WO2002064794A2 - Gene cible d'herbicides et procedes correspondants - Google Patents

Gene cible d'herbicides et procedes correspondants Download PDF

Info

Publication number
WO2002064794A2
WO2002064794A2 PCT/EP2002/000188 EP0200188W WO02064794A2 WO 2002064794 A2 WO2002064794 A2 WO 2002064794A2 EP 0200188 W EP0200188 W EP 0200188W WO 02064794 A2 WO02064794 A2 WO 02064794A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
protein
gene
plant
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2002/000188
Other languages
English (en)
Other versions
WO2002064794A3 (fr
Inventor
Joshua Zvi Levin
Gregory Joseph Budziszewski
Glover Lyn Wegrich
George W. Aux
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
Original Assignee
Syngenta Participations AG
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Syngenta Participations AG filed Critical Syngenta Participations AG
Publication of WO2002064794A2 publication Critical patent/WO2002064794A2/fr
Anticipated expiration legal-status Critical
Publication of WO2002064794A3 publication Critical patent/WO2002064794A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to genes isolated from Arabidopsis that code for proteins essential for seedling growth.
  • the invention also includes the methods of using these proteins as herbicide targets, based on the essentiality of the genes for normal growth and development.
  • the invention is also useful as a screening assay to identify inhibitors that are potential herbicides.
  • the invention may also be applied to the development of herbicide tolerant plants, plant tissues, plant seeds, and plant cells.
  • Herbicides that exhibit greater potency, broader weed spectrum, and more rapid degradation in soil can also, unfortunately, have greater crop phytotoxicity.
  • One solution applied to this problem has been to develop crops that are resistant or tolerant to herbicides. Crop hybrids or varieties tolerant to the herbicides allow for the use of the herbicides to kill weeds without attendant risk of damage to the crop. Development of tolerance can allow application of a herbicide to a crop where its use was previously precluded or limited (e.g. to pre-emergence use) due to sensitivity of he crop to the herbicide.
  • U.S. Patent No. 4,761,373 to Anderson et al. is directed to plants resistant to various imidazolinone or sulfonamide herbicides.
  • acetohydroxyacid synthase (AHAS) enzyme confers the resistance.
  • U.S. Patent No. 4,975,374 to Goodman et al. relates to plant cells and plants containing a gene encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that were known to inhibit GS, e.g. phosphinothricin and methionine sulfoximine.
  • U.S. Patent No. 5,013,659 to Bedbrook et al. is directed to plants expressing a mutant acetolactate synthase that renders the plants resistant to inhibition by sulfonylurea herbicides.
  • U.S. Patent No. 5,162,602 to Somers et al. discloses plants tolerant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The tolerance is conferred by an altered acetyl coenzyme A carboxylase (ACCas
  • a feature of the invention is the identification of genes in Arabidopsis, herein referred to as the ET6497 gene, which encodes a protein with sequence similarity to translation release factor (RF-1) (Craigen and Caskey (1987) Biochimie 69: 1031-1041; Craigen et al. (1990) Mol. Micro. 4: 861-865; Zhouravleva et al. (1995) EMBO J. 14: 4065- 4072; Kisselev and Frolova (1995) Biochem. Cell. Biol. 73: 1079-1086; Stansfield et al. (1997) Eur. J. Biochem.
  • RF-1 translation release factor
  • the GT1773 gene which encodes a protein that appears to be bi-functional and has sequence similarity to diaminohydroxyphosphoribosylaminopyrimidine deaminases and 5-amino-6-(5- phosphoribosylamino) uracil reductases (Fuller and Mulks (1995) J. Bacteriol. 177: 7265- 7270; Richter et al. (1997) J. Bacteriol. 179: 2022-2028; Begley et al. (1998) Topics Curr. Chem.
  • the GT0992 gene which encodes a protein with no known function, but may function as a transport protein
  • the ET6233 gene which encodes a protein with similarity to the Bacillus megaterium cbiX protein required for cobyric acid biosynthesis (Raux et al. (1998) Biochem. J. 335: 159-166; Raux et al. (1998) Biochem. J. 335: 167-173; Raux et al. (1996) J. Bacteriol. 178: 753-767)
  • the ET0763 gene which encodes a protein with similarity to 30S ribosomal protein S 1 (Sugita et al.
  • One object of the present invention is to provide essential genes in plants for assay development for inhibitory compounds with herbicidal activity. Genetic results show that when either the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes are mutated in Arabidopsis, the resulting phenotype is seedling lethal in the homozygous state. This suggests a critical role for the gene products encoded by each of these genes.
  • the inventors of the present invention have demonstrated that the activity encoded by the Arabidopsis ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes (herein referred to as ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity) is essential in Arabidopsis seedlings. This implies that chemicals that inhibit the function of any one of these proteins in plants are likely to have detrimental effects on plants and are potentially good herbicide candidates.
  • the present invention therefore provides methods of using a purified protein encoded by any one of the gene sequences described below to identify inhibitors thereof, which can then be used as herbicides to suppress the growth of undesirable vegetation, e.g. in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • the present invention discloses a nucleotide sequence derived from Arabidopsis, designated the ET6497 gene.
  • the nucleotide sequence of the cDNA clone is set forth in SEQ ID NO: l, and the corresponding amino acid sequence is set forth in SEQ ID NO:2.
  • the nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO: 17.
  • the present invention discloses a nucleotide sequence derived from Arabidopsis, designated the GT1773 gene.
  • the nucleotide sequence of the cDNA clone is set forth in SEQ ID NO:3, and the corresponding amino acid sequence is set forth in SEQ ID NO:4.
  • the nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO: 18.
  • the present invention discloses a nucleotide sequence derived from Arab/do/-.-?/.-', designated the GT0992 gene.
  • the nucleotide sequence of the cDNA clone is set forth in SEQ ID NO:5, and the corresponding amino acid sequence is set forth in SEQ ID NO: 6.
  • the nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO: 19.
  • the present invention discloses a nucleotide sequence derived from Arabidopsis, designated the ET6233 gene.
  • the nucleotide sequence of the cDNA clone is set forth in SEQ ID NO:7, and the corresponding amino acid sequence is set forth in SEQ ID NO:8.
  • the nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO:20. Furthermore, the present invention discloses a nucleotide sequence derived from Arabidopsis, designated the ET0763 gene. The nucleotide sequence of the cDNA clone is set forth in SEQ ID NO:9, and the corresponding amino acid sequence is set forth in SEQ ID NO: 10. The nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO:21. Furthermore, the present invention discloses a nucleotide sequence derived horn Arabidopsis, designated the ET5848 gene.
  • the nucleotide sequence of the cDNA clone is set forth in SEQ ID NO: 11
  • the corresponding amino acid sequence is set forth in SEQ ID NO: 12.
  • the nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO:22.
  • the present invention discloses a nucleotide sequence derived from Arabidopsis, designated the GT5062 gene.
  • the nucleotide sequence of the cDNA clone is set forth in SEQ ID NO: 13, and the corresponding amino acid sequence is set forth in SEQ ID NO: 14.
  • the nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO:23.
  • the present invention discloses a nucleotide sequence derived from Arabidopsis, designated the ET5036 gene.
  • the nucleotide sequence of the cDNA clone is set forth in SEQ ID NO: 15, and the corresponding amino acid sequence is set forth in SEQ ID NO: 16.
  • the nucleotide sequence of the genomic DNA sequence is set forth in SEQ ID NO:24.
  • the present invention also includes nucleotide sequences substantially similar to those set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO: 13, and SEQ ID NO: 15.
  • the present invention also encompasses plant proteins whose amino acid sequence are substantially similar to the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16.
  • Such proteins can be used in a screening assay to identify inhibitors that are potential herbicides.
  • the present invention relates to a method for identifying chemicals having the ability to inhibit ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity in plants preferably comprising the steps of: a) obtaining transgenic plants, plant tissue, plant seeds or plant cells, preferably stably transformed, comprising a non-native nucleotide sequence encoding an enzyme having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, and capable of overexpressing an enzymatically active ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene product (either full length or truncated but still active), respectively; b) applying a chemical to the transgenic plants, plant cells, tissues or parts and to the isogenic non- transformed plants, plant cells, tissues or parts; c) determining the growth or viability of
  • the enzyme having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity is encoded by a nucleotide sequence derived from a plant, preferably a monocotyledonous or a dicotyledonous plant, preferably a dicotyledonous plant, preferably Arabidopsis thaliana, desirably identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO: 13, or SEQ ID NO: 15, respectively.
  • the enzyme having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity is encoded by a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, or SEQ ID NO: 16, respectively.
  • the enzyme having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, or SEQ ID NO: 16, respectively.
  • the present invention further embodies plants, plant tissues, plant seeds, and plant cells that have modified ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity and that are therefore tolerant to inhibition by a herbicide at levels normally inhibitory to naturally occurring ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • Herbicide tolerant plants encompassed by the invention include those that would otherwise be potential targets for normally inhibiting herbicides, particularly the agronomically important crops mentioned above.
  • plants, plant tissue, plant seeds, or plant cells are transformed, preferably stably transformed, with a recombinant DNA molecule comprising a suitable promoter functional in plants operatively linked to a nucleotide coding sequence that encodes a modified ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene that is tolerant to inhibition by a herbicide at a concentration that would normally inhibit the activity of wild- type, unmodified ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene product, respectively.
  • Modified ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity may also be conferred upon a plant by increasing expression of wild-type herbicide-sensitive ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein by providing multiple copies of wild-type ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes, respectively, to the plant or by overexpression of wild-type ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes, respectively, under control of a stronger-than-wild-type promoter.
  • transgenic plants, plant tissue, plant seeds, or plant cells thus created are then selected by conventional selection techniques, whereby herbicide tolerant lines are isolated, characterized, and developed. Alternately, random or site-specific mutagenesis may be used to generate herbicide tolerant lines.
  • the present invention provides a plant, plant cell, plant seed, or plant tissue transformed with a DNA molecule comprising a nucleotide sequence isolated from a plant that encodes an enzyme having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, wherein the DNA expresses the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, and wherein the DNA molecule confers upon the plant, plant cell, plant seed, or plant tissue tolerance to a herbicide in amounts that normally inhibits naturally occurring ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • the enzyme having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO: 13, or SEQ ID NO: 15, respectively, or has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, respectively.
