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EP1114168A2 - Uracil permease from arabidopsis as herbicidal target gene - Google Patents

Uracil permease from arabidopsis as herbicidal target gene

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Publication number
EP1114168A2
EP1114168A2 EP99947332A EP99947332A EP1114168A2 EP 1114168 A2 EP1114168 A2 EP 1114168A2 EP 99947332 A EP99947332 A EP 99947332A EP 99947332 A EP99947332 A EP 99947332A EP 1114168 A2 EP1114168 A2 EP 1114168A2
Authority
EP
European Patent Office
Prior art keywords
plant
activity
seq
permease
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99947332A
Other languages
German (de)
English (en)
French (fr)
Inventor
Joshua Zvi Levin
Sharon Lee Potter Lewis
Gregory Joseph Budziszewski
Eric Russell Ward
Paul Bernasconi
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 EP1114168A2 publication Critical patent/EP1114168A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the invention relates to a gene isolated from Arabidopsis that codes for a protein essential for seedling growth.
  • the invention also includes the methods of using this protein as an herbicide target, based on the essentiality of the gene 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 the crop to the herbicide.
  • U.S. Patent No. 4,761 ,373 to Anderson et al. is directed to plants resistant to various imidazoiinone or sulfonamide herbicides.
  • U.S. Patent No. 4,975,374 to Goodman et al. relates to plant cells and plants containing a gene encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that were known to inhibit GS, e.g. phosphinothricin and methionine sulfoximine.
  • GS glutamine synthetase
  • U.S. Patent No. 5,013,659 to Bedbrook et al. is directed to plants expressing a mutant acetolactate synthase that renders the plants resistant to inhibition by sulfonylurea herbicides.
  • Chimeric is used to indicate that a DNA sequence, such as a vector or a gene, is comprised of more than one DNA sequences of distinct origin which are fused together by recombinant DNA techniques resulting in a DNA sequence, which does not occur naturally,
  • 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 introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly.
  • the DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule.
  • the shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.
  • Enzyme activity means herein the ability of an enzyme to catalyze the conversion of a substrate into a product.
  • a substrate for the enzyme comprises the natural substrate of the enzyme but also comprises analogues of the natural substrate which can also be converted by the enzyme into a product or into an analogue of a product.
  • the activity of the enzyme is measured for example by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time.
  • the activity of the enzyme is also measured by determining the amount of an unused co-factor of the reaction remaining in the reaction mixture after a certain period of time or by determining the amount of used co-factor in the reaction mixture after a certain period of time.
  • the activity of the enzyme is also measured by determining the amount of a donor of free energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine) remaining in the reaction mixture after a certain period of time or by determining the amount of a used donor of free energy or energy-rich molecule (e.g. ADP, pyruvate, acetate or creatine) in the reaction mixture after a certain period of time.
  • a donor of free energy or energy-rich molecule e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine
  • Expression refers to the transcription and/or translation of an endogenous gene or a transgene in plants.
  • expression may refer to the transcription of the antisense DNA only.
  • Gene refers to a coding sequence and associated regulatory sequences wherein the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
  • regulatory sequences are promoter sequences, 5' and 3' untranslated sequences. Further elements that may be present are, for example, introns.
  • ...gene of interest refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability.
  • the "gene of interest” may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.
  • Herbicide a chemical substance used to kill or suppress the growth of plants, plant cells, plant seeds, or plant tissues.
  • Heterologous DNA Sequence a DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • Homologous DNA Sequence a DNA sequence naturally associated with a host cell into which it is introduced.
  • BLAST Basic Local Alignment Search Tool
  • Version BLAST 2.0 Garnier BLAST
  • the scores assigned in a BLAST search have a well-defined statistical.
  • Inhibitor a chemical substance that inactivates the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the plant.
  • an inhibitor is a chemical substance that inactivates the enzymatic activity of 4788 from a plant.
  • Isogenic plants which are genetically identical, except that they may differ by the presence or absence of a transgene.
  • an isolated DNA molecule or an isolated enzyme in the context of the present invention, is a DNA molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • Marker gene a gene encoding a selectable or screenable trait.
  • Mature protein protein which is normally targeted to a cellular organelle, such as a chloroplast, and from which the transit peptide has been removed.
  • Minimal Promoter promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
  • Modified Enzyme Activity enzyme activity different from that which naturally occurs in a plant (i.e. enzyme activity that occurs naturally in the absence of direct or indirect manipulation of such activity by man), which is tolerant to inhibitors that inhibit the naturally occurring enzyme activity.
  • a regulatory DNA sequence is said to be "operably linked to” or “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence.
  • Plant refers to any plant, particularly to seed plants
  • Plant cell structural and physiological unit of the plant, comprising a protoplast and a cell wall.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
  • Plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
  • Pre-protein protein which is normally targeted to a cellular organelle, such as a chloroplast, and still comprising its transit peptide.
  • Recombinant DNA molecule a combination of DNA sequences that are joined together using recombinant DNA technology recombinant DNA technology: procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  • Selectable marker a gene whose expression in a plant cell gives the cell a selective advantage.
  • the selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells.
  • the selective advantage possessed by the transformed cells, compared to non- transformed cells may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source.
  • Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant cell gives the cell both, a negative and a positive selective advantageSignificant Increase: an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
  • the term "substantially similar”, when used herein with respect to a nucleotide sequence means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g.
  • 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.
