JP2001511004A - GlutRNA-Gln-Amidotransferase-A Novel Essential Translation Component - Google Patents

Abstract

(57) [Summary] The method of the present invention provides an amino acid sequence of GlutRNAGln amide transferase (AdT), which is a protein essential for protein translation, and a nucleic acid sequence encoding the same. The AdT proteins and nucleic acid molecules encoding the same described herein can be used as targets for the identification of translation blocking agents. Such an agent can be used as an antibacterial, antifungal or herbicide.

Description

DETAILED DESCRIPTION OF THE INVENTION         GlutRNA^GlnAmidotransferases-new essential translational components Related application citations   This application is incorporated by reference herein on February 3, 1997, which is incorporated herein by reference in its entirety. The present application relates to U.S. Provisional Application No. 60 / 037,275. Field of the invention   The invention relates to the field of inhibitors of protein translation, in particular microorganisms and organelles. Translation of proteins in LA. The present invention relates to newly identified polynucleotides. And polypeptides, and their production and use, and their modification. Variants, agonists and antagonists, and their uses. Special In these and other respects, the present invention provides a Glu-tRNA^GlnAmidotransfer New polynucleotides and polypeptides of the Lase family In the detailed text, the following "Glu-tRNA^GlnAdT "or" AdT "). The present invention The same applies to protein translation inhibitors as antibacterial, antifungal and herbicides. Provided are methods and compositions for use in the determination and use. Background of the Invention   Before being incorporated into proteins, amino acids are called transfer RNAs (tRNAs) It is chemically linked to a small RNA molecule. For each of the 20 different amino acids The specific enzyme catalyzes the ligation of the specific tRNA molecule to the 3 'end. Protein raw Although the general mechanism of synthesis (the translation process) is preserved throughout the living world, Glnt RNA^GlnThere are two different pathways for the formation of GlntRNA^GlnTwo paths of formation Is evolutionarily conserved, but it is not yet known why different pathways exist . In the cytoplasm of gram-negative eubacteria and eukaryotic cells, the enzyme glutamini -TRNA synthetase (GlnRS) converts glutamine directly to cognate tRNA. Silylated GlntRNA^GlnI will provide a. Interestingly, GlnRS is Detect by system Not issued. Archaea, Gram-positive eubacteria, mitochondria, and chloroplasts In certain organisms and organelles, GlntRNA^GluAmi, a different pathway of formation The transamidation pathway is functioning (Curnow et al. (1996) Nature 382) : 589-590; Curnow et al. (1997) Proc. Natl. Acad. Sci. USA94 (22): 11819-11826 Sch6n et al. (1988) Biochimie 70 (3): 391-394; Wilcox and Nirenberg (196 1: 187-190). This pathway (shown in FIG. 1)^GlnProducing a glutamyl -tRNA by tRNA synthetase (GluRS)^GlnInitiated by incorrect acylation of . The mischarged tRNA is then replaced by GlutRNA^GlnAmidotransferase (AdT ) By GlntRNA^GlnIs converted to Glutamate is a tRNA^GlnIs covalently bound to Only in cases, AdT catalyzes the amidation of glutamic acid to glutamine. Partial spirit GlutRNA derived from Bacillus megaterium^GlnAmidotransferase activity Is ATP, Mg⁺⁺And GlntRNA in the presence of an amide-nitrogen donor (glutamine)^{G ln} GlutRNA to^GlnHave been shown to effect amidation of (Wilcox and Nirenber g 1968). Subsequent work has shown that in vitro, this amidation can be activated by the activation intermediate (phospho-G lutRNA^Gln(Wilcox (1969) Cold Spring Har b. Symp. Quant. Biol. 34: 521-528; Wlicox (1969) Eur J. Biochem 11 (3): 405. ~ 412). GlutRNA, the first aminoacylation product^GlnIs the wrong protein translation It is correctly charged because it is toxic to cells by the fact that it causes translation. Must be converted to tRNA.   This pathway regulates GlntRNA in these cells.^GlnGlutami is a major source of It appears to be able to function as a regulator of metabolism. Glutamine is the last evolution Has been suggested to be an amino acid formed in Therefore, transamination It is hypothesized that cells performing the pathway produced AdT using the gene encoding GluRS. obtain. Similarly, in cells that act on the direct glutaminylation pathway, the enzyme Gl J. Mol. Evol. 40 (5) 476-481). Both enzymes are tRNA^GlnAnd free glutamate This is reasonable, as it is required to specifically recognize and bind proteins. I However, database searches and, in particular, only those sequenced and published to date One gram-positive organism, the mycoplasma genome (Fraser et al. (1995) Scienc e 270 (5235): 397-403), the sequence information currently available indicates that Gl No significant homology to uRS and GlnRS was shown. Therefore, amidotran Spherases have significant homology to aminoacyl-tRNA synthetases. Maybe not. Undoubted evolution and life in understanding this system Despite its chemical importance, very little research has been done on this enzyme to date. (Wilcox and Nirenberg, 1968; Wilcox, 1969; Strauch et al., (1988 J.). Bacteriol. 170: 916-920; and Jahn (1990) J. Am. Biol. Chem. 265 (14): 805 9-64). Summary of the Invention   The present invention relates, in part, to GlutRNA^GlnGlntRNA from^GlnAdT involved in the generation of Based on the isolation and characterization of a heterotrimeric protein designated as. Departure Ming is also GlutRNA^GlnHomology between the amino acid sequence of AdT and the known amino acid sequence Sex provides polypeptides identified as novel AdT polypeptides.   The invention further provides a polynucleotide encoding an AdT polypeptide. . In particular, the present invention provides a GlutRNA comprising the sequence shown in FIG. 3 (SEQ ID NO: 1).^GlnAdT, Or a variant thereof (eg, a naturally occurring allelic variant of AdT) The polynucleotide sequence, and the polypeptide encoded thereby I do. Thus, the present invention is directed to the AdT polynucleotides, particularly under stringent conditions. And a polynucleotide that hybridizes to the nucleotide sequence.   The present invention relates to the nucleotide sequence of FIG. 3 (SEQ ID NOs: 1, 3, 5 and 7). B. having the amino acid sequence encoded by GlutRNA from subtilis^GlnAdT protein And biologically, diagnostically, prophylactically, clinically, or therapeutically useful modifications thereof Provided are bodies and compositions containing them. Particularly preferred variants are the AdT gene AdT polypeptides encoded by the naturally occurring alleles of E. coli. Above Also provided by the invention is a method of producing the AdT polypeptide of the invention.   The invention also relates to AdT, in particular B.I. subtilis AdT (mRNA, cDNA, and genomic DNA Isolated nucleic acid molecules (biological, diagnostic, prophylactic, clinical, Or therapeutically useful variants thereof) and compositions comprising them. .   According to yet another aspect of the present invention, it is useful as an antibacterial, antifungal, and herbicide. Inhibitors to such AdT polypeptides are provided. Therefore The present invention relates to the identification of agonists and antagonists of the AdT protein. Provided are compositions and methods of use.   The present invention provides compositions and methods for: (i) assessing AdT expression; Treatment of a disease (eg, a disease associated with an excess or deficiency of the amount of available AdT), (iii ) Assessment of genetic variation, and (iv) the AdT polypeptide or polynucleotide, Administration to a cell or multicellular organism to elicit an immune response. This station of the present invention In certain preferred embodiments, antibodies against the AdT polypeptide Is provided.   The present invention also provides compositions and methods for protecting plants, especially crop plants. I do. For example, the invention provides AdT antagonists, useful as herbicides. Also provided are herbicidal compositions comprising such inhibitors of AdT. The present invention It is also resistant to inhibition from certain herbicides, especially those containing inhibitors of AdT A non-inhibitory mutant of AdT and a functional derivative thereof. Non-inhibited Polynucleotides encoding sex AdT are placed in plants by various transformation methods And thereby render the plant resistant to certain herbicides, including inhibitors of AdT. Gender or resistance. Therefore, transgenes containing non-inhibitory variants of AdT How to treat weeds by applying AdT inhibitors to nick plants Also included in the present invention.   Various changes and modifications within the spirit and scope of the disclosed invention will be apparent from the following description. By reading and by reading the other parts disclosed herein, one of ordinary skill in the art will appreciate It will be obvious. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows GlntRNA^Gln1 shows the transamination pathway for the formation of. FIG. 2 shows the gene arrangement of the AdT gene. FIG. 1 shows the nucleic acid sequence of the AdT protein from subtilis. Description of the invention I. General description   The present invention relates, in part, to heterotrimers, which cause GlntRNA in cells. Based on the identification and characterization of the protein (herein AdT protein) Follow. The invention particularly relates to three subunits of AdT protein isolated from subtilis The amino acid sequence of each knit, and the nucleoside encoding the AdT protein Provide the tide sequence. AdT protein molecules and AdT nucleic acid molecules are used for protein synthesis. To identify drugs used to inhibit steroids, especially antibacterial, antifungal, and herbicides Can serve as a target in the method. II. Specific embodiments A. AdT protein   Prior to the present invention, GlutRNA was used in the art.^GlnGlntRNA^GlnInvolved in the transition to It was taught that the enzyme was present. However, it is not responsible for the production of GlntRNA. The isolation and characterization of the protein remained unknown. The present invention Is B. subtilis Provides the amino acid sequence of the three subunits of the AdT protein You. Quite unexpectedly, this AdT protein is a heterotrimeric protein Something has been found.   In one embodiment, a cloned nucleic acid component as described herein By using an amino acid sequence or by using a protein having the amino acid sequence disclosed herein. By synthesizing proteins, the present invention produces previously unknown proteins Provide the ability to   As used herein, AdT protein refers to the polynucleotide of FIG. B. a protein having the amino acid sequence of subtilis AdT; Allele variants, and conservative substitutions thereof having AdT activity. AdT Tan The protein is composed of three subunits: A subunit (SEQ ID NO: 4), B Subunit (SEQ ID NO: 6) and C subunit (SEQ ID NO: 8) (in the present specification) So, collectively, the aAdT subunit, bAdT subunit, and cAdT subunit Unit). For convenience, the collective subunit is referred to as the AdT protein, or It is called the AdT protein of the present invention. A single subunit is mentioned in context Or the aggregate protein is referred to by those skilled in the art. I can do it.   