  • the invention also provides a method for suppressing the growth of a plant comprising the step of applying to the plant a chemical that inhibits the naturally occurring ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity in the plant.
  • the present invention is directed to a method for selectively suppressing the growth of undesired vegetation in a field containing a crop of planted crop seeds or plants, comprising the steps of: (a) optionally planting herbicide tolerant crops or crop seeds, which are plants or plant seeds that are tolerant to a herbicide that inhibits the naturally occurring ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity; and (b) applying to the herbicide tolerant crops or crop seeds and the undesired vegetation in the field a herbicide in amounts that inhibit naturally occurring ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, wherein the herbicide 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:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.
  • the nucleotide sequence encodes an amino acid sequence substantially similar to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
  • the nucleotide sequence is SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l 1 , SEQ ID NO: 13, or SEQ ID NO: 15.
  • the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
  • the nucleotide sequence is a plant nucleotide sequence, which preferably encodes a polypeptide having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • the invention further provides:
  • a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: l , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO: 13, or SEQ ID NO: 15.
  • the amino acid sequence is encoded by SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO- 1 1, SEQ ID NO:13, or SEQ ID NO: 15.
  • the polypeptide is encoded by SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO- 1 1, SEQ ID NO:13, or SEQ ID NO: 15.
  • the polypeptide is encoded by SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:
  • -1- comprises an amino acid sequence substantially similar to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, respectively.
  • amino acid sequence is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
  • the amino acid sequence preferably has ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • the amino acid sequence comprises at least 20 consecutive amino acid residues of the amino acid sequence encoded by SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 , respectively.
  • the amino acid sequence comprises at least 20 consecutive amino acid residues of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, respectively.
  • the invention further provides:
  • An expression cassette comprising a promoter operatively linked to a DNA molecule according to the present invention, a 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • the invention further provides:
  • a process for making nucleotides sequences encoding gene products having altered ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity comprising: a) shuffling an unmodified nucleotide sequence of the present invention, b) expressing the resulting shuffled nucleotide sequences, and c) selecting for altered ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, as compared to the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, of the gene product of said unmodified nucleotide sequence.
  • the unmodified nucleotide sequence is identical or substantially similar to SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO:13, or SEQ ID NO:15, respectively, 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • the invention further provides:
  • a method for selecting compounds that interact with the protein encoded by SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l 1, SEQ ID NO: 13, or SEQ ID NO: 15, respectively comprising: a) expressing a DNA molecule comprising SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, respectively, or a sequence substantially similar to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, respectively, 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity comprising: a) introducing a DNA molecule comprising a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, respectively, and having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET
  • 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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.
  • 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
  • a donor of free energy or energy-rich molecule e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine
  • E6497 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO:2, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO: l or is substantially similar to SEQ ID NO: l.
  • GT1773 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO:4, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO: 3 or is substantially similar to SEQ ID NO:3.
  • GT0992 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO:6, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO: 5 or is substantially similar to SEQ ID NO:5.
  • E6233 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO:8, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO:7 or is substantially similar to SEQ ID NO:7.
  • E0763 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO: 10, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO:9 or is substantially similar to SEQ ID NO:9.
  • E5848 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO: 12, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO: 1 1 or is substantially similar to SEQ ID NO: l l.
  • GT5062 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO: 14, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO: 13 or is substantially similar to SEQ ID NO:13.
  • E5036 Gene refers to a DNA molecule comprising a nucleotide sequence encoding SEQ ID NO: 16, or a nucleotide sequence substantially similar thereto.
  • the nucleotide sequence is set forth in SEQ ID NO: 15 or is substantially similar to SEQ ID NO: 15.
  • 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; and genetic constructs wherein an otherwise homologous DNA sequence is operatively linked to a non-native sequence.
  • Homologous DNA Sequence a DNA sequence naturally associated with a host cell into which it is introduced.
  • Inhibitor a chemical substance that causes abnormal growth, e.g., by inactivating the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the plant.
  • a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the plant.
  • an inhibitor is a chemical substance that alters the enzymatic activity encoded by the ET6497, GT1773, GT0992, ET6233, ET0763,
  • ET5848, GT5062, or ET5036 gene from a plant More generally, an inhibitor causes abnormal growth of a host cell by interacting with the gene product encoded by the ET6497,
  • 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 that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, 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.
  • Pre-protein protein which is normally targeted to a cellular organelle, such as a chloroplast, and still comprising its transit peptide.
  • an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
  • the 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 at least 60% identical, still more preferably at least 70% identical, still more preferably at least 80% identical, still more preferably at least 90% identical, still more preferably at least 95% identical, yet still more preferably at least 99% identical to SEQ ID NO:2; in the context of the "GT1773 gene”, “substantially similar” refers to nucleotide sequences that encode a protein at least 46% identical, more preferably at least 55% identical, still more preferably at least 65% identical, still more preferably at least 75% identical , still more preferably at least 85% identical, still more preferably at least 95% identical, yet still more preferably at least 99% identical to SEQ ID NO:4; in the context of the "GT0992 gene”, “substantially similar” refers to nucleotide sequences that encode a protein at least 43% identical, more preferably at least 55% identical, more preferably at least 65% identical,
  • a nucleotide sequence "substantially similar" to the reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1 % SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.1
  • “Homologs of the ET6497 gene” include nucleotide sequences that encode an amino acid sequence that is at least 30% identical to SEQ ID NO:2, more preferably at least 34% identical, yet still more preferably at least 39% identical, yet still more preferably at least 43% identical to SEQ ID NO:2, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the ET6497 protein.
  • “Homologs of the GT1773 gene” include nucleotide sequences that encode an amino acid sequence that is at least 26% identical to SEQ ID NO:4, more preferably at least 32% identical, still more preferably at least 38% identical, still more preferably at least 43% identical to SEQ ID NO:4, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the GT1773 protein.
  • “Homologs of the GT0992 gene” include nucleotide sequences that encode an amino acid sequence that is at least 29% identical to SEQ ID NO:6, still more preferably at least 33% identical, yet still more preferably at least 37% identical, yet still more preferably at least 38% identical, yet still more preferably at least 39% identical to SEQ ID NO:6, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the GT0992 protein.
  • “Homologs of the ET6233 gene” include nucleotide sequences that encode an amino acid sequence that is at least 27% identical to SEQ ID NO:8, still more preferably at least 34% identical to SEQ ID NO:8, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the ET6233 protein.
  • “Homologs of the ET0763 gene” include nucleotide sequences that encode an amino acid sequence that is at least 24% identical to SEQ ID NO: 10, more preferably at least 25% identical, yet still more preferably at least 26% identical to SEQ ID NO: 10, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the ET0763 protein.
  • “Homologs of the ET5848 gene” include nucleotide sequences that encode an amino acid sequence that is at least 25% identical to SEQ ID NO: 12, more preferably at least 29% identical, still more preferably at least 49% identical to SEQ ID NO: 12, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the ET5848 protein.
  • “Homologs of the GT5062 gene” include nucleotide sequences that encode an amino acid sequence that is at least 29% identical to SEQ ID NO: 14, more preferably at least 31% identical, yet still more preferably at least 41 % identical, yet still more preferably at least 54% identical to SEQ ID NO: 14, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the GT5062 protein.
  • “Homologs of the ET5036 gene” include nucleotide sequences that encode an amino acid sequence that is at least 50% identical to SEQ ID NO: 16, more preferably at least 60% identical, still more preferably at least 70% identical to SEQ ID NO: 16, as measured, using the GAP parameters described below, wherein the amino acid sequence encoded by the homolog has the biological activity of the ET5036 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 protein or amino acid sequence and the reference protein or amino acid sequence is at least 60%, more preferably at least 70%, still more preferably at least 80%, still more preferably at least 90%, still more preferably at least 95%, yet still more preferably at least 99%, as determined using default GAP analysis parameters with the University of Wisconsin GCG, SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453).
  • the percentage of identity between the substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is at least 46%, more preferably at least 55%, still more preferably at least 65%, still more preferably at least 75%, still more preferably at least 85%, still more preferably at least 95%, yet still more preferably at least 99%.
  • the percentage of identity between the substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is at least 43%, more preferably at least 55%, more preferably at least 65%, still more preferably at least 75%, still more preferably at least 85%, still more preferably at least 95%, yet still more preferably at least 99%.
