WO1998033925A1 - Glutrnagln amidotransferase: un nouveau composant essentiel de la traduction - Google Patents

Glutrnagln amidotransferase: un nouveau composant essentiel de la traduction Download PDF

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Publication number
WO1998033925A1
WO1998033925A1 PCT/US1998/001860 US9801860W WO9833925A1 WO 1998033925 A1 WO1998033925 A1 WO 1998033925A1 US 9801860 W US9801860 W US 9801860W WO 9833925 A1 WO9833925 A1 WO 9833925A1
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WIPO (PCT)
Prior art keywords
adt
seq
leu
protein
polynucleotide
Prior art date
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PCT/US1998/001860
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English (en)
Inventor
Dieter SÖLL
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Yale University
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 Yale University filed Critical Yale University
Priority to AU62591/98A priority Critical patent/AU6259198A/en
Priority to CA002279490A priority patent/CA2279490A1/fr
Priority to JP53313298A priority patent/JP2001511004A/ja
Priority to EP98904799A priority patent/EP1015602A1/fr
Publication of WO1998033925A1 publication Critical patent/WO1998033925A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of inhibitors of protein translation
  • invention relates to newly identified polynucleotides and polypeptides, and their
  • the invention relates to novel
  • Glu-tRN A Gln AdT Gln AdT
  • AdT AdT
  • translation inhibitors as antibacterial, antifungal or herbicidal agents.
  • amino acids Prior to their incorporation into protein, amino acids are chemically linked to
  • RNA molecules called transfer RNA (tRNA).
  • tRNA transfer RNA
  • GlnRS glutaminyl-tRNA synthetase
  • GluRS glutamyl-tRNA synthetase
  • AdT amidotransferase
  • the enzyme GlnRS may have
  • amidotransferase may not have significant amino acids available sequence information. Thus, the amidotransferase may not have significant amino acids available sequence information. Thus, the amidotransferase may not have significant amino acids available sequence information. Thus, the amidotransferase may not have significant amino acids available sequence information. Thus, the amidotransferase may not have significant amino acids available sequence information. Thus, the amidotransferase may not have significant amino acids.
  • the present invention is based, in part, on the isolation and characterization of
  • This invention further provides polypeptides that have been
  • This invention further provides polynucleotides that encode AdT
  • polypeptides are polypeptides.
  • this invention provides the polynucleotide sequence
  • polypeptides encoded thereby are polypeptides encoded thereby.
  • this invention provides polynucleotides that
  • AdT polynucleotide sequences hybridize to AdT polynucleotide sequences, particularly under stringent conditions.
  • This invention provides GlutRNA Gln AdT protein from B. subtilis comprising
  • NOS:l, 3, 5 and 7 as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • polypeptides are also provided by this invention.
  • the invention also provides isolated nucleic acid molecules encoding AdT,
  • B. subtilis AdT including niRNAs, cDNAs, and genomic DNAs
  • inhibitors to such AdT polypeptides useful as antibacterial agents, antifungal agents
  • the present invention provides compositions and methods for
  • AdT polypeptide or polynucleotide administered to a cell or to a multicellular
  • This invention also provides compositions and methods for protecting plants,
  • this invention provides antagonists of AdT which are useful as herbicides, as well as the herbicidal compositions which include
  • This invention also provides non-inhibited mutants of AdT
  • coding for the non-inhibited AdT can be placed in plants by various transformation
  • AdT inhibitors to transgenic plants containing the non-inhibited mutants of AdT are:
  • Figure 1 shows the transamidation pathway for the formation of GlntRNA Gln .
  • Figure 2 shows the gene arrangement ofthe AdT gene.
  • Figure 3 shows the nucleic acid sequence ofthe AdT protein from B. subtilis.
  • the present invention specifically provides a cell, herein after the AdT protein.
  • the AdT protein and nucleic acid molecules can serve as targets in methods for
  • the present invention provides, in part, the amino acid sequences ofthe
  • the present invention provides the ability to produce a
  • AdT protein refers to a protein that has the amino acid
  • A SEQ ID NO:4
  • B SEQ ID NO:6
  • C C
  • AdT protein referred to as the AdT protein or the AdT protein ofthe present invention.
