CA2385875A1 - Gmp synthetase derived from plants - Google Patents

Abstract

The invention relates to a DNA that encodes a polypeptide that has GMP
synthetase (EC 6.3.5.2) activity. The invention further relates to the use of said nucleic acid in the production of a test system.

Description

' CA 02385875 2002-03-27 GhlP SYaSE DE1~IVED F1~D1~! PLANTS
The present invention relates to the identification of plant GMP
synthetase (guanosine-monophosphate synthetase) as novel target for herbicidal agents. The present invention further relates to DNA sequences coding for a polygeptide having GMP synthetase (EC 6.3.5.2) activity. The invention moreover relates to the use of a nucleic acid coding for a protein having GMP synthetase activity of plant origin far producing an assay system for identifying inhibitors of GMP synthetase having a herbicidal action, and to inhibitors of plant GMP synthetase identified using this assay system. The invention further relates to the use of the nucleic acid coding for plant GMP synthetase for producing plants with increased resistance to inhibitors of GMP synthetase, and for producing plants with a modified content of guanosine nucleotides. The invention additionally relates to a method for eliminating unwanted plant growth, which comprises treating the plants to be eliminated with a compound which specifically binds to GMP synthetase encoded by a DNA sequence SEQ-ID No. 1 or a DNA
sequence hybridizing with the latter, and inhibits the function thereof.
Plants are able to synthesize the-it cellular components fronn carbon dioxide, water and inorganic salts.
This prt~cess is possible only through the use of biochemical reactions to synthesize organic substances. It is necessary for plants to synthesize de novo the nucleotides as constituents of nucleic acids.
Especially in rapidly growing plant tissues it is necessary for nucleotides as constituents of the nucleic acids DNA and RNA to be synthesized by multistage metabolic pathways. Nucleotides are moreover linked in with virtually all metabolic pathways.
Nucleoside triphosphates, especially ATP, drive many energy-expending reactions in cells. Adenine nucleotide additionally occurs as component in essential coenzymes such as coenzym8 A and nicotinamide and flavin coenzymes, which are involved in many cellular conversions. Guanasine nucleotides give a reaction direction to various cellular processes such as protein translation, microtubule assembly, vesicular transport, signal transduction and cell division. In addition, nucleotides are the starting metabolites far the biosynthesis of methylxanthines such as caffeine and theobramine, especially in the Rubiaceae and Theaceae families of plants.

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00'Jr~~5~777 CA 02385875 2002-03-27 Purine nucleotides are formed in microorganisms, animals and plants de novo in the same way starting from phosphoribosyl pyrophosphate (PRPP). IMP is synthesized in a 10-stage reaction sequence. IMP can be converted in subsequent reactions by adenylosuccinate synthetase and adenylosuccinate lyase into AMP.
In the synthesis of GMP there is initial conversion of IMP by IMP
dehydrogenase into XMP which is aminated by GMP synthetase to give GMP, see Fig. 1.
Genes which code for GMP sythetase [sic] have been isolated from various organisms.
The compartmentation of the purine biosynthetic pathway in plants has not to date been extensively investigated. The nitrogen which is fixed in the form of glutamine and aspartate in the root nodules of legumes is firstly courted via the de navo synthetic pathway into purines. This pathway is localized in the plastids in the root nodules of Glycine max and Vigna unguiculata L.
(Boland and Schubert, Arch. Biochem. Bivphys. 220 (1983), 179-187; Shelp et al., Arch. Biochem. Biophys. 224 (1983), 429-441). However, more recent investigations have Shawn that enzyme activitites of the purine biosynthetic pathway are also to be found in mitochondria in the ro~ort nodules of Vigna ungiculata [sic] (Atkins et al., Plant Physiology 113 (1997), 127-135; Smith et al., Plant Molecular Biology 36 (1998), 811-820).
The regulation of this synthetic pathway has to date been investigated only in microorganisms and animals and comprises transcription control, end-product inhibition and allosteric regulation. The enzyme PRPP ami.dotransfera-se (PRPP ATAse) of the second reaction step is attributEd with a key position in the animal as well as the plant system and is subject to allosteric regulation by the end products IMP, AMP and GMP (Reynolds et al., Archives of Biochemistry and Biophysics 229 (1984), 623-631).
GMP-Synthstase also plays a part in relation to the balanced synthesis of guanosine nucleotides and adenosine nucleotides because ATP is a substrate of GMP synthetase.
Since plants are dependent on a functioning nucleotide metabolism, this metabolism is obviously a possible target of novel herbicides. In fact, agents with an inhibitory effect on enzymes of de navo purine biosynthesis have already been described. An example which may be mentioned is 5'-phosphohydantocidin which inhibits an enzyme of plant purine metabolism, adenylosucc-inate synthetase (ASS) (Siehl et al., Plant Physiol. 110 (1996), 753-758). Inhibitors for enzymes of ~