  • the percentage of identity between the substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is at least 38%, more preferably at least 45%, more preferably at least 55%, yet still more preferably at least 65%, yet still more preferably at least 75%, yet still more preferably at least 85%, yet still more preferably at least 95%, yet still more preferably at least 99%.
  • the percentage of identity between the substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is at least 34%, more preferably at least 44%, more preferably at least 55%, yet still more preferably at least 65%, still more preferably at least 75%, still more preferably at least 85%, still more preferably at least 95%, yet still more preferably at least 99%.
  • the percentage of identity between the substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is at least 52%, more preferably at least 65%, still more preferably at least 75%, still more preferably at least 85%, still more preferably at least 95%, yet still more preferably at least 99%.
  • the percentage of identity between the substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is at least 80%, more preferably at least 90%, still more preferably at least 92%, still more preferably at least 95%, yet still more preferably at least 99%.
  • the percentage of identity between the substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is at least 50%, more preferably at least 60%, still more preferably at least 65%, still more preferably at least 75%, yet still more preferably at least 85%, yet still more preferably at least 95%, yet still more preferably at least 99% .
  • ET6497 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO: l .
  • "Homologs of the ET6497 protein” are amino acid sequences that are at least 30% identical to SEQ ID NO:2, more preferably at least 34% identical, yet still more preferably at least 39%, yet still more preferably at least 43% identical to SEQ ID NO:2, as measured using the GAP parameters described above, wherein the homologs of the ET6497 protein have the biological activity of the ET6497 protein.
  • GT1773 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO: 3.
  • "Homologs of the GT1773 protein” are amino acid sequences that are at least 26% identical to SEQ ID NO:4, more preferably at least 32% identical, still more preferably at least 38% identical, yet still more preferably at least 43% identical to SEQ ID NO:4, as measured using the GAP parameters described above, wherein the homologs of the GT1773 protein have the biological activity of the GT1773 protein.
  • GT0992 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO:5.
  • "Homologs of the GT0992 protein” are amino acid sequences that are at least 29% identical to SEQ ID NO:6, still more preferably at least 33% identical, yet still more preferably at least 37% identical, yet still more preferably at least 38% identical, yet still more preferably at least 39% identical to SEQ ID NO:6, as measured using the GAP parameters described above, wherein the homologs of the GT0992 protein have the biological activity of the GT0992 protein.
  • ET6233 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO:7.
  • "Homologs of the ET6233 protein” are amino acid sequences that are at least 27% identical to SEQ ID NO:8, still more preferably at least 34% identical to SEQ ID NO:8, as measured using the GAP parameters described above, wherein the homologs of the ET6233 protein have the biological activity ofthe ET6233 protein.
  • ET0763 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO:9.
  • "Homologs of the ET0763 protein” are amino acid sequences that are at least 24% identical to SEQ ID NO: 10, still more preferably at least 25% identical, yet still more preferably at least 26% identical to SEQ ID NO: 10, as measured using the GAP parameters described above, wherein the homologs of the ET0763 protein have the biological activity of the ET0763 protein.
  • E5848 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO: 1 1.
  • “Homologs of the ET5848 protein” are amino acid sequences that are at least 25% identical to SEQ ID NO: 12, still more preferably at least 29% identical, yet still more preferably at least 49% identical to SEQ ID NO: 12, as measured using the GAP parameters described above, wherein the homologs of the ET5848 protein have the biological activity of the ET5848 protein.
  • GT5062 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO: 13.
  • "Homologs of the GT5062 protein” are amino acid sequences that are at least 29% identical to SEQ ID NO: 14, still more preferably at least 31% identical, yet still more preferably at least 41% identical, yet still more preferably at least 54% identical to SEQ ID NO: 14, as measured using the GAP parameters described above, wherein the homologs of the GT5062 protein have the biological activity of the GT5062 protein.
  • ET5036 protein refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO: 15.
  • "Homologs of the ET5036 protein” are amino acid sequences that are at least 50% identical to SEQ ID NO: 16, still more preferably at least 60% identical, yet still more preferably at least 70% identical to SEQ ID NO: 16, as measured using the GAP parameters described above, wherein the homologs of the ET5036 protein have the biological activity of the ET5036 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.
  • Transformation a process for introducing heterologous DNA into a cell, tissue, or plant.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • Transgenic stably transformed with a recombinant DNA molecule that preferably comprises a suitable promoter operatively linked to a DNA sequence of interest.
  • SEQ ID NO:2 amino acid sequence encoded by the Arabidopsis ET6497 nucleotide sequence shown in SEQ ID NO:l SEQ ID NO:3 cDNA coding sequence for the Arabidopsis GT1773 gene
  • SEQ ID NO:6 amino acid sequence encoded by the Arabidopsis GT0992 nucleotide sequence shown in SEQ ID NO: 5 SEQ ID NO:7 cDNA coding sequence for the Arabidopsis ET6233 gene
  • SEQ ID NO:9 cDNA coding sequence for the Arabidopsis ET0763 gene
  • SEQ ID NO: 10 amino acid sequence encoded by the Arabidopsis ET0763 nucleotide sequence shown in SEQ ID NO: 9
  • SEQ ID NO: 13 cDNA coding sequence for the Arabidopsis GT5062 gene
  • SEQ ID NO: 14 amino acid sequence encoded by the Arabidopsis GT5062 nucleotide sequence shown in SEQ ID NO: 13
  • SEQ ID NO: 15 cDNA coding sequence for the Arabidopsis ET5036 gene
  • SEQ ID NO: 17 genomic sequence of the Arabidopsis ET6497 gene
  • SEQ ID NO: 18 genomic sequence of the Arabidopsis GT1773 gene
  • SEQ ID NO: 19 genomic sequence of the Arabidopsis GT0992 gene
  • SEQ ID NO:20 genomic sequence of the Arabidopsis ET6233 gene
  • Arabidopsis insertional mutant lines segregating for seedling lethal mutations are identified as a first step in the identification of essential proteins.
  • Ds transposon insertion lines were produced as described in Sundareson et al. (1995) Genes and Dev., 9: 1797-1810), incorporated herein by reference. Starting with F3 or F4 seeds collected from single F2 or F3 kanamycin-resistant plants containing Ds insertions in their genomes (see Figure 3 of Sundareson et al. (1995) Genes and Dev., 9:1797-1810), those lines segregating homozygous seedling lethal seedlings are identified.
  • Inviable phenotypes include altered pigmentation or altered morphology. These phenotypes are observed either on plates directly or in soil following transplantation of seedlings.
  • a line When a line is identified as segregating a seedling lethal, it is determined if the resistance marker in the Ds transposon insertion co-segregates with the lethality (Errampalli et al. (1991) The Plant Cell, 3:149-157). Co-segregation analysis is done by placing the seeds on media containing the selective agent and scoring the seedlings for resistance or sensitivity to the agent. Examples of selective agents used are kanamycin, hygromycin, or phosphinothricin. About 35 resistant seedlings are transplanted to soil and their progeny are examined for the segregation of the seedling lethal.
  • the Arabidopsis ET6497 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line # ET6497. A region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 3, clone T20O10, GenBank accession # AL163816). The inventors are the first to demonstrate that the ET6497 gene product is essential for normal growth and development in plants, as well as defining the function of the ET6497 gene through protein homo logy. The present invention discloses the cDNA coding nucleotide sequence of the Arabidopsis ET6497 gene as well as the amino acid sequence of the Arabidopsis ET6497 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:l, wherein said amino acid sequence has ET6497 activity.
  • the sequence of the ET6497 gene shows similarity to translation release factor 1 (R- Fl), from Caenorhabditis elegans (GenPept Accession # AAB94190), Schizosaccharomyces pombe (GenPept Accession # CAA90504), Thermus thermophilus (GenPept Accession # BAA 13349.1), Escherichia coli (GenPept Accession # AAC43437), Neisseria meningitidis (GenPept Accession # AAF42034), Synechocystis sps (GenPept Accession # BAA18826).
  • R- Fl translation release factor 1
  • the Arabidopsis GT1773 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line # GT1773.
  • a region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 3, clone T21L8, GenBank accession number AL096860).
  • the inventors are the first to demonstrate that the GT1773 gene product is essential for normal growth and development in plants, as well as defining the function of the GT1773 gene through protein homology.
  • the present invention discloses cDNA coding nucleotide sequences of the Arabidopsis GT1773 gene as well as the amino acid sequence of the Arabidopsis GT1773 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 3, wherein said amino acid sequence has GT1773 activity.
  • the sequence of the GT1773 gene shows similarity to diaminohydroxyphosphoribosylaminopyrimidine deaminases, 5-amino-6-(5- phosphoribosylamino) uracil reductases, and a hypothetical protein from Arabidopsis thaliana (GenPept Accession # CAB79096), Methanococcus jannaschii (GenPept Accession # AAB98665), Escherichia coli (GenPept Accession # AAB40170 and SWISS PROT Accession # P30176), Thermotoga maritima (GenPept Accession # AAD36891), Streptomyces coelicolor (GenPept Accession # CAB92254), and Synechocystis sps (GenPept Accession # BAA10295).
  • the Arabidopsis GT0992 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line #GT0992.
  • a region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 5, clone MXC9, GenBank accession number AB007727).
  • the inventors are the first to demonstrate that the GT0992 gene product is essential for normal growth and development in plants, as well as defining the function of the GT0992 gene through protein homology.
  • the present invention discloses the cDNA coding nucleotide sequence of the Arabidopsis GT0992 gene as well as the amino acid sequence of the Arabidopsis GT0992 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:5, wherein said amino acid sequence has GT0992 activity.
  • the sequence of the GT0992 gene shows similarity to proteins of unknown function, which confer tellurium resistance and may function as transmembrane transport proteins, from Rickettsia prowazekii (GenPept Accession # CAA15212), Vibrio cholerae (GenPept Accession # AAF96448), Escherichia coli (GenPept Accession # AAA57890), Streptomyces coelicolor (GenPept Accession # CAB38153), Oryza sativa (GenPept Accession # B AA90517), and Neisseria meningitidis (GenPept Accession # AAF40491 ).
  • the Arabidopsis ET6233 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line # ET6233.
  • a region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 1, clone F14I3, GenBank accession number AC007980).
  • the inventors are the first to demonstrate that the ET6233 gene product is essential for normal growth and development in plants, as well as defining the function of the ET6233 gene through protein homology.
  • the present invention discloses the cDNA coding nucleotide sequence of the
  • Arabidopsis ET6233 gene as well as the amino acid sequence of the Arabidopsis ET6233 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:7, wherein said amino acid sequence has ET6233 activity.
  • sequence of the ET6233 gene shows similarity to the Bacillus megaterium cbiX protein, which is required for cobyric acid biosynthesis (GenPept Accession # CAA04308), and other related proteins from Methanococcus jannaschii (GenPept Accession # AAB98975) and Bacillus halodurans (GenPept Accession # BAB05215.1).
  • the Arabidopsis ET0763 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line # ET0763.