  • polypeptides ofthe invention include the polypeptides encoded by SEQ ID NO: 1
  • AdT have the biological activity of AdT and also those which have at least 70% sequence
  • SEQ LD NO: 1 SEQ LD NO: 1 and also include portions of such polypeptides.
  • AdT proteins ofthe present invention include the specifically identified
  • allelic variants allelic variants, conservative substitution variants and homologues that can be isolated/generated and characterized
  • AdT proteins will be collectively referred to as the AdT proteins
  • AdT proteins includes all naturally occurring allelic variants ofthe
  • AdT protein will have a slightly different amino acid sequence than that
  • allelic variants ofthe AdT protein will contain conservative amino acid
  • AdT AdT
  • AdT proteins ofthe present invention are preferably in isolated form.
  • a protein is said to be isolated when physical, mechanical or chemical
  • Example 1 purification methods to obtain an isolated AdT protein.
  • One purification scheme is outlined in Example 1. The nature and degree of isolation will depend on the
  • Fragments ofthe AdT proteins can be generated using standard peptide
  • recombinant methods can be used to generate nucleic acid molecules that encode a
  • the present invention further provides antibodies that selectively bind one or
  • AdT proteins ofthe present invention or to a specific subunit of an AdT
  • fragments containing the antigen binding domain and/or one or more complement are fragments containing the antigen binding domain and/or one or more complement
  • Antibodies are generally prepared by immunizing a suitable mammalian host
  • immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other
  • reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be
  • Administration of an AdT immunogen is conducted generally by injection
  • monoclonal antibody may be prepared using the standard method of Kohler and
  • antibodies are screened by immunoassay in which the antigen is the AdT protein or
  • the cells can be cultured either in vitro or by production
  • polyclonal antisera which contain the immunologically significant portion can be used
  • fragments such as the Fab, Fab', of F(ab')2 fragments is often preferable, especially in
  • the antibodies or fragments may also be produced, using current technology,
  • transporter can also be produced in the context of chimeric or CDR grafted antibodies
  • anti- AdT antibodies are useful as modulators of AdT
  • the present invention is based, in part, on isolating nucleic acid
  • the present invention further provides nucleic acid molecules that
  • AdT AdT protein
  • nucleic acid molecules the AdT genes, or AdT.
  • AdT polypeptides having an amino acid sequence encoded by SEQ ID NO:l, and
  • polynucleotides which are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 80%
  • polynucleotides complementary thereto are complementary thereto.
  • the invention further relates to variants ofthe herein above described
  • Variants that are fragments ofthe polypeptides ofthe invention may be any variants of the polypeptides ofthe invention.
  • invention may be used to synthesize full-length polynucleotides ofthe invention.
  • a fragment is a variant polypeptide having an amino acid sequence that
  • AdT polypeptides fragments may be "free-standing,"
  • SEQ ID NO:l in which several, a few, 10 to 15, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no
  • amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not
  • nucleic acid molecule is defined as an RNA or DNA
  • genomic DNA sequences herein disclosed. Specifically contemplated are genomic DNA,
  • nucleic acid molecules are defined further as
  • nucleic acid molecule is said to be "isolated" when the
  • nucleic acid molecule is substantially separated from contaminant nucleic acid
  • the present invention further provides fragments ofthe AdT encoding nucleic acid
  • encoding nucleic acid molecule refers to a small portion ofthe entire protein encoding sequence. The size ofthe fragment will be determined by its intended use. For
  • fragment length * is to be used as a nucleic acid probe or PCR primer
  • PCR polymerase chain reaction
  • AdT proteins can easily be synthesized by chemical techniques, for example, the
  • AdT encoding nucleic acid molecules ofthe present invention may further comprise
  • probes can be used to identify nucleic acid molecules encoding
  • probes can be used to diagnose the presence of a AdT protein as a means for
  • Suitable labels include, but are not limited to, biotin,
  • radiolabeled nucleotides biotin, and the like.