  • the percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 65%, more desirably at least 75%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, yet still more preferably at least 99%. Sequence comparisons are carried out using a Smith-Waterman sequence alignment algorithm (see e.g. Waterman, M.S.
  • a nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1 % SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M 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
  • substantially similar when used herein with respect to a protein, means a protein corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, e.g. where only changes in amino acids sequence not affecting the polypeptide function occur.
  • the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is at least 65%, more desirably at least 75%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, yet still more preferably at least 99%.
  • a substrate is the molecule that the enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function, or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.
  • Tolerance the ability to continue normal growth or function when exposed to an inhibitor or herbicide in an amount sufficient to suppress the normal growth or function of native, unmodified plants.
  • Transformation a process for introducing heterologous DNA into a cell, tissue, or plant.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • Transgenic stably transformed with a recombinant DNA molecule that preferably comprises a suitable promoter operatively linked to a DNA sequence of interest.
  • a feature of the invention is the identification of a putative permease gene in Arabidopsis. Another feature of the invention is the discovery that the putative permease gene is essential for seedling growth and development. An advantage of the present invention is that the newly discovered essential gene containing a novel herbicidal mode of action enables one skilled in the art to easily and rapidly identify novel herbicides.
  • One object of the present invention is to provide an essential gene in plants for assay development for inhibitory compounds with herbicidal activity. Genetic results show that when the putative permease gene is mutated in Arabidopsis, the resulting phenotype is seedling lethal in the homozygous state. This suggests a critical role for the gene product encoded by the mutated gene.
  • the inventors of the present invention have demonstrated that the activity is essential in Arabidopsis seedlings. This implies that chemicals which inhibit the function of the protein 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 the gene sequence described below to identify inhibitors thereof, which can then be used as herbicides to suppress the growth of undesirable vegetation, e.g.
  • crops in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
  • the present invention discloses a novel nucleotide sequence derived from Arabidopsis, designated the 4788 gene.
  • the nucleotide sequence of the genomic clone is set forth in SEQ ID NO:1
  • the nucleotide sequence of the corresponding cDNA clone is set forth in SEQ ID NO:2
  • the amino acid sequence of the Arabidopsis 4788 protein is set forth in SEQ ID NO:3.
  • the present invention also includes nucleotide sequences substantially similar to those set forth in SEQ ID NO:1 and SEQ ID NO: 2.
  • the present invention also encompasses nucleotide sequences substantially similar to those set forth in SEQ ID NO:1 and SEQ ID NO: 2, wherein said nucleotide sequence is a plant nucleotide sequence.
  • Preferred is a nucleotide sequences substantially similar to those set forth in SEQ ID NO:1 and SEQ ID NO: 2, wherein said nucleotide sequence is an Arabidopsis thaliana nucleotide sequence.
  • the present invention also encompasses proteins whose amino acid sequence are substantially similar to the amino acid sequences set forth in SEQ ID NO:3.
  • amino acid sequence comprising and amino acid sequence substantially similar to SEQ ID NO:3.
  • amino acid sequence comprising and amino acid sequence which is SEQ ID NO:3.
  • amino acid sequence comprising amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO:1 or SEQ ID NO:2., wherein the protein has permease activity. More preferred is an amino acid sequence comprising amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO:1 or SEQ ID NO:2, wherein the protein has purine or pyrimidine permease activity. Particularly preferred is an amino acid sequence comprising amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO:1 or SEQ ID NO:2., wherein the protein has uracil permease.
  • amino acid sequence comprising at least 20 consecutive amino acid residues of the amino acid sequence encoded by SEQ ID NO: 1 or SEQ ID NO: 2.
  • amino acid sequence comprising at least 20 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 3.
  • An further embodiment is a chimeric gene comprising a promoter operatively linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO:2.
  • a recombinant vector comprising a chimeric gene comprising a promoter operatively linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO:2, wherein said vector is capable of being stably transformed into a host cell.
  • a host cell comprising a vector comprising a chimeric gene comprising a promoter operatively linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO:2, wherein said vector is capable of being stably transformed into a host cell and wherein said nucleotide sequence is expressible in said cell.
  • a preferred host cell according to the invention is an eukaryotic cell, more preferred is a host cell selected from the group consisting of an insect cell, a yeast cell, and a plant cell.
  • a further preferred host cell is a prokaryotic cell, more preferred is a bacterial cell.
  • a plant comprising a vector comprising a chimeric gene comprising a promoter operatively linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO:2, wherein said vector is capable of being stably transformed into a plant cell; also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • a plant according to the invention wherin said plant being tolerant to an inhibitor of permease activity; also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • a further embodiment of the invention is a process for making nucleotides sequences encoding gene products having altered permease activity comprising, a) shuffling a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO:2, b) expressing the resulting shuffled nucleotide sequences and c) selecting for altered permease activity as compared to the permease activity of the gene product of said unmodified nucleotide sequence.
  • nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 2. More preferred is a process according to the invention, wherein said permease activity is purine or pyrimidine permease activity. Particularly preferred is a process according to the invention, wherein said permease activity is uracil permease activity.
  • a shuffled DNA molecule obtainable by the process for making nucleotides sequences encoding gene products having altered permease activity comprising, a) shuffling a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO:2, b) expressing the resulting shuffled nucleotide sequences and c) selecting for altered permease activity as compared to the permease activity of the gene product of said unmodified nucleotide sequence.
  • a further embodiment of the invention is a shuffled DNA molecule produced by a process according to the invention.