  • a region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 1, clone F14O23, GenBank accession number AC012654).
  • the inventors are the first to demonstrate that the ET0763 gene product is essential for normal growth and development in plants, as well as defining the function of the ET0763 gene through protein homology.
  • the present invention discloses the cDNA coding nucleotide sequence of the Arabidopsis ET0763 gene as well as the amino acid sequence of the Arabidopsis ET0763 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:9, wherein said amino acid sequence has ET0763 activity.
  • the sequence of the ET0763 gene shows similarity to 30S ribosomal protein S 1 from Escherichia coli (GenPept Accession # CAA23644), Streptomyces coelicolor (GenPept Accession # CAB52054), Spinacia oleracea (GenPept Accession # AAA34045), Porphyra purpurea (GenPept Accession # AAC08231), Mycobacterium tuberculosis (GenPept Accession # CAB08883), and Synechococcus sps (GenPept Accession # BAA05946).
  • the Arabidopsis ET5848 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line # ET5848. A region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 1, clone F17O7, GenBank accession number AC003671). The inventors are the first to demonstrate that the ET5848 gene product is essential for normal growth and development in plants, as well as defining the function of the ET5848 gene through protein homology.
  • the present invention discloses the cDNA coding nucleotide sequence of the Arabidopsis ET5848 gene as well as the amino acid sequence of the Arabidopsis ET5848 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 11 , wherein said amino acid sequence has ET5848 activity.
  • the sequence of the ET5848 gene shows similarity to proteins of unknown function from Arabidopsis thaliana (GenPept Accession numbers AAF27677, CAB61960, AAF25966, AAC18801, AAD55510; GenBank accession number AC016162.3).
  • the Arabidopsis GT5062 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line # GT5062.
  • a region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 3, clone F5K20, GenBank accession number AL132960).
  • the inventors are the first to demonstrate that the GT5062 gene product is essential for normal growth and development in plants, as well as defining the function of the GT5062 gene through protein homology.
  • the present invention discloses the cDNA coding nucleotide sequence of the Arabidopsis GT5062 gene as well as the amino acid sequence of the Arabidopsis GT5062 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 13, wherein said amino acid sequence has GT5062 activity.
  • the sequence of the GT5062 gene shows similarity to uracil phosphoribosyltransferase (also known as UMP pyrophosphorylase) from Schizo saccharomyces pombe (GenPept Accession # CAB 11230), Methanobacterium thermoantotrophicum (GenPept Accession # AAB85603), Thermotoga maritima (GenPept Accession # AAD35803), Escherichia coli (GenPept Accession # BAA16386), Synechocystis sps (GenPept Accession # BAA 16768), and Nicotiana tabacum (SWISS PROT Accession # P93394).
  • uracil phosphoribosyltransferase also known as UMP pyrophosphorylase
  • the Arabidopsis ET5036 gene is identified by isolating DNA flanking the Ds transposon border from the tagged seedling-lethal line # GT5062.
  • a region of the Arabidopsis DNA flanking the Ds transposon border corresponds to Arabidopsis genomic sequence (chromosome 1, clone T10O24, GenBank accession number AC007067).
  • the inventors are the first to demonstrate that the GT5062 gene product is essential for normal growth and development in plants, as well as defining the function of the GT5062 gene through protein homology.
  • the present invention discloses the cDNA coding nucleotide sequence of the Arabidopsis GT5062 gene as well as the amino acid sequence of the Arabidopsis GT5062 protein.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 15, wherein said amino acid sequence has ET5036 activity.
  • sequence of the ET5036 gene does not show similarity to any genes in public sequence databases.
  • a nucleotide sequence encoding a protein having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, is inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced.
  • SEQ ID NO: l nucleotide sequences substantially similar to SEQ ID NO: 1 , or homologs of the ET6497 gene are used for the recombinant production of a protein having ET6497 activity.
  • SEQ ID NO:3, nucleotide sequences substantially similar to SEQ ID NO:3, or homologs of the GT1773 gene are used for the recombinant production of a protein having GT1773 activity.
  • SEQ ID NO:5, nucleotide sequences substantially similar to SEQ ID NO:5, or homologs of the GT0992 gene are used for the recombinant production of a protein having GT0992 activity.
  • SEQ ID NO:7, nucleotide sequences substantially similar to SEQ ID NO:7, or homologs of the ET6233 gene are used for the recombinant production of a protein having ET6233 activity.
  • SEQ ID NO:9 nucleotide sequences substantially similar to SEQ ID NO:9, or homologs of the ET0763 gene are used for the recombinant production of a protein having ET0763 activity.
  • SEQ ID NO: 11 nucleotide sequences substantially similar to SEQ ID NO: 11, or homologs of the ET5848 gene are used for the recombinant production of a protein having ET5848 activity.
  • SEQ ID NO: 13 nucleotide sequences substantially similar to SEQ ID NO: 13, or homologs of the GT5062 gene are used for the recombinant production of a protein having GT5062 activity.
  • SEQ ID NO: 15 nucleotide sequences substantially similar to SEQ ID NO: 15, or homologs of the ET5036 gene are used for the recombinant production of a protein having ET5036 activity.
  • 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, may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E.
  • baculovirus expression vectors e.g., those derived from the genome of Autographica califomica nuclear polyhedrosis virus (AcMNPV).
  • a preferred baculovirus/insect system is pAcHLT (Pharmingen, San Diego, CA) used to transfect Spodoptera frugiperda Sf9 cells (ATCC) in the presence of linear Autographa califomica baculovirus DNA (Pharmingen, San Diego, CA). The resulting virus is used to infect HighFive Tricoplusia ni cells (Invitrogen, La Jolla, CA).
  • the nucleotide sequence encoding a protein having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity is derived from an eukaryote, such as a mammal, a fly or a yeast, but is preferably derived from a plant, preferably a monocotyledonous or a dicotyledonous plant.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: l , or encodes a protein having ET6497 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 identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:3, or encodes a protein having GT1773 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:4.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:5, or encodes a protein having GT0992 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:6.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:7, or encodes a protein having ET6233 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:8.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:9, or encodes a protein having ET0763 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 10.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 1 , or encodes a protein having ET5848 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 12.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 13, or encodes a protein having GT5062 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 14.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 15, or encodes a protein having ET5036 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 16.
  • the nucleotide sequence encoding a protein having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively is derived from a prokaryote. Recombinantly produced protein having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity is isolated and purified using a variety of standard techniques.
  • Recombinantly produced proteins having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity are useful for a variety of purposes.
  • they can be used in in vitro assays to screen known herbicidal chemicals whose target has not been identified to determine if they inhibit ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036, respectively.
  • Such in vitro assays may also be used as more general screens to identify chemicals that inhibit such enzymatic activity and that are therefore novel herbicide candidates.
  • recombinantly produced proteins having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity may be used to elucidate the complex structure of these molecules and to further characterize their association with known inhibitors in order to rationally design new inhibitory herbicides as well as herbicide tolerant forms of the enzymes.
  • 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. (Thornwood, NY). Ligand binding is determined by changes in the diffusion rate of the protein.
  • SMDI Surface-Enhanced Laser Desorption/Ionization
  • 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 the products essential plant genes comprises the steps of: a) reacting an ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein, respectively, and a substrate thereof in the presence of a suspected inhibitor of the protein's respective function; b) comparing the rate of enzymatic activity of the protein 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein, respectively.
  • the inhibitory effect on ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity may be determined by a reduction or complete inhibition of ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, 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. XI. In Vivo Inhibitor Assay
  • 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity uses transgenic plants, plant tissue, plant seeds or plant cells capable of overexpressing a nucleotide sequence having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, wherein the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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: l, or encodes an enzyme having ET6497 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.
  • the nucleotide sequence is 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:3, or encodes an enzyme having GT1773 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:4.
  • the nucleotide sequence is derived from a prokaryote.
  • the nucleotide sequence is 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:5, or encodes an enzyme having GT0992 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:6.
  • the nucleotide sequence is derived from a prokaryote.
  • the nucleotide sequence is 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:7, or encodes an enzyme having ET6233 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:8.
  • the nucleotide sequence is derived from a prokaryote.
  • the nucleotide sequence is 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:9, or encodes an enzyme having ET0763 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 10.
  • the nucleotide sequence is derived from a prokaryote.
  • the nucleotide sequence is 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: 11, or encodes an enzyme having ET5848 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 12.
  • the nucleotide sequence is derived from a prokaryote.
  • the nucleotide sequence is 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: 13, or encodes an enzyme having GT5062 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 14.
  • the nucleotide sequence is derived from a prokaryote.
  • the nucleotide sequence is derived from an eukaryote, such as a yeast, but is preferably derived from a plant.
  • nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 15, or encodes an enzyme having ET5036 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 16.
  • 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity.
  • the present invention is further directed to plants, plant tissue, plant seeds, and plant cells tolerant to herbicides that inhibit the naturally occurring ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity in these plants, wherein the tolerance is conferred by an altered ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively.
  • Altered ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity may be conferred upon a plant according to the invention by increasing expression of wild-type herbicide-sensitive ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene, respectively, for example by providing additional wild-type ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes and/or by overexpressing the endogenous ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene, for example by driving expression with a strong promoter.