  • biotin and the like.
  • AdT homologues The preferred source of the AdT homologues are pathogenic
  • microorganisms such as bacteria and fungi, as well as plants in which it is desirable to
  • the most preferred sources are gram positive bacteria, pathogenic
  • fungi and plant organelles such as chloroplasts.
  • subtilis AdT protein to generate antibody probes to screen expression libraries
  • polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies
  • sequence can be expressed as a fusion protein, expressed directly using its own
  • control sequences or expressed by constructing an expression cassette using control
  • This method can be used to identify and isolate altered and
  • pairs of oligonucleotide primers can be prepared for use in a
  • PCR polymerase chain reaction
  • nucleic acid molecule or fragment thereof.
  • the preferred primers will flank one or more ofthe subunit
  • B. subtilis sequences under high stringency Such sequences will typically contain at
  • the present invention further provides recombinant DNA molecules that
  • AdT AdT encoding sequences herein described, or a fragment of
  • nucleic acid molecules As used herein, an recombinant DNA
  • molecule is a DNA molecule that has been subjected to molecular manipulation in
  • AdT subunit is operably linked to one or more expression control
  • the recombinant DNA molecule can encode either a single subunit ofthe AdT protein, or can encode an operon that contains all
  • AdT encoding sequences ofthe present invention is operably linked depends directly,
  • invention is at least capable of directing the replication or insertion into the host
  • Expression control elements that are used for regulating the expression of an
  • operably linked protein encoding sequence include, but are not limited to, but are
  • inducible promoters not limited to, inducible promoters, constitutive promoters, secretion signals,
  • inducible promoter that is readily controlled, such as being responsive to a nutrient in
  • the host cell's medium is used.
  • the vector containing an AdT encoding nucleic acid in one embodiment, the vector containing an AdT encoding nucleic acid
  • molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability
  • a prokaryotic host cell such as a bacterial host cell
  • vectors that include a prokaryotic replicon may also include a gene whose expression confers
  • a detectable marker such as a drug resistance.
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or
  • viral promoter capable of directing the expression (transcription and translation) ofthe
  • AdT encoding sequence in a bacterial host cell such as E. coli.
  • a promoter is an
  • expression control element formed by a DNA sequence that permits binding of RNA
  • hosts are typically provided in plasmid vectors containing convenient restriction sites
  • plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories
  • Expression vectors compatible with eukaryotic cells preferably those
  • vectors are provided containing convenient restriction sites for
  • pKSV-10 Pulcosa
  • pBPV-l/pML2d International Biotechnologies, Inc.
  • pTDTl ATCC, #31255
  • molecules ofthe present invention may further include a selectable marker that is
  • a drug resistance selection marker preferably a drug resistance selection marker.
  • preferred drug resistance marker is the gene whose expression results in neomycin
  • the selectable marker can be present on
  • the present invention further provides host cells transformed with a nucleic acid
  • heterotrimeric protein or one or more subunits The host cell can be either prokaryotic
  • Eukaryotic cells useful for expression of an AdT protein are not
  • Preferred eukaryotic host cells include, but are not limited to, yeast, insect and
  • mammalian cells preferably vertebrate cells such as those from a mouse, rat, monkey
  • human fibroblastic cell line the most preferred being cells that do not naturally
  • AdT protein Any prokaryotic host can be used to express an AdT-encoding recombinant
  • the preferred prokaryotic host is E. coli.
  • vertebrate cells with vectors containing recombinant DNAs, electroporation, cationic
  • lipid or salt treatment methods are typically employed, see, for example, Graham et
  • molecule ofthe present invention can be identified by well known techniques.
  • inventions can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence ofthe recombinant
  • the present invention further provides methods for producing an AdT protein
  • AdT protein the production of a recombinant AdT protein typically involves the following
  • a nucleic acid molecule is obtained that encodes an AdT protein, such as
  • AdT encoding nucleic acid molecule is then preferably placed in an operable
  • the expression unit containing the AdT encoding sequence.
