967).
  • the primary titer of the Arabidopsis antisense cDNA library established in Agrobacterium is approximately 2 X 10 5 .
  • Example 3 Generation of the Random Antisense Transgenic Plant Pool
  • Arabidopsis thaliana (L.) Heynh (WS-0 ecotype) is used for transformation as follows.
  • the Agrobacterium culture is grown at 28°C to a density of 0.8-1.0 (OD 6 oo) and harvested by centrifugation at 5,000 rpm for 10 min at 25°C.
  • the bacterial pellet is resuspended in IM (Infiltration Media: 1X MS salt, 1 X B5 vitamin, 5% sucrose, 0.005% Silwet L-77) to a final OD 600 of 2.0-2.4.
  • IM Infiltration Media: 1X MS salt, 1 X B5 vitamin, 5% sucrose, 0.005% Silwet L-77
  • T1 Transgenic plants are selected by soaking the seeds in 0.1% basta solution and then growing the plants in soil. The transformation efficiency is 0.5%.
  • Example 4 Identification of the Lethal Mutant Line 35-191 at the T1 Generation
  • the basta-sensitive and non-transgenic seedlings do not grow beyond the seedling stage, whereas transgenic, basta-resistant seedlings grow beyond the seedling stage. Since antisense effect is primarily dominant, some antisense mutants may be found in the T1 generation.
  • the antisense transgenic lines that survived beyond the seedling stage approximately 1 % of the T1 transgenic plants show lethality or sterility at the T1 generation.
  • a line designated 35-191 shows severe bleaching of the rosette leaves and cauline leaves. Later, the floral part is degenerated and thus the plants do not set seeds. Finally, the plants die much earlier than wild type plants.
  • cDNA clones from the 35-191 mutant lines are isolated by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • 50 ⁇ l of the reaction mixture is prepared to contain 25 ⁇ l of the quick start PCR mix (TaKaRa), 100 ng of genomic DNA and 200 ng each of the primers.
  • PCR is performed for 35 cycles (30 sec at 94°C, 30 sec at 52°C, and 1 min at 68°C) with the primer set P1 (5'-TTC GCA AGA CCC TTC CTC TA -3': SEQ ID NO:6) and primer P4 (5'-CTT ATC TGG GAA CTA CTC-3': SEQ ID NO:7).
  • the size of the cloned cDNA insert from line 35-191 is 1.0 kb.
  • the PCR product is cloned into pGEM-T Easy vector (Promega Biotech).
  • the partial sequences of the cDNA clone are determined from both ends. Sequencing is performed with double-stranded DNA by the dideoxy chain termination method using a ThermoSequenase kit (Amersham). SP6 and T7 primers are used to determine the sequences from the 5' and 3' ends of the clone, respectively. The sequence is then compared against the sequences in the databases by the Blast program.
  • the nucleotide sequence corresponding to the 5' portion of the cDNA clone (SEQ ID NO:3), and the nucleotide sequence corresponding to the 3' portion of the cDNA clone (SEQ ID NO:4), are identical to the 5' and 3' ends of the Arabidopsis alanine.-glyoxylate aminotransferase mRNA (Genbank accession #: AF063901 , SEQ ID NO:1) except one base pair. The difference is likely due to the differences in the ecotypes of plants used in our experiment (WS-O) from that in the Genbank database (Col-O).
  • the cDNA fragment isolated from the mutant line is recloned and introduced into wild type plants.
  • the cDNA clone is digested with Not I and Eco RI, and cloned into pNB96 vector for plant transformation.
  • 8 plants out of 20 plants that survive beyond the seedling stage after basta selection show the same mutant phenotype as the original mutation, although there is some variability in severity of mutant phenotypes.
  • This result confirms that antisense expression of the cDNA clone is responsible for the mutant phenotype and that the amino acid: glyoxylate aminotransferase is an essential gene.
  • Example 8 Expression of Recombinant Amino acid:glyoxylate aminotransferase Protein in E. coli
  • the coding region of the protein corresponding to the cDNA clone SEQ ID NO:1 , is subcloned into an appropriate expression vector, and transformed into E. coli using the manufacturer's conditions.
