EP0994952A1 - Atp-phosphoribosyl transferase vegetale et adn la codant - Google Patents

Atp-phosphoribosyl transferase vegetale et adn la codant

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Publication number
EP0994952A1
EP0994952A1 EP98938705A EP98938705A EP0994952A1 EP 0994952 A1 EP0994952 A1 EP 0994952A1 EP 98938705 A EP98938705 A EP 98938705A EP 98938705 A EP98938705 A EP 98938705A EP 0994952 A1 EP0994952 A1 EP 0994952A1
Authority
EP
European Patent Office
Prior art keywords
plant
aprt
atp
leu
dna molecule
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Withdrawn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
EP98938705A
Other languages
German (de)
English (en)
Inventor
Ko Fujimori
Masaharu Mizutani
Daisaku Ohta
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
Novartis Erfindungen Verwaltungs GmbH
Novartis 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 Novartis Erfindungen Verwaltungs GmbH, Novartis AG filed Critical Novartis Erfindungen Verwaltungs GmbH
Publication of EP0994952A1 publication Critical patent/EP0994952A1/fr
Withdrawn legal-status Critical Current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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • 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

Definitions

  • the invention relates generally to a plant enzymatic activity involved in the biosynthesis of L-histidine (L-His).
  • the invention relates to the plant enzyme which catalyzes the first reaction in the histidine biosynthesis, the condensation of adenosine 5'-triphosphate (ATP) and 5-phosphoribosyl 1 -pyrophosphate (PRPP) to form N'- -phosphoribosyl-ATP (PR-ATP); and gene coding therefor.
  • the invention further relates to various utilities including the recombinant production of this enzyme in a heterologous host, incorporation into an assay system for screening chemicals for herbicidal activity, and the development of genetic markers in plants.
  • APRT ATP- phosphoribosyl transferase
  • the present invention provides an isolated DNA molecule encoding the ATP- phoshoribosyl transferase (APRT) enzyme from a plant source.
  • APRT ATP- phoshoribosyl transferase
  • a DNA coding sequence for an APRT enzyme in Arabidopsis thaliana and wheat is provided in SEQ ID NOS: 3, 5, 7 and 9.
  • the DNA coding sequence for APRT enzyme(s) from any plant source may be obtained using standard methods.
  • the present invention also embodies the recombinant production of the APRT enzyme, and methods for using recombinantly produced APRT.
  • the present invention provides methods of using purified APRT in an assay system to screen for novel inhibitors of APRT activity which may be used as herbicides to control undesirable vegetation in fields where crops are grown, particularly agronomicaliy 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, tobacco, and soybeans.
  • agronomicaliy 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, tobacco, and soybeans.
  • the present invention embodies an assay system for screening chemicals for herbicidal activity using APRT.
  • a method for assaying a chemical for the ability to inhibit the activity of a APRT enzyme from a plant comprising
  • a method for assaying a chemical for the ability to inhibit the activity of a APRT enzyme from a plant comprising
  • the present invention is further directed to probes and methods for detecting the presence and form of the APRT gene and quantitating levels of APRT transcripts in an organism. These methods may be used to diagnose plant disease conditions which are associated with an altered form of the APRT enzyme or altered levels of expression of the APRT enzyme.
  • the present invention is directed to an isolated DNA molecule which encodes a plant ATP-phosphoribosyl transferase (referred to herein as APRT), the enzyme which catalyzes a step in the biosynthesis of L-His.
  • APRT plant ATP-phosphoribosyl transferase
  • the invention relates to DNA molecules encoding the plant ATP-phosphoribosyl transferase from dicotyledonous plants, but especially from Arabidopsis plants, such as those given in SEQ ID NO: 7and 9 respectively.
  • the invention further relates to an isolated DNA molecule encoding the plant ATP-phosphoribosyl transferase from a dicotyledonous plant, wherein said protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 10. Also comprised is an isolated DNA molecule encoding the plant ATP-phosphoribosyl transferase from a monocotyledonous plant, wherein said protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 6.
  • the DNA coding sequence for the APRT enzyme may be isolated from the genome of any plant species according to the invention using standard methods.
  • the present invention provides probes capable of specifically hybridizing to a plant DNA sequence encoding APRT enzyme activity to the respective mRNA and methods for detecting the said DNA sequences in eucaryotic organisms using the probes according to the invention.
  • the present invention further embodies expression cassetts and recombinant vectors comprising the said expression cassetts comprising essentially a promoter, but especially a promoter that is active in a plant, operably linked to a DNA molecule encoding the APRT enzyme from a eukaryotic organism according to the invention.
  • the expression cassette according to the invention may in addition further comprise a signal sequence operably linked to said DNA molecule, wherein said signal sequence is capable of targeting the protein encoded by said DNA molecule into a suitable cellular compartement, but preferably into the chloroplast or the mitochondria.
  • the present invention provides plants, plant tissues and plant seeds with altered APRT activity which are resistant or at least tolerant to inhibition by a herbicide at levels which normally are inhibitory to the naturally occurring APRT activity in the plant.
  • the invention embodies plants, wherein the altered APRT activity is conferred by over-expression of the wild-type APRT enzyme or by expression of a DNA molecule encoding a herbicide tolerant APRT enzyme.
  • the said herbicide tolerant APRT enzyme may be a modified form of a APRT enzyme that naturally occurs in a eukaryote or a prokaryote; or a modified form of a APRT enzyme that naturally occurs in said plant; or the said herbicide tolerant APRT enzyme may naturally occur in a prokaryote.