  • the expression unit is used to transform
  • a suitable host and the transformed host is cultured under conditions that allow the
  • AdT protein production of the AdT protein.
  • AdT protein is isolated from the AdT protein.
  • desired coding sequences may be obtained from genomic fragments and used directly
  • control sequences The control sequences, expression vectors, and transformation
  • Suitable restriction sites can, if not normally available, be
  • AdT AdT encoding sequences
  • agonists and antagonists of an AdT protein can be identified by the
  • an AdT protein is mixed with an agent.
  • the mixture is analyzed to determine if the agent bound the AdT protein.
  • antagonists are identified as being able to bind to an AdT protein. Alternatively or
  • AdT activity can be directly assessed as a means
  • the AdT protein used in the above assay can be: an isolated and fully
  • characterized protein a single subunit of an AdT protein, a partially purified protein, a
  • the AdT protein can be the entire AdT
  • AdT protein a specific fragment of the AdT protein or a single subunit ofthe AdT protein.
  • agent binding e.g., by a shift in molecular weight or activity, as
  • the AdT protein is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the source ofthe AdT protein will be based on the intended use ofthe
  • microbial AdT protein is used to identify AdT
  • assay can be used to determine whether an agent binds to a soluble fragment of an AdT protein.
  • immunodetection and biochip technologies can be used to determine whether an agent binds to a soluble fragment of an AdT protein.
  • Agents can be further tested for the ability to modulate the activity of an AdT
  • Example 1 provides a cell-free assay system or a cellular assay system.
  • an agent is said to antagonize AdT activity when the agent
  • the preferred antagonist will selectively antagonize AdT, not
  • the preferred antagonist will reduce AdT activity by more than 50%, more
  • AdT activity preferably by more than 90%, most preferably eliminating all AdT activity.
  • an agent is said to agonize AdT activity when the agent
  • AdT activity increases AdT activity.
  • the preferred agonist will increase AdT activity by more than
  • the preferred antagonists and agonists will be selective for a specific species
  • Agents can be screened using one
  • AdT protein or a combination of AdT proteins, to aid in identifying agents for target
  • Agents that are assayed in the above method can be randomly selected or
  • sequences ofthe AdT protein are sequences ofthe AdT protein.
  • An example of randomly selected agents is the use a
  • an agent is said to be rationally selected or designed when the
  • agent is chosen on a nonrandom basis that takes into account the sequence ofthe
  • Agents can be any substance that can be used to promote target site and/or its conformation in connection with the agent's action.
  • Agents can be any combination of
  • a rationally selected peptide agent can be a
  • the agents ofthe present invention can be, as examples, peptides, small molecules
  • agents of the present invention are peptide agents
  • Small peptide agents can serve as competitive inhibitors of AdT protein
  • the peptide agents ofthe invention can be prepared using standard solid phase
  • DNA encoding these peptides may be synthesized using commercially available
  • Another class of agents ofthe present invention are antibodies immunoreactive
  • protein translation particularly protein translation in gram positive microorganisms
  • Agents that bind an AdT protein and act as an agonist or antagonist can be used to modulate translation in these
  • protein translation that requires AdT can be modulated by administering to an
  • an organism can be any organism, so long as it is desirable to
  • the invention provides an infectious agent in a mammalian subject or to act as an herbicide agent.
  • protein translation that requires AdT refers to protein
  • an agent is said to modulate AdT meditated protein translation when the
  • agent reduces the degree of protein translation.
  • AdT modulating agents will be based primarily on the target
  • AdT protein used to identify the agent as well as the activity/selectivity ofthe agent.
  • an AdT inhibitory agent that is used as an antimicrobial agent, is an AdT inhibitory agent.
  • Herbicide agent will be any suitable herbicide that will cause apoptosis.
  • Herbicide agent will be any suitable herbicide.
  • an AdT inhibitory agent can be administered via parenteral,
  • administration may be by the oral route.