this metabolic pathway also exist from animals and microorganisms. Folate analogs inhibit various folate-dependent reactions, inter olio the enzyme GAR transformylase and have antiproliferative, antiinflammatory and immunosuppressant effects. Mycophenolate (MPA), as an inhibitor of IMP
dehydrogenase, has antimicrobial, antiviral and immunosupgressant effects. (Kitchin et al., Journal of the American Academy of Dermatology 37 (1997), 445-449).
Demonstration of the suitability of an enzyme as herbicide target can be shown, far example, by reducing the enzyme activity by means of the antisense technique in transgenic plants. If reduced growth is brought about in this way, it can be concluded that the enzyme whose activity has been reduced is a suitable site of action of herbicidal agents. This has been shown by way of example for acetolactate synthase on transonic potato plants (Hofgen et al., Plant Physiology 107 (1995), 469-477).
It is an object of the pre~nt invention to prove that GMP
synthetase in plants is a suitable herbicidal target, to isolate a complete plant cDNA ceding for the enzyme GMP synthetase and functional expression thereof in bacterial or eukaryotic cells, and to produce an efficient and simple GMP synthetase assay system for carrying out the inhibitor-enzyme binding studies.

We have found that this object is achieved by isolation of a gene coding for the plant enzyme GMP synthetase, the production of antisense constructs of GMP synthetase, and functional expression of the GMP synthet-ase in bacterial or eukaryvtic cells.
One aspect of the present invention relates to the isolation of a full-length cDNA coding for a functional glutamine-hydrolyzing GMP synthetase (EC 6.3.5..2.) from tobacco (Nicotiaaa tabacnm).
A first aspect of the present invention is a DNA sequence SEQ-ID
NO:1 comprising the coding region of a plant GMP synthetase from tobacco, see Example 1.
Another aspect of the i~ntion is a DNA sequence SEQ-ID No. 3 comprising a portion of the coding region of a plant GMP
synthetase from Physcrnnitrella patens, see Example 2.
Further aspects of the irroention are DNA sequences which are derived from SEQ-ID NO: 1 or SEQ-ID No: 3 or hybridize with one of these sequences and code for a protein which has the biological activity of a GMP synthetase.

0~5~~Sd~/..~ CA 02385875 2002-03-27 Tobacco plants of the line Nicotiana tabacum co. Samsun NN
harboring an antisense construct of GMP synthetase have been characterized in detail. The plants show growth retardation to differing extents. The trarrsgenic lines, and the progeny as 1st and 2nd generation showed reduced growth in soil. In plants with reduced growth it was possible to detect a reduced, comparEd with the wild-type, GMP-7M RNA amount in the Northern hybridization.
It was also possible in a Western blot experiment to detect a reduced amount, comgar~ed with wild-type plants, of GMP synthetase in the transgenic lines, see Example 7. A correlation can be found in the growth retardation and reduction in the amount of GMP synthetase protein. This clear association demonstrates for the first time that GMP is unambiguously a suitable target protein for herbicidal agents.
In order to be able to find efficient inhibitors of plant GMP
synthetase, it is neces-s-ary to provide suitable assay systems wih which inhibitor-enzyme binding studies can be carried out. For this purpose, for example, the complete cDNA sequence of the GMP
synthetas~e from tobacco is clorned in an expression vector (pQE, Qiagen) and is expressed in E. coli, see Example 4.
An alternative possibility is, however, to express the expression cassette comprising a DNA sequence SEQ-ID No. 1 for example in other bacteria, in yeasts, fungi, algae, plant cells, insect cells or maimaalian cells, see Example 5.
The GMP synthetase protein expressed with the aid of the expression cassette ac~c~arding to the invention is particularly suitable for finding inhibitors specific far GMP synthetase.
For this purpose, the plant GMP synthetase can be employed, far example, in an enzyme assay in which the activity of the GMP
synthetase is measured in the presence and absence of the agent to be tested. Qualitative and quantitative information about the inhibitory characteristics of the agent to be tested is obtainable from comparison of the two activity determinations, see Example 8.
The assay system according to the invention can be used for rapid and simple testing of a large number of chemical compounds for herbicidal properties. The method allows reproducible selection from a large number of substances specifically of those having a potent effect in order then to carry out other, mare intensive tests which are familiar to the skilled worker on these substances.