  • Plants encompassed by the invention include monocotyledonous and dicotyledonous plants, but especially those which would be potential targets for APRT inhibiting herbicides, particularly agronomicaliy 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, tobacco, sunflower, sugar cane, sugar beet, oilseed rape, and soybeans.
  • herbicides particularly agronomicaliy 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, tobacco, sunflower, sugar cane, sugar beet, oilseed rape, and soybeans.
  • a DNA molecule encoding a modified ATP- phosphoribosyl transferase comprising a plant ATP-phosphoribosyl transferase having at least one amino acid modification, wherein said modified plant ATP-phosphoribosyl transferase is tolerant to a herbicide in amounts which inhibit said a plant ATP- phosphoribosyl transferase.
  • the invention embodies a DNA molecule according to the invention which is part of a plant genome.
  • the present invention is further directed to methods for the production of plants, plant tissues, and plant seeds which contain a APRT enzyme resistant to, or tolerant of inhibition by a herbicide at a concentration which inhibits the naturally occurring APRT activity.
  • the said resistance or tolerance may be obtained by expressing in the said transgenic plants either a DNA molecule encoding a modified form of a APRT enzyme that naturally occurs in a eukaryote, or a modified form of a APRT enzyme that naturally occurs in said plant, or a APRT enzyme that naturally occurs in a prokaryot, or a APRT enzyme which is a modified form of a protein which naturally occurs in a prokaryote.
  • One specific embodiment of the invention is directed to the preparation of transgenic maize plants, maize tissue or maize seed which have been stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operably linked to a structural gene encoding an unmodified prokaryotic APRT enzyme which is resistant to the herbicide.
  • the invention is further directed to the preparation of transgenic plants, plant tissue and plant seed which has been stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operably linked to a structural gene encoding an unmodified eukaryotic APRT enzyme. This results in over-expression of the unmodified APRT in the plant sufficient to overcome inhibition of the enzyme by the herbicide.
  • the present invention also embodies the production of plants which express an altered APRT enzyme tolerant of inhibition by a herbicide at a concentration which normally inhibits the activity of wild-type, unaltered APRT.
  • the plant may be stably transformed with a recombinant DNA molecule comprising a structural gene encoding the resistant APRT, or prepared by direct selection techniques whereby herbicide resistant lines are isolated, characterized and developed.
  • the present invention is further directed to a method for controlling the growth of undesired vegetation which comprises applying to a population of a plant with altered APRT activity which is resistant to inhibition by a herbicide at levels which normally are inhibitory to the naturally occurring APRT activity in the said plant, an effective amount of a APRT - inhibiting herbicide.
  • Plants to be protected in the described way are especially those which would be potential targets for APRT inhibiting herbicides, particularly agronomicaliy important crops such as, for example, 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, tobacco, sunflower and soybeans.
  • agronomicaliy important crops such as, for example, 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, tobacco, sunflower and soybeans.
  • Herbicides that qualify as APRT inhibitors are those selected from the group consisting of aryluracil, diphenylether, oxidiazole, imide, phenyl pyrazole, pyridine derivative, phenopylate and O- phenylpyrrolidino- and piperidinocarbamate analogs of said phenopylate.
  • the present invention also embodies the recombinant production of the APRT enzyme, and methods for using recombinantly produced APRT.
  • the invention thus further embodies host cells, but especially cells selected from the group consisting of plant cells, animal cells, bacterial cells, yeast cells and insect cells, stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in the respective host cell operably linked to a structural gene encoding an unmodified or modified plant APRT enzyme, wherein said host cell is capable of expressing said DNA molecule.
  • the present invention further provides methods of using purified APRT to screen for novel herbicides which affect the activity of APRT, and to identify herbicide-resistant APRT mutants.
  • step (b) selecting those cells from step (a) whose growth is not inhibited;
  • step (c) isolating and identifying the APRT enzyme present in the cells selected from step (b).
  • Genes encoding altered APRT can be used as selectable markers in plant cell transformation methods.
  • the present invention thus further embodies a method of selecting plants, plant tissue or plant cells transformed with a transgene of interest from non-transformed plants, comprising the steps of:
  • the present invention is further directed to probes and methods for detecting the presence and form of the APRT gene and quantitating levels of APRT transcripts in an organism. These methods may be used to diagnose disease conditions which are associated with an altered form of the APRT enzyme or altered levels of expression of the APRT enzyme.
  • Plant APRT coding sequences may be isolated according to well known techniques based on their structural sequence homology to the Arabidopsis thaliana and wheat APRT enzymes. In these techniques all or part of the known APRT coding sequence is used as a probe which selectively hybridizes to other APRT coding sequences present in genomic or cDNA libraries from a chosen organism. Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g.. Sambrook et al., Molecular Cloning , eds., Cold Spring Harbor Laboratory Press.
  • the isolated plant APRT sequences may be manipulated according to standard genetic engineering techniques to suit any desired purpose.
  • the entire APRT sequence or portions thereof may be used as probes capable of specifically hybridizing to APRT coding sequences and messenger RNAs.
  • probes include sequences that are unique among APRT coding sequences and are preferably at least 20 nucleotides in length, and most preferably at least 50 nucleotides in length.
  • Such probes may be used to amplify and analyze APRT coding sequences from a chosen organism via the well known process of polymerase chain reaction (PCR). This technique may be used to isolate additional APRT coding sequences from a desired organism or as a diagnostic assay to determine the presence of APRT coding sequences in an organism.