  • AdT protein or agonist of an AdT protein that is identified by the methods herein described.
  • compositions ofthe present invention are present in addition to the AdT modulating agent, the compositions ofthe present invention.
  • invention may contain other ingredients, such as suitable pharmaceutically acceptable
  • Suitable formulations for parenteral administration include aqueous
  • solutions ofthe active compounds in water-soluble variant for example, water-soluble
  • injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles may be administered.
  • fatty oils for example, sesame oil, or synthetic fatty acid esters, for example,
  • Aqueous injection suspensions may contain substances
  • the suspension may include carboxymethyl cellulose, sorbitol, and/or dintran.
  • the suspension may include
  • Liposomes can also be used to encapsulate the agent for
  • inventions may be formulated for enteral, parenteral or topical administration. Indeed,
  • Suitable formulations for oral administration include hard or soft gelatin
  • antimicrobial agents As used herein, two agents are said to be administered in
  • the present invention further provides methods for identifying cells or
  • the present invention are particularly useful in the determining the presence of
  • an AdT protein can be any substance that influences the expression of an AdT protein.
  • an AdT protein can be any substance that influences the expression of an AdT protein.
  • AdT protein identified by determining whether an AdT protein, or nucleic acid encoding an AdT
  • AdT protein is expressed.
  • the expression of an AdT protein can be used as a means for
  • AdT protein is expressed/produced in a particular cell or sample.
  • an extract containing nucleic acid molecules or an extract containing proteins is prepared.
  • the extract is then assayed to determine whether an AdT protein, or an
  • AdT encoding nucleic acid molecule is produced in the cell.
  • a diagnostic test based on nucleic acid molecules For example, to perform a diagnostic test based on nucleic acid molecules, a
  • suitable nucleic acid sample is obtained and prepared using conventional techniques.
  • DNA can be prepared, for example, simply by boiling a sample in SDS.
  • extracted nucleic acid can then be subjected to amplification, for example by using the
  • PCR polymerase chain reaction
  • the presence of an AdT protein can be identified.
  • a suitable protein sample is
  • Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance obtained and prepared using conventional techniques. Protein samples can be any substance
  • the extracted protein can then be analyzed to
  • AdT protein determines the presence of an AdT protein using known methods. For example, the presence of specific sized or charged variants of a protein can be identified using
  • antibodies can be used for detection
  • AdT expression can also be used in methods to identify agents
  • Agents that activate AdT expression can be used as
  • the transamidation pathway is operative in chloroplasts.
  • AdT inhibitors which specifically inhibit plastid isoforms of
  • AdT can be useful in designing herbicides that are not toxic or harmful to humans and
  • AdT cytoplasm exclusive of organelles
  • human AdT would provide a new form of
  • chloroplasts which are not limited to the chloroplasts may also find utility in use as an herbicide.
  • An identified compound which inhibits function ofthe wild-type AdT enzyme is an identified compound which inhibits function ofthe wild-type AdT enzyme
  • the active ingredient is utilized as an active ingredient in an herbicide.
  • the active ingredient is normally
  • acceptable carriers and can be applied to the crop area or plant to be treated,
  • compounds can include fertilizers, other herbicides, fungicides, bactericides,
  • the herbicide may be any herbicide that has the herbicide characteristics, but not limited to, butyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N
  • the active ingredient ofthe present invention or an
  • agrochemical composition which contains at least one ofthe active ingredients ofthe
  • Resistant mutants to the AdT-inhibiting compound can be identified by:
  • Mutagenesis can be
  • chemical mutatgenesis e.g., ethyl methanesulfonate
  • ultraviolet radiation e.g., X-ray exposure
  • gamma radiation see, e.g., Watson et al, Recombinant DNA, Second
  • mutant AdT is isolated, and the DNA sequence ofthe mutant gene is determined and
  • the coding DNA sequence for the mutant AdT can be introduced into the
  • Examples of such methods include micro injection, electroporation, Agrobacterium-
  • AdT herbicide resistance genes ofthe references. Since AdT performs its function in the chloroplast, it may be particularly relevant to use a plastid transit sequence to ensure
  • husbandry and plant breeding can be used to maintain and increase the transformed
  • the transformed plants can also be used in conventional hybridization
  • AdT can be grown in soil and the herbicide containing the AdT inhibitor can be
  • the weeds will be susceptible to the herbicide containing the AdT inhibitor.