  • Altered ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity also may be accomplished by expressing nucleotide sequences that are substantially similar to SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11 , SEQ ID NO: 13, or SEQ ID NO: 15, respectively, or homologs in a plant.
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity is conferred on a plant by expressing modified herbicide-tolerant ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes, respectively, in the plant. Combinations of these techniques may also be used.
  • 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity through increased expression results in a level of ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, in the plant cell at least sufficient to overcome growth inhibition caused by the herbicide when applied in amounts sufficient to inhibit normal growth of control plants.
  • the level of expressed enzyme generally is at least two times, preferably at least five times, and more preferably at least ten times the natively expressed amount.
  • Increased expression may be due to multiple copies of a wild-type ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene; multiple occurrences of the coding sequence within the gene (i.e. gene amplification) or a mutation in the non-coding, regulatory sequence of the endogenous gene in the plant cell.
  • Plants having such altered gene activity can be obtained by direct selection in plants by methods known in the art (see, e.g. U.S. Patent No. 5,162,602, and U.S. Patent No. 4,761,373, and references cited therein). These plants also may be obtained by genetic engineering techniques known in the art.
  • Increased expression of a herbicide-sensitive ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene can also be accomplished by transforming a plant cell with a recombinant or chimeric DNA molecule comprising a promoter capable of driving expression of an associated structural gene in a plant cell operatively linked to a homologous or heterologous structural gene encoding the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein, respectively, or a homolog thereof.
  • the transformation is stable, thereby providing a heritable transgenic trait.
  • plants, plant tissue, plant seeds, or plant cells are stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operatively linked to a coding sequence encoding a herbicide tolerant form of the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein.
  • a herbicide tolerant form of the enzyme has at least one amino acid substitution, addition or deletion that confers tolerance to a herbicide that inhibits the unmodified, naturally occurring form of the enzyme.
  • the transgenic plants, plant tissue, plant seeds, or plant cells thus created are then selected by conventional selection techniques, whereby herbicide tolerant lines are isolated, characterized, and developed.
  • One general strategy involves direct or indirect mutagenesis procedures on microbes.
  • 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.
  • the microbe selected for mutagenesis contains a normal, inhibitor-sensitive ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene, or nucleotide sequence substantially similar thereto, which encodes a protein having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene product activity, and is dependent upon the activity conferred by this gene for growth.
  • 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.
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes conferring tolerance to the inhibitor are isolated from these colonies, 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene involves direct selection in plants.
  • the effect of a mutagenized ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene on the growth inhibition of plants such as Arabidopsis, soybean, or maize is determined by plating seeds sterilized by art-recognized methods on plates on a simple minimal salts medium containing increasing concentrations of the inhibitor.
  • Such concentrations are in the range of 0.001, 0.003, 0.01 , 0.03, 0.1, 0.3, 1, 3, 10, 30, 1 10, 300, 1000 and 3000 parts per million (ppm).
  • the lowest dose at which significant growth inhibition can be reproducibly detected is used for subsequent experiments. Determination of the lowest dose is routine in the art.
  • Mutagenesis of plant material is utilized to increase the frequency at which resistant alleles occur in the selected population.
  • Mutagenized seed material is derived from a variety of sources, including chemical or physical mutagenesis or seeds, or chemical or physical mutagenesis or pollen (Neuffer, In Maize for Biological Research Sheridan, ed. Univ. Press, Grand Forks, ND., pp. 61-64 (1982)), which is then used to fertilize plants and the resulting Mi mutant seeds collected.
  • M2 seeds (Lehle Seeds, Arlington, AZ), which are progeny seeds of plants grown from seeds mutagenized with chemicals, such as ethyl methane sulfonate, or with physical agents, such as gamma rays or fast neutrons, are plated at densities of up to 10,000 seeds/plate (10 cm diameter) on minimal salts medium containing an appropriate concentration of inhibitor to select for tolerance. Seedlings that continue to grow and remain green 7-21 days after plating are transplanted to soil and grown to maturity and seed set. Progeny of these seeds are tested for tolerance to the herbicide.
  • 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 can be either Arabidopsis plants or any other plant whose growth is susceptible to the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 inhibitors.
  • the inserted ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes are mapped relative to known restriction fragment length polymorphisms (RFLPs) (See, for example, Chang et al. Proc. Natl.
  • the tolerance trait maps to a position indistinguishable from the position of the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene.
  • Another method of obtaining herbicide-tolerant alleles of a ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene is by selection in plant cell cultures. Explants of plant tissue, e.g. embryos, leaf disks, etc.
  • Those alleles identified as conferring herbicide tolerance may then be engineered for optimal expression and transformed into the plant.
  • plants can be regenerated from the tissue or cell cultures containing these alleles.
  • Still another method involves mutagenesis of wild-type, herbicide sensitive plant ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes in genetically manipulatable microbes, followed by culturing the microbe on medium that contains inhibitory concentrations (i.e. sufficient to cause abnormal growth, inhibit growth or cause cell death) of the inhibitor, and then selecting those colonies that grow normally in the presence of the inhibitor.
  • a plant cDNA such as the Arabidopsis cDNA encoding the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein
  • a plant cDNA is cloned into a microbe that is dependent on ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene product activity, respectively, for growth, or that otherwise lacks the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity.
  • the transformed microbe is then subjected to in vivo mutagenesis or to in vitro mutagenesis by any of several chemical or enzymatic methods known in the art, e.g. sodium bisulfite (Shortle et al, Methods Enzymol 100:451-468 (1983); methoxylamine (Kadonaga et al, Nucleic Acids Res. 73:1733-1745 (1985); oligonucleotide-directed saturation mutagenesis (Hutchinson et al, Proc. Natl. Acad. Sci. USA, #3:710-714 (1986); or various polymerase misincorporation strategies (see, e.g. Shortle et al., Proc. Natl. Acad.
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity.
  • DNA shuffling also leads to the recombination and rearrangement of sequences within a ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene or to recombination and exchange of sequences between two or more different of ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes.
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes are produced of millions of mutated ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 coding sequences.
  • the mutated genes, or shuffled genes are screened for desirable properties, e.g.
  • a mutagenized ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene is formed from at least one template ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene, wherein the template ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ohgonucleotides, wherein said ohgonucleotides comprise an area of identity and an area of heterology to the double-stranded random fragments
  • 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes are mutagenized in vitro by a staggered extension process (StEP), as described e.g. in Zhao et al. (1998) Nature Biotechnology 16: 258-261.
  • StEP staggered extension process
  • the two or more ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes, e.g.
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes whereby the different ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes used in the PCR reaction are preferably comprised in separate vectors.
  • the primers desirably anneal to sequences located less than 500 bp away from ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 sequences, preferably less than 200 bp away from the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 sequences, more preferably less than 120 bp away from the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 sequences.
  • the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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.
  • GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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.
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene or any combination of ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes, or homologs thereof, is used for in vitro recombination in the context of the present invention, for example, a ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene derived from a plant, such as, e.g. Arabidopsis thaliana, e.g.
  • ET6497 gene set forth in SEQ ID NO:l a GT1773 gene set forth in SEQ ID NO:3, a GT0992 gene set forth in SEQ ID NO:5, a ET6233 gene set forth in SEQ ID NO:7, a ET0763 gene set forth in SEQ ID NO:9, a ET5848 gene set forth in SEQ ID NO: 11, a GT5062 gene set forth in SEQ ID NO: 13, and a ET5036 gene set forth in SEQ ID NO:15.
  • Whole ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 genes or portions thereof are used in the context of the present invention.
  • the library of mutated ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene product activity for growth.
  • Host cells transformed with the vectors comprising the library of mutated ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene that is tolerant to an inhibitor may be performed in the same manner as the assay to identify inhibitors of the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity (Inhibitor Assay, above) with the following modifications: First, a mutant ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein is substituted in one of the reaction mixtures for the wild-type ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein of the inhibitor assay.
  • 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
  • Mutated activity is any measure of activity of the mutated enzyme while in the presence of a suitable substrate and the inhibitor.
  • Unmutated activity is any measure of activity of the wild-type enzyme while in the presence of a suitable substrate and the inhibitor.
  • genes encoding herbicide-tolerant ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 protein can also be used as selectable markers in plant cell transformation methods.
  • plants, plant tissue, plant seeds, or plant cells transformed with a heterologous DNA sequence can also be transformed with a sequence encoding an altered ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity capable of being expressed by the plant.
  • the transformed cells are transferred to medium containing an inhibitor of the enzyme in an amount sufficient to inhibit the growth or survivability of plant cells not expressing the modified coding sequence, wherein only the transformed cells will grow.