  • PCR polymerase chain reaction
  • APRT specific hybridization probes may also be used to map the location of the native APRT gene(s) in the genome of a chosen plant using standard techniques based on the selective hybridization of the probe to genomic APRT sequences. These techniques include, but are not limited to, identification of DNA polymorphisms identified or contained within the APRT probe sequence, and use of such polymorphisms to follow segregation of the APRT gene relative to other markers of known map position in a mapping population derived from self fertilization of a hybrid of two polymorphic parental lines (see e.g. Helentjaris et al., Plant Mol. Biol. 5: 109 (1985). Sommer et al.
  • APRT sequence is contemplated to be useful as a probe for mapping APRT genes
  • preferred probes are those APRT sequences from plants more closely related to the chosen plant, and most preferred probes are those APRT sequences from the chosen plant.
  • Mapping of APRT genes in this manner is contemplated to be particularly useful for breeding purposes. For instance, by knowing the genetic map position of a mutant APRT gene that confers increased production of histidine, flanking DNA markers can be identified from a reference genetic map (see, e.g., Helentjaris, Trends Genet. 3: 217 (1987)). During introgression of this trait into a new breeding line, these markers can then be used to monitor the extent of APRT-linked flanking chromosomal DNA still present in the recurrent parent after each round of back- crossing.
  • APRT specific hybridization probes may also be used to quantitate levels of APRT mRNA in a plant using standard techniques such as Northern blot analysis. This technique may be used as a diagnostic assay to detect altered levels of APRT expression.
  • DNA molecules which hybridizes to a DNA molecule according to the invention as defined hereinbefore, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said APRT enzyme at least 10 nucleotides in length, under moderately stringent conditions.
  • T m melting temperature T m which can be easily calculated according to the formula provided in DNA PROBES, George H. Keller and Mark M. Manak , Macmillan Publishers Ltd, 1993, Section one: Molecular Hybridization Technology; page 8 ff.
  • the preferred hybridization temperature is in the range of about 25°C below the calculated melting temperature T m and preferably in the range of about 12-15°C below the calculated melting temperature T m and in the case of oligonucleotides in the range of about 5-10°C below the melting temperature T m .
  • the plant APRT coding sequence may be inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced.
  • the choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the routineer in the art.
  • the resultant molecule, containing the individual elements linked in proper reading frame may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli (see, e.g. Studier and Moffatt, J. Mol. Biol.
  • plasmids such as pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, San Diego, CA), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV).
  • a preferred baculovirus/insect system is pVL 1392/Sf21 cells (Invitrogen, San Diego, CA).
  • Recombinantly produced plant APRT enzyme can be isolated and purified using a variety of standard techniques. The actual techniques which may be used will vary depending upon the host organism used, whether the APRT enzyme is designed for secretion, and other such factors familiar to the skilled artisan (see, e.g. chapter 16 of Ausubel, F. et al., Current Protocols in Molecular Biology, pub. by John Wiley & Sons, Inc. (1994) .
  • Recombinantly produced plant APRT enzyme is useful for a variety of purposes. For example, it may be used to supply APRT enzyme for an in vitro assay to screen known herbicidal chemicals whose target has not been identified to determine if they inhibit APRT. Such an in vitro assay may also be used as a more general screen to identify chemicals which inhibit APRT activity and which are therefore herbicide candidates. Alternatively, recombinantly produced APRT may be used to elucidate the complex structure of this enzyme. Such information regarding the structure of the APRT enzyme may be used, for example, in the rational design of new inhibitory herbicides.
  • the amount of APRT enzyme present in a plant or plant cell is increased by introducing into the plant or plant cell a chimeric gene capable of expressing APRT enzyme in a plant cell.
  • a chimeric gene will comprise a promoter capable of regulating gene expression in a plant, operably linked to a DNA sequence which encodes a APRT enzyme, followed by a transcriptional terminator and polyadenylation signal.
  • Coding sequences for APRT enzymes may be genetically engineered for optimal expression in a particular crop plant. 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); Koziel er a/., Bio/technol. 11: 194 (1993)).
  • a DNA sequence coding for a APRT enzyme may be inserted into an expression cassette designed for plants to construct a chimeric gene according to the invention using standard genetic engineering techniques.
  • the choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the achieving the desired pattern and level of expression in the chosen plant host is within the level of skill of the routineer in the art.
  • the resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into a host plant cell.
  • promoters capable of functioning in plants or plant cells include the cauliflower mosaic virus (CaMV) 19S or 35S promoters and CaMV double promoters; nopaline synthase promoters; pathogenesis-related (PR) protein promoters; small subunit of ribulose bisphosphate carboxylase (ssuRUBISCO) promoters, and the like.
  • CaMV cauliflower mosaic virus
  • PR pathogenesis-related
  • ssuRUBISCO small subunit of ribulose bisphosphate carboxylase
  • Signal or transit peptides may be fused to the APRT coding sequence in the chimeric DNA constructs of the invention to direct transport of the expressed APRT to the desired site of action.
  • signal peptides include those natively linked to the plant pathogenesis-related proteins, e.g. PR-1 , PR-2, and the like. See, e.g., Payne et al., Plant Mol. Biol. 11:89-94 (1988).
  • Examples of transit peptides include the chloroplast transit peptides such as those described in Von Heijne et al., Plant Mol. Biol. Rep. 9:104-126 (1991); Mazur et al., Plant Physiol.