  • E. coli BL21 (DE3) harboring pABC were incubated overnight in
  • Buffer A 25 mM Hepes-KOH, pH 7.5, 25 mM KC1
  • the cells were lysed by sonication (4 x 15
  • pellet was resuspended in 100 mM NaCl, incubated overnight at 4°C and ethanol
  • the tRNAGln was purified by a two-step anion exchange
  • nucleic acids were recovered by ethanol precipitation followed by centrifugation at 10,000 x g for 15 minutes at 4°C and resuspended in
  • GlntRNA Gln was adapted from Jahn, D. et al. (1990). Unless otherwise noted, these
  • amidotransferase assay mixtures either directly or following water saturated phenol
  • DTT dithiothreitol
  • the amino acid sequence of B. subtilis AdT is encoded by the nucleotide
  • the molecular weights ofthe three subunits is computed to be: 53.039 Kd, A
  • AdT trimeric protein
  • the antiserum was shown to contain
  • the assay used to identify inhibitors of AdT activity comprises:
  • step (b) measuring an uninhibited reactivity ofthe AdT from step (a);
  • step (d) measuring an inhibited reactivity ofthe AdT from step (c); and, (e) comparing the inhibited reactivity to the uninhibited reactivity ofthe
  • Inhibitors of AdT identified using this process are utilized as antibacterial,
  • inhibitor-resistant AdT mutants comprises :
  • second sample comprising an AdT inhibitor
  • step (b) measuring an unmutated reactivity ofthe AdT from step (a);
  • step (d) measuring a mutated reactivity ofthe mutated AdT from step (c);
  • Nucleic acids for diagnosis are obtained from cells or tissues. Genomic DNA
  • PCR may be used directly for dectection or may be amplified enzymatically by using PCR
  • genomic DNA can be any DNA sequence known in the art.
  • the genomic DNA can be any sequence known in the art.
  • Deletions and insertions can be detected by a change in size ofthe amplified
  • Point mutations can be identified by
  • RNASE RNASE
  • DNA sequence differences may be made by digestion or by differences in melting temperatures. DNA sequence differences may be made by deletion or by differences in melting temperatures. DNA sequence differences may be made by deletion or by deletion of DNA sequence differences.
  • nuclease protection assays such as
  • RNase and SI protection or a chemical cleavage method.
  • RTPCR reverse transcriptase reverse transcriptase
  • RTPCR reverse transcriptase
  • RNA molecules conduction with automated detection systems, such as, for example, GeneScan.
  • automated detection systems such as, for example, GeneScan.
  • cDNA may also be used for the same purpose, PCR or RTPCR.
  • PCR RTPCR
  • PCR primers complementary to the nucleic acid encoding AdT can be used to identify and analyze mutations. These primers may be used for amplifying AdT DNA isolated
  • the invention also provides these primers
  • the primers may be 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end.
  • the primers may
  • AdT polynucleotide Increased or decreased expression of AdT polynucleotide can be measured
  • AdT protein over-or under- expression of AdT protein compared to normal control tissue samples.