The polypeptide of the present invention is a polypeptide encoded by SEQ ID NO: 1 (FIG. 3). And the biological activity of polypeptides and fragments, especially AdT. Polypeptides and fragments, and also SEQ ID NO: 1 or related parts At least 70% sequence identity to the polypeptide encoded by the At least 80% identity to the polypeptide encoded by SEQ ID NO: 1, and More preferably at least 90% of the polypeptide encoded by SEQ ID NO: 1 Similarity (more preferably at least 90% identity), and even more preferably Has at least 95% similarity to the polypeptide encoded by SEQ ID NO: 1. More preferably polypeptides and flags having at least 95% identity) And portions of such polypeptides as well.   The AdT proteins of the present invention are described herein, and in particular, identified and characterized. Performing undue experimentation in accordance with the modified variants and the methods outlined below Allelic variants, conservative substitution mutations that can be isolated / produced and characterized Includes body and homologs. For convenience, all AdT proteins are collectively It is referred to as protein or the AdT protein of the present invention.   The term “AdT protein” is used to describe B. elegans having normal AdT activity. subtilis AdT tampa Includes all naturally occurring allelic variants of protein. Generally, AdT Tan An allelic variant of a protein is a protein specifically encoded by SEQ ID NO: 1. It has a slightly different amino acid sequence compared to protein, but converts GlutRNA to GlntRNA. Can be replaced. Allelic variants are those that differ slightly from the amino acid sequence described above. No acid sequence, but has the ability to produce GlntRNA. Typically, AdT tamper An allelic variant of a protein is a conservative amino acid substitution in the AdT sequence described herein. Or the AdT homolog (B. AdT isolated from organisms other than subtilis Amino acid substitution at the corresponding position of the protein).   The AdT protein of the present invention is preferably in an isolated form. In this specification As used, usually using physical, mechanical, or chemical methods, When the AdT protein is removed from cellular components associated with The quality is said to be isolated. One of ordinary skill in the art will be able to use standard methods to obtain an isolated AdT protein. A simple purification method can be easily used. One purification scheme is outlined in Example 1. single The nature and extent of the release will depend on the intended use.   Cloning of a nucleic acid molecule encoding AdT defines the AdT protein of the present invention. Allows the generation of fragment fragments. As discussed below, the AdT of the present invention Protein fragments are used to generate subunit specific antibodies, AdT protein Identification of agents that bind to B. For isolation of homologs of subtilis AdT protein This is particularly useful.   Fragments of the AdT protein can be obtained using standard peptide synthesis techniques and herein. Can be generated using the amino acid sequences disclosed in US Pat. Alternatively, if the recombination method is Used to generate nucleic acid molecules encoding fragments of the AdT protein obtain.   Fragments of the AdT protein subunit containing particularly interesting structures Genogenicity analysis, Chou-Fasman analysis, Garnier-Robson analysis, Kyte-Doolittle analysis, Eisen A field such as berg analysis, Karplus-Schultz analysis, or Jameson-Wolf analysis Can be identified using known methods. Fragments containing such residues are It is particularly useful for generating anti-AdT antibodies specific to buunit.   As discussed below, AdT family member proteins are useful for: The purpose of use is not limited thereto: 1) blocking AdT activity. Target for the identification of drugs that block or stimulate, 2) bind the AdT protein Target or decoy for the identification and isolation of binding partners 3) AdT protein Identification of drugs that block or stimulate the activity of proteins, and 4) Targets for the identification of mediated activity or disease. B. Anti-AdT antibody   The invention further relates to one or more AdT proteins of the invention, or an AdT protein of the invention. Antibodies that selectively bind to specific subunits of protein are provided. Most preferred Antibodies bind to the complete heterotrimeric AdT protein but are isolated from subunits. Does not bind to the knit or binds to the isolated subunit, but Either does not bind to the established trimer protein. Specific intent Anti-AdT antibodies include monoclonal and polyclonal antibodies, Fragments comprising the native binding domain and / or one or more complements of these antibodies Includes a foot determination area.   Antibodies are generally isolated or immunocomplexed in the form of an AdT protein, or Prepared by immunizing a suitable mammalian host with (Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). Immunogenicity Regions of the AdT protein that show structural integrity are readily identified using methods known to those of skill in the art. Can be Other important regions and domains can be identified by protein analysis methods known to those skilled in the art. And can be easily identified using protein comparison methods.   Fragments containing these residues may produce specific classes of anti-AdT antibodies. Particularly suitable for and.   Methods and carriers for preparing proteins for use as immunogens -(Eg, BSA, KLH, or other carrier protein) and protein Methods for preparing epidemiological conjugates are well known to those skilled in the art. In some situations For example, direct conjugation using carbodiimide reagents can be used; In the examples, ligation reagents (eg, Pierce Chemical Co. Offered by Rockford, IL Can be useful.   Administration of the AdT antigen generally involves injection over an appropriate period of time and And the use of a suitable adjuvant as generally understood in US Pat. Immunity During the optimization schedule, antibody titers are measured to determine the adequacy of antibody formation. obtain.   The polyclonal antiserum produced by this method has several applications, pharmaceutical compositions Although it may be sufficient for the product, monoclonal antibody preparations are preferred. Desired Immortalized cell lines that secrete monoclonal antibodies are based on Kohler and Milstein standards. Method or immortalization of lymphocytes or spleen cells as generally known Can be prepared using modifications that result in Immortalization secreting the desired antibody Cell lines are immunoassays where the antigen is an AdT protein or peptide fragment. Screened by b. Appropriate immortalized cell culture secreting the desired antibody If nutrients are identified, the cells can be produced in vitro or in ascites fluid. It can be cultured either raw.   The desired monoclonal antibody is then recovered from the culture supernatant or from the ascites supernatant. It is. Monoclonal or polyclonal blood containing immunologically important parts Clear fragments can be used as antagonists and whole antibodies. Exemption Use of epidemiologically reactive fragments (eg, Fab, Fab 'of F (ab') 2 fragment) Are often preferred, especially in therapeutic settings. Because these flags Because they are generally less immunogenic than whole immunoglobulins.   Antibodies or fragments can also be produced using current techniques by recombinant means. I can do it. Regions that specifically bind to the desired region of the transporter may also be multi-occurring. In the context of a source chimeric or CDR-grafted antibody, it can be produced.   As described below, anti-AdT antibodies are useful as modulators of AdT activity. , An immunoassay to determine AdT expression / activity and B.I. subtilis AdT protein Useful for purifying quality homologs. C. AdT-encoding nucleic acid molecule   As described above, the present invention relates, in part, to the three subunits of the AdT protein. B. Based on isolating nucleic acid molecules from subtilis. Therefore, The present invention further relates to an AdT protein (preferably simply) as defined herein. Isolated form). For convenience, code all AdTs The nucleic acid molecule to be loaded is identified as a nucleic acid molecule encoding AdT, the AdT gene, or AdT. Done. B. encoding each AdT subunit subtilis nucleic acid molecule The otide sequence is provided in SEQ ID NO: 1. Each subunit A (SEQ ID NO: 4) , Subunit B (SEQ ID NO: 6) and subunit C (SEQ ID NO: 8) All start and stop codons are the nucleic acids for AdT provided in FIG. It is shown in the otide sequence.   A further preferred embodiment of the present invention relates to an amino acid encoded by SEQ ID NO: 1. Over the entire length of the polynucleotide encoding the AdT polypeptide having the sequence. A polynucleotide that is at least 70% identical in sequence, and A polynucleotide that is complementary to a nucleotide. Or the most highly preferred The polynucleotide is a polynucleotide encoding an AdT polypeptide; and Regions that are at least 80% identical over their entire length to their complements It is a polynucleotide containing. In this regard, the full length of the same polynucleotide Polynucleotides that are at least 90% identical across are particularly preferred. And Among these particularly preferred polynucleotides, those polynucleotides that are at least 95% identical Nucleotides are particularly preferred. In addition, polynucleotides that are at least 97% identical Otides are highly preferred among polynucleotides that are at least 95% identical, And polynucleotides that are at least 98% and at least 99% identical in these Otides are particularly highly preferred, and polynucleotides that are at least 99% identical are Is more preferable.   The present invention further provides for the modification of a polypeptide having the deduced amino acid sequence of SEQ ID NO: 1. It relates to variants of the polynucleotides described herein which encode the body.   Variants that are fragments of the polypeptides of the present invention may be modified by peptide synthesis. These modifications can be used to produce the corresponding full-length polypeptides; The body can be used as an intermediate to produce a full-length polypeptide. The present invention A variant which is a fragment of the polynucleotide of the present invention is a full-length polynucleotide of the present invention. It can be used to synthesize otide. Fragment of the polynucleotide of the present invention Variant is used to synthesize the full-length polynucleotide of the invention. obtain. Such methods are widely available (eg, WO 97/26340 and WO 97/26340). 97/38716).   A fragment is identical to part of the amino acid sequence of a polypeptide described above. Is a variant polypeptide having an amino acid sequence that is not the same as all. AdT A polypeptide fragment can be "free-standing" or Or larger polypeptides in which the AdT polypeptide forms part or region And most preferably a single continuous region, a single larger polypeptide As do.   