  • Also comprised by the invention is a shuffled DNA molecule obtained a process according to the invention, wherein said shuffled DNA molecule encodes a gene product having enhanced tolerance to an inhibitor of permease activity.
  • a further embodiment of the invention is a chimeric gene comprising a promoter operatively linked to a shuffled DNA molecule produced by a process according to the invention.
  • a further embodiment of the invention is a recombinant vector comprising a chimeric gene comprising a promoter operatively linked to a shuffled DNA molecule produced by a process according to the invention, wherein said vector is capable of being stably transformed into a host cell.
  • a host cell comprising a vector according to claim comprising a chimeric gene comprising a promoter operatively linked to a shuffled DNA molecule produced by said process, wherein said vector is capable of being stably transformed into a host cell and wherein said nucleotide sequence is expressible in said cell.
  • a host cell according to the invention which is an eukaryotic cell, more preferred wherein said host cell is selected from the group consisting of an insect cell, a yeast cell, and a plant cell.
  • a host cell according to the invention which is a prokaryotic cell, more preferred is a host cell according to the invention which is a bacterial cell.
  • a further embodiment is a plant comprising a plant cell comprising a vector according according to the invention comprising a chimeric gene comprising a promoter operatively linked to a shuffled DNA molecule produced by the process according to the invention, wherein said vector is capable of being stably transformed into a plant cell and wherein said nucleotide sequence is expressible in said cell; also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • a vector according according to the invention comprising a chimeric gene comprising a promoter operatively linked to a shuffled DNA molecule produced by the process according to the invention, wherein said vector is capable of being stably transformed into a plant cell and wherein said nucleotide sequence is expressible in said cell; also including the progeny and seed for such a plant, which seed is optionally treated (e.g.
  • a further embodiment is a plant according to the invention which is tolerant to an inhibitor of permease activity; also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • an inhibitor of permease activity also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • a process of identifying compounds having herbicidal activity comprising: a) combining a protein comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2. and a compound to be tested for the ability to bind to said protein, under conditions conducive to binding, b) selecting a compound identified in step (a) that is capable of binding said protein, c) applying identified compound in step (b) to a plant to test for herbicidal activity, and d) selecting compounds having herbicidal activity.
  • a process of identifying an inhibitor of permease activity having herbicidal activity comprising: a) combining a permease and a compound to be tested for the ability to inhibit the activity of said permease, under conditions conducive to such inhibition, b) selecting a compound identified in step (a) that is capable of inhibiting said permease activity, c) applying identified compound in step (b) to a plant to test for herbicidal activity, and d) selecting compounds having herbicidal activity.
  • permease is a purine or pyrimidine permease, more preferred, wherein said permease is a uracil permease.
  • a further embodiment of the invention is a compound having herbicidal activity identifiable according to the process according to the invnetion.
  • a further embodiment is a process of identifying an inhibitor of permease activity comprising, a) introducing SEQ ID NO: 1 or SEQ ID NO:2, or nucleotide sequences substantially similar thereto into a permease-deficient host cell, such as E. coli uraA such that said sequence is functionally expressable; b) combining said host cell containing said nucleotide sequence, with a minimal inhibitory concentration of 5-fluorouracil, and with a compound to be tested for the ability to inhibit the activity of said permease, under conditions conducive to such inhibition, c) measure host cell growth under the conditions of step (b); and d) selecting said compound that inhibits host cell growth in step (c).
  • a method for suppressing the growth of a plant comprising, applying to said plant a compound that inhibits the activity of the amino acid sequence comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2 in an amount sufficient to suppress the growth of said plant.
  • a process of identifying compounds having herbicidal activity comprising: a) combining a protein of claim 10 and a compound to be tested for the ability to bind to said protein, under conditions conducive to binding, b) selecting a compound identified in step (a) that is capable of binding said protein, c) applying identified compound in step (b) to a plant to test for herbicidal activity, and d) selecting compounds having herbicidal activity. and the compounds having herbicidal activity identifiable according to the process according to the invention.
  • a method of improving crops comprising, applying to a herbicide tolerant plant or seed selected from the group consisting of the plant or seed according to the invention wherein said plant is tolerant to an inhibitor of permease, a compound according to the invention having herbicidal activity in an amount that inhibits the growth of undesired vegetation without significantly suppressing the growth of the herbicide tolerant plant or seed.
  • the present invention also includes methods of using the 4788 gene product as an herbicide target, based on the essentiality of the gene for normal growth and development. Furthermore, the invention can be used in a screening assay to identify inhibitors that are potential herbicides.
  • the present invention describes a method for identifying chemicals having the ability to inhibit 4788 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 4788 activity and capable of overexpressing an enzymatically active 4788 gene product; 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 transgenic and non-transformed plants, plant cells, tissues after application of the chemical; d) comparing the growth or viability of the transgenic and non- transformed plants, plant cells, tissues after application of the chemical; and e) selecting chemicals suppress the viability or growth of the non-transgenic plants, plant cells, tissues or parts, without significantly suppressing the growth of the viability or growth of the isogenic transgenic plants, plant cells, tissues or parts.
  • the enzyme having 4788 activity is encoded by a nucleotide sequence derived from a plant, preferably Arabidopsis thaliana, desirably identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
  • the enzyme having 4788 activity is encoded by a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO:3.
  • the enzyme having 4788 activity has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:3.
  • the present invention further embodies plants, plant tissues, and plant cells that have modified 4788 activity and that are therefore tolerant to inhibition by a herbicide at levels normally inhibitory to naturally occurring 4788 activity; also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use.