  • the method is applicable to any plant cell capable of being transformed with a modified ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene, and can be used with any heterologous DNA sequence of interest.
  • Expression of the heterologous DNA sequence and the modified gene can be driven by the same promoter functional in plant cells, or by separate promoters.
  • a wild type or herbicide-tolerant form of the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene, or homologs thereof, can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting a DNA molecule encoding the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene into an expression system to which the DNA molecule is heterologous (i.e., not normally present) using standard cloning procedures known in the art.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences in a host cell containing the vector.
  • a large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.
  • the components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions, nucleotide optimization or other modifications may be employed.
  • Expression systems known in the art can be used to transform virtually any crop plant cell under suitable conditions.
  • a heterologous DNA sequence comprising a wild-type or herbicide-tolerant form of the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene is preferably stably transformed and integrated into the genome of the host cells.
  • the heterologous DNA sequence comprising a wild-type or herbicide-tolerant form of the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene located on a self-replicating vector.
  • self-replicating vectors are viruses, in particular gemini viruses.
  • Transformed cells can be regenerated into whole plants such that the chosen form of the ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene confers herbicide tolerance in the transgenic plants.
  • Gene sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants.
  • the expression cassettes may also comprise any further sequences required or selected for the expression of the heterologous DNA sequence.
  • Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
  • the selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the heterologous DNA sequence in the plant transformed with this DNA sequence.
  • Selected promoters will express heterologous DNA sequences 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.
  • 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 heterologous DNA sequence and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledonous and dicotyledonous plants.
  • intron sequences such as introns of the maize Adhl gene have been shown to enhance expression, particularly in monocotyledonous cells.
  • non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
  • the coding sequence of the selected gene 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).
  • Methods for modifying coding sequences by taking into account codon usage in plant genes and in higher plants, green algae, and cyanobacteria are well known (see table 4 in: Murray et al. Nucl. Acids Res. 17: 477-498 (1989); Campbell and Gowri Plant Physiol. 92: 1- 11(1990).
  • the cDNAs encoding these products can also be manipulated to effect the targeting of heterologous products encoded by DNA sequences to these organelles.
  • sequences have been characterized which cause the targeting of products encoded by DNA sequences to other cell compartments.
  • Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769- 783 (1990)).
  • amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
  • Patent Nos. 4,940,935 and 5,188,642 disclose 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 pCIBlO and hygromycin selection derivatives thereof. (See, for example, U.S. Patent No. 5,639,949). 2.
  • 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).
  • Transformation Techniques Once the coding sequence of interest has been cloned into an expression system, it is transformed into a plant cell. Methods for transformation and regeneration of plants are well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells.
  • Transformation techniques for dicotyledons are well known in the art and include ⁇ grob ⁇ cter-Mm-based techniques and techniques that do not require Agrobacterium.
  • Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art. Transformation of most monocotyledon species has now also become routine.
  • Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, particle bombardment into callus tissue, as well as Agrobacterium-mediated transformation. D.
  • a nucleotide sequence encoding a polypeptide having ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 rpsl2 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).
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene of the present invention can be utilized to confer herbicide tolerance to a wide variety of plant cells, including those of gymnosperms, monocots, and dicots.
  • the gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • crop plant cells such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear
  • the high-level expression of a wild-type ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene and/or the expression of herbicide-tolerant forms of a ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene conferring herbicide tolerance in plants, in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding approaches and techniques known in the art.
  • ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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.
  • Arabidopsis genomic DNA is isolated from line ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 using the Nucleon PhytoPureTM Plant DNA Isolation Kit (Amersham International pic, Buckinghamshire, England). Fragments of genomic DNA flanking the borders of the transposon are isolated using the TAIL-PCR technique (Liu et al. (1995) The Plant Journal, 8:457-463; Liu and Whittier (1995), Genomics, 25: 674-681). Three sets of 12 TAIL-PCR reactions, referred to as the primary, secondary and tertiary reactions, are performed. In each reaction, one arbitrary degenerate primer and one transposon-specific primer are used.
  • the arbitrary degenerate primer is chosen from among six primers, LWAD1, CA51, CA52, CA53, CA54, and CA55 (Table 1), which are used to prime the genomic DNA flanking the insertion. These degenerate primers are used in combination with two sets of three, nested, transposon-specific primers (Table 2). These primers are homologous to regions of the Ds elements which lie at the outermost ends of the transposons, DS5 at the 5' end (primers 5A, 5B, and 5C) and DS3 at the 3' end (primers 3A, 3B, and 3C).
  • PCR primers specific to the flanking genomic region are designed and used in conjunction with the tertiary nested primer in a PCR reaction, to confirm the transposon insertion point within the genomic DNA. Finding a PCR product of the appropriate size, based on the sequence of the TAIL-PCR clone confirms a valid rescue.
  • Example 2 Sequence Analysis of Tagged Seedling Lethal Line ET6497
  • PCR products are obtained from the Ds5 border.
  • the preliminary sequences, obtained from the TAIL-PCR, are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds5 end indicates that the transposon has inserted with the Ds5 end adjacent to Arabidopsis genomic DNA (base numbers 3162 and lower of Arabidopsis chromosome 3, BAC T20O10, GenBank accession number AL163816).
  • the transposon insertion region of BAC T20O10 is annotated as encoding a putative Prokaryotic-type class I peptide chain release factor (GenPept accession number CAB87736).
  • ORF complete open reading frame
  • 5' RACE primers are designed in the 3' portion of the predicted ORF, which is present in three Arabidopsis ESTs (GenBank accession numbers AV558327, AV558301, T41756).
  • PCR is performed using template DNA from a Gene Racer (Invitrogen) cDNA library prepared from seedling tissue.
  • a resulting PCR product is TA-cloned (Original TA-Cloning kit, Invitrogen) and sequenced.
  • the cDNA sequence (SEQ ID NO: l) differs from the sequence predicted in the GenBank annotation, thus identifying for the first time the actual ORF. Analysis of the cDN A sequence from this gene reveals a high degree of similarity to the Prokaryotic-type class I peptide chain release factor (see homolog table ET6497).
  • % ID percent identity relative to the protein encoded by the Arabidopsis thaliana ET6497 gene
  • Example 3 Sequence Analysis of Tagged Seedling Lethal Line GT1773
  • PCR products are obtained from the Ds3 border and from the Ds5 border.
  • the preliminary sequences obtained from the TAIL-PCR are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds3 end indicates that the transposon has inserted with the Ds3 end adjacent to Arabidopsis genomic DNA (base numbers 63475 and lower of BAC T21L8 Arabidopsis chromosome 3, GenBank accession number AL096860).
  • the initial sequence of the region bordering the Ds5 end indicates that the transposon has inserted with the Ds5 end adjacent to Arabidopsis genomic DNA (base numbers 63474 and higher of Arabidopsis BAC T21L8 on chromosome 3).
  • This region of BAC T21L8 is annotated as having similarity to the riboflavin biosynthesis protein ribG of Synechocystis sp., PIR2:S74377 (GenPept accession number CAB51211).
  • 5' and 3' RACE primers are designed to the predicted ORF.
  • PCR is performed using template from a Gene Racer (Invitrogen) cDNA library prepared from seedling tissue.
  • the resulting PCR products are TA-cloned (Original TA-Cloning kit, Invitrogen) and sequenced.
  • the cDNA sequence (SEQ ID NO:3) differs from the cDNA sequence predicted in the GenBank annotation, thus identifying for the first time the actual ORF.
  • a second cDNA PCR product is identified, TA cloned, and sequenced (SEQ ID NO:37).
  • the resulting sequence is analyzed and may represent an incompletely spliced mRNA transcript of the above mentioned cDNA.
  • the higher molecular weight clone (1796 nucleotides) contains 5 extra bases corresponding to nucleotides 61969-61973 of BAC T21L8, when compared to the lower molecular weight cDNA clone (1791 nucleotides).
  • % ID percent identity relative to the protein encoded by the Arabidopsis thaliana GT1773 gene
  • PCR products are obtained from the Ds3 border and from the Ds5 border.
  • the preliminary sequences obtained from the TAIL-PCR are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds3 end indicates that the transposon has inserted with the Ds3 end adjacent to Arabidopsis genomic DNA (base numbers 28738 and higher of Arabidopsis PI clone MXC9 on chromosome 5, GenBank accession number AB007727).
  • the initial sequence of the region bordering the Ds5 end indicates that the transposon has inserted with the Ds5 end adjacent to Arabidopsis genomic DNA (base numbers 28735 and lower of Arabidopsis PI clone MXC9 on chromosome 5).
  • This region of MXC9 is annotated as encoding a putative protein with similarity to transmembrane transport proteins. (GenPept accession number BAB10031).
  • 5' RACE primers are designed to in the 3' portion of the predicted ORF, which is present in two Arabidopsis ESTs (GenBank accession numbers AV556256, AV557070).
  • PCR is performed using template DNA from a Gene Racer (Invitrogen) cDNA library prepared from seedling tissue..
  • the resulting PCR product is TA- cloned (Original TA-Cloning kit, Invitrogen) and sequenced.
  • the identified cDNA (SEQ ID NO:5) differs from the cDNA sequence predicted in the GenBank annotation, thus identifying for the first time the actual ORF. Analysis of the cDNA sequence from this gene reveals a high degree of sequence similarity to tellurium resistance proteins from several species of bacteria (see homolog table GT0992).
  • % ID percent identity relative to the protein encoded by the Arabidopsis thaliana GT0992 gene
  • transposant line ET6233 PCR products are obtained from the Ds3 border and from the Ds5 border.
  • the preliminary sequences obtained from TAIL-PCR are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds3 end indicates that the transposon has inserted with the Ds3 end adjacent to Arabidopsis genomic DNA (base numbers 94094 and lower of BAC clone F14I3 Arabidopsis chromosome 1, GenBank accession number AC007980).
  • the initial sequence of the region bordering the Ds5 end indicates that the transposon has inserted with the Ds5 end adjacent to Arabidopsis genomic DNA (base numbers 94085 and higher of BAC clone F14I3 Arabidopsis chromosome 1).
  • Analysis of the border sequences reveals a nine base pair duplication that occurred during the transposon insertion, corresponding to bases 94085 through 94083 of BAC F14I3.