  • E. coli includes plasmids such as pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), and pTrcHis (Invitrogen, La Jolla, CA).
  • E. coli is cultured, and expression of amino acid:glyoxylate aminotransferase activity is confirmed. Protein conferring amino acid:glyoxylate aminotransferase activity is isolated using standard techniques.
  • the nucleotide sequence of SEQ ID NO:1 is amplified by PCR.
  • 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) for use in bacteria, and transformed into a bacterial strain deficient in amino acid:glyoxylate aminotransferase activity by electroporation using the BioRad Gene Pulser and the manufacturer's conditions.
  • the transformed bacteria are grown on medium that contains inhibitory concentrations of an inhibitor of amino acid:glyoxyiate aminotransferase activity 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.
  • the DNA fragments are cloned into expression vectors for transient or stable transformation into plant cells, which are screened for differential survival and/or growth in the presence of an inhibitor of amino acid:glyoxylate aminotransferase activity.
  • PCR- amplified DNA fragments comprising the Arabidopsis amino acid:glyoxylate aminotransferase gene encoding the protein and PCR-amplified DNA fragments derived from or comprising another amino acid:glyoxylate aminotransferase gene are recombined in vitro and resulting variants with improved tolerance to the inhibitor are recovered as described above.
  • Example 10 In vitro Recombination of Amino acid:glyoxylate aminotransferase Genes by Staggered Extension Process
  • the Arabidopsis amino acid:glyoxylate aminotransferase gene and another amino acid:glyoxylate aminotransferase gene, or homologs thereof, or fragments thereof, 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 amino acid:glyoxylate aminotransferase genes are screened as described in Example 9.
  • Recombinant amino acid:glyoxylate aminotransferase protein is obtained, for example, according to Example 8.
  • the protein is immobilized on chips appropriate for ligand binding assays using techniques which are well known in the art.
  • the protein immobilized on the chip is exposed to sample compound in solution according to methods well know in the art. While the sample compound is in contact with the immobilized protein measurements capable of detecting protein-ligand interactions are conducted. Examples of such measurements are SELDI, biacore and FCS, described above. Compounds found to bind the protein are readily discovered in this fashion and are subjected to further characterization.
  • the alanine:glyoxylate aminotransferase activity assay is derived from Stintjes et al. (1992) Anal. Biochem. 206, 334-343.
  • the reaction volumes are preferably the ones described below, but can be varied depending on the experimental requirements.
  • the production of pyruvate is determined preferably according to Stintjes ef al. (1992) Anal.
  • Biochem. 206, 334-343 by adding 5 ⁇ L of 20mM o-phenylenediamine in 0.6 M hydrochloric acid. Fluorescence intensity is measured for the solution with an excitation wavelength of 410 ⁇ 10nm and an emission wavelength of 535 ⁇ 10nm. Alternatively, the absorbance of the solution may be measured with a wavelength of 410 ⁇ 10nm.
  • pyruvate formation is quantitated by a coupled reaction procedure.
  • 0.5 units of lactate dehydrogenase and 0.2 mM NADH are added and the fluorescence intensity of the solution is measured with an excitation wavelength of 340 ⁇ 10nm and an emission wavelength of 410 ⁇ 10nm.
  • the absorbance of the solution may be measured at 340 nm.

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Abstract

L'invention concerne des gènes d'origine végétale d' « acide aminé » glyoxylate aminotransférase d'origine végétale. L'invention concerne aussi la production, par des techniques de recombinaison, de ces enzymes végétales d' « acide aminé » glyoxylate aminotransférase dans des hôtes hétérologues, la recherche d'une activité herbicide de produits chimiques au moyen de ces enzymes produites par des techniques de recombinaison, et l'utilisation des produits chimiques herbicides ainsi identifiés pour supprimer la croissance de végétation indésirable. De plus, l'invention concerne des procédés permettant de développer une tolérance aux herbicides chez des plantes, dans des tissus végétaux, des semences végétales et des cellules végétales au moyen des gènes d' « acide aminé » glyoxylate aminotransférase de l'invention.
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