  • 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 4788 gene that is tolerant to inhibition by a herbicide at a concentration that would normally inhibit the activity of wild-type, unmodified 4788 gene product.
  • Modified 4788 activity may also be conferred upon a plant by increasing expression of wild-type herbicide-sensitive 4788 protein by providing multiple copies of wild-type 4788 genes to the plant or by overexpression of wild-type 4788 genes 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, or plant tissue transformed with a DNA molecule comprising a nucleotide sequence isolated from a plant that encodes an enzyme having 4788 activity, also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use, wherein the enzyme has 4788 activity and wherein the DNA molecule confers upon the plant, plant cell, plant seed, or plant tissue tolerance to a herbicide in amounts that normally inhibits naturally occurring 4788 activity.
  • a DNA molecule comprising a nucleotide sequence isolated from a plant that encodes an enzyme having 4788 activity, also including the progeny and seed for such a plant, which seed is optionally treated (e.g., primed or coated) and/or packaged, e.g. placed in a bag or other container with instructions for use, wherein the enzyme has 4788 activity and wherein the DNA molecule confers upon the plant
  • the enzyme having 4788 activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, or has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:3.
  • 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 4788 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 4788 activity; and (b) applying to the crops or crop seeds and the undesired vegetation in the field a herbicide in amounts that inhibit naturally occurring 4788 activity, wherein the herbicide suppresses the growth of the weeds without significantly suppressing the growth of the crops.
  • Arabidopsis insertional mutant lines segregating for seedling lethal mutations are identified as a first step in the identification of essential proteins. Starting with T2 seeds collected from single T1 plants containing T-DNA insertions in their genomes, those lines segregating homozygous seedling lethal seedlings are identified. These lines are found by placing seeds onto minimal plant growth media, which contain the fungicides benomyl and maxim, and screening for inviable seedlings after 7 and 14 days in the light at room temperature. 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 T-DNA 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 hygromycin or phosphinothricin. About 35 resistant seedlings are transplanted to soil and their progeny are examined for the segregation of the seedling lethal.
  • Southern analysis is performed as an initial step in the characterization of the molecular nature of each insertion. Southerns are done with genomic DNA isolated from heterozygotes and using probes capable of hybridizing with the T-DNA vector DNA. Often, the T-DNA insertion in a given plant is shown to contain multiple copies of the T-DNA vector that insert at a single genetic locus. Using the results of the Southern, appropriate restriction enzymes are chosen to perform plasmid rescue in order to molecularly clone Arabidopsis genomic DNA flanking one or both sides of the T-DNA insertion.
  • Plasmids obtained in this manner are analyzed by restriction enzyme digestion to sort the plasmids into classes based on their digestion pattern. For each class of plasmid clone, the DNA sequence is determined. The resulting sequences are analyzed for the presence of non-T- DNA vector sequences. When such sequences are found, they are used to search DNA and protein databases using the BLAST and BLAST2 programs (Altschul et al. (1990) J Mol. Biol. 215: 403-410; Altschul et al (1997) Nucleic Acid Res. 25:3389-3402). Additional genomic and cDNA sequences for each gene are identified by standard molecular biology procedures. II. Sequence of the Arabidopsis 4788 Gene
  • the Arabidopsis 4788 gene is sequenced by isolating DNA flanking the T-DNA border from the tagged seedling-lethal line #4788.
  • Arabidopsis DNA flanking the T-DNA border is identical to a internal region of a sequenced BAC of Arabidopsis (BAC T9J22, chromosome 2).
  • This BAC clone contains 115,851 bp of sequence, of which a very small portion corresponds to the genomic region that contains the 4788 gene. Nothwithstanding the BAC information, the inventors are the first to establish definitively the entire gene sequence, and to demonstrate for the first time that the 4788 gene product is essential for normal growth and development, as well as defining the function of the 4788 gene product.
  • the present invention discloses the nucleotide sequence of the Arabidopsis 4788 gene as well as the amino acid sequence of the Arabidopsis 4788 protein.
  • the nucleotide sequence corresponding to the genomic clone is set forth in SEQ ID NO:1
  • the corresponding cDNA clone is set forth in SEQ ID NO:2
  • the amino acid sequence encoding the mature protein is set forth in SEQ ID NO:3.
  • the present invention also encompasses an isolated amino acid sequence derived from a plant, wherein said amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, wherein said amino acid sequence has 4788 activity.
  • the sequence of the 4788 gene shows similarity to uracil permeases.
  • the 4788 gene is also a member of a gene family in Arabidopsis.
  • This gene family consists of at least six members (Genbank Ace. #s: AC002505 (2739376), BAC T9J22; AC004481 (3337350), BAC F13P17; AC001229 (2190545), BAC F5I14; AB009053 (n/a), clone MQB2 (13 th ORF); U83501 (1791307); and AA712474 (EST clone 194H6T7) as well as AA605567 (EST clone 205J16XP).
  • a nucleotide sequence encoding 4788 protein is inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced.
  • the choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the routineer in the art.
  • the resultant molecule, containing the individual elements 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 californica 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 californica baculovirus DNA (Pharmigen, 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 4788 activity is derived from an eukaryote, such as a mammal, a fly or a yeast, but is preferably derived from a plant.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, or encodes a protein having 4788 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:3.
  • the nucleotide sequence set forth in SEQ ID NO:2 encodes the Arabidopsis 4788 protein, whose amino acid sequence is set forth in SEQ ID NO:3.
  • the nucleotide sequence is derived from a prokaryote, preferably a bacteria, e.g. the uraA gene in E. coli (Andersen et al. (1995) J. Bacteriol. 177: 2008-2013).
  • Recombinantly produced 4788 is isolated and .purified using a variety of standard techniques. The actual techniques that may be used will vary depending upon the host organism used, whether the protein is designed for secretion, and other such factors familiar to the skilled artisan (see, e.g. chapter 16 of Ausubel, F. et al., "Current Protocols in Molecular Biology", pub. by John Wiley & Sons, Inc. (1994).
  • Recombinantly produced 4788 proteins are useful for a variety of purposes. For example, they can be used in in vitro assays to screen known herbicidal chemicals whose target has not been identified to determine if they inhibit 4788. Such in vitro assays may also be used as more general screens to identify chemicals that inhibit such enzymatic activity and that are therefore novel herbicide candidates Alternatively, recombinantly produced 4788 proteins 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.
  • Nucleotide sequences substantially similar to SEQ ID NO 1 or SEQ ID NO.2 and proteins substantially similar to SEQ ID NO:3 from any source, including microbial sources, can be used in the assays exemplified herein Desirably such nucleotide sequences and proteins are derived from plants More desirably, they are derived from dicot plants. Alternatively, such nucleotide sequences and proteins are derived from non-maize sources, alternatively from non-monocot sources.
  • the 4788 gene product is believed to function as a permease, more specifically as a uracil permease similar to the maize Lpe1 protein (Schultes et al (1996) The Plant Cell, 8 463- 475)
  • the leaf permeasel gene product of maize has similarity to purine and pyrimidine permeases from bacteria and fungi, and mutations in Ipel are known to adversely affect chloroplast development. Therefore, the /pe7-encoded protein is a potential herbicide target, which is further supported by the demonstration herein of the essentiality of the Arabidopsis 4788 gene product.
  • a novel assay can be developed based on expression of the plant protein in a bacterial host lacking the corresponding activity (Andersen et al (1995) J. Bacte ⁇ ol. 177: 2008-2013).
  • a simple assay can be developed to screen for compounds that affect normal functioning of the plant-encoded activity. Such compounds are promising in vitro leads that can be tested for in vivo herbicidal activity.
  • One assay consists of growing E. coli uraA harboring and functionally expressing the 4788 gene in minimal medium in the presence of a minimal inhibitory concentration of 5-fluorourac ⁇ l. This is accomplished in a 96-well format for automated high-throughput screening. Compounds that are effective in blocking function of the 4788 protein inhibits the ability of the cells to take up 5-FU, and bacterial growth results This growth is measured by simple turbidomet ⁇ c means.
  • the next step is to develop an assay that allows screening large number of chemicals to determine which ones interact with the protein.
  • an assay that allows screening large number of chemicals to determine which ones interact with the protein.
  • FCS Fluorescence Correlation Spectroscopy
  • SELDI Surface-Enhanced Laser Desorption/ionization
  • 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 ul 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).
  • 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 4788 activity uses transgenic plants, plant tissue, plant seeds or plant cells capable of overexpressing a nucleotide sequence having 4788 activity, wherein the 4788 gene product is enzymatically active in the transgenic plants, plant tissue, plant seeds or plant cells.
  • the nucleotide sequence is preferably derived from an eukaryote, such as a yeast, but is preferably derived from a plant.
  • the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, or encodes an enzyme having 4788 activity, whose amino acid sequence is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:3.
  • the nucleotide sequence is derived from a prokaryote, preferably a bacteria, e.g. the uraA gene of E. coli.
  • 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 4788 activity.
  • the present invention is further directed to plants, plant tissue, plant seeds, and plant cells tolerant to herbicides that inhibit the naturally occurring 4788 activity in these plants, wherein the tolerance is conferred by an altered 4788 activity.
  • Altered 4788 activity may be conferred upon a plant according to the invention by increasing expression of wild-type herbicide-sensitive 4788 by providing additional wild-type 4788 genes to the plant, by expressing modified herbicide-tolerant 4788 genes in the plant, or by a combination of these techniques.
  • Representative plants include any plants to which these herbicides are applied for their normally intended purpose.
  • Achieving altered 4788 activity through increased expression results in a level of a 4788 in the plant cell at least sufficient to overcome growth inhibition caused by the herbicide.
  • the level of expressed enzyme generally is at least two times, preferably at least five times, and more preferably at least ten times the natively expressed amount.
  • Increased expression may be due to multiple copies of a wild-type 4788 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.
  • Increased expression of a herbicide-sensitive 4788 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 4788 protein.
  • 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 4788.
  • a herbicide tolerant form of the enzyme has at least one amino acid substitution, addition or deletion that confers tolerance to a herbicide that inhibits the unmodified, naturally occurring form of the enzyme.
  • the transgenic plants, plant tissue, plant seeds, or plant cells thus created are then selected by conventional selection techniques, whereby herbicide tolerant lines are isolated, characterized, and developed. Below are described methods for obtaining genes that encode herbicide tolerant forms of 4788:
  • a genetically manipulatable microbe such as E. coli or S. cerevisiae may be subjected to random mutagenesis in vivo with mutagens such as UV light or ethyl or methyl methane sulfonate.
  • mutagens such as UV light or ethyl or methyl methane sulfonate.
  • Mutagenesis procedures are described, for example, in Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972); Davis et al, Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1980); Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983); and U.S. Patent No. 4,975,374.
  • the microbe selected for mutagenesis contains a normal, inhibitor-sensitive 4788 gene and is dependent upon the activity conferred by this gene.
  • the mutagenized cells are grown in the presence of the inhibitor at concentrations that inhibit the unmodified gene. Colonies of the mutagenized microbe that grow better than the unmutagenized microbe in the presence of the inhibitor (i.e. exhibit resistance to the inhibitor) are selected for further analysis. 4788 genes from these colonies are isolated, either by cloning or by PCR amplification, and their sequences are elucidated. Sequences encoding altered gene products are then cloned back into the microbe to confirm their ability to confer inhibitor tolerance.
  • a method of obtaining mutant herbicide-tolerant alleles of a plant 4788 gene involves direct selection in plants.
  • the effect of a mutagenized 4788 gene on the growth inhibition of plants such as Arabidopsis, soybean, or maize is determined by plating seeds sterilized by art-recognized methods on plates on a simple minimal salts medium containing increasing concentrations of the inhibitor.
  • concentrations are in the range of 0.001 , 0.003, 0.01 , 0.03, 0.1 , 0.3, 1 , 3, 10, 30, 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 inserted 4788 genes are mapped relative to known restriction fragment length polymorphisms (RFLPs) (See, for example, Chang et al. Proc. Natl. Acad, Sci, USA 85: 6856-6860 (1988); Nam et al., Plant Cell 1 : 699-705 (1989), cleaved amplified polymorphic sequences (CAPS) (Konieczny and Ausubel (1993) The Plant Journal, 4(2): 403-410), or SSLPs (Bell and Ecker (1994) Genomics, 19: 137-144).
  • RFLPs restriction fragment length polymorphisms
  • CAS cleaved amplified polymorphic sequences
  • SSLPs Bell and Ecker (1994) Genomics, 19: 137-144.
  • the 4788 inhibitor tolerance trait is independently mapped using the same markers. When tolerance is due to a mutation in that 4788 gene, the tolerance trait maps to a position indistinguishable from the position of the 4788 gene.
  • Another method of obtaining herbicide-tolerant alleles of a 4788 gene is by selection in plant cell cultures.
  • Explants of plant tissue e.g. embryos, leaf disks, etc. or actively growing callus or suspension cultures of a plant of interest are grown on medium in the presence of increasing concentrations of the inhibitory herbicide or an analogous inhibitor suitable for use in a laboratory environment. Varying degrees of growth are recorded in different cultures. In certain cultures, fast-growing variant colonies arise that continue to grow even in the presence of normally inhibitory concentrations of inhibitor. The frequency with which such faster-growing variants occur can be increased by treatment with a chemical or physical mutagen before exposing the tissues or cells to the inhibitor.
  • Putative tolerance-conferring alleles of the 4788 gene are isolated and tested as described in the foregoing paragraphs. Those alleles identified as conferring herbicide tolerance may then be engineered for optimal expression and transformed into the plant. Alternatively, plants can be regenerated from the tissue or cell cultures containing these alleles.
  • Still another method involves mutagenesis of wild-type, herbicide sensitive plant 4788 genes in bacteria or yeast, followed by culturing the microbe on medium that contains inhibitory concentrations of the inhibitor and then selecting those colonies that grow in the presence of the inhibitor. More specifically, a plant cDNA, such as the Arabidopsis cDNA encoding the 4788 is cloned into a microbe that otherwise lacks the selected gene's activity. The transformed microbe is then subjected to in vivo mutagenesis or to in vitro mutagenesis by any of several chemical or enzymatic methods known in the art, e.g. sodium bisulfite (Shortle et al., Methods Enzymol.
  • Colonies that grow in the presence of normally inhibitory concentrations of inhibitor are picked and purified by repeated restreaking. Their plasmids are purified and tested for the ability to confer tolerance to the inhibitor by retransforming them into the microbe lacking 4788 gene activity. The DNA sequences of cDNA inserts from plasmids that pass this test are then determined.
  • Herbicide resistant 4788 proteins are also obtained using methods involving in vitro recombination, also called DNA shuffling.
  • DNA shuffling mutations, preferably random mutations, are introduced in 4788 genes.
  • DNA shuffling also leads to the recombination and rearrangement of sequences within an 4788 gene or to recombination and exchange of sequences between two or more different of 4788 genes. These methods allows for the production of millions of mutated 4788 genes.
  • the mutated genes, or shuffled genes are screened for desirable properties, e.g. improved tolerance to herbicides and for mutations that provide broad spectrum tolerance to the different classes of inhibitor chemistry. Such screens are well within the skills of a routineer in the art.
  • a mutagenized 4788 gene is formed from at least one template 4788 gene, wherein the template 4788 gene has been cleaved into double- stranded random fragments of a desired size, and comprising the steps of adding to the resultant population of double-stranded random fragments one or more single or double- stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded random fragments; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polyn
  • the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1 % by weight of the total DNA.
  • the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides.
  • the size of the double-stranded random fragments is from about 5 bp to 5 kb.
  • the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles. Such method is described e.g. in Stemmer et al. (1994) Nature 370: 389-391 , in US Patent 5,605,793 and in Crameri et al. (1998) Nature 391 : 288-291 , Cherry et al. (1999) Nature Biotechnology, 17: 379-384, as well as in WO 97/20078, and these references are incorporated herein by reference.
  • any combination of two or more different 4788 genes are mutagenized in vitro by a staggered extension process (StEP), as described e.g. in Zhao et al. (1998) Nature Biotechnology 16: 258-261.