  • This region of BAC clone F14I3 on chromosome 1 is annotated as encoding a hypothetical protein (GenPept accession number AAD50047).
  • primers are designed to the 5' and 3' ends of the predicted ORF.
  • PCR is performed using template DNA from a cDNA library prepared from seedling tissue.
  • the resulting PCR product is TA-cloned (Original TA-Cloning kit, Invitrogen) and sequenced.
  • the cDNA sequence (SEQ ID NO: 7) differs from the sequence predicted in the GenBank annotation, thus identifying for the first time the actual ORF. Analysis of the cDNA sequence from this gene reveals that it is similar to the Bacillus megaterium cbiX gene, which is necessary for cobyric acid biosynthesis (see homolog table ET6233).
  • % ID percent identity relative to the protein encoded by the Arabidopsis thaliana ET6233 gene
  • Example 6 Sequence Analysis of Tagged Seedling Lethal Line ET0763
  • PCR products are obtained from the Ds3 border and from the Ds5 border.
  • the preliminary sequences obtained from TAIL-PCR are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds3 end indicates that the transposon has inserted with the Ds3 end adjacent to Arabidopsis genomic DNA (base numbers 43150 and higher of BAC clone F14O23 Arabidopsis chromosome 1, GenBank accession number AC012654).
  • the initial sequence of the region bordering the Ds5 end indicates that the transposon has inserted with the Ds5 end adjacent to Arabidopsis genomic DNA (base numbers 43160 and lower of BAC clone F14O23 Arabidopsis chromosome 1 , GenBank accession number AC012654).
  • BAC F14O23 This region of BAC clone F14O23 on chromosome 1 is annotated as encoding a hypothetical protein that contains a putative SI RNA binding domain. (GenPept accession number AAF43225).
  • primers are designed to the 5' and 3' ends of the predicted ORF. PCR is performed using template DNA from a cDNA library prepared from seedling tissue. The resulting PCR product is TA-cloned (Original TA-Cloning kit, Invitrogen) and sequenced.
  • the cDNA sequence (SEQ ID NO:9) is the same as the sequence predicted in the GenBank annotation, thus confirming for the first time the actual ORF. Analysis of the cDNA sequence from this gene reveals that it is similar to 30S Ribosomal Protein SI in several other species (see homolog table ET0763).
  • % ID percent identity relative to the protein encoded by the Arabidopsis thaliana ET0763 gene
  • transposant line ET5848 PCR products are obtained from the Ds3 border.
  • the preliminary sequences obtained from TAIL-PCR are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds3 end indicates that the transposon has inserted with the Ds3 end adjacent to Arabidopsis genomic DNA (base numbers 27530 and higher of BAC clone F1707 Arabidopsis chromosome 1 , GenBank accession number AC003671).
  • This region of BAC clone F1707, on chromosome 1, is annotated as encoding a hypothetical protein. (GenPept accession number AAC18802).
  • primers are designed to the 5' and 3' ends of the predicted ORF.
  • PCR is performed using template DNA from a cDNA library prepared from seedling tissue.
  • the resulting PCR product is TA-cloned (Original TA-Cloning kit, Invitrogen) and sequenced.
  • the cDNA sequence (SEQ ID NO: 11) is the same as the sequence predicted in the GenBank annotation, thus confirming for the first time the actual ORF.
  • Analysis of the cDNA sequence from this gene reveals that it is similar to a large family of Arabidopsis genes, including some putative myb transcription factors and some with unknown function (see homolog table ET5848).
  • % ID percent identity relative to the protein encoded by the Arabidopsis thaliana ET5848 gene
  • PCR products are obtained from the Ds3 border and from the
  • Ds5 border The preliminary sequences obtained from TAIL-PCR are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds3 end indicates that the transposon has inserted with the Ds3 end adjacent to Arabidopsis genomic DNA (base numbers 60591 and lower of BAC clone F5K20 Arabidopsis chromosome 3, GenBank accession number ALl 32960).
  • the initial sequence of the region bordering the Ds5 end indicates that the transposon has inserted with the Ds5 end adjacent to Arabidopsis genomic DNA (base numbers 60592 and higher of BAC clone F5K20 Arabidopsis chromosome 3).
  • This region of BAC clone F5K20 on chromosome 3 is annotated as encoding a hypothetical protein that shows similarity to a uracil phosphoribosyltransferase (GenPept accession number CAB88352).
  • primers are designed to the 5' and 3' ends of the predicted ORF.
  • PCR is performed using template DNA from a cDNA library prepared from seedling tissue.
  • the resulting PCR product is TA-cloned (Original TA-Cloning kit, Invitrogen) and sequenced.
  • the cDNA sequence (SEQ ID NO: 13) is the same as the sequence predicted in the GenBank annotation, thus confirming for the first time the actual ORF.
  • Analysis of the cDNA sequence from this gene reveals that it is similar to uracil phosphoribosyltransferases from other species (see homolog table GT5062).
  • % ID percent identity relative to the protein encoded by the Arabidopsis thaliana GT5062 gene
  • transposant line ET5036 PCR products are obtained from the Ds3 border.
  • the preliminary sequences obtained from TAIL-PCR are used in BLASTn searches against nucleotide databases (Altschul et al. (1990) J Mol. Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the initial sequence of the region bordering the Ds3 end indicates that the transposon has inserted with the Ds3 end adjacent to Arabidopsis genomic DNA (base numbers 51094 and lower of BAC clone T10O24 Arabidopsis chromosome 1, GenBank accession number AC007067).
  • This region of BAC clone T10O24, on chromosome 1 is annotated as encoding a hypothetical protein. (GenPept accession number AAD39574).
  • the coding region of the protein, corresponding to the cDNA clone SEQ ID NO: l is subcloned into an appropriate expression vector, and transformed into E. coli using the manufacturer's conditions.
  • Specific examples include 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 the ET6497 activity is confirmed. Protein conferring ET6497 activity is isolated using standard techniques.
  • the coding region of the protein, corresponding to the cDNA clone SEQ ID NO:3 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 the GT1773 activity is confirmed. Protein conferring GT1773 activity is isolated using standard techniques.
  • the coding region of the protein is subcloned into an appropriate expression vector, and transformed into E. coli using the manufacturer's conditions.
  • Specific examples include 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 the GT0992 activity is confirmed. Protein conferring GT0992 activity is isolated using standard techniques.
  • the coding region of the protein, corresponding to the cDNA clone SEQ ID NO:7 is subcloned into an appropriate expression vector, and transformed into E.
  • E. coli is cultured, and expression of the ET6233 activity is confirmed. Protein conferring ET6233 activity is isolated using standard techniques.
  • the coding region of the protein corresponding to the cDNA clone SEQ ID NO:9, 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 the ET0763 activity is confirmed. Protein conferring ET0763 activity is isolated using standard techniques.
  • the coding region of the protein corresponding to the cDNA clone SEQ ID NO: 1 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 the ET5848 activity is confirmed. Protein conferring ET5848 activity is isolated using standard techniques.
  • the coding region of the protein, corresponding to the cDNA clone SEQ ID NO: 13, is subcloned into an appropriate expression vector, and transformed into E. coli using the manufacturer's conditions. Specific examples include 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 the GT5062 activity is confirmed. Protein conferring GT5062 activity is isolated using standard techniques.
  • the coding region of the protein, corresponding to the cDNA clone SEQ ID NO: 15, is subcloned into an appropriate expression vector, and transformed into E.
  • E. coli is cultured, and expression of the ET5036 activity is confirmed. Protein conferring ET5036 activity is isolated using standard techniques.
  • Example 1 1 In vitro Recombination of ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 Genes by DNA Shuffling
  • the nucleotide sequence of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity, respectively, 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 ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 activity.
  • PCR-amplified DNA fragments comprising the Arabidopsis ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene encoding the protein and PCR-amplified DNA fragments derived from or comprising another ET6497, GT1773, GT0992, ET6233, ET0763, ET5848, GT5062, or ET5036 gene are recombined in vitro and resulting variants with improved tolerance to the inhibitor are recovered as described above.
  • Example 12 In vitro Recombination of ET6497, GT1773, GT0992, ET6233, ET0763,
  • the Arabidopsis ET6497 gene encoding the protein and another ET6497 gene, or homolog thereof, or fragment thereof, are each cloned into the polylinker of a pBluescript vector.
  • Amplified PCR fragments are digested with appropriate restriction enzymes and cloned into pTRC99a and mutated ET6497 genes are screened as described in Example 10. The same procedure is carried out with genes encoding GT1773, GT0992, ET6233, ET0763, ET5848,
  • Example 13 In Vitro Binding Assays Recombinant ET6497 protein is obtained, for example, according to Example 10.
  • 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.
  • Recombinant GT1773 protein is obtained, for example, according to Example 10.
  • 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.
  • Recombinant GT0992 protein is obtained, for example, according to Example 10.
  • 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.
  • Recombinant ET6233 protein is obtained, for example, according to Example 10.
  • 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.
  • Recombinant ET0763 protein is obtained, for example, according to Example 10.
  • 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.
  • Recombinant ET5848 protein is obtained, for example, according to Example 10.
  • 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.
  • Recombinant GT5062 protein is obtained, for example, according to Example 10.
  • 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.
  • Recombinant ET5036 protein is obtained, for example, according to Example 10. 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.
  • Example 14 Plastid Transformation Transformation vectors
  • plastid transformation vector pPH143 or pPH145 (WO 97/32011) is used; and this reference is incorporated herein by reference.
  • the nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence.
  • This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance.
  • the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.
  • Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per plate in a 1" circular array on T agar medium and bombarded 12-14 days after sowing with 1 ⁇ m tungsten particles (M10, Biorad, Hercules, CA) coated with DNA from plasmids pPH143 and pPH145 essentially as described (Svab, Z. and Maliga, P. (1993) Proc. Natl Acad. Sci. USA 90, 913- 917).
  • Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 ⁇ mol photons/m 2 /s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530) containing 500 ⁇ g/ml spectinomycin dihydrochloride (Sigma, St. Louis, MO). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, allowed to form callus, and secondary shoots isolated and subcloned.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des gènes isolés depuis Arabidopsis et codant pour des protéines essentielles pour la croissance de semis. Elle concerne également les procédés d'utilisation de ces protéines afin de rechercher de nouveaux herbicides, qui sont basés sur le caractère essentiel de ces gènes pour une croissance et un développement normaux. On peut également mettre l'invention en application dans un essai de criblage afin d'identifier des inhibiteurs représentant des herbicides potentiels. Elle s'applique également au développement de plantes tolérantes aux herbicides, de tissus végétaux, de semences et de cellules végétales.
PCT/EP2002/000188 2001-01-11 2002-01-10 Gene cible d'herbicides et procedes correspondants Ceased WO2002064794A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26096701P 2001-01-11 2001-01-11
US60/260,967 2001-01-11