  • the chimeric DNA construct(s) of the invention may contain multiple copies of a promoter or multiple copies of the coding sequence for a APRT enzyme.
  • the construct(s) may include coding sequences for markers and coding sequences for other peptides such as signal or transit peptides, each in proper reading frame with the other functional elements in the DNA molecule. The preparation of such constructs are within the ordinary level of skill in the art.
  • Useful markers include peptides providing herbicide, antibiotic or drug resistance, such as, for example, resistance to hygromycin, kanamycin, G418, gentamycin, lincomycin, methotrexate, glyphosate, phosphinothricin, or the like. These markers can be used to select cells transformed with the chimeric DNA constructs of the invention from untransformed cells.
  • Other useful markers are peptidic enzymes which can be easily detected by a visible reaction, for example a color reaction, for example luciferase, ⁇ -glucuronidase, or ⁇ -galactosidase.
  • Chimeric genes designed for plant expression such as those described herein can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant (i.e. monocot or dicot) and/or organelle (i.e. nucleus, chloroplast, mitochondria) targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., BioTechniques 4.320-334 (1986)), electroporation (Riggs et al, Proc. Natl. Acad. Sci.
  • a chimeric gene encoding a APRT enzyme may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.
  • the coding sequence for a APRT enzyme may be isolated, genetically engineered for optimal expression and then transformed into the desired variety.
  • progeny' is understood to embrace both, “asexually” and “sexually” generated progeny of transgenic plants. This definition is also meant to include all mutants and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.
  • Another object of the invention concerns the proliferation material of transgenic plants.
  • transgenic plants are defined relative to the invention as any plant material that may be propagated sexually or asexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, pollen, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.
  • Parts of plants such as for example flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed by means of the process of the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention. It is thus a further object of the present invention to provide plant propagation material for cultivated plants, but especially plant seed that is treated with an seed protectant coating customarily used in seed treatment.
  • the seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds.
  • the bag, container or vessel may be designed for either short term or long term storage, or both, of the seed.
  • a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal.
  • the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type.
  • the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed.
  • the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering.
  • water absorbent materials are placed between or adjacent to packaging material layers.
  • the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.
  • An example of such treatment is sterilization, for example by chemical means or by exposure to radiation.
  • the invention relates to a bag of seeds comprising seed according to the invention.
  • a bag of seeds comprising seed of a transgenic plant comprising a DNA encoding a protein having a plant ATP-phosphoribosyl transferase activity together with lable instructions.
  • a bag of seeds comprising seed of a transgenic plant comprising a DNA encoding a DNA molecule encoding a modified ATP- phosphoribosyl transferase comprising a plant ATP-phosphoribosyl transferase having at least one amino acid modification, wherein said modified plant ATP-phosphoribosyl transferase is tolerant to a herbicide in amounts which inhibit said plant ATP- phosphoribosyl transferase together with lable instructions.
  • Such bags containing seed to breed progeny from plants transformed according to the method of the present invention a method such as that which follows may be used: maize plants produced as described in the examples set forth below are grown in pots in a greenhouse or in soil, as is known in the art, and permitted to flower. Pollen is obtained from the mature tassel and used to pollinate the ears of the same plant, sibling plants, or any desirable maize plant. Similarly, the ear developing on the transformed plant may be pollinated by pollen obtained from the same plant, sibling plants, or any desirable maize plant. Transformed progeny obtained by this method may be distinguished from non- transformed progeny by the presence of the introduced gene(s) and/or accompanying DNA (genotype), or the phenotype conferred.
  • the transformed progeny may similarly be selfed or crossed to other plants, as is normally done with any plant carrying a desirable trait.
  • tobacco or other transformed plants produced by this method may be selfed or crossed as is known in the art in order to produce progeny with desired characteristics.
  • other transgenic organisms produced by a combination of the methods known in the art and this invention may be bred as is known in the art in order to produce progeny with desired characteristics.
  • the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants.
  • said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting.
  • Specialized processes such as hydroponics or greenhouse technologies can also be applied.
  • measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.
  • Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering.
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken.
  • the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
  • Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means.
  • Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
  • the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties.
  • new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment" , yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.
  • the protectant coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation.
  • other methods of application to plants are possible, eg treatment directed at the buds or the fruit.
  • Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD ⁇ ), methaiaxyl (Apron ® ), and pirimiphos-methyl (Actellic ® ). If desired these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.
  • the protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit. It is a further aspect of the present invention to provide new agricultural methods such as the methods examplified above which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.
  • SEQ ID NO: 2 Peptide #2 VGDFGGPASAF
  • SEQ ID NO: 3 DNA molecule encoding the wheat (Triticum aestivum) ATP-phosphoribosyl transferase.
  • SEQ ID NO 4 Amino acid sequence of wheat (Triticum aestivum) ATP-phosphoribosyl transferase,) encoded by the DNA sequence provided in SEQ ID No:3.
  • SEQ ID NO: 5 DNA molecule encoding the wheat APRT-phosphoribosyl transferase.
  • SEQ ID NO: 6 Amino acid sequence of wheat APRT-phosphoribosyl transferase encoded by the DNA sequence provided in SEQ ID NO: 5:.
  • SEQ ID NO: 7 DNA molecule encoding the Arabidopsis ATP-phosphoribosyl transferase
  • SEQ ID NO: 8 Amino acid sequence of Arabidopsis ATP-phosphoribosyl transferase encoded by the DNA sequence provided in SEQ ID NO: 7:.
  • SEQ ID NO: 9 DNA molecule encoding the Arabidopsis ATP-phosphoribosyl transferase
  • SEQ ID NO: 10 Amino acid sequence of Arabidopsis ATP-phosphoribosyl transferase encoded by the DNA sequence provided in SEQ ID NO: 9:.
  • SEQ ID NO: 11 Degenerate oligonucletide 5'-TAYATHTTYGAYGARGARAC-3 ⁇ designed as a hybridization probe from the peptide sequence determined from the purified wheat germ APRT set forth in SEQ ID NO: 1
  • SEQ ID NO: 12 antisense, 5'-GTCTCCTCGTCAAATATGTA-3 ⁇ primer designed from the amino acid sequence determined from the purified APRT shown in SEQ ID NO: 1.
  • SEQ ID NO: 13 Primer: 5'-CGGGATCCATGAAGCGTGACCAGATTCGTCTTG -3'
  • SEQ ID NO: 14 Primer: 5'-GCTCTAGAAGCTTCAGCATATGCATCTTCC-3'.
  • SEQ ID NO: 15 Double-stranded DNA fragment comprised of oligonucleotide of sequence
  • Double-stranded DNA fragment comprised of oligonucleotide of sequence
  • EXAMPLE 1 Purification of ATP-Phosphoribosyl Transferase (APRT) from Wheat Germ
  • Wheat germ purchased from Sigma is used as an enzyme source. Wheat germ is homogenized with acetone in a methanol/dry ice bath using a homogenizer. The acetone- insoluble materials are dried under reduced pressure, and then extracted in ice-cold buffer A (0.1 M potassium phosphate pH 7.5, 0.1 M NaCI, 1 mM L-His, 5 mM EDTA, 30 mM 2- mercaptoethanol and 10% (w/w) polyvinylpyrrolidone). The extract is passed through 4 layers cheese cloth and centrifuged at 10,000 x g for 15 min to remove insoluble materials.
  • ice-cold buffer A 0.1 M potassium phosphate pH 7.5, 0.1 M NaCI, 1 mM L-His, 5 mM EDTA, 30 mM 2- mercaptoethanol and 10% (w/w) polyvinylpyrrolidone.
  • the extract is passed through 4 layers cheese cloth and centrifuged at 10,000 x g for 15 min to remove insoluble
  • the proteins in the supernatant are precipitated with ammonium sulfate (80% saturation) by centrifugation at 10,000 x g for 15 min.
  • the precipitate is redissolved in buffer B (0.1 M Tris- HCI pH 7.5, 30 mM 2-mercaptoethanol) and desalted using a Sephadex G-25 column (PD 10, Pharmacia).
  • the combined active fractions is dialyzed against 10 L of 50 mM Tris-HCI pH 7.5, and applied to a DEAE-Toyopearl 650M column (5 x 25 cm, Tosoh, Tokyo, Japan) equilibrated with the same buffer. Proteins are eluted with a gradient of NaCI (0-0.5 M) in the same buffer.
  • the active fractions are concentrated and dialyzed against 10 L of 20 mM potassium phosphate buffer pH 7.5.
  • the dialyzed solution is applied to a Heparin- Sepharose CL-6B column (2.5 x 8 cm, Pharmacia) equilibrated with potassium phosphate pH 7.5, and elution is performed by increasing the buffer concentration from 0.02 M to 0.4 M.
  • the active fractions from the Heparin-Sepharose column are applied to a HiLoad 26/60 Superdex 200 pg column (Pharmacia) equilibrated with 20 mM Tris-HCI (pH 7.5) containing 0.2 M NaCI and proteins are eluted with the same buffer.
  • the buffer is changed to 20 mM Tris-HCI pH 7.5 by using a PD10 column (Pharmacia).
  • the enzyme solution is applied to a 1 mL HiTrap Blue column (Pharmacia) and eluted isocratically with 20 mM Tris-HCI (pH 7.5).
  • the purified APRT protein so obtained from wheat germ is digested with lysyl endopeptidase, and the resulting digest is separated by reverse phase HPLC.
  • the resulting peptides are subjected to automated Edman degradation (Strickler et a;, Anal Biochem. 140: 553-566 (1984)) with an Applied Biosystems (Foster City., CA) 470A protein sequencer, and peptide sequences are determined:
  • the standard assay mixture (175 ⁇ L) contains 1 1.4 mM Tris-HCI (pH 9.0), 22.8 mM MgCl2, 85.7 mM KCI, 5.7 mM ATP, 0.57 mM phosphoribosyl pyrophosphate (PRPP), and enzyme.
  • the reaction is started by the addition of phosphoribosyl pyrophosphate, and the mixture is incubated at 30°C for 15 min. The reaction is stopped by adding 50 ⁇ l 1 N HCI.
  • the enzyme assay is performed in the standard reaction mixture supplemented with 10 ⁇ l of the extract from a Salmonella typhimurium strain SB3095 (hisG46, pepH102, fla-2055) that is lacking APRT activity (Ames et al., J. Biol. Chem., 2019-2026, 236, 1961 ).
  • This assay mixture contains the plant APRT and the other enzymes of the His biosynthetic enzymes derived from strain SB3095, and 5-amino-1-ribosyl-4-imidazole carboxamide (AICAR) is stoicheometrically released during the L-His biosynthesis as a byproduct.
  • AICAR 5-amino-1-ribosyl-4-imidazole carboxamide
  • AICAR is determined photometrically with the Bratton-Marshall method as described by Ames et al. (Ames et al., J. Biol. Chem., 2019-2026, 236, 1961). This method is used for the determination of enzyme activity both in crude plant extracts and during purification. 10 ⁇ M solution of AICAR gives an absorbance of 0.270 at 550 nm in this assay method (Ames et al., J. Biol. Chem., 2019-2026, 236, 1961). APRT is also spectrophotomerically assayed in the standard reaction mixture without the S. typhimurium extract.
  • APRT activity is determined by monitoring the PRPP- and ATP-dependent production of PRATP, which can be estimated by following the absorbance increase at 290 nm using an extinction coefficient of 3.6 x 10 3 for PRATP (Smith and Ames J. Biol. Chem. 240, 3056-3063, 1965).
  • Purified APRT may be used in assays to discover novel inhibitors of the enzyme, which inhibitors potentially would function as commercially viable herbicides.
  • the inhibitory effect of a chemical on IGPD is determined in the enzyme assay methods described above.
  • Total RNA is prepared from 7-d-old wheat seedlings by phenol/chloroform extraction followed by lithium chloride precipitation.
  • Poly(A)+ RNA is isolated from the total RNA using a poly(A)+ Quick mRNA isolation kit (Stratagene, LaJolla, CA).
  • a cDNA library is constructed from the poly(A)+ RNA in the bacteriophage vector lambda ZAPII (Stratagene) using the Uni-ZAP XR Gigapack II Gold cloning kit (Stratagene) as described in the manufacturers' instruction.
  • a phage or plasmid cDNA library is plated at a density of approximately 10,000 plaques on a 10 cm Petri dish, and filter lifts of the plaques are made after overnight growth of the plates at 37 °C.
  • a degenerate oligonucletide, 5'- TAYATHT ⁇ GAYGARGARAC-3', (SEQ ID NO 11) is designed as a hybridization probe from the peptide sequence determined from the purified wheat germ APRT set forth in SEQ op
  • plaque lifts are probed with the probe labeled with P-ATP by MEGALABEL (TAKARA Shuzo, Kyoto, Japan)
  • Hybridization conditions are 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04 pH 7.0, 1 mM EDTA at 40 °C.
  • SDS sodium dodecyl sulfate
  • the filters are washed with 2X SSC, 1 % SDS. Positively hybridizing plaques are detected by autoradiography. After purification to single plaques, cDNA inserts are isolated, and their sequences determined by the chain termination method using dideoxy terminators labeled with fluorescent dyes (Applied Biosystems, Inc., Foster City, CA).
  • SEQ ID NOS:3 and 4 The sequence thus obtained for the wheat APRT cDNA and the protein it encodes are provided in SEQ ID NOS:3 and 4.
  • Peptide #1 (SEQ ID NOS: 1) exactly matches the predicted protein sequence (SEQ ID NO: 4) determined from the wheat APRT cDNA.
  • Additional wheat APRT cDNA clone is isolated from a wheat cDNA library using the cDNA fragment (SEQ ID NO: 3) as a hybridization probe.
  • the DNA is labeled with [ ⁇ Pj-dCTP by the random priming labeling method and used as a probe to screen 600,000 plaques from the wheat cDNA library.
  • the wheat APRT DNA coding sequence elucidated from the clone thus isolated is provided in SEQ ID NO: 5.
  • the amino acid sequence encoded by this DNA sequence is provided in SEQ ID NO: 6.
  • the isolation of the two DNA sequences coding for individual protein sequences is likely due to the presence of multiple isoforms encoded by different genes in wheat genome. Wheat, with its hexaploid genome, may have even more than two APRT genes.
  • the protein sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 6 are compared in Table I.
  • EXAMPLE 3 Isolation of Additional APRT Genes Based on the Sequence Homology to Known APRT Sequences
  • Total RNA is prepared from 7-d-old Arabidopsis seedlings by phenol/chloroform extraction followed by lithium chloride precipitation.
  • Poly(A)+ RNA is isolated from the total RNA using a poly(A)+ Quick mRNA isolation kit (Stratagene, LaJolla, CA).
  • a cDNA library is constructed from the poly(A)+ RNA in the bacteriophage vector lambda ZAPII (Stratagene) using the Uni-ZAP XR Gigapack II Gold cloning kit (Stratagene) as described in the manufacturers' instruction.
  • Lambda phage is collected from the Arabidopsis cDNA library, and phage DNA is in a solution containing 0.1 % (w/v) sodium dodecyl sulfate and 20 mM EDTA (pH 8.0), phage DNA is prepared by phenol/chloroform extraction and a part of this is used as a template for PCR with a set of SK primer, 5'-TCTAGAACTAGTGGATC-3' (Stratagene) and an antisense, 5'-GTCTCCTCGTCAAATATGTA-3' (SEQ ID 12), primer designed from the amino acid sequence determined from the purified APRT shown in SEQ ID NO: 1.
  • the PCR is carried out in 50 ⁇ l of a reaction mixture consisting of 20 mM of Tris- HCI (pH 8.4) containing 10 pmol of the primers, 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dTTP, 200 ⁇ M dGTP, 2 mM MgCl2 50 mM KCI, 1.25 units/mL Taq DNA polymerase (GIBCO-BRL).