A more particularly preferred embodiment is SEQ ID NO: 1 (where substituted, deleted Or, the amino acid to be added is any number, several, 10-15, 5- 10, 1-5, 1-3, 2, 1, or none) A polynucleotide encoding an AdT variant having the amino acid sequence of the peptide. You. Particularly preferred among these are silent substitutions, additions and deletions. Do not alter the properties and activities of AdT.   As used herein, a “nucleic acid molecule” is a paper as defined above. It is defined as an RNA or DNA molecule that encodes a peptide, or Complementary to the nucleic acid sequence encoding the peptide. Particularly preferred nucleic acid molecules are described herein. B. disclosed in this document. nucleotide sequence identical or complementary to the subtilis DNA sequence With columns. Particularly contemplated nucleic acid molecules include genomic DNA, polycistronic mRNA and And antisense molecules, as well as alternative backbone-based or alternative bases (Whether derived from natural sources or synthesized). Only However, such nucleic acid molecules are described in Non-AdT proteins isolated from organisms other than subtilis Novel and non-obvious to any prior art nucleic acid molecule that encodes a protein Is further defined as   As used herein, a nucleic acid molecule encodes a polypeptide other than AdT A nucleic acid molecule is "isolated" when it is substantially separated from contaminating nucleic acid molecules. Be told. One skilled in the art can use nucleic acid isolation to obtain isolated AdT-encoding nucleic acid molecules. The procedure can be used easily.   The present invention further provides fragments of the AdT-encoding nucleic acid molecules of the invention. You. As used herein, a fragment of a nucleic acid molecule encoding AdT Refers to a small portion of the sequence that encodes the entire protein. Of the fragment Size is determined by its intended use. For example, this fragment Active portion of AdT protein (eg, active domain or effector binding site) This fragment, if selected to encode the It needs to be made large enough to code the potential region. Fragment If the sample is to be used as a nucleic acid probe or PCR primer, Fragment length should be such that relatively few false positives are obtained during probing / priming. Is selected. Particularly useful as a selective hybridization probe or PCR B. A fragment of a subtilis AdT nucleic acid molecule can be prepared by methods known in the art. It can be easily determined using.   Genetics for polymerase chain reaction (PCR) or encoding AdT protein The present invention is used as a probe or a specific primer to synthesize a nucleotide sequence. Flag of nucleic acid molecule encoding light AdT (ie, synthetic oligonucleotide) Are described by chemical techniques (eg, Matteucci et al., J Am Chem Soc (1981) 103: 3185. -3191 phosphotriester method) or automated synthesis method It can be easily synthesized. In addition, larger DNA segments can be prepared by well-known methods (eg, , A group of oligonucleotides that define various modular segments of the AdT gene Oligonucleotides for synthesis followed by assembly of the fully modified AdT gene Can be easily prepared.   Nucleic acid molecules encoding AdT of the present invention may be used for diagnostic and probe purposes. It can be further modified to include a detectable label. As mentioned above, such a Probes encode other allelic variants or homologues of the AdT protein Can be used to identify nucleic acid molecules. And as described below Such probes are useful for diagnosing pathological conditions caused by AdT-mediated translation. As a method, it can be used to diagnose the presence of the AdT protein. A variety of this Such labels are known to those of skill in the art and may encode the AdT described herein. Can be used easily with children. Suitable labels are biotin, radiolabeled nucleotides, Including, but not limited to, biotin. One skilled in the art can Any art-known label may be used to obtain the nucleic acid molecule to be loaded. D. Isolation of other AdT-encoding nucleic acid molecules   B. Subtilis-derived AdT proteins and their corresponding nucleic acid molecules have been identified in B. sub It enables the identification and isolation of AdT proteins from organisms other than tilis, Collectively hereinafter referred to as AdT homologs. The preferred source of the AdT homolog is bacterial And pathogenic microorganisms such as fungi, and plants where controlled growth is desired is there. The most preferred sources are gram-positive bacteria, pathogenic fungi and plant organelles. LA (for example, chloroplast).   In essence, those skilled in the art will easy use of the amino acid sequence of the subtilis AdT protein To generate antibody probes and to screen expression libraries prepared from cells. Can learn. Typically, a mouse immunized with a purified protein (as described below) Use polyclonal antiserum or monoclonal antibodies from mammals such as herons Probe the expression library prepared from the target organism, Appropriate coding sequences for quality homologs can be obtained. This cloned cD The NA sequence can be expressed as a fusion protein or directly using its own regulatory sequences. Suitable for the particular host used to express the enzyme or to express the enzyme It can be expressed by constructing an expression cassette using control sequences.   Alternatively, a portion of the AdT coding sequence described herein can be synthesized and . Encodes a member of the AdT family of proteins from organisms other than subtilis It can be used as a probe to recover DNA. Contains about 18-20 nucleotides Oligomers (encoding about 6-7 amino acid stretches) are prepared and Screen genomic DNA or cDNA libraries using Sufficient stringency to eliminate transient conditions or excessive levels of false positives Hybridization is obtained under mild conditions. This method is Can be used to identify and isolate alternatives or variants of the nucleotide sequence.   Further, the pair of oligonucleotide primers may be an AdT-encoding nucleic acid molecule or Polymerase chain reaction to selectively amplify / clone the fragment (PCR). To use such PCR primers PCR denaturation / annealing / extension cycles are well known in the art, and other AdT Can be readily adapted for use in isolating nucleic acid nucleic acid molecules. Probe or ply B. Particularly suitable for use as a mer The region of the subtilis AdT gene is easily identified. Can be Generally, preferred primers are B. subtilis AdT gene Adjacent to the subunit code area.   The homologs of the AdT proteins disclosed herein share homology. General In addition, the nucleic acid molecule encoding the AdT homolog can be converted to B under highly stringent conditions. . Hybridizes with the subtilis sequence. Such sequences are typically B. subtili s At least 70% homology to the sequence, preferably at least 80% homology, most Also preferably contain at least 90% homology. E. FIG. Recombinant DNA molecules containing AdT-encoding nucleic acid molecules   The present invention further provides a recombinant D comprising one or more of the AdT coding sequences described herein. Provided are NA molecules, or fragments of the nucleic acid molecules described herein. This specification As used herein, recombinant DNA molecules are subjected to molecular manipulation in vitro. DNA molecule. Methods for producing recombinant DNA molecules are well known in the art and include, for example, See, eg, Sambrook et al., Molecular Cloning (1989). Preferred recombinant DNA content An AdT-encoding DNA sequence encoding an AdT protein or an AdT subunit; The sequence is operably linked to one or more expression control sequences and / or vector sequences. It is. Can a recombinant DNA molecule encode a single subunit of the AdT protein? Or an operon containing all three AdT subunits.   Vectors and / or genes into which one of the AdT coding sequences of the invention is operably linked. The choice of the current control sequence depends on the desired functional property (e.g., Protein expression) as well as directly on the host cell to be transformed. Intended by the present invention The vector to be replicated contains a copy of the AdT coding sequence contained in the recombinant DNA molecule, or It is at least possible to direct the insertion into the chromosome, and preferably it is Can also be directed.   Used to regulate the expression of an operably linked protein coding sequence Expression control elements are known in the art and include inducible promoters. Promoter, secretory signal, enhancer, transcription terminator, And other regulatory elements, including but not limited to Easily regulated inducible promoters, e.g., that respond to nutrients in the host cell's medium. Such a promoter is used.   In one embodiment, the vector comprising the AdT-encoding nucleic acid molecule is a prokaryotic replica. A prokaryotic host cell (e.g., a bacterial cell) transformed with the DNA sequence. The autonomous replication and maintenance of the recombinant DNA molecule within the chromosome Contains DNA sequence. Such replicons are well-known in the art. Furthermore, prokaryotic Vectors containing biological replicons also contain a marker (eg, (Eg, drug resistance). Typical bacterial drug resistance genes are This gene confers resistance to ampicillin or tetracycline.   Vectors containing prokaryotic replicons can also be used in bacterial host cells (e.g., E. coli). coli) Prokaryotic or viral that can direct the expression (transcription and translation) of the AdT coding sequence It may further include a promoter. The promoter is the promoter formed by the DNA sequence. Current regulatory elements, which cause RNA polymerase binding and transcription You. A promoter sequence compatible with the bacterial host is typically a DNA segment of the invention. Provided in a plasmid vector that contains convenient restriction sites for insertion of the plasmid. This Typical of vector plasmids such as BioRad Laboratories (Richimond, CA) PUC8, pUC9, pBR322 and pBR329, Pharmacia, Piscataway, NJ available from PPL and pKK233 available from the company.   Expression vectors suitable for eukaryotic cells, preferably those suitable for vertebrate cells. Can also be used for variant recombinant DNA molecules containing the AdT coding sequence . Eukaryotic cell expression vectors are well known in the art, and Available from sources. Typically, for insertion of a desired DNA segment Vectors are provided that contain convenient restriction sites. Typical of such vectors Are PSVL and pKSV-10 (Pharmacia), pBPV-1 / pML2d (International Biotechnol ogies, Inc. ), PTDTI (ATCC, # 31255), such as the vector pCDM8 described herein. It is a eukaryotic expression vector.   Eukaryotic cell expression vectors used to construct the recombinant DNA molecules of the present invention are furthermore In addition, a selection marker effective in eukaryotic cells, preferably a drug resistance selection marker May be included. Preferred drug resistance markers are those that show neomycin resistance when expressed. The gene is the neomycin phosphotransferase (neo) gene. Sou thern et al., J. Mol Anal Genet (1982) 1: 327-341. Alternatively, the selectable marker is The two vectors may be present on a plasmid, and these two vectors Of the appropriate drug, introduced by It is selected by culturing in the presence. F. Host cells containing exogenously recruited AdT-encoding nucleic acid molecules   The present invention further provides a heterotrimeric protein of the AdT protein of the present invention or A host transformed with a nucleic acid molecule encoding any one or more of the subunits Provide cells. Host cells can be either prokaryotic or eukaryotic. Ad Eukaryotic cells useful for the expression of T proteins are those whose cell line is compatible with the culture method, As long as it is compatible with the growth of the expression vector and the expression of the AdT gene, No. Preferred eukaryotic host cells are yeast, insect and mammalian cells, preferably spinal Vertebrate cells (e.g., cells from a mouse, rat, monkey or human fibroblast cell line) However, it is not limited to these, and most preferably AdT protein is naturally used. Cells that do not express.   Any prokaryotic host can be used for expression of the AdT-encoding recombinant DNA molecule. Preferred The prokaryotic host is E. coli.   Transformation of a suitable cell host with a recombinant DNA molecule of the present invention is accomplished by well known methods. This is typically achieved by determining the type of vector used and the Depends on the host system. For transformation of prokaryotic host cells, electroporation And salt treatment methods are typically used. For example, Cohen et al., Proc Acad Sci  USA (1972) 69: 2110; and Maniatis et al., Molecular Cloning, A Laboratory Manu. al, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). thing. For transformation of vertebrate cells with vectors containing recombinant DNA, see Lectroporation, cationic lipid or salt treatment methods are typically used. For example, Graham et al., Virol (1973) 52: 456; Wigler et al., Proc Natl Acad Sci USA (197 9) See 76: 1373-76.   Successfully transformed cells, that is, cells containing a recombinant DNA molecule of the present invention, Can be identified by well-known techniques. For example, the result of introducing the recombinant DNA of the present invention The resulting cells are cloned to give a single colony. These colonies The cells of origin can be collected, lysed, and their DNA content determined for the presence of recombinant DNA. Southen, J Mol Biol (1975) 98: 503, or Berent et al., Biotech (1985) 3:20. The protein can be tested using methods such as those described in Quality can be assayed by immunological methods. G. FIG. Production of AdT protein using recombinant DNA molecules   The invention further provides a method for the production of an AdT protein, the method comprising: One of the AdT-encoding nucleic acid molecules described herein is used. In general terms, recombinant Ad Production of a T protein typically involves the following steps.   First, a nucleic acid molecule encoding an AdT protein or AdT subunit, for example, The nucleic acid molecule shown in FIG. 3 (SEQ ID NO: 1) is obtained. Then, an AdT-encoding nucleic acid molecule is preferred. Alternatively, as described above, the AdT can be placed in operable linkage with a suitable control sequence. Generate an expression unit containing the coding sequence. A suitable inn using this expression unit Conditions for transforming the host and producing the transformed host for AdT protein Incubate underneath. Optionally, isolating AdT protein from the medium or cells; The recovery and purification of carbonaceous material may be useful in some instances where some impurities can be tolerated. It is not necessary.   Each of the preceding steps can be performed in various ways. For example, the desired coding sequence And can be used directly in a suitable host. Various Construction of an expression vector operable in a host involves the appropriate combination of replicons and control sequences. Achieved using combination. Control sequences, expression vectors, and transformation methods Depending on the type of host cell used to express the gene, and Detailed. Excisable genes are provided for insertion into these vectors. Appropriate restriction sites (if not obtained correctly) It can be added to the end of a row. One skilled in the art will recognize that the AdT protein can be used with the AdT coding sequence. Any host / expression system known in the art can be readily adapted to produce quality . H. Identification of drugs that bind to AdT protein   Another embodiment of the present invention relates to an AdT protein agonist or agonist as described herein. Or methods for identifying agents that are or antagonists. In particular, AdT protein agonists and antagonists And / or the ability to inhibit AdT activity. A Activity assays for dT activity and binding assays using the AdT protein Suitable for use in high throughput screening methods.   In particular, in one embodiment, the AdT protein is mixed with an agent. Ad After mixing under conditions that allow the T protein to associate with the drug, the mixture is separated. To determine if the drug is associated with the AdT protein. Agonist And antagonists are identified as being able to bind to the AdT protein. Alternatively or continuously, as described below, the AdT activity may be an agonism of AdT activity. Can be evaluated directly as a means to identify the target and antagonist.   The AdT protein used in the above assay may be: And a fully characterized protein, a single subunit of the AdT protein, A partially purified protein, a cell modified to express the AdT protein Vesicles or fractions of cells that have been modified to express the AdT protein. In addition, AdT The protein can be the entire AdT protein, a specific fragment of the AdT protein, or It can be a single subunit of the AdT protein. AdT protein for drug binding Then, as described in the Examples, for example, by changes in molecular weight or activity, It will be clear to those skilled in the art that the assays of the present invention can be used as long as You. AdT proteins are particularly suitable for high-throughput screening methods.   The source of the AdT protein is based on the intended use of the modulator. For example, microorganism A dT protein is used to identify AdT inhibitors with bacterial activity On the other hand, the chloroplast-derived AdT protein is the same as an AdT inhibitor having herbicidal activity. Used to determine   The method used to identify whether a drug binds to the AdT protein The method is mainly based on the nature of the AdT protein used. For example, gel delay Say determines if drug binds to soluble fragment of AdT protein Can be used to Alternatively, immunodetection and biochip technology are It can be employed for use with proteins. One skilled in the art will recognize that certain drugs may be Many techniques known in the art for determining whether to bind to a protein are available. Easy to use.   The drug also provides a cell-free assay for the ability to modulate the activity of the AdT protein Systems or cell assay systems. Example 1 describes an AdT activity One such method is provided that can be used in assays.   As used herein, a drug refers to a drug that reduces AdT activity It is said that AdT activity is antagonized. A preferred antagonist is A Selectively antagonizes dT without affecting any other cellular proteins Does not affect other proteins involved in translation, in particular. Furthermore, the preferred en Tagonists reduce AdT activity by more than 50%, and more preferably by more than 90%. And most preferably remove all AdT activity.   As used herein, an agent means that it increases AdT activity. Is said to agonize the AdT activity when Preferred agonists are Increase ADT activity by more than 50%, more preferably by more than 90%, Preferably, the AdT activity is increased more than 2-fold.   Preferred antagonists and agonists include specific species, genera, families, orders, Selective to the world. To help identify drugs for target specificity, Drugs are screened using one AdT protein or a combination of AdT proteins. Can be trained. For example, using several different microbial AdT proteins, Common antimicrobial agents can be identified, while removal using chloroplast-derived AdT protein. Herbicides can be identified.   Drugs assayed in the above method can be selected randomly or can be rationally selected. Can be selected or designed. As used herein, a drug refers to the drug When the agent is randomly selected without considering the specific sequence of the AdT protein Is said to be randomly selected. An example of a randomly selected drug is Use library or peptide combinatorial libraries, or Or a growth broth of a plant extract.   As used herein, a drug refers to the sequence or sequence of a target site. And / or non-random considering its conformation in relation to the activity of the drug It is said that if selected based on appropriate criteria, it is rationally selected or designed. medicine Agents are rationally selected or synthesized using the peptide sequences that make up the AdT protein. Can be designed logically. For example, a rationally selected peptide agent may have its amino acid It may be a peptide whose sequence is identical to a fragment of the AdT protein.   Agents of the invention include, for example, peptides, small molecules, and vitamin derivatives, and It can be a carbohydrate. One skilled in the art will recognize that the agents of the present invention have limitations with respect to structural properties. You can easily recognize that there is nothing. One class of drugs of the invention is peptide drugs. Therefore, the amino acid sequence was selected based on the amino acid sequence of the AdT protein. Things. Small peptide agents are competitive inhibitors of AdT protein construction Can work.   The peptide agent of the present invention may be a standard solid phase (or solution phase) as is known in the art. It can be prepared using peptide synthesis methods. In addition to these peptides, DNA to be loaded can be synthesized using a commercially available oligonucleotide synthesizer, and It can be produced recombinantly using standard recombinant production systems. Not coded by gene If amino acids are to be included, production using solid phase peptide synthesis is required. You.   Another class of agents of the invention is the critical position of the AdT protein. Antibodies that are immunoreactive with As noted above, antibodies can be used to target a suitable mammalian subject. An antigenic region that is part of the AdT protein that is intended to be targeted by the antibody Obtained by immunization with a peptide containing the region. Important areas are shown in Figure 2. Including domains that are Such drugs can be used in competitive binding studies to generate second generation inhibition An agent can be identified. K. Use of drugs that bind to AdT protein   As shown in the background section, AdT protein is protein-translated, especially Gram-positive Involved in protein translation of microbial, fungal and cellular organelles, especially chloroplasts. Binds to AdT protein and acts as an agonist or antagonist Different drugs can be used to regulate translation in these organisms, and It can act as a fungicide or as a main component of a herbicide. For details, the AdT protein is required. Required protein translation is binding to AdT protein and an agonist of AdT activity Or by administering an agent that acts as an antagonist to the organism. Can be   As used herein, an organism refers to protein translation in that organism. Modulation (e.g., controlling the growth of an infectious agent in a mammalian subject, or May be any organism as long as the desired action is desired. The present invention provides for the growth of microorganisms. It is particularly useful for treating human subjects for control.   As used herein, protein translation requiring AdT is defined as active A Refers to protein translation that does not occur in the absence of dT protein. In this specification When used in certain applications, some drugs reduce the extent of protein translation. If so, it is said to regulate AdT-mediated protein translation.   The use of AdT modulators is primarily due to the target AdT protein used to identify the drug. Quality, as well as the activity / selectivity of the drug. For example, an AdT inhibitor (this is (Used as a biological agent) is preferably one or more microbial AdT proteins. Isolated by using The herbicide preferably targets the chloroplast AdT protein Identified using L. Administration of AdT protein agonists and antagonists   The administration of AdT protein agonists and antagonists is Depends on For example, for the regulation of microbial growth in a mammalian subject, AdT Inhibitors may be administered by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. It can be administered throughout. Alternatively or simultaneously, administration is by the oral route. Can get. The dose administered will depend on the age, health, and weight of the recipient, If any, it depends on the type, frequency of treatment, and the nature of the effect desired. For example, for the treatment of a microbial infection, an agent that modulates AdT activity is treated It is administered systemically or locally to the individual. As described below, such drugs There are a number of ways that can be readily adapted for the administration of   The present invention further relates to the AdT protein identified by the methods described herein. Compositions comprising an antagonist or agonist are provided. Effective amount of each component Determination of the appropriate area is within the skill of the art, and is based on the intended use.   In addition to the AdT modulator, the compositions of the present invention may contain other ingredients, for example, a suitable pharmaceutically acceptable agent. Can include a carrier that can be acceptable, including excipients and adjuvants; Facilitates processing of the compound into a formulation, which can be delivered to the site of action Can be used pharmaceutically. Suitable formulations for parenteral administration include water-soluble variants, For example, aqueous solutions of the active compounds in water-soluble salts are included. In addition to this, the active compound And, if appropriate, oily injection suspensions. Appropriate The lipophilic solvent or vehicle can be a fatty oil, such as sesame oil, or a synthetic fatty acid ester. And tere, for example, ethyl oleate or triglycerides. Aqueous injection Suspensions can contain substances that increase the viscosity of the suspension, such as, for example, carboxy. Sodium methylcellulose, sorbitol, and / or dintr an). If desired, the suspension may also contain stabilizers. Liposomes also It can be used to encapsulate drugs for delivery into cells.   Pharmaceutical formulations for systemic administration according to the present invention may be enteral, parenteral or topical. May be formulated for local administration. In fact, all three types of these formulations are It can be used simultaneously to achieve systemic administration of the active ingredient.   Suitable formulations for oral administration include hard gelatin capsules or soft gelatin capsules. Pills, pills, tablets (including coated tablets), elixirs, suspensions, syrups or Inhalants, and controlled release variants thereof. M. Combination therapy   An agent of the invention that modulates AdT activity can be provided alone, or It can be used in combination with synthetic microorganisms, fungi or other agents that regulate plant growth. For example, The agent of the present invention that reduces microbial AdT activity is administered in combination with other antimicrobial agents. obtain. As used herein, two drugs are the same when the two drugs are the same. Sometimes administered separately or in such a way that the drugs act simultaneously When administered in combination, it is said to be administered in combination. N. Methods for identifying the presence of an AdT protein or gene   The present invention further provides cells or organisms that express the AdT protein or the AdT gene. A method for identifying is provided. Such a method expresses the AdT protein It can be used to diagnose the presence of an organism. The method of the present invention can be used to detect the presence of It is particularly useful for determining Specifically, the presence of the AdT protein Determine if nucleic acid encoding protein or AdT protein is expressed Can be identified by AdT protein expression depends on AdT-mediated translation Used to diagnose the presence of an organism.   Determine if AdT protein is expressed / produced in a particular cell or sample Various immunological and molecular genetic techniques can be used to determine. General Next, an extract containing nucleic acid molecules or an extract containing proteins is prepared. Next Assay the extract of AdT protein or AdT-encoding nucleic acid molecule in cells. Determine if it is produced.   For example, in order to make a diagnosis based on nucleic acid molecules, an appropriate nucleic acid sample is used in the prior art. And prepared using DNA, for example, simply boil the sample in SDS It can be prepared by boiling. The extracted nucleic acid is then, for example, standard The AdT-encoding nucleic acid can be subjected to amplification using the polymerase chain reaction (PCR) The offspring or fragments thereof are selectively amplified. Specific amplified fragment Gel size or presence (typically after restriction endonuclease cleavage) Determined by electrophoresis or determine nucleotide sequence of fragment (Eg, Weber and May Am J. et al. Hum Genet (1989) 44: 388-339; Davis, J. et al. Et al., Nature (194) 371: 130-136). Next, the resulting fragment The size or sequence of the fragment, e.g., using a hybridization probe, Compare to known AdT protein coding sequence. Using this method, the AdT protein Can be identified.   Proper protein samples have traditionally been used to perform protein-based diagnostic tests. Obtained and prepared using techniques. Protein samples can be Prepared by simply mixing the protein with SDS and then salting out the protein fraction. Can be The extracted protein is then analyzed and the AdT protein is identified using known methods. Determine the presence of the substrate. For example, a variant of a protein of a particular size or charge Can be identified using mobility in an electric field. Alternatively, antibodies for detection purposes Can be used. One of skill in the art will determine whether the sample contains the AdT protein. Known protein analysis methods can be readily adapted to determine.   Alternatively, AdT expression also identifies agents that reduce the level of expression of the AdT gene. Method. For example, cells or groups that express the AdT protein The tissue can be contacted with a test agent to determine the effect of that agent on AdT expression. AdT An agent that activates expression can be used as an agonist of AdT activity, while Agents that reduce the activity can be used as antagonists of AdT activity. O. Preparation and use of herbicides   As discussed herein, the transamination pathway is effective in chloroplasts. Ad The ability to identify AdT inhibitors that specifically inhibit the plastid isoform of T Can be useful in designing herbicides that are not toxic or dangerous to humans and animals . Therefore, it inhibits only the chloroplast isoform of the enzyme (e.g. In other words, the herbicide that does not inhibit AdT nor human AdT in the cytoplasmic fluid except organelles) Is a new form of herbicide that is highly effective and less toxic to humans I will provide a. However, Adt inhibitors, not limited to chloroplasts, are also herbicides. Utility in all uses.   Identified compounds that inhibit the function of the wild-type AdT enzyme are identified as active ingredients in herbicides. Used. The active ingredient is usually combined with one or more agriculturally acceptable carriers. Applied in the form of a product and produced simultaneously or sequentially with further compounds. It can be applied to object areas or plants to be treated. These additional compounds are fertilizers , Other herbicides, fungicides, fungicides, nematocides, or any of these preparations Some mixtures may be combined with additional carriers, surfactants or application-enhancing adjuvants May be included. Herbicide is applied as seed coating, spraying on the ground It can be obtained, incorporated into the soil, or applied directly to plants. Preferably, An agricultural chemical containing at least one of the active ingredient of the present invention or the active ingredient of the present invention. The composition is applied as a leaf preapration. Preparation of herbicides and Methods of application and application are well known to those skilled in the art.   A mutant resistant to an AdT inhibitor compound mutagenizes a cell or organism, and The mutagenized population is at a concentration sufficient to inhibit the growth of wild-type cells or organisms. Grow in the presence of inhibitors and grow faster than wild-type cells or organisms The cells or organisms that can be propagated can be identified by selecting from the population. Mutation induction The departure may be accomplished by any one of the means well known to those skilled in the art, including : Chemical mutagenesis (eg, ethyl methanesulfonate); UV irradiation; X-ray exposure; And gamma-irradiation (eg, Waston et al., Recombinant DNA, 2nd edition (1992) (See Chapter 11: 191-211; Freifelder, Molecular Biology (1987) Chapter 11; 293-313) . Ability to tolerate or resist normal levels of toxicity of the inhibitor The mutant individual having the mutant AdT is genetically purified, and the gene encoding the mutant AdT is isolated. To determine the DNA sequence of the mutant gene and translate it into the predicted amino acid sequence . Amino acids that differ between the wild-type and mutant AdT enzymes are It is presumed to be responsible for the variant inhibitor resistance phenotype.   The coding DNA sequence for a mutated AdT can be prepared by many different methods well known to those skilled in the art. It can be introduced into plant cells. Examples of such methods include microinjection , Electroporation, Agrobacterium-mediated transformation, direct inheritance Child transfer, and microprojectile bombardment. Weeding Plants by incorporating DNA that encodes and expresses an enzyme that is resistant to the drug Techniques for producing herbicide tolerance in rice are well known (eg, US Pat. No. 5,145,777: (See U.S. Pat.No. 5,290,926), such that the protein product is transported into chloroplasts. Techniques for adding a chloroplast transport sequence upstream from a herbicide gene (Comai et al., Nature (1985) 313: 741-744; U.S. Patent No. 4,940,835; U.S. Patent No. 5,188,642). You. In the same manner, other herbicides in these references with the gene encoding the mutant AdT One of the resistance genes may be replaced. Since AdT performs its function in chloroplasts, To ensure expression in chloroplasts or other plastids as known in the art In particular, it may be particularly appropriate to use a plastid transport sequence.   A mutated AdT gene is introduced into a plant cell, and the transformed plant is transformed into such a cell. After regeneration from the plant, using conventional methods of plant husbandry and plant breeding And maintain and increase transformed plants. The transformed plant is transformed into a conventional hybrid Used in a dilation scheme, this also contains a new mutant AdT gene. It is also possible to produce regular plant types (eg, Fehr and Hadley, Hybri dization of Crop Plants (1980); Jensen, Plant Breeding Methodology (1988); A llard, Principles of Plant Breeding (1960)).   Plants expressing genes that are tolerant or resistant to inhibitors of AdT Can be grown in soil and apply a herbicide containing an AdT inhibitor to inhibit weed growth. Can harm. Since weed plants do not carry the mutated AdT gene, weeds are Sensitive to herbicides containing   The following examples are intended to illustrate aspects of the invention, but are not intended to limit aspects of the invention. Not determined.                                  Example   The present invention is further described by reference to the following detailed examples. this These examples are provided for illustrative purposes only and are intended to be limiting unless otherwise indicated. do not do.Experimental procedure   Recombinant Bacillus subtilis GlntRNA^GlnPreparation of amide transferase and Purification. E. coli BL21 (DE3) containing pABC was added overnight to 3 mL of LB medium (10 g bactotryptone (10 g). (bactotryptone), 5 g yeast extract, 10 g NaCl) at 37 (with g / mL ampicillin). Incubated at ° C. Scale up the culture to 1 L and again at 37 ° C Incubated overnight. Harvest cells by centrifugation (4000 xg, 5 minutes, 4 ° C) 20 mL of Buffer A (25 mM HepesKOH, pH 7.5, 25 mM KCl, 10 mM MgCl_TwoAnd 1 mM (DTT). This step and all the following steps are performed unless otherwise indicated. C. was performed. Cells are lysed by sonication (4 × 15 seconds) and 100,000 × g for 1 hour Centrifuged. The enzyme is then purified by a series of chromatography using the Pharmacia FPLC system. Process until homogeneity as determined by SDS polyacrylamide gel electrophoresis. Was purified. The supernatant is first applied to a Q Sepharose (HR16 / 10) column (strong anion exchange). The activity was eluted with a linear gradient of 0 to 1 M NaCl in buffer A. this The active fraction from the column is applied to a Superdex-200 (HR26 / 100) column (gel filtration), and The activity was isocratically eluted in buffer A. From this column containing activity Fractions are pooled and applied to a MonoQ (HRI0 / 10) column, and the activity is measured in buffer A. Eluted with a linear gradient of 150 to 300 mM NaCl in. Purify the active fraction from this column And dialyzed against buffer A + 200 mM NaCl in 50% glycerol for 12 hours And stored at 70 ° C.   Bacillus subtilis tRNA expressed in vivo^GlnIsolation and purification. E.coli Add 3 mL of a culture of DH5a / pGP1-2 / pBTT (encoding tRNAGln) to LB medium (10 g bactotrip Tons, 5 g yeast extract, 10 g NaCl) at 50 (g / mL ampicillin and 10 (g / mL Incubated with namycin at 37 ° C. overnight. Scale up the culture to 1 L And repeated the overnight incubation. Cells at 4000 xg for 5 minutes at 4 ° C Harvest by centrifugation in 10 mL of lysis buffer (20 mM Tris-HCl, pH 7.4 and And 20mM MgCl_Two). 2 consecutive runs of all nucleic acids with an equal volume of water-saturated phenol The aqueous phase was then precipitated with isopropanol. Nucleic acid pellets were collected by centrifugation at 10,000 xg for 15 minutes at 4 ° C. Peret Resuspend the cells in 5 mL of 200 mM Tris-OAc, pH 9.0 and incubate at 37 ° C for 1 hour. To ensure complete deacylation of the tRNA. Nucleic acids are precipitated by ethanol precipitation. And then centrifuged at 10,000 xg for 15 minutes at 4 ° C. Pellet 100 mM N resuspended in aCl, incubated at 4 ° C overnight, and precipitated with ethanol . tRNAGln was purified by a two-step anion exchange chromatography protocol. Nuclear The acid was resuspended in 5 mL of Buffer 1 (140 mM NaOAc, pH 4.5) and 1 gm DE52 resin / 10 0 OD260 was added. Resin was added to 200 mL of Buffer A and 150 mL of Buffer 2 (140 mM NaOAc, p Wash with H4.5 + 300 mM NaCl) and transfer the tRNA to 100 mL Buffer 3 (140 mM NaOAc, pH 4.5 + 1M NaCl). Nucleic acids are recovered by ethanol precipitation and 10,000 xg for 15 Centrifuge at 4 ° C for 10 minutes, and 10 mM Tris-HCl, pH 7.4, 1 mM MgCl_Two, And 1 mM Resuspended in DTT and applied to a Pharmacia MonoQ (HR10 / 10) column. 10m tRNA M Tris ・ HCl, pH7.4, 1mM MgCl_TwoAnd a gradient of 450 to 750 mM NaCl in 1 mM DTT Eluted. TRNAGl based on its ability to be aminoacylated at both Glu and Gln n-containing fractions are pooled and the groups in the amidotransferase assay Used as quality.   Aminoacylation reaction. Radiolabeled GlntRNA^GlnThe procedure for the formation of is described in Jahn, D .; (1990). Unless otherwise indicated, these reactions were performed at 37 ° C with 10 mM ATP. , 50mM HepesKOH pH7.0, 25mM KCl, 15mM MgCl_Two, And a buffer consisting of 5 mM DTT Made in liquid. E. coli DH5a containing plasmids pGP12 and pBTT (see above), and Yo The concentration of tRNAGln recovered from L14C (U) -glutamate (300 mCi / mMol) was 10 (M Was. GluRS was isolated from B. subtilis and then DEAE-Sepharose chromatography Partially purified by luffy. Reactions were allowed to proceed at different times depending on the assay . An aliquot from this mixture is then used for the amidotransferase assay mixture. Direct or water-saturated phenol extraction, ethanol precipitation, and amino Added after resuspension in silylation buffer.   Amidotransferase reaction. GlntRNA^GlnRadiolabeled GlntRNA from^GlnForm Jahn, D .; (1990). Unless otherwise indicated, these Reaction at 37 ° C, 1 mM ATP, 5 mM HepesKOH pH 7.0, 2.5 mM KCl, 1.5 mM MgCl2, And 0.5 mM dithiothreitol (DTT). L14C (U) -Glut RNA^GlnWas 1 M and the concentration of L-glutamine was 1 mM. Enzyme purification Add aliquots 0-20 (L) from the fractions obtained during and assay the mixture Incubate for different lengths of time, then wash with 10 L, 3 M NaOAc, pH 5.0. Enchanted. The mixture is extracted with an equal volume of water-saturated phenol, and the aqueous and organic phases are combined. Was separated by centrifugation at 15,000 xg for 60 seconds at room temperature. Remove the aqueous phase 3x volume of ethanol was added and the tRNA was precipitated at 70 ° C for 15 minutes. Settling The obtained tRNA was collected by centrifugation at 15,000 × g at 4 ° C. for 15 minutes. The resulting pellet Resuspended in 50 L of 0.01 N KOH and deaminoacylated at 65 ° C. for 10 minutes. base Was neutralized to pH (6-7) with 1.3 L, 0.1 N NCl and the solution was dried completely under reduced pressure. The dried pellet was resuspended in 3 L of double distilled H2O, and TLC plates (cellulose , Aldrich). 3.5-2 hours at front, two solvent system (A. 20: 1) : 5 isopropyl alcohol: formic acid: water or B.I. 2: 1: 6: 6 ammonia: (Water: chloroform: methanol). Dry the plate at 85 ° C Dry and expose to activated phosphoroimaging plates ((12:00 And images were analyzed using MacBas v2.0. In this way, Glu to Gln Conversion to was measured.                                 Example 1AdT Of DNA fragment encoding   3 genes; 1 transcript of appropriate size hybridizing with 3 probes The objects are provided in FIG. 3 (SEQ ID NO: 1). Open readies for each subunit A signaling frame is provided.                                 Example 2B.subtilis AdT Protein characterization   The amino acid sequence of B. subtilis AdT is based on the nucleotide sequence of FIG. 3 (SEQ ID NO: 1). Loaded.   The molecular weights of the three subunits are calculated as follows: 53.039Kd, A subunit Kit; 53.314 Kd, B subunit; and 10.859 Kd, C subunit. Sub-unit A, B and C have the amino acid sequences provided in SEQ ID NOs: 4, 6 and 8, respectively. Is done.   Subunit size is confirmed by polyacrylamide gel electrophoresis .                                 Example 3Preparation of polyclonal antiserum containing anti-AdT antibody   Polyclonal antiserum containing anti-AdT antibody was ligated to rabbit recombinant AdT (trimeric protein). Quality) was administered to rabbits according to known methods. Antiserum with AdT tan When incubated with B. subtilis extract containing protein, AdT activity is complete. Totally inhibited.   Using polyacrylamide gel electrophoresis, the antiserum was assembled with AdT It has been shown to contain antibodies immunoreactive with the protein. Polyclonal anti Serum is markedly immunoreactive with individual unassembled subunits Was low.                                 Example 4AdT Production and purification                                 Example 5AdT Identification of inhibitors of activity   Purified Amidotransferase Assay to Identify Inhibitors of AdT Activity Used for The assay used to identify inhibitors of AdT activity is Less than,   (a) incubating a first sample of AdT and its substrate;   (b) measuring the uninhibited reactivity of AdT from step (a);   (c) a second sample containing an inhibitor compound, a first sample of AdT and its substrate Incubating in the presence of   (d) measuring the inhibited reactivity of AdT from step (c); and   (e) comparing the inhibited reactivity of AdT with the uninhibited reactivity Is included.   Inhibitors of AdT identified using this process include antibacterial, antifungal, And used as herbicides.                                 Example 6Identification of inhibitor-resistant AdT mutants   Purified amide transferase and AdT identified inhibitors Used in assays to identify bitter resistant AdT variants. Inhibitor resistant AdT mutation The assays used for body identification are:   (a) a first sample of AdT and its substrate is prepared from a second sample containing an AdT inhibitor; Incubating in the presence;   (b) measuring the unmutated reactivity of AdT from step (a);   (c) a first sample of the mutant AdT and its substrate, a second sample containing an AdT inhibitor; Incubating in the presence of a protein;   (d) measuring the mutated reactivity of the mutated AdT from step (c); and   (e) comparing the mutated reactivity of AdT with the unmutated reactivity Is included.   Inhibitor-resistant AdT variants identified using this process are AdT inhibitors It is used to produce cells and organisms that are resistant to bacteria.                                 Example 7Diagnostic assays   Nucleic acids for diagnosis are obtained from cells or tissues. Genomic DNA for detection Can be used directly or enzymatically enriched using PCR or other amplification techniques prior to analysis Can be widthed. The genomic DNA is converted to an amide transfer as provided in SEQ ID NO: 1. Comparable to a polynucleotide encoding an enzyme. Amplification compared to SEQ ID NO: 1 Deletions and insertions can be detected by a change in the size of the product. Point mutations are amplified By hybridizing the labeled DNA with the labeled AdT polynucleotide sequence. Can be determined. Perfectly matched sequences are obtained by RNASE cleavage from mismatched duplexes. Or by differences in melting temperatures. DNA sequence differences Changes in the electrophoretic mobility of DNA fragments in gels with or without Or by direct DNA sequencing. specific Sequence changes at positions can be detected by nuclease protection assays, such as RNase and S1 protection. Alternatively, it can be revealed by a chemical cleavage method.   Cells having a mutation or polymorphism in the gene of the present invention also Technique. For example, the mutation can be detected using RTPCR. RTPCR It is particularly preferred to use in combination with an automated detection system, for example GeneScan. No. RNA or cDNA can also be used for the same purpose, PCR or RTPCR. An example Identify mutations using PCR primers complementary to the nucleic acid encoding AdT And analyze. These primers are used to isolate Ad- Can be used to amplify T DNA. The invention also relates to 1, 2, 3 or 4 nucleosides. These primers are provided in which the tide has been removed from the 5 'and / or 3' end. The primers can be used to amplify genes isolated from infected individuals. That As a result, this gene can be subjected to various techniques for elucidation of the DNA sequence. like this In the meantime, mutations in the AdT DNA sequence can be detected, and diagnosis of infection and serotypes. Or for the classification of infectious agents.   An increase or decrease in AdT polynucleotide expression is a measure of polynucleotide quantification. Any method known in the art for the amplification, PCR, RTPCR, RNase protection Measurements using protection, Northern blotting and other hybridization methods Can be done.   Furthermore, the present invention for detecting overexpression or underexpression of AdT protein Diagnostic assays were compared to normal control tissue samples. Host-derived Assay techniques that can reliably determine the level of AdT protein in a sample are those of skill in the art. It is well known. Such assays include radioimmunoassays, competition Binding assays, Western blot analysis and ELISA assays.                                 Example 8Production of transgenic plants   A mutated AdT-encoding DNA sequence that confers resistance to an AdT inhibitor is ligated with an inhibitor. Isolated from mutant-resistant AdT mutants using mutagenesis and isolation techniques well known to those skilled in the art. You. The coding sequence for the mutant AdT gene is then introduced into the plant cells and Many different techniques well known to those skilled in the art from transformed plant cells Regenerate using Using the transformed plants in a conventional plant breeding scheme, the mutant AdT It produces new varieties of plants that also carry the gene. Crop plant with mutant AdT gene Grow and apply a herbicide containing an AdT inhibitor to the crop to control weeds. I will.   Although the present invention has been described in detail with reference to the above embodiment, the spirit of the present invention may be deviated. It is understood that various modifications can be made without delay. Everything mentioned in this specification Is incorporated herein by reference.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61K 48/00 45/00 A61P 31/04 48/00 C07K 16/40 A61P 31/04 C12N 1/15 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/00 1/21 C12Q 1/52 5/10 1/68 A 9/00 C12N 15/00 ZNAA C12Q 1/52 5 / 00 A 1/68 A61K 37/52

Claims (1)

[Claims]   1. An isolated amide transferase (AdT) protein encoding Polynucleotide.   2. Comprising a polynucleotide sequence selected from the group consisting of: Polynucleotide:   (A) a polynucleotide encoding the polypeptide encoded by SEQ ID NO: 1 A polynucleotide having at least 70% identity to the tide;   (B) a polynucleotide that is complementary to the polynucleotide of (a); and   (C) at least 15 contiguous bases of the polynucleotide of (a) or (b) A polynucleotide comprising 3. The polynucleotide according to claim 2, wherein the polynucleotide is DNA. 4. The polynucleotide according to claim 2, wherein the polynucleotide is RNA. 5. 4. The polypeptide of claim 3, comprising nucleotides 103 to 3306 set forth in SEQ ID NO: 1. Renucleotide. 6. A vector comprising the DNA according to claim 3. 7. A host cell comprising the vector of claim 6. 8. The polypeptide encoded by the DNA may be a host cell according to claim 7. A process for producing a polypeptide, comprising the step of expressing 9. A process for producing a cell that expresses a polypeptide, the cell comprising: The polypeptide encoded by the DNA contained in the vector according to claim 6. Do Transforming or transfecting the cells with the vector to express A process comprising steps. 10. Amidotransferase polypeptide or amidotransferase A process for producing a fragment of the polypeptide or the polypeptide. Culturing the host cell according to claim 7 under conditions sufficient for the production of a fragment. A process comprising steps. 11. Polypeptide encoded by nucleotides 103 to 3306 shown in SEQ ID NO: 1. A polypeptide comprising an amino acid sequence at least 70% identical to a peptide. 12. An antibody, or a fragment of an antibody comprising an antigen binding site, wherein the antibody is An antibody or an antigen-binding site that binds to the polypeptide of claim 11. Antibody fragment. 13. An antagonist that inhibits the activity of the polypeptide of claim 11. 14． A composition for treating an individual in need of an amide transferase. A composition comprising a therapeutically effective amount of the polypeptide of claim 11. 15. A composition for treating an individual in need of an amide transferase. Thus, the composition comprises a DNA encoding the polypeptide of claim 11, Here, the DNA develops the polypeptide when the composition is administered to the individual. A composition that manifests itself. 16. Treating an individual in need of inhibition of an amidotransferase polypeptide A composition for the treatment of an antagonist according to claim 13 in a therapeutically effective manner. A composition comprising an amount. 17． A method for diagnosing a disease associated with expression of the polypeptide according to claim 11. A nucleic acid sequence encoding the polypeptide. Process. 18. In a sample derived from a host, the presence of the polypeptide according to claim 11 is detected. A diagnostic process comprising the step of analyzing for 19. 12. Binding to the polypeptide of claim 11 and the activity of said polypeptide. A method for identifying a compound that inhibits the following, comprising:   (A) a step of incubating a first sample of the polypeptide and its substrate About;   (B) measuring the uninhibited reactivity of the polypeptide from step (a) Process;   (C) using a first sample of the polypeptide and its substrate as an inhibitor compound Incubating in the presence of a second sample comprising:   (D) measuring from step (c) the inhibited reactivity of the polypeptide; And   (E) comparing the inhibited reactivity of the polypeptide with the uninhibited reactivity Process to compare A method comprising: 20. 12. Binding to the polypeptide of claim 11 and the activity of said polypeptide. A method for identifying a compound that inhibits the following, comprising:   (A) transferring cells expressing the binding site of the polypeptide on the cell surface to the binding site Contacting with the compound to be screened under conditions that allow binding of Wherein the binding is a signal detectable in response to binding of the compound to the binding site. Steps associated with a second component that can provide:   (B) the presence or absence of a signal resulting from the interaction of the compound with the binding site. By detecting the presence, the compound binds to the bond and activates the bond. The step of deciding whether to sexualize or inhibit A method comprising: 21. A method for identifying an inhibitor-resistant AdT mutant, comprising: :   (A) A first sample of wild-type AdT and its substrate were prepared using a first sample containing an AdT inhibitor. Incubating in the presence of two samples;   (B) measuring the unmutated reactivity of the AdT from step (a);   (C) the first sample of mutated AdT and its substrate containing an AdT inhibitor; Incubating in the presence of the second sample;   (D) measuring the mutated reactivity of the mutated AdT from step (c); Love   (E) comparing the mutated reactivity with the unmutated reactivity of wild-type AdT A method comprising: 22. An immunological composition for inducing an immune response in a mammal, comprising: Amidotransfer suitable for producing antibodies to protect the animal from disease An immunological composition comprising the enzyme, or a fragment or variant thereof. 23. An immunological composition for inducing an immune response in a mammal, comprising: In order to induce an immune response that produces antibodies to protect the animal from disease, In vivo, the amidotransferase, or a fragment or variant thereof, is Amidetransferase, a fragment or variant thereof for expression An immunological composition comprising the encoding gene. 24. Amidotransferase polynucleotide or encoded therefrom An immunological composition comprising DNA encoding and expressing a protein, comprising: When introduced into a mammal, the amide transfer Response to Roseose Polynucleotide or Protein Encoded Therefrom An immunological composition that induces 25. Comprising a polynucleotide sequence selected from the group consisting of: Polynucleotide:   (A) for a polynucleotide encoding a polypeptide comprising SEQ ID NO: 4 A polynucleotide having at least 70% identity;   (B) a polynucleotide that is complementary to the nucleotide of (a); and   (C) at least 15 contiguous bases of the polynucleotide of (a) or (b) A polynucleotide comprising 26. A vector comprising the polynucleotide of claim 25. 27. A host cell comprising the vector of claim 26. 28. The polypeptide encoded by the polynucleotide according to claim 27, A process for producing a polypeptide comprising expressing from a host cell as described. Seth. 29. An amino acid sequence that is at least 70% identical to the amino acid set forth in SEQ ID NO: 4. A polypeptide comprising a sequence. 30. An antibody or an antibody fragment comprising an antigen-binding site, wherein the antibody is An antibody, or an antibody comprising an antigen-binding site, which binds to the polypeptide of claim 29. Body fragment. 31. An antagonist that inhibits the activity of the polypeptide of claim 29. 32. Comprising a polynucleotide sequence selected from the group consisting of: Polynucleotide:   (A) for a polynucleotide encoding a polypeptide comprising SEQ ID NO: 6 A polynucleotide having at least 70% identity;   (B) a polynucleotide that is complementary to the polynucleotide of (a); and   (C) at least 15 contiguous bases of the polynucleotide of (a) or (b) A polynucleotide comprising 33. A vector comprising the polynucleotide of claim 32. 34. A host cell comprising the vector of claim 33. 35. The polypeptide encoded by the polynucleotide, according to claim 34, A method for producing a polypeptide, comprising expressing from a host cell as described. Roses. 36. An amino acid sequence that is at least 70% identical to the amino acid set forth in SEQ ID NO: 6. A polypeptide comprising a sequence. 37. An antibody or an antibody fragment comprising an antigen-binding site, wherein the antibody is An antibody, or an antibody comprising an antigen-binding site, which binds to the polypeptide of claim 36. Body fragment. 38. An antagonist that inhibits the activity of the polypeptide of claim 36. 39. Comprising a polynucleotide sequence selected from the group consisting of: Polynucleotide:   (A) for a polynucleotide encoding a polypeptide comprising SEQ ID NO: 8 A polynucleotide having at least 70% identity;   (B) a polynucleotide that is complementary to the polynucleotide of (a); and   (C) at least 15 consecutive salts of the polynucleotide of (a) or (b) A polynucleotide comprising a group. 40. A vector comprising the polynucleotide of claim 39. 41. A host cell comprising the vector of claim 40. 42. The polypeptide encoded by the polynucleotide, according to claim 41, A process for producing a polypeptide comprising expressing from a host cell as described. Seth. 43. An amino acid sequence that is at least 70% identical to the amino acid set forth in SEQ ID NO: 8. A polypeptide comprising a sequence. 44. An antibody or an antibody fragment comprising an antigen-binding site, wherein the antibody is An antibody, or an antibody comprising an antigen-binding site, which binds to the polypeptide of claim 43. Body fragment. 45. An antagonist that inhibits the activity of the polypeptide of claim 43. 46. Isolated including subunit A, subunit B and subunit C A heterotrimeric protein, where:   The subunit A comprises SEQ ID NO: 4, an allelic variant of SEQ ID NO: 4, SEQ ID NO: No. 4 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of:   The subunit B comprises SEQ ID NO: 6, an allelic variant of SEQ ID NO: 6, SEQ ID NO: No. 6 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of:   The subunit C comprises SEQ ID NO: 8, an allelic variant of SEQ ID NO: 8, SEQ ID NO: No. 8 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of An isolated heterotrimeric protein. 47. An antibody or an antibody fragment comprising an antigen-binding site, wherein the antibody is An antibody which binds to the protein of claim 46, or an antibody comprising an antigen-binding site Fragment. 48. An isolated nucleic acid molecule encoding the protein of claim 46. 49. 49. A recombinant host that has been modified to include the nucleic acid molecule of claim 48. 50. 50. The host of claim 49 under conditions in which the introduced nucleic acid molecule is expressed. A method for producing an AdT protein, comprising the step of culturing 51. A method for identifying an agent that blocks translation, comprising: Process:   (A) contacting the drug with the AdT protein or a subunit thereof; And   (B) determining whether the agent binds to the AdT protein or the subunit Defining; , Here, the agent that blocks translation is the AdT protein or the subunit. A method identified as being able to bind to. 52. The AdT protein comprises a subunit A, a subunit B and a subunit. 52. The method of claim 51, comprising a heterotrimeric protein consisting of C. What here:   The subunit A comprises SEQ ID NO: 4, an allelic variant of SEQ ID NO: 4, SEQ ID NO: No. 4 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of:   The subunit B comprises SEQ ID NO: 6, an allelic variant of SEQ ID NO: 6, SEQ ID NO: No. 6 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of:   The subunit C comprises SEQ ID NO: 8, an allelic variant of SEQ ID NO: 8, SEQ ID NO: No. 8 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme A method having an amino acid sequence selected from the group consisting of: 53. 53. The method of claim 52, wherein a single subunit of the AdT protein is used. The method described above, and:   When subunit A is used, subunit A comprises SEQ ID NO: 4, SEQ ID NO: No. 4 allele variant, conservative substitution variant of SEQ ID NO: 4, and SEQ ID NO: 4 Hybridizes to the encoding nucleic acid molecule under stringent conditions, and Nucleic acid component encoding a subunit of a heterotrimeric amide transferase An amino acid sequence selected from the group consisting of Do;   When subunit B is used, subunit B is represented by SEQ ID NO: 6, SEQ ID NO: No. 6 allele variant, conservative substitution variant of SEQ ID NO: 6, and SEQ ID NO: 6 Hybridizes to the encoding nucleic acid molecule under stringent conditions, and Nucleic acid component encoding a subunit of a heterotrimeric amide transferase An amino acid sequence selected from the group consisting of Do;   When subunit C is used, subunit C comprises SEQ ID NO: 8, SEQ ID NO: No. 8 allelic variant, a conservative substitution variant of SEQ ID NO: 8, and SEQ ID NO: 8 Hybridizes to the encoding nucleic acid molecule under stringent conditions, and Nucleic acid component encoding a subunit of a heterotrimeric amide transferase An amino acid sequence selected from the group consisting of how to. 54. The agent further describes its ability to block the activity of the AdT protein. 52. The method of claim 51, wherein the method is tested. 55. 55. The method of claim 54, wherein the AdT activity is tested in a cell-free system. The method described. 56. 55. The method of claim 54, wherein said AdT activity is tested in a cell line. 57. A method for identifying an agent that blocks translation, comprising: Process of:   (A) contacting the agent with one or more subunits of the AdT protein;   (B) the three subunits of the AdT protein Incubation under conditions to form soluble AdT protein, Wherein at least one of said subunits is from step (a);   (C) determining whether the agent blocks the association of the three subunits Step; Contains Here, the agent that blocks the translation is a member of the subunit of the AdT protein. A method identified as being able to block a match. 58. The AdT protein comprises a subunit A, a subunit B and a subunit. 58. The method of claim 57, comprising a heterotrimeric protein consisting of C. What here:   The subunit A comprises SEQ ID NO: 4, an allelic variant of SEQ ID NO: 4, SEQ ID NO: No. 4 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of:   The subunit B comprises SEQ ID NO: 6, an allelic variant of SEQ ID NO: 6, SEQ ID NO: No. 6 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of:   The subunit C comprises SEQ ID NO: 8, an allelic variant of SEQ ID NO: 8, SEQ ID NO: No. 8 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme A method having an amino acid sequence selected from the group consisting of: 59. The agent further describes its ability to block the activity of the AdT protein. 58. The method of claim 57, wherein the method is tested. 60. 6. The activity of the AdT protein is tested in a cell-free system. 10. The method according to 9. 61. 60. The method of claim 59, wherein the activity of the AdT protein is tested in a cell line. Method. 62. A method for identifying an agent that blocks translation, comprising: Process:   (A) contacting the drug with the AdT protein;   (B) determining whether the agent blocks the activity of the AdT protein About A method comprising: 63. The AdT protein comprises a subunit A, a subunit B and a subunit. 63. The method of claim 62 comprising a heterotrimeric protein consisting of C What here:   The subunit A comprises SEQ ID NO: 4, an allelic variant of SEQ ID NO: 4, SEQ ID NO: No. 4 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of Erase Having an amino acid sequence selected from the group consisting of:   The subunit B comprises SEQ ID NO: 6, an allelic variant of SEQ ID NO: 6, SEQ ID NO: No. 6 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme Having an amino acid sequence selected from the group consisting of:   The subunit C comprises SEQ ID NO: 8, an allelic variant of SEQ ID NO: 8, SEQ ID NO: No. 8 conservatively substituted variants, and a nucleic acid molecule encoding SEQ ID NO. Under heterogeneous conditions and amide transfection of the heterotrimer. Amino acid sequence encoded by a nucleic acid molecule encoding a subunit of the enzyme A method having an amino acid sequence selected from the group consisting of: 64. 7. The activity of the AdT protein is tested in a cell-free system. 3. The method according to 3. 65. 64. The method of claim 63, wherein the activity of the AdT protein is tested in a cell line. Method. 66. A method of blocking protein translation in a cell, comprising: Binding to the T protein or its subunits, sufficient to block the translation Contacting the drug with an appropriate amount of a drug. 67. The agent binds to a subunit of the AdT and 67. The method of claim 66, wherein the association is blocked. 68. 67. The method of claim 66, wherein said agent is used as an antibacterial agent. 69. 67. The method of claim 66, wherein the agent is used as an antifungal agent. 70. 67. The method of claim 66, wherein the agent is used as a herbicide. 71. Encodes a mutant AdT that confers resistance to wild-type AdT inhibitors , An isolated polynucleotide. 72. A vector comprising the polynucleotide of claim 71. 73. A host cell comprising the vector of claim 72. 74. 74. The host cell of claim 73, wherein said host cell comprises a plant cell. 75. The polypeptide encoded by the polynucleotide, according to claim 73, A process for producing a polypeptide comprising expressing from a host cell as described. Seth. 76. A process for producing a cell that expresses a polypeptide, said cell comprising: Is encoded by the polynucleotide contained in the vector according to claim 73. The cells are transformed or transformed with the vector to express the polypeptide. A process comprising the step of transfecting. 77. Process for producing plants containing resistance genes to AdT inhibitors 75. A process for regenerating a plant from a plant cell according to claim 74, wherein the process comprises: Process that encompasses the process. 78. The process of plant agriculture, comprising:   (A) planting a plant containing a gene resistant to an AdT inhibitor;   (B) A step of applying a herbicide containing an AdT inhibitor Including the process.