Publications (2)

Publication Number Publication Date
WO2002064794A2 true WO2002064794A2 (fr) 2002-08-22
WO2002064794A3 WO2002064794A3 (fr) 2003-10-02

Family

ID=22991408

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/000188 Ceased WO2002064794A2 (fr) 2001-01-11 2002-01-10 Gene cible d'herbicides et procedes correspondants

Country Status (1)

Country Link
WO (1) WO2002064794A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2392444A (en) * 2002-08-29 2004-03-03 Syngenta Participations Ag Nucleic acid molecules encoding proteins essential for plant growth and development and uses thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2290900A (en) * 1999-01-15 2000-08-01 Syngenta Participations Ag Herbicide target gene and methods
EP1033405A3 (fr) * 1999-02-25 2001-08-01 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments
AU3286500A (en) * 1999-03-05 2000-09-28 Syngenta Participations Ag Herbicide target genes and methods

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2392444A (en) * 2002-08-29 2004-03-03 Syngenta Participations Ag Nucleic acid molecules encoding proteins essential for plant growth and development and uses thereof

Also Published As

Publication number Publication date
WO2002064794A3 (fr) 2003-10-02

Similar Documents

Publication Publication Date Title
AU2290900A (en) Herbicide target gene and methods
US20080256666A1 (en) Methods of identifying and creating rubisco large subunit variants with improved rubisco activity, compositions and methods of use thereof
US6387637B1 (en) Herbicide target genes and method
WO2000053782A2 (fr) Genes cibles d'herbicide et procedes correspondants
EP1621614A2 (fr) Gènes cibles d'herbicides et procédés
AU6082399A (en) Uracil permease from arabidopsis as herbicidal target gene
AU744487B2 (en) Riboflavin biosynthesis genes from plants and uses thereof
US6300091B1 (en) Herbicide target genes and methods
US20020120963A1 (en) Herbicide target genes and methods
EP1240185A2 (fr) Genes cibles d'herbicide et procedes correspondants
WO2002064794A2 (fr) Gene cible d'herbicides et procedes correspondants
EP1093520A1 (fr) Hmp. kinase et tmp-ppase provenant d'arabidopsis thaliana et leur utilisation dans le criblage d'herbicides
US20020127537A1 (en) Herbicide target genes and methods
US6294345B1 (en) Genes encoding proteins essential for plant growth and methods of use
US20020157142A1 (en) Herbicide target genes and methods
EP1276883A2 (fr) Genes d'acide amine:glyoxylate aminotransferase d'origine vegetale et utilisations de ceux-ci
US20030200569A1 (en) Amino acid:glyoxylate aminotransferase genes from plants and uses thereof
US6271445B1 (en) Nucleic acid molecules encoding 5′-phosphoribosyl-5-aminoimidazole (AIR) synthetase
WO1999067402A2 (fr) Procedes de criblage de composes herbicides utilisant air synthetase d'arabidopsis thaliana

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP