JPH10510714A - Plant adenylosuccinate synthetase and DNA encoding the same - Google Patents

Abstract

  (57) [Summary] The present invention provides novel plant DNA sequences that encode natural adenylosuccinate synthetase (ADSS). Methods of using the complete or partial ADSS coding sequence as a probe for diagnostics, mapping (mapping) and other purposes are taught. The creation of transformed host cells capable of expressing ADSS is also taught. Methods of using the transformed host cells are taught, including methods for the recombinant production of ADSS enzymes. Also provided are methods of using plant ADSS enzymes to screen for inhibitors of ADSS activity.

Description

DETAILED DESCRIPTION OF THE INVENTION Plant adenylosuccinate synthetase and DNA encoding the same   The present invention generally relates to adenosine 5'-monophosphate biosynthesis in plants. Enzyme activity. In particular, the present invention relates to a plant enzyme that catalyzes the synthesis of adenylosuccinic acid. Element and the gene encoding the enzyme. In one aspect, the invention It relates to the recombinant production of the above enzymes in a host. In another aspect, the present invention provides a novel Applies to herbal identification. In yet another aspect, the invention relates to a genetic marker in a plant. Regarding the development of traits.   Adenosine 5'-monophosphate (AMP, also known as adenylic acid) Adenosine 5'-triphosphate is a key energy transfer molecule for all life systems It is a precursor of (ATP). First identified enzyme in AMP biosynthesis The steps are inosine 5'-monophosphate (IMP, inosinic acid) and aspartate Is the synthesis of adenylosuccinic acid. The enzyme that catalyzes this step is Acid synthetase (IMP: L-aspartate ligase (GDP formation), EC6 . 3.4.4, referred to herein as "ADSS").   In E. coli, ADSS is a dimer of the same 48 kD subunit. Body. Its three-dimensional structure has been determined to a resolution of 2.8Å (Poland et al., J. . Biol. Chem. 268: 25334-25342 (1993)). In mammalian cells, The ADSS enzyme exists as two isoforms. The acidic form present in non-muscle tissue is A It is thought to be involved in the de novo (new) production of MP. Salt present in muscle tissue The base is accompanied by the end result of deamination of aspartate to fumarate Acts as part of the purine nucleotide cycle involving interconversion of IMP and AMP (Lehninger, Biochemistry. Worth Publishers, NY (1975 ), P. 743; Lowenstein, Int. J. Sports Med. 11: S36-S46 (1990).   The gene encoding the ADSS enzyme may be derived from various species, including: Isolated: E. coli (Wolfe and Smith, J. Biol. Chem. 263: 19147-19153). (1988)), Dee. Discoidum (D. discoideum) (Weismuller et al. Biol. Chem. 266: 2480-2485 (1991)), mouse (Guicherit et al., J. Biol. Chem. . 266: 22582-22587 (1991); Guicherit et al. Biol. Chem. 269: 4488-4496 (19 94)), Bacillus subtilis (Maentsaelae) And Zalkin, J .; Bacteriol. 174: 1881-1890 (1992)), human (Powell et al., FEBS)  Lett. 303: 4-10 (1992); Cerevisiae (Geneva) No. L22185) and Kaenorhabditis elegans (Ca enorhabditis elegans (EST; Genebank accession number M75738). Only However, the gene encoding the ADSS enzyme has never been obtained from any plant species. Separation It has not been.   Currently plant ADSS enzymes and ADSS enzymes / genes isolated from other organisms Little is known about the relationship between the plant and the plant ADSS enzyme To isolate ADSS-encoding genes from any plant species using known research methods I can't.   Methods for isolating genes are based on knowledge of the structure of the proteins they encode However, it cannot be applied to plant ADSS enzymes. Because, plant AD This is because much less is known about SS enzymes. Metabolic enzymes, For example, ADSS is typically purified from plants because of its extremely low abundance. Very difficult. In addition, various phenolic and hydrocarbon compounds in plants May hinder the isolation of pure enzymes having natural activity.   In the absence of direct structural information, many standard techniques are And their corresponding genes. Such standard technologies are Corresponds to nucleic acid hybridization and conserved amino acid sequence motifs By polymerase chain reaction (PCR) using oligonucleotide primers Amplification. Unfortunately, these technologies are not publicly available if they have the same functionality. Remarkable structural similarities to intellectual proteins and genes (ie, amino acid sequence and And DNA sequences) for the isolation of plant ADSS genes It would not be expected to be useful. Known ADSS remains from organisms other than plants There is no significant structural similarity even between genes and proteins (eg, Po well et al., FEBS Lett. 303: 4-10 (1992)). It is unlikely to share significant structural similarities with the SS protein.   Used to isolate biosynthetic genes in other metabolic pathways from higher eukaryotes Another approach that has been developed is the complementarity of microbial mutants lacking the activity. You. Vectors that can direct the expression of cDNA in a microbial host A library of cDNAs from higher eukaryotes is cloned in the reactor. Said The vector is then transformed or otherwise introduced into the mutant microorganism And colonies whose phenotype is no longer mutant are selected.   This strategy removes genes from higher eukaryotes involved in several metabolic pathways: Histidine biosynthesis (eg, by reference herein). International Patent Application WO 94/26909, incorporated in its entirety into (Eg, Frisch et al., Mol. Gen. Genet. 228: 287 (1991)), purine biosynthesis (eg, For example, Aimi et al. Biol. Chem. 265: 9011 (1990)) and tryptophan biosynthesis (For example, Niyogi et al., Plant Cell 5: 1011 (1993)). This strategy also applies to corn Kosiglutamine synthase (Snustad et al., Genetics 120: 1111-1114 (1988 )), Soy-pyrroline-5-carboxylate reductase (Delauney et al., Mo. l. Genet. 221: 299-305 (1990)), corn dihydrodipicolinatecin Tase (Frisch et al., Mol. Gen. Genet. 228: 287-293 (1991)), oilseed rape Body 3-isopropylmalate dehydrogenase (Ellerstrom et al., Plant Mol. Bio l. 18: 557-566 (1992); Elledge et al., Proc. Natl. Acad. Sci. USA 88: 1731-1 735 (1991)) and dihydroorotate dehydrogenase (Minet et al., Plant J. . 2; 417-422 (1992)) to isolate plant genes, including those encoding Have been used.   Microbial mutants suspected of lacking ADSS activity are available (Eg E. coli PurA mutant designated as CGCS5408 and Yale University E. coli Genetic Stock Center E. coli strains CGCS4431 and 7039 from Dorfman, Genetics 61: 3. 77-389 (1969)). However, this Encodes ADSS enzyme activity, despite the availability of these mutants The application of supplemental techniques to isolate cDNA is described in birds (Powell et al., FEBS Lett. 303 : 4-10 (1992)) and Bacillus subtilis ADSS (Maentsaelae and Za lkin, J .; Bacteriol. 174: 1881-1890 (1992)) Have been.   This complementarity strategy, when applied to ADSS, especially eukaryotic ADSS genes There are several reasons that could explain the failure. First, for example, the use of microbial hosts Sudden eukaryotic ADSS cDNA sequences may be It cannot be expressed to a moderate level in a mutant microorganism. Second, for example If the activity requires post-translational modifications not performed by the microorganism, for example, glycosylation If the primary translation product from the cloned eukaryotic coding sequence is The inability to produce a polypeptide. Third, a heterologous tag expressed in E. coli Can be lethal to cells in which the protein is expressed, thus making its isolation impossible That is. Fourth, for example, certain organelles specifically lacking in a microbial host If proteins are usually targeted to the membrane system, eukaryotic proteins will Mainly in that it cannot exhibit its active conformation. This last possibility Sex is determined by organelles that are not present in the microbial host used in the complementation assay. (Schubert, Annu. Rev. Plant Physiol. 37:53). 9-574 (1986)), and the N-terminal introduced sequence and unique characteristics of the mature polypeptide. Organelle system likely reached as a result of a post-translational targeting mechanism encompassing both sexes (Eg, Kohorm and Tobin, Ptant Cell 1: 159 (1989); Li et al., Plant Cel. l 3: 709 (1991); Li et al. Biol. Chem. 267: 18999 (1992)) Plant ADSS enzyme Seems particularly applicable to It is. In addition, two other purine biosynthesis genes isolated from plants, 5 ' -Phosphoribosyl-5-aminoimidazole synthetase (Senecoff and Maeg her, Plant Physiol. 102; 387-399 (1993) and glycinamide synthetase (Schnorr et al., Plant J. 6: 113-121 (1994) also describe chloroplast-targeted It is thought to code quality.   Therefore, the adenylosuccinate synthetase (ADSS) enzyme from plant sources is Identification and isolation of the encoding DNA molecule is one of the main objects of the present invention. You. This object can be achieved within the scope of the present invention.   Accordingly, the present invention relates to adenylosuccinate synthetase (ADS) from plant sources. S) providing an isolated DNA molecule encoding the enzyme.   Arabidopsis thaliana, Zea maze ys) and the DNA coding sequence for the ADSS enzyme in wheat, respectively. It is shown in SEQ ID NOs: 1, 3 and 5. Using the information provided by the present invention And adenylosuccinate synthetase (ADSS) enzymes from any plant source DNA coding sequences for can now be obtained using standard methods.   The present invention therefore provides a plant having adenylosuccinate synthetase (ADSS) activity. From a plant, preferably from dicotyledonous or monocotyledonous plants, more preferably from Arabidopsis Species, Isolated DNA encoding a protein from a corn or wheat plant About molecules.   In particular, the invention relates to Arabidopsis thaliana, zea maize and wheat. An isolated DNA molecule comprising a coding sequence for an ADSS enzyme in The prime consists of the amino acid sequences shown in SEQ ID NOs: 2, 4 and 6, respectively.   The present invention further provides a plant having adenylosuccinate synthetase (ADSS) activity. From a plant, preferably from dicotyledonous or monocotyledonous plants, more preferably from Arabidopsis DNA encoding proteins from corn seeds or corn and wheat plants It relates to an expression cassette consisting of a promoter operably linked to a molecule.   Another subject of the present invention is a recombinant vector containing the above expression cassette. The vector can be stably transformed into a host cell. In addition, the vector Also routinely transformed host cells are included, wherein the host cells are preferably bacterial. A cell selected from the group consisting of a cell, a yeast cell and an insect cell; and Furthermore, the DNA molecule according to the present invention can be expressed.   The invention also encompasses recombinant production of the ADSS enzyme. In particular, the present invention (A) adenylosuccinate in an expression cassette designed for the selected host; Insert a DNA sequence encoding a protein having an intestase (ADSS) activity And; (B) transferring the resulting molecule containing the individual elements, linked in an appropriate reading frame, to a host Inserting into a vector that can be transformed into cells; (C) growing the host cell thus transformed in a suitable culture medium; And (D) proteins from transformed cells and / or culture media Isolate the product and purify it Having adenylosuccinate synthetase (ADSS) activity Quality in a host organism.   Also, a method using ADSS produced by recombination is encompassed in the present invention. . In particular, the present invention screens for novel herbicides that affect the activity of ADSS The use of purified ADSS to provide   Preferred are chemicals for their ability to inhibit the activity of ADSS enzymes from plants. A method for assaying quality, comprising: (A) the first reaction mixture contains the ADSS enzyme and the ADSS enzyme Combining under conditions that can catalyze the synthesis of succinic acid; (B) combining the chemical and the ADSS enzyme in a second reaction mixture with the first reaction mixture; Combined under the same conditions as in the reaction mixture of (D) produced in the first and second reaction mixtures Compare the amount of adenylosuccinic acid Wherein the amount of adenylosuccinic acid in the second reaction mixture is higher. If the amount is significantly less than the amount of adenylosuccinic acid in the first reaction mixture, Chemicals can inhibit the activity of the ADSS enzyme.   The present invention further provides a plant adenylosuccinate synthetase gene or mRNA. For a probe capable of specifically hybridizing, the probe may have a length To an adenylosuccinate synthetase enzyme from a plant of at least 10 nucleotides Contains adjacent portions of the coding sequence.   Another embodiment of the present invention relates to a method for measuring adenylosuccinate synthetase (ADSS) activity. For producing a DNA molecule containing a DNA portion encoding a protein having the same And (A) specifically high in plant adenylosuccinate synthetase gene or mRNA; A nucleotide probe capable of hybridizing, said probe having a length Adenylosuccinate synthetase enzyme from a plant having at least 10 nucleotides Preparing said probe containing adjacent portions of the coding sequence for: (B) using the nucleotide probe prepared according to step (a), Genomic DNA or cDNA fragments from cloned organisms Probe other ADSS coding sequences of; (C) a protein having adenylosuccinate synthetase (ADSS) activity The DNA molecule containing the DNA portion to be loaded Consisting of   The present invention further provides methods for detecting the presence and form of the ADSS gene, and And a method for quantifying the level of ADSS transcripts in plants and organisms (organs). this These methods involve altered forms of the ADSS enzyme or altered expression of the ADSS enzyme. It can be used to diagnose disease states associated with levels.   In one aspect, the invention catalyzes the synthesis of adenylosuccinic acid from IMP The enzyme adenylosuccinate synthetase (herein referred to as "ADSS") An isolated DNA molecule encoding a eukaryotic form of E. coli. Arabidopsis The DNA coding sequence for the ADSS enzyme from Thalia and the corresponding amino acid The acid sequences are provided as SEQ ID NOs: 1 and 2, respectively. corn( Maze) The DNA coding sequence for the ADSS enzyme and the corresponding amino acid sequence are They are presented as SEQ ID NOs: 3 and 4, respectively. Against wheat ADSS enzyme The corresponding DNA coding sequence and the corresponding amino acid sequence are SEQ ID NO: 5 and SEQ ID NO: 5, respectively. And 6 are presented.   DNA encoding the ADSS enzyme may be obtained from any plant species desired in accordance with the present invention. Can be isolated from the genome. Taught to isolate plant ADSS coding sequences One way Is presented in Example 1. In this method, c encoding the ADSS enzyme The DNA clone is a mutant host organism that lacks ADSS enzyme activity. CDNA clones derived from the eukaryote based on their ability to supply Identified from the library. Suitable host organisms for use in this method are cDN A that can be used to screen an expression library, Mutants lacking ADSS activity are available, or generally Can be produced. Such host organisms include, but are not limited to, E. coli and yeast. Not limited.   Also, the plant ADSS coding sequence may be a Arabidopsis. Thaliana (SEQ ID NO: 1), Zea Maze (SEQ ID NO: 3) or wheat (SEQ ID NO: 3) No .: 5) Fully public based on their sequence homology to the ADSS coding sequence. It can be isolated according to known techniques. In these technologies, known ADSS code distributions are used. All or part of the sequence may be a cloned genomic DNA fragment from the selected organism or Is present in a collection of cDNA fragments (ie, a genomic or cDNA library). That selectively hybridize to other ADSS coding sequences present. Used as a probe. One such technique is the use of plated DNA libraries. Hybridization screening (plaques or colonies; eg Samb rook et al., “Molecular Cloning”, Cold Spring Harbor Labo ratory Press, (1989)) and known ADSS amino acid sequences. PCR using oligonucleotide primers corresponding to the sequence domain Amplification (eg, Innis et al., “PCR Protocols, a Guide to Methods and Applicatio ns ", Academic Press (1990)). Especially for the isolation of ADSS coding sequences from organisms closely related to the organism from which Well suited. Thus, the code for Arabidopsis, There Maze or Wheat The application of the above method using the sequence as a probe is It is expected that it will be particularly well-suited for isolating nucleic acid sequences.   The isolated plant ADSS sequence taught by the present invention can be used for any desired purpose. It can be manipulated according to any suitable standard genetic engineering technique. For example, the entire ADSS Sequences or parts of them are unique to the ADSS coding sequence and messenger RNA. It can be used as a probe that can hybridize differently. Various conditions In order to achieve specific hybridization underneath, the probe is ADS Include sequences that are unique among the S coding sequences and have at least 10 Otide length, preferably at least 20 nucleotides in length, and most preferably Or at least 50 nucleotides in length. The above probe is a polymerase The ADSS code sequence is obtained by a well-known method of chain reaction (replication chain reaction, PCR). Column Can be used to amplify and / or analyze. This technology is In order to isolate additional ADSS coding sequences, and in organisms, ADSS coding sequences May be useful as a diagnostic assay to determine the presence of This technology May also be associated with certain conditions, such as herbicide tolerance, AMP deficiency, poor health, etc. Used to detect the presence of the altered ADSS coding sequence in plants. Can be   ADSS-specific hybridization probes also contain genomic ADSS sequences. Using standard techniques based on the selective hybridization of probes to Map the location of the native ADSS gene in the genome of the selected plant Can be used to These techniques identify or encapsulate within the ADSS probe sequence. Identification of DNA polymorphisms involved and self-receipt of parental hybrids of the two polymorphisms Other known map (map) locations in mapping aggregates derived from flour Use of the above polymorphism to track the separation of the ADSS gene for a marker But not limited thereto (eg, Helentjaris et al., Plant Mol. B. iol. 5: 109 (1985); Sommer et al., Biotechniques 12: 82 (1992); D'Ovidio et al., Pl. ant Mol. Biol. 15: 169 (1990)). All plant ADSS sequences are ADSS inherited It is considered useful as a probe for mapping offspring, but is preferred Probes from plant species more closely related to the selected plant species And the most preferred probe is from a selected plant species. ADSS sequence. The mapping of ADSS gene by this method is special for breeding purpose. It is considered to be useful. For example, a mutant ADSS residue that confers herbicide resistance Knowing the location of the gene on the genetic map allows flanking DNA markers to be referenced. (Eg, Helentjaris, Trends Genet. 3: 217 (19) 87)). During the transfer of the herbicide tolerance trait to the new breeding line, these markers Kerr is an ADSS-linked franchise that is still present in the recurrent parent after each round of backcrossing. Can be used to track the extent of chromosomal DNA.   ADSS-specific hybridization probes can also be obtained by standard techniques, for example, Northern blot analysis was used to quantify the level of ADSS mRNA in plants. Can be used for This technique can be used for certain conditions, such as adenylosuccinic acid deficiency. Altered or AMP levels of ADSS expression or ADSS that may be relevant Diagnostic assay to detect enhanced tolerance to herbicides targeting Can be useful.   The plant ADSS coding sequence was selected for recombinant production of the enzyme in the host organism. Inserted into an expression cassette designed for the selected host, and Into a more produced host. Specific regulatory sequences, eg, the selected host Promoters and signals that are suitable for The selection of sequences, 5 'and 3' untranslated sequences and enhancers is well known to those of ordinary skill in the art. Within the level of knowledge. Individual elements (elements) linked in the appropriate reading frame (G) is contained in a vector that can be transformed into a host cell. May be inserted. For recombinant production of suitable expression vectors and proteins Are used in host organisms such as E. coli (eg, Studier and Moffatt, J. Mol. Bio. l. 189: 113 (1986); Brosius, DNA 8: 759 (1989)), yeast (eg, Schneide r and Guarente, Meth. Enzymol. 194: 373 (1991)) and insect cells (eg See, for example, Luckow and Summers, Bio / Technol. 6:47 (1988)) Is knowledge. Particular examples are plasmids such as pBluescript (Stratagene , La Jolla, CA), pFLAG (International Biotechnologies, Inc., New H aven, CT), pTrcHis (Invitrogen, La Jolla, CA) and Baculo Ils expression vectors such as Autographa californica nuclear polyhedrosis And those derived from the genome of Rus (AcMNPV). Preferred Vacu The rovirus / insect system is pVI11392 / Sf21 cells (Invitrogen, La Jolla , CA).   Recombinantly produced plant ADSS enzymes can be isolated and isolated using a variety of standard techniques. And can be purified. The actual technique that can be used depends on the host organism used, the ADSS yeast Whether the element is designed for secretion and is well known to those skilled in the art. May vary depending on other such factors (eg, Ausubel, F., etc. "Current Protocols in Molecular Biology", John Wiley & Sons, Inc. Published ( 1994) Chapter 16).   Recombinantly produced plant ADSS enzymes are useful for a variety of purposes. For example, providing ADSS enzyme activity to synthesize adenylosuccinic acid in vitro Can be used to In vitro synthesis of adenylosuccinic acid is based on IMP, GTP and And aspartate with a divalent cation such as Mg²⁺A suitable buffer containing In the presence of an ADSS enzyme (eg, Baugher et al.) Biochem. Biophys. Res. Commun. 94: 123-129 (1988); Stayton et al., Curr. Top . Cell. Regul. 22: 103-141 (1983); Bass et al., A-rch. Biochem. Biophys. 256: 335-342 (1987)). The adenylosuccinic acid produced is commercially available from other sources Is a useful reagent that can be used as a substitute for purified adenylosuccinic acid.   The recombinantly produced plant ADSS enzyme has no target identified and Screening of known herbicides that have not been determined to inhibit DSS It may also be useful in in vitro assays for In such a test tube The assay also inhibits ADSS activity and is therefore a candidate herbicide It can be used as a more general screen to identify chemicals. Ma The recombinantly produced ADSS is It can also be used to elucidate the complex structure of enzymes. Regarding the structure of ADSS enzyme Such information can be used, for example, in the rational design of new inhibitory herbicides. .   Typically, the inhibitory effect on ADSS is that of adenylate in an in vitro assay. Determined by reduction or complete inhibition of succinic acid synthesis (eg, Baugher et al., Bio chem. Biophys. Res. Commun. 94: 123-129 (1980); Stayton et al., Curr. Top. Ce ll. Regul. 22: 103-141 (1983); Bass et al., Arch. Biochem. Biophys. 256: 335- 342 (1987)). Such a determination can be tested in the presence and absence of candidate inhibitors By simply comparing the amount of adenylosuccinic acid synthesized in an in vitro assay Can be done. Of adenylosuccinate synthetase synthesized in the presence of chemicals If the amount is significantly less than the amount synthesized in the absence, the chemical is ADSS Identified as an inhibitor. The expression "significantly less" refers to the error of the measurement technology. Understood to mean a decrease in the amount of ADSS less than the area (margin) Should.   The present invention is further described with reference to the following detailed examples. These examples are Presented for explanation and intended to be limiting unless otherwise specified is not.   The standard recombinant DNA and molecular cloning techniques used here are And is well known in T. Maniatis, E.F.Fritsch and J. Sambrook, M. olecula r Cloning: A Laboratory manual, Cold Spring Harbor Laboratory, Cold Spri ng Harbor, NY (1982) and T.J.Silhavy, M.L.Berman and L.W.Enquist, Expe riments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Ha rbor, NY (1984) and Ausubel, F.M.M, etc., Current Protocols in Molecular Bi ology, Greene Publishing Assoc. and published by Wiley-Interscience (1987) Have been. Example 1: Encoding ADSS gene by functional complementation of E. coli mutant Isolation of Arabidopsis cDNA   Plasmid vector pFL61 (Minet et al., PLant J. 2: 417-422 (1992)) An Arabidopsis thaliana (Landsberg) cDNA library was obtained, And it was amplified. E. coli purA mutant PC0543 (CGSC # 5408; E. coli Genetic Stock Center, Yale University, New York, Connecticut -Haven) was obtained and maintained on N-Agar. The above plasmid live Rally is Bio-Rad Gene Pulser and manufacture Into CGSC # 5408 by electroporation using commercial conditions Transformed. Cells were treated with 100 mg / ml ampicillin and 0.4% Minimal E agar containing nonoic acid (Vogel and Bonner, J. Biol. Chem. 218: 97-1). 06 (1956)) at a concentration of about 10,000,000 transformants / 10 cm plate. Plate culture was performed. Adenine Prototrophy Body is 1/6 × 10 from pFL61 library⁷Collected at a frequency of Plasmi DNA was isolated from colonies for sequence analysis. Purified plasmid DNA It has been shown that CGSC # 5408 is frequently transformed into purine prototrophy. The purified plasmid contains two additional E. coli purA mutants: ES4 (CGSC # 4 431; E. coli Genetic Stock Center, Yale University, Connecticut New Haven, Ga.) And TX595 (CGSC # 7039; E. coli Geneti) Supplementary Information on Stock Center, Yale University, New Haven, CT) And further confirmed that they encode a functional ADSS enzyme.   Restriction digestion indicated that the cDNA insert was greater than 3 kB. Sequence analysis is cD NA is chimeric and contains 1512 bp preceded by a poly A region at the 3 'end showed that. This 1512 bp region contains the mature protein sequence and partial potential Encodes an incomplete ADSS containing a chloroplast transit peptide. GAP blog Rum (Deveraux et al., Nucleic Acids Res. 12: 387-395 (1984)) Search shows homology with ADSS from Saccharomyces cerevisiae. 2 types Are 70% similar and 51% identical to regions of high homology. Protein The quality is 65% similar and 44% identical to E. coli ADSS.   ADSS in pBluescript SK vector -1 is the United States, Illinois 61604, Peoria, North University Agricultural Research Services, Paty Street 1815, Patent ・ Culture Collection (NRRL), Northern Regional Research ・ Center [Agricultural Reseach Service, Patent Culture Collection (NRRL ), Northern Regional Reseach Center] to pWDC-6 (NRRL # B-21). 328) was deposited on September 22, 1994.   The complete Arabidopsis cDNA sequence encoding ADSS-1 is SEQ ID NO: 1. Is shown in Except for the first four nucleotides, this sequence is pWDC-6 It is included in. Amino acid sequence of ADSS-1 encoded by this cDNA The columns are shown in SEQ ID NO: 2. Example 2: ADSS inheritance based on sequence homology to Arabidopsis ADSS Isolation of maize cDNA encoding offspring   Custom-made Unizap Zea mays (cultivar Blizzar) d) cDNA library was purchased from Clontech. About 160000 pfu Phage library plated at a concentration of 8000 plaques per 10 cm Petri dish Cultured, and two filter lifts are applied to the nitrocellulose membrane (Scheiller and And Scheull) at 37 ° C. for about 7 hours after growth. This filter Is a random primer method (Life Technol ogies, Bethesda, MD)³²Arabidop labeled with P-dCTP Probed with PCR amplified fragment of cis ADSS cDNA. Hybridization And washing conditions are described in Church and Gilbert, 1984 [Proc. Natl. Acad. Sci. USA 81: 1991-1995 (1984)]. Positive hive After purification of the redidation plaques to single, the plasmid is cleaved in vivo. And the cDNA insert is a dideoxy terminator labeled with a fluorescent dye. (Applied Biosystems, Inc., Foster City, CA). Column determined. The corn ADSS cDNA thus obtained and The sequences for the protein it encodes are provided in SEQ ID NOs: 3 and 4, respectively. It is shown. Plasmid containing this corn ADSS cDNA insert Was named pWDC-9 (NRRL # B-21349) on October 24, 1994. Was deposited. Example 3: ADSS gene based on sequence homology to maize ADSS Of Wheat cDNA Encoding Wheat   Custom-made Unizap Triticumaestivu m) (Cultivar Kanzler) cDNA library was purchased from Clontech. About 5 The 0000 pfu phage library is 5,000 pfu per 10 cm Petri dish. Plate to the concentration of Two filter lifts with nitrocellulose membrane (Scheiller and Scheull) Created after about 7 hours growth at 37 ° C. This filter lift is random Primer method (Life Technologies, Bethesda, MD)³²P-dC 1005 bases from 5 'end of corn ADSS cDNA labeled with TP Probed with EcoRI, XbaI restriction fragment. Hybridization and washing Conditions were 50 ° C. as described by Church and Gilbert, (1984), supra. After purifying the positive hybridization plaques to single The cDNA insert is cleaved within and the cDNA insert is labeled with a fluorescent dye Minator (Applied Biosystems, Inc., Foster City, CA) Was sequenced using. The thus obtained wheat ADSS cDNA and And the sequences for the proteins they encode are SEQ ID NOs: 5 and 6, respectively. Has been presented. This wheat ADSS cDNA is not full-length, but Based on the information obtained by N-terminal sequencing of the mature protein purified from SEQ ID NO: 6 for mature ADSS protein beginning near amino acid 35 at 6 The coding sequence of Based on its homology to maize ADSS Wheat ADSS cDNA is an expected leaf not present in the mature protein The coding sequence for the nine amino acids of the chloroplast transit peptide is missing. This com Giant ADSS cDNA insert Plasmid containing pWDC-10 (NRRL @ B 21505). Example 4: Other ADSSs based on sequence homology to known ADSS coding sequences Gene isolation   The phage or plasmid library is approximately 10,000 on a 10 cm Petri dish. Plate to plaque concentration and filter lift of plaque at 37 ° C. Plate after overnight growth. This plaque lift has SEQ ID NO: 1, High sequence conservation in the cDNA shown in 3,5 or the elucidated plant ADSS sequence Was probed with a portion of the above cDNA. cDNA DNA probe Prime Time kit (International Biotechnologies, Inc.) , New Haven, CT) by random primer method³²Pd Labeled with CTP. Hybridization conditions are 50 ° C and 7% dodecyl sulfate. Sodium (SDS), 0.5M NaPO_Four pH 7.0 with 1 mM EDTA is there. After overnight hybridization, the filters were 2x SSC, 1% SD Washed with S. Positive hybridization plaques are autoradiographic Detected by the After purifying the plaque to a single point, the cDNA insert was isolated. And their sequences are labeled with a fluorescent dye, dideoxy terminator ( Applied Biosystems, Inc., Foster City, CA) Determined by the chain termination method.   The standard protocol described above applies to all other eukaryotes, especially other higher plant species. To obtain an ADSS gene whose sequence is homologous to the known ADSS coding sequence of Can be used by those skilled in the art.   Amino acid sequence of Arabidopsis, corn and wheat proteins (which The alignments of SEQ ID NOs: 2, 4 and 6) are shown in Table 1. this Nucleotide sequences encoding these proteins (SEQ ID NOs: 1, 3 and 3, respectively) Table 2 shows the alignments of (5) and (5). For each alignment, A bidopsis sequence is used as a reference sequence. To get the best alignment Spaces inserted in the sequence are indicated by dashes. Corn and Sequences identical to the Arabidopsis sequence in the wheat sequence are indicated by periods. , And non-identical sequences have been described.                                 Deposit   Deposited within the scope of the present invention are the United States, Illinois 61604, Peoria, Agricultural Research at University University Street 1815 Services, Patent Culture Collection (NRRL), Northern Lee Johnal Research Center [Agricultural Reseach Service, Patent Cultu re Collection (NRRL), Northern Regional Reseach Center].   The Arabidopsis ADSS-1 cDNA insert, presented as SEQ ID NO: 1, The contained plasmid was pWDC-6 (NRRL # B-September 22, 1994). 21328).   Contains the maize ADSS cDNA insert presented as SEQ ID NO: 3 The plasmid was prepared on October 24, 1994 with pWDC-9 (NRRL # B-21). 349).   A plasmid containing the wheat ADSS cDNA insert presented as SEQ ID NO: 6 Sumid was released on November 3, 1995 on pWDC-10 (NRRL # B-21505). ).                   _{★★★★★★★★★★★★★★★★★★}   Various modifications of the invention described herein will be apparent to those skilled in the art. Like that Such modifications are intended to fall within the scope of the appended claims.

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Claims (1)

[Claims]   1. Tan from plants having adenylosuccinate synthetase (ADSS) activity An isolated DNA molecule encoding a protein.   2. 2. The isolated DNA molecule of claim 1, wherein said plant is a dicotyledon.   3. 3. The isolated DN of claim 2, wherein said dicotyledonous plant is of the species Arabidopsis. A molecule.   4. 4. The protein according to claim 3, wherein the protein comprises the amino acid sequence of SEQ ID NO: 2. Isolated DNA molecule.   5. An isolated DNA molecule according to claim 4, consisting of the sequence set forth in SEQ ID NO: 1. .   6. 2. The isolated DNA molecule of claim 1, wherein said plant is a monocotyledonous plant.   7. A monocotyledonous plant selected from the group consisting of corn and wheat; 7. The isolated DNA molecule of claim 6.   8. The isolated DNA component according to claim 7, wherein the monocotyledonous plant is corn. Child.   9. 9. The protein according to claim 8, wherein the protein comprises the amino acid sequence of SEQ ID NO: 4. Isolated DNA molecule. 10. 10. The isolated DNA molecule of claim 9, consisting of the sequence set forth in SEQ ID NO: 3. . 11. 8. The isolated DNA molecule of claim 7, wherein said monocotyledonous plant is wheat. 12. The protein comprises the amino acid sequence of SEQ ID NO: 6. An isolated DNA molecule as described. 13. 13. The isolated DNA fragment according to claim 12, consisting of the sequence of SEQ ID NO: 5. Child. 14． An operably linked DNA molecule according to any one of claims 1 to 13. An expression cassette containing a modified promoter. 15. The expression cassette according to claim 14, which can be stably transformed into a host cell. Recombinant vector. 16. The stable transformation with the vector according to claim 15, which is capable of expressing the DNA molecule. Host cells. 17． Claims selected from the group consisting of bacterial cells, yeast cells and insect cells Item 17. The host cell according to Item 16. 18. Assess chemicals for their ability to inhibit the activity of ADSS enzymes from plants A method for     (A) The ADSS enzyme is added to the first reaction mixture. Combining under conditions that can catalyze the synthesis of nirosuccinic acid;     (B) combining the chemical and the ADSS enzyme in a second reaction mixture, Combined under the same conditions as in the first reaction mixture; and     (D) the adenylosuccinic acid produced in the first and second reaction mixtures Compare quantities     The amount of adenylosuccinic acid in the second reaction mixture. Is the first reaction mixture. If the amount is significantly less than the amount of deniro succinic acid, the chemical may be the ADSS enzyme. The above method, which is capable of inhibiting elemental activity. 19. Highly specific for plant adenylosuccinate synthetase gene or mRNA A nucleotide probe capable of hybridizing, said probe having a length Adenylosuccinate synthetase enzyme from a plant having at least 10 nucleotides The nucleotide probe as described above, which contains the adjacent portion of the coding sequence for 20. (A) Adenilocha in an expression cassette designed for the selected host DNA sequence encoding a protein having citrate synthetase (ADSS) activity Insert;     (B) obtaining the resulting molecule containing the individual elements linked in an appropriate reading frame, Inserting into a vector that can be transformed into a host cell;     (C) growing the transformed host cells in a suitable culture medium. Let; and     (D) tamper from the transformed cells or culture medium or both. Isolate the protein product and purify it     Having adenylosuccinate synthetase (ADSS) activity A method for producing protein in a host organism. 21. Adenylosuccinate synthetase (ADSS) activity For producing a DNA molecule containing a DNA portion encoding a protein having the same And     (A) Specific to plant adenylosuccinate synthetase gene or mRNA A nucleotide probe capable of hybridizing, the probe comprising: Adenylosuccinate synthetase from plants at least 10 nucleotides in length Preparing said probe containing adjacent portions of the coding sequence for the enzyme;     (B) selecting using the nucleotide probe prepared according to step (a) In a collection of cloned genomic DNA or cDNA fragments from the isolated organism Probe other ADSS coding sequences; and     (C) a protein having adenylosuccinate synthetase (ADSS) activity A DNA molecule containing the DNA portion encoding     The above method, comprising: 22. AD from organisms selected by the method of the polymerase chain reaction (PCR) 20. The nucleonucleotide of claim 19 for amplifying and / or analyzing the SS coding sequence. How to use a probe. 23. For selective hybridization of probe to genomic ADSS sequence Of the native ADSS gene in the genome of a plant selected based on standard techniques A method of using the nucleotide probe of claim 19 to map a position. .