  • the reaction is performed through 35 cycles of 1 min at 94 °C, 1 min at 50 °C and 90 sec at 72 °C using a thermal cycler (Perkin Elmer/Cetus, model 9600). PCR products are separated by agarose gel (1%) electrophoresis.
  • a major band (0.4 kb), which represents the PCR-amplified fragment derived from Arabidopsis APRT, is isolated from the gel and cloned into a pCRII vector using a TA cloning kit (Invitorgen). The DNA insert is used to isolate its corresponding full-length clone from the Arabidopsis cDNA library.
  • the DNA is labeled with [ P]-dCTP by random priming labeling method and used as a probe to screen 600,000 plaques from the Arabidopsis cDNA library.
  • the APRT DNA coding sequences elucidated from this clone are provided in SEQ ID NOS: 7 and 9.
  • the amino acid sequence encoded by these DNA sequences are provided in SEQ ID NOS: 8 and 10, respectively.
  • the APRT protein is expressed using the baculovirus expression vector system according to the method described previously (Summers and Smith, 1987), using a baculovirus transfer vector pVL1392 (Invitrogen, San Diego, CA), Spodoptera furugiperda 21 (Sf21) cells (Invitrogen) and an infectious BaculoGold Baculovirus DNA (Pharmingen, San Diego, CA).
  • Sf21 cells are maintained at 27 9 C as a monolayer culture in a Grace's medium supplemented with 0.33% TC yeastolate, 0.33% lactoalbumin, 10% fetal bovine serum, and 50 ⁇ g/ml of gentamycin sulfate.
  • the expressed APRT protein is purified from the infected Sf21 cells. Briefly, the infected cells are sonicated in buffer A containing 0.1 M potassium phosphate (pH 7.5), 0.1 M NaCI, 1 mM L-His, 5 mM EDTA, 30 mM 2-mercaptoethanol and 10% (w/w) polyvinylpyrrolidone) and centrifuged at 10,000 x g for 15min. The recombinantly produced APRT is purified by the method provided by EXAMPLE 1.
  • EXAMPLE 6 Heterologous Expression in E.coli
  • the coding region without putative chloroplast transit sequence is amplified by PCR under a condition: 95 °C for 5 min, 30 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72°C, using a set of primers: 5'-CGGGATCCATGAAGCGTGACCAGATTCGTCTTG -3' (SEQ ID 13) and 5'-GCTCTAGAAGCTTCAGCATATGCATCTTCC-3' (SEQ ID NO: 14).
  • the DNA fragment thus amplified is subcloned into a PCRII vector using a TA cloning kit (Invitrogen), and then inserted into an expression plasmid pMAL-C2 vector (New England Biolabs, Inc. MA, USA).
  • the pMAL-C2 carrying the insert of the APRT DNA is used for the transformation of an E. coli strain JM109.
  • the APRT protein is recombinantly produced in the E. coli cells as a fusion protein with a maltose binding protein, which is purified through a one-step amylose resin affinity column chromatography as described by the manufacturer.
  • EXAMPLE 7 Selecting for plant APRT genes resistant to APRT-inhibitory herbicides in the E. coli expression system.
  • APRT plasmids into an available E. coli APRT defective mutant, (e.g. strain JC411 , which carries the hisG1 mutation (Delorme et al., J. Bacteriol. 174: 6571 -6579 (1992)) and histidine prototrophic colonies are selected on M9-0.2% (w/v) glucose minimal plates supplemented with 100 mg/ml ampicillin and amino acids except for histidine (Sambrook J, Fritsch EF, Maniatis T, ed. eds. Molecular cloning: A Laboratory Manual. 2nd edition.
  • the plant APRT plasmids are mutagenized in a variety of ways, using published procedures for chemical (e.g. sodium bisulfite (Shortle et al., Methods Enzymol. 700:457-468 (1983); methoxylamine (Kadonaga et al., Nucleic Acids Res. 73:1733-1745 (1985); oligonucleotide-directed saturation mutagenesis (Hutchinson et al., Proc. Natl. Acad. Sci. USA, 83:710-714 (1986); or various polymerase misincorporation strategies (see, e.g. Shortle et al., Proc. Natl. Acad. Sci.
  • Any plant APRT gene expressing herbicide resistance in the bacterial system may be engineered for optimal expression and transformed into plants using standard techniques as described herein. The resulting plants may then be treated with herbicide to confirm and quantitate the level of resistance conferred by the introduced APRT gene.
  • EXAMPLE 8 Production of herbicide-tolerant plants by overexpression of plant APRT genes
  • pCGN1761 ENX which is derived from pCGN1761 as follows.
  • pCGN1761 was digested at its unique EcoRI site, and ligated to a double-stranded DNA fragment comprised of two oligonucleotides of sequence 5' AAT TAT GAC GTA ACG TAG GAA TTA GCG GCCC GCT CTC GAG T 3' (SEQ ID NO: 15) and 5' AAT TAC TCG AGA GCG GCC GCG AAT TCC TAC GTT ACG TCA T 3' (SEQ ID NO: 16).
  • the resulting plasmid, pCGN1761 ENX contained unique EcoRI, Notl, and Xhol sites that lie between a duplicated 35S promoter from cauliflower mosaic virus (Kay et al., Science 236:1299-1302 (1987)) and the 3' untranslated sequences of the tml gene of Agrobacterium tumefaciens.
  • This plasmid is digested and ligated to a fragment resulting from restriction enzyme digestion of one of the plasmids bearing a APRT cDNA, such that it carries the complete APRT cDNA.