  • the two or more 4788 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 4788 coding regions to be mutagenized but desirably over 40 cycles, more desirably over 60 cycles, preferably over 80 cycles are used.
  • Optimal extension conditions and the optimal number of PCR cycles for every combination of 4788 genes are determined as described in using procedures well-known in the art.
  • the other parameters for the PCR reaction are essentially the same as commonly used in the art.
  • the primers for the amplification reaction are preferably designed to anneal to DNA sequences located outside of the coding sequence of the 4788 genes, e.g. to DNA sequences of a vector comprising the 4788 genes, whereby the different 4788 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 4788 coding sequences, preferably less than 200 bp away from the 4788 coding sequences, more preferably less than 120 bp away from the 4788 coding sequences.
  • the 4788 coding sequences are surrounded by restriction sites, which are included in the DNA sequence amplified during the PCR reaction, thereby facilitating the cloning of the amplified products into a suitable vector.
  • fragments of 4788 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 4788 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.
  • Herbicide resistant proteins are also obtained using methods involving in situ modification of a target gene.
  • a technology for targeting and mutating genes in vivo can be used, based on self-complementary chimeric oligonucleotides. This approach is being developed for the modification of endogenous genes in a site-specific and genetically inheritable manner (Beetham et al. (1999) Proc. Natl. Acad. Sci. 96: 8774-8778; U.S. Patent No. 5,756,325; U.S. Patent No. 5,871 ,984; U.S. Patent 5,731 ,181 ), and these references are incorporated herein by reference.
  • any 4788 gene or any combination of 4788 genes is used for in vitro recombination in the context of the present invention, for example, an 4788 gene derived from a plant, such as, e.g. Arabidopsis thaliana, e.g. an 4788 gene set forth in SEQ ID NO:1 or SEQ ID NO:2, an 4788 gene from a bacteria, such as Bacillus caldolyticus (Ghim and Neuhard (1994) J. Bacteriol. 176: 3698-3707) or E. coli (Andersen et al. (1995) J. Bacteriol. 177: 2008-2013), a 4788 gene from Zea mays (Schultes et al.
  • a plant such as, e.g. Arabidopsis thaliana
  • an 4788 gene from a bacteria such as Bacillus caldolyticus (Ghim and Neuhard (1994) J. Bacteriol. 176
  • Host cells transformed with the vectors comprising the library of mutated 4788 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 4788 gene that is tolerant to an inhibitor may be performed in the same manner as the assay to identify inhibitors of the 4788 activity (Inhibitor Assay, above) with the following modifications: First, a mutant 4788 is substituted in one of the reaction mixtures for the wild-type 4788 of the inhibitor assay. Second, an inhibitor of wild-type enzyme is present in both reaction mixtures. Third, mutated activity (activity in the presence of inhibitor and mutated enzyme) and unmutated activity (activity in the presence of inhibitor and wild-type enzyme) are compared to determine whether a significant increase in enzymatic activity is observed in the mutated activity when compared to the unmutated activity.
  • Mutated activity is any measure of activity of the mutated enzyme while in the presence of a suitable substrate and the inhibitor.
  • Unmutated activity is any measure of activity of the wild-type enzyme while in the presence of a suitable substrate and the inhibitor.
  • a significant increase is defined as an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by at least 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by at least 5-fold or greater, most preferably an increase by at least 10-fold or greater.
  • genes encoding herbicide tolerant 4788 can also be used as selectable markers in plant cell transformation methods.
  • plants, plant tissue, plant seeds, or plant cells transformed with a transgene can also be transformed with a gene encoding an altered 4788 capable of being expressed by the plant.
  • the transformed cells are transferred to medium containing an inhibitor of the enzyme in an amount sufficient to inhibit the survivability of plant cells not expressing the modified gene, wherein only the transformed cells will survive.
  • the method is applicable to any plant cell capable of being transformed with a modified 4788-encoding gene, and can be used with any transgene of interest. Expression of the transgene and the modified gene can be driven by the same promoter functional in plant cells, or by separate promoters.
  • a wild-type or herbicide-tolerant form of the 4788 gene can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting a DNA molecule encoding the 4788 into an expression system to which the DNA molecule is heterologous (i.e., not normally present) using standard cloning procedures known in the art.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences in a host cell containing the vector.
  • a large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.
  • the components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed.
  • transgene comprising a wild-type or herbicide-tolerant form of the 4788 gene is preferably stably transformed and integrated into the genome of the host cells.
  • the transgene comprising a wild-type or herbicide-tolerant form of the 4788 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 4788 gene confers herbicide tolerance in the transgenic plants.
  • A. Requirements for Construction of Plant Expression Cassettes Gene sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants.
  • the expression cassettes may also comprise any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described infra. The following is a description of various components of typical expression cassettes. 1. Promoters
  • the selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters known in the art can be used.
  • the CaMV 35S promoter for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter may be used.
  • the chemically inducible PR-1 promoter from tobacco or Arabidopsis may be used (see, e.g., U.S. Patent No. 5,689,044).
  • transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledonous and dicotyledonous plants.
  • the coding sequence of the selected gene may be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest. Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (see, e.g. Perlak et al., Proc. Natl. Acad. Sci. USA 88: 3324 (1991 ); and Koziel et al., Bio/technol. 77: 194 (1993)).
  • Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail.
  • the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)).
  • Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411-418 (1989)).
  • the cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles.
  • sequences have been characterized which cause the targeting of gene products to other cell compartments.
  • Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
  • transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors.
  • the selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al.
  • vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001 , as well as the binary vector pCIB10 and hygromycin selection derivatives thereof. (See, for example, U.S. Patent No. 5,639,949).
  • Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non- Agrobacterium transformation include pCIB3064, pSOG19, and pSOG35. (See, for example, U.S. Patent No. 5,639,949).
  • the coding sequence of interest Once the coding sequence of interest has been cloned into an expression system, it is transformed into a plant cell.
  • Methods for transformation and regeneration of plants are well known in the art.
  • Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles.
  • bacteria from the genus Agrobacterium can be utilized to transform plant cells.
  • Transformation techniques for dicotyledons are well known in the art and include Agrobacte ⁇ um-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-me ⁇ ate ⁇ transformation.
  • the wild-type or altered form of a 4788 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.
  • a wild-type 4788 gene and/or the expression of herbicide- tolerant forms of a 4788 gene conferring herbicide tolerance in plants can be incorporated into plant lines through breeding approaches and techniques known in the art.
  • a herbicide tolerant 4788 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.
  • the invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
  • the plasmid rescue technique is used to molecularly clone Arabidopsis genomic DNA flanking one or both sides of T-DNA insertions resulting from T-DNA mutagenesis. Plasmids obtained in this manner are analyzed by restriction enzyme digestion to sort the plasmids into classes based on their digestion pattern. For each class of plasmid clone, the DNA sequence is determined. The resulting sequences are analyzed for the presence of non-T-DNA vector sequences. The plasmids recovered from the plasmid rescue protocol are sequenced using the slp346 primer. Primer slp346 provides information on the flanking sequence immediately adjacent to the left T-DNA border.
  • Plasmid rescue is validated by PCR of genomic DNA from a heterozygote for the 4788 mutation.
  • This PCR experiment uses a primer anchored in the predicted flanking sequence and the slp346 primer (anchored in the T-DNA insertion). Finding a PCR product of the size expected based on the sequence of the plasmid rescued clone confirms a valid rescue.
  • the sequence obtained from the above clone is used in an NCBI WWW blastn search against nucleotide sequence databases (Altschul et al. (1990) J Mol. Biol. 215: 403-410; Altschul et al (1997) Nucleic Acids Res. 25: 3389-3402).
  • the search results show that the recovered sequence is identical to genomic DNA from Arabidopsis chromosome II BAC T9J22 (Genbank Ace. AC002505 (2739376)).
  • the region of genomic DNA where the insertion event occurred is annotated as encoding a putative permease (ACCESSION # 2739376).
  • Primers are then designed to the 5' and 3' ends of predicted mRNA, and PCR is performed using DNA from an Arabidopsis cDNA library as the template.
  • the resulting PCR product is TA-ligated and cloned (Original TA Cloning Kit, Invitrogen), and sequenced.
  • the cDNA sequence is the same as the sequence predicted in the Genbank annotation, thus validating for the first time the putative open reading frame annotation.
  • the coding region of the putative mature protein is subcloned into previously described expression vectors, 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).
  • the A. thaliana 4788 gene encoding the 4788 protein 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 ) and transformed into E.coli uraA strain (Andersen et al.
  • thaliana 4788 gene encoding the protein and PCR-amplified DNA fragments comprising the E.coli uraA gene are recombined in vitro and resulting variants with improved tolerance to the inhibitor are recovered as described above.
  • Example 4 In vitro Recombination of 4788 Genes by Staggered Extension Process
  • the A. thaliana 4788 gene encoding the 4788 protein and the E.coli uraA gene are each cloned into the polylinker of a pBluescript vector.
  • a PCR reaction is carried out essentially as described (Zhao et al. (1998) Nature Biotechnology 16: 258-261 ) using the "reverse primer” and the "M13 -20 primer” (Stratagene Catalog). Amplified PCR fragments are digested with appropriate restriction enzymes and cloned into pTRC99a and mutated 4788 genes are screened as described in Example 3.
  • Recombinant 4788 protein is obtained, for example according to Example 2.
  • the protein is immobilized on chips appropriate for ligand binding assays.
  • 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, for e.g. according to Example 6, below.
  • a simple assay is developed to screen for compounds that affect normal functioning of the 4788 activity. Such compounds are promising in vitro leads that can be tested for in vivo herbicidal activity.
  • the assay consists of growing E. coli uraA harboring and functionally expressing the 4788 gene in minimal medium in the presence of a minimal inhibitory concentration of 5-fluorouracil. This is accomplished in a 96-well format for automated high- throughput screening. Compounds that are effective in blocking function of the 4788 protein inhibit the ability of the cells to take up 5-FU, and bacterial growth results. This growth is measured by simple turbidometric means.
  • the invention relates to a process of identifying an inhibitor of permease activity comprising: a) introduce SEQ ID NO: 1 or SEQ ID NO:2, or nucleotide sequences substantially similar thereto into a permease-deficient host cell, such as E.
  • step (e) coli uraA such that said sequence is functionally expressable; b) combining said host cell containing said nucleotide sequence, with a minimal inhibitory concentration of 5-fluorouracil, and with a compound to be tested for the ability to inhibit the activity of said permease, under conditions conducive to such inhibition, c) measure host cell growth under the conditions of step (b); and d) selecting said compound that inhibits host cell growth in step (c), and optionally e) applying identified compound in step (d) to a plant to test for herbicidal activity, and f) selecting compounds having herbicidal activity in step (e).

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