  • radioimmunoassay examples include radioimmunoassay, competitive-binding assays, Western Blot analysis and
  • inhibitors is isolated from an inhibitor-resistant AdT mutant using mutagenesis and
  • mutant AdT gene is then introduced into a plant cell and whole transformed plants are
  • Crop plants carrying the mutant AdT gene are grown in
  • ADDRESSEE MORGAN, LEWIS & BOCKIUS LLP
  • GGT ATG CCG ATC GGC GTA AAA GAT AAT ATC GTA ACA AAA GGG CTG CGC 672 Gly Met Pro He Gly Val Lys Asp Asn He Val Thr Lys Gly Leu Arg 210 215 220 ACA ACA TGC TCC AGC AAA ATT CTC GAA AAC TTT GAT CCG ATT TAC GAT 720 Thr Thr Cys Ser Ser Lys He Leu Glu Asn Phe Asp Pro He Tyr Asp 225 230 " 235
  • GGT TCA AGC GGC GGA TCT GCA GCT GCG GTT GCT GCG GGA GAA GTT CCG 912 Gly Ser Ser Gly Gly Ser Ala Ala Ala Val Ala Ala Gly Glu Val Pro 290 295 300
  • ATC CGC ACG CCG GAA GAA NCG TAC GCA TAT CTT GAA AAG CTG AAA TCC 2394 He Arg Thr Pro Glu Glu Xaa Tyr Ala Tyr Leu Glu Lys Leu Lys Ser 780 785 790 795

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Abstract

Le présent procédé a pour objet la séquence d'acides aminés et la séquence nucléotidique codante de la GlutRNAGln amidotransférase (AdT), une protéine essentielle à la traduction des protéines. Les protéines AdT et les molécules d'acide nucléique codantes décrites peuvent être utilisées pour identifier des agents bloquant les traductions. De tels agents peuvent s'utiliser comme agent antimicrobien, antifongique, ou herbicide.
PCT/US1998/001860 1997-02-03 1998-02-03 Glutrnagln amidotransferase: un nouveau composant essentiel de la traduction WO1998033925A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU62591/98A AU6259198A (en) 1997-02-03 1998-02-03 Glutrnagln amidotransferase - a novel essential translational component
CA002279490A CA2279490A1 (fr) 1997-02-03 1998-02-03 Glutrnagln amidotransferase: un nouveau composant essentiel de la traduction
JP53313298A JP2001511004A (ja) 1997-02-03 1998-02-03 ＧｌｕｔＲＮＡ▲上Ｇｌｎ▼アミドトランスフェラーゼ−新規な必須の翻訳成分
EP98904799A EP1015602A1 (fr) 1997-02-03 1998-02-03 Glutrna gln amidotransferase: un nouveau composant essentiel de la traduction

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US3727597P 1997-02-03 1997-02-03
US60/037,275 1997-02-03

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1021560A1 (fr) * 1997-10-02 2000-07-26 Smithkline Beecham Corporation Criblage de medicaments antimicrobiens au moyen d'une cellule recombinee renfermant un gene d'amidotransferase dependant de l'arn
WO2001027248A1 (fr) * 1999-10-11 2001-04-19 Basf Aktiengesellschaft Prpp-amidotransferase obtenue a partir de plantes
WO2002000703A2 (fr) * 2000-06-27 2002-01-03 Bayer Aktiengesellschaft Regulation de l'enzyme de type glutamyl-arnt (gln) amido-transferase humaine
WO2005054453A1 (fr) 2003-12-02 2005-06-16 Basf Aktiengesellschaft 2-methyl-6-solanylbenzoquinone methyltransferase utilisee comme cible pour des herbicides
US7319013B2 (en) 2002-03-20 2008-01-15 Basf Aktiengesellschaft Serine hydroxymethyltransferase as a target for herbicides

Citations (2)

* Cited by examiner, † Cited by third party
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EP0191221A1 (fr) * 1984-10-24 1986-08-20 The Regents Of The University Of California Vecteurs pour la transformation d'ascomycètes
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus

Patent Citations (2)

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EP0191221A1 (fr) * 1984-10-24 1986-08-20 The Regents Of The University Of California Vecteurs pour la transformation d'ascomycètes
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus

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Title
CURNOW, A.W., ET AL.: "Bacillus subtilis Glu-tRNAGln amidotransferase subunits C (gatC). A (gatA) and B (gatB) genes complete cds.", EMBL ACCESSION NO.AF008553, 18 November 1997 (1997-11-18), XP002066351 *
CURNOW, ALAN W. ET AL: "Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation", PROC. NATL. ACAD. SCI. U. S. A. (1997), 94(22), 11819-11826, XP002066350 *
CURNOW, ALAN W. ET AL: "tRNA-dependent amino acid transformations", NUCLEIC ACIDS SYMP. SER. (1997), 36(SYMPOSIUM ON RNA BIOLOGY, II. RNA: TOOL AND TARGET, 1997), 2-4, XP002066352 *
CURNOW, ALAN W. ET AL: "tRNA-dependent asparagine formation", NATURE (LONDON) (1996), 382(6592), 589-590, XP002066356 *
JAHN D ET AL: "PURIFICATION AND FUNCTIONAL CHARACTERIZATION OF THE GLUTAMINE GLUTAMINYL TRANSFER RNA AMIDOTRANSFERASE FROM CHLAMYDOMONAS- REINHARDTII.", J BIOL CHEM 265 (14). 1990. 8059-8064., XP002066355 *
KIM, S.I., ET AL.: "Bacillus subtilis PET112-like protein gene,complete cds", EMBL ACCESSION NO. U49790, 8 June 1996 (1996-06-08), XP002066349 *
SCHOEN, ASTRID ET AL: "Protein biosynthesis in organelles requires misaminoacylation of tRNA", NATURE (LONDON) (1988), 331(6152), 187-90, XP002067757 *
STRAUCH, M.A., ET AL.: "Characterization of the glutamyl-tRNAGln-to-glutaminyl-tRNAGln amidotransferase reaction of Bacillus subtilis", JOURNAL OF BACTERIOLOGY, vol. 170, 1988, pages 916 - 922, XP002066354 *
TABATA, S.: "Synechocystis sp. PCC6803 complete genome, 9/27, 1056467-1188885", EMBL ACCESSION NO. D90907, 31 October 1996 (1996-10-31), XP002067229 *
WENGENDER, P.A., ET AL.: "Identification of a putP proline permease gene homolog from Staphlyococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 61, no. 1, January 1995 (1995-01-01), pages 252 - 259, XP002067230 *
WILCOX, M., ET AL.: "Transfer RNA as a cofactor coupling amino acid synthesis with that of protein", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 61, 1968, WASHINGTON US, pages 229 - 236, XP002066353 *
ZALKIN, HOWARD: "Glu-tRNAGln amidotransferase", METHODS ENZYMOL. (1985), 113(GLUTAMATE, GLUTAMINE, GLUTATHIONE, RELAT. COMPD.), 303-5, XP002066357 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1021560A1 (fr) * 1997-10-02 2000-07-26 Smithkline Beecham Corporation Criblage de medicaments antimicrobiens au moyen d'une cellule recombinee renfermant un gene d'amidotransferase dependant de l'arn
EP1021560A4 (fr) * 1997-10-02 2003-07-23 Smithkline Beecham Corp Criblage de medicaments antimicrobiens au moyen d'une cellule recombinee renfermant un gene d'amidotransferase dependant de l'arn
WO2001027248A1 (fr) * 1999-10-11 2001-04-19 Basf Aktiengesellschaft Prpp-amidotransferase obtenue a partir de plantes
WO2002000703A2 (fr) * 2000-06-27 2002-01-03 Bayer Aktiengesellschaft Regulation de l'enzyme de type glutamyl-arnt (gln) amido-transferase humaine
WO2002000703A3 (fr) * 2000-06-27 2002-06-27 Bayer Ag Regulation de l'enzyme de type glutamyl-arnt (gln) amido-transferase humaine
US7319013B2 (en) 2002-03-20 2008-01-15 Basf Aktiengesellschaft Serine hydroxymethyltransferase as a target for herbicides
US7662603B2 (en) 2002-03-20 2010-02-16 Basf Aktiengesellschaft Nucleic acid encoding a polypeptide with the biological activity of a serine hydroxymethyltransferase
WO2005054453A1 (fr) 2003-12-02 2005-06-16 Basf Aktiengesellschaft 2-methyl-6-solanylbenzoquinone methyltransferase utilisee comme cible pour des herbicides

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