  • Kanamycin-resistant shoots from 15 independent leaf disks are transferred to rooting medium, then transplanted to soil and the resulting plants grown to maturity in the greenhouse. Seed from these plants are collected and germinated on MS agar medium containing kanamycin. Multiple individual kanamycin resistant seedlings from each independent primary transformant are grown to maturity in the greenhouse, and their seed collected. These seeds are germinated on MS agar medium containing kanamycin. Plant lines that give rise to exclusively kanamycin resistant seedlings are homozygous for the inserted gene and are subjected to further analysis. Leaf disks of each of the 15 independent transgenic lines are excised with a paper punch and placed onto MS agar containing various increasing concentrations of a APRT inhibitory herbicide.
  • RNA is extracted from leaves of each of these lines. Total RNA from each independent homozygous line, and from non-transgenic control plants, is separated by agarose gel electrophoresis in the presence of formaldehyde (Ausubel et al., Current Protocols in Molecular Biology. Wiley & Sons, New York (1987)). The gel is blotted to nylon membrane (Ausubel et al., supra.) and hybridized with the radiolabeled Arabidopsis APRT cDNA. Hybridization and washing conditions are as described by Church and Gilbert, Proc. Natl. Acad. Sci. USA 87:1991-1995 (1984). The filter is autoradiographed, and intense RNA bands corresponding to the APRT transgene are detected in all herbicide-tolerant transgenic plant lines.
  • MOLECULE TYPE peptide
  • rTynPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AAA CTG ACC TAC ATA TTT GAC GAG GAG ACT CCT AGG TGG CGC AAG CTT 480 Lys Leu Thr Tyr He Phe Asp Glu Glu Thr Pro Arg Trp Arg Lys Leu 145 150 155 160
  • GGCCATGCCC TCCGCTAGAA TGGACCGTCT CAGTGAGCAT CTGAACTTAT GCT03CTGTA 638
  • AACCTATTCC CCTGGACAGG CAGTGGTTGG TTTATCCCTT TTATCTACCA ⁇ _ACTCGATA 818
  • Trp Arg Lys Leu Leu Ala Glu Leu Gly Met 180 185
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AAT CCA CGA CAA TAT GTT GCT CAA ATT CCT CAG TTA CCA
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA to mRNA
  • HYT ⁇ THETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA to mRNA
  • HYTrOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA to mRNA
  • HYrrXDTHETICAL NO
  • ANTI-SENSE NO

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Abstract

La présente invention concerne une molécule d'ADN isolée codant l'enzyme ATP-phosphoribosyl transférase (APRT) à partir d'une source végétale ou des formes modifiées de cette enzyme tolérantes aux herbicides. Suivant les informations fournies par la présente invention, on peut obtenir, selon les méthodes classiques, la séquence codant l'ADN pour un ou des enzymes APRT à partir de toute source végétale. La présente invention concerne également la production par recombinaison de l'enzyme APRT, et des procédés d'utilisation des APRT produits par recombinaison. La présente invention concerne, en outre, des sondes et des procédés permettant de détecter la présence et la forme du gène APRT, ainsi que de quantifier les taux de transcrits d'APRT dans un organisme. La présente invention concerne, par ailleurs, des cassettes d'expression et des vecteurs recombinés renfermant lesdites cassettes d'expression comprenant essentiellement un promoteur, mais en particulier un promoteur actif dans une plante, fixé de manière fonctionnelle à une molécule d'ADN codant l'enzyme APRT à partir d'un organisme eucaryote, conformément à l'invention. L'invention concerne aussi la préparation de plantes transgéniques, de tissus végétaux et de graines végétales transformés de manière stable avec une molécule d'ADN recombinée comprenant un promoteur approprié, fonctionnel dans des plantes et fixé de manière fonctionnelle à un gène de structure codant une enzyme APRT eucaryote modifiée ou non modifiée.
EP98938705A 1997-07-28 1998-07-24 Atp-phosphoribosyl transferase vegetale et adn la codant Withdrawn EP0994952A1 (fr)

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GBGB9715905.7A GB9715905D0 (en) 1997-07-28 1997-07-28 Plant phosphoribosyl transferase and DNA coding thereof
GB9715905 1997-07-28
PCT/EP1998/004652 WO1999005286A1 (fr) 1997-07-28 1998-07-24 Atp-phosphoribosyl transferase vegetale et adn la codant

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AU5532700A (en) * 1999-06-15 2001-01-02 Syngenta Participations Ag Herbicide target genes and methods
US6294345B1 (en) 1999-07-27 2001-09-25 Syngenta Participations Ag Genes encoding proteins essential for plant growth and methods of use
EP1240185A2 (fr) * 1999-12-16 2002-09-18 Syngenta Participations AG Genes cibles d'herbicide et procedes correspondants
AU8769701A (en) * 2000-08-18 2002-02-25 Advanta Seeds Bv Inhibition of generative propagation in genetically modified herbicide resistantgrasses
EP1458630A1 (fr) 2001-12-21 2004-09-22 Nektar Therapeutics Emballage a capsules a membrane etanche
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US6008045A (en) * 1991-11-15 1999-12-28 Board Of Trustees Of The Leland Stanford University Nucleic acid encoding human DNA polymerase α
US5541310A (en) * 1993-05-13 1996-07-30 Ciba-Geigy Corporation Herbicide resistant plants
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