A further aspect of the invention is a method far identifying substances having a herbicidal action, which inhibit the GMP
synthetase activity in plants, consisting of 5 a) preparat-ion of trawsge is plants, plant tissues, or plant cells which comprise an additional DNA sequence ceding for an enzymE having GMP synthetase activity and are able to overexpress an enzymatically active GMP synthetase;
b) application of a substance to transgEnic plants, plant cells, plant tissues or plant parts and to untransfarmed plants, plant cells, plant tissues or plant parts;
c) determination of the grDwth or survivability of the transgenic and untransfarmed plants, plant cells, plant 'tissues or plant parts after application of the chemical substance; and d) comparison of the growth or survivability of the transgenic and untransfarme~l plants, plant cells, plant tissues or plant parts after application of the chemical substance;
where suppression of the growth or survivability of the untransfarmed plants, plant cells, plant tissues or plant parts without, however, greatly suppressing the growth or the survivability of the transgenic plants, plant cells, plant tissues or plant parts demonstrates that the substance from b) shows herbicidal activity and inhibits the GMP synthetase enzymie activity in plants.
A further aspect of the invention is a method far identifying inhibitors of plant GMP synthetases, with potential herbidical action, by cloning the gene of a plant GMP synthetase, bringing about ovErexgression in a suitable expression cassette - for example in insect cells, opening the cells and employing the cell extract directly or after cancerrtrat.ion or isolation of the enzyme GMP synthetases in an assay system for measuring the enzymic activity in the presence of low molecular weight chemical compounds.
A further aspect of the invention comprises compounds having a herbicidal action which can be identified using the assay system described above.
A further aspect of the invention is a method for eliminating unwanted plant growth, which comprises treating the plants to be eliminated with a compound which specifically binds to plant GMP

~

synthetase and inhibits the function thereof.
Inhibitors of GMP synthetase with a herbicidal action can be used as defoliants, desiccant , haulm destroyers and, in particular, weedkillers. Weeds mean in the widest sense all plants which grow where they are unwanted. Whether the agents found with the aid of the assay system according to the invention act as total or selective herbicides depends inter olio on the application rate.
Inhibitors of GMP synthetase with a herbicidal action can be used, for example, to control the following weeds:
Dicotyledonous weeds of the genera:
Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenapodium, Urtica, Senecio, Amaranthus, Partulaca, Xanthium, Convalvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilan, Emex, Datura, Viola, Galeopsis, Popover, Centaurea, Trifalium, Ranunculus, Taraxacum.
Monocotyledonous weeds of the genera:
Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bramus, Avsna, Cygerus, Sorghum, Agropyron, Cynodon, Monocharia, Fimbristylis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrastis, Alapecurus, Apera.
Another aspect of the invention comprises expression cassettes whose sequence codes for a GMP synthetase from tobacco or functional equivalent thereof. The nucleic acid sequence may in this connection be, far example, a DNA or a cDNA sequence.
Another aspect of the invention is an expression cassette comprising a DNA sequeTtce SEQ-ID No. 3 ceding for a portion of the plant GMP synthetase from Physcamitrella patens.
The expression cas-settes according to the invention additionally comprise regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. In a preferred embodiment, an expression cassette according to the invention comprises a promoter upstream, i.e. at the 5' end of the coding sequence, and a polyadenylatian signal downstream, i.e. at the 3' end, and, where appropriate, further regulatory elements which are operatively linked to the GMP synthetase gene coding sequence lying between them. An operative linkage means tha sequential arrangement of promoter, coding sequence, terminator and, where appropriate, further regulatory elements in ~~5~~5Q777 CA 02385875 2002-03-27 such a way that each of the regulatory elements is able to perform its function as intended on expression of the coding sequence.
An expression cass8tte aacarding to the invention is produced by fusing a suitable promoter with a suitable GMP synthetase DNA
sequence and a polyadenylation signal by conventional recombination and cloniirg techniques as described, for example, in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
Another aspect of the invention comprises functionally equivalent DNA sequence's which code far a GMP synthetase and which have, based on the complete length of the DNA sequence, a sequence homology with the DNA sequence SEQ-ID NO: 1 or SEQ-ID No. 3 of from 40 to 100%.
A preferred aspect of the invention comprises functionally equivalent DNA sequences which code for a GMP synthetase and which have, based on the complete length of the DNA sequence, a sequence homology with the DNA sequence SEQ-ID NO: 1 or SEQ-ID
No. 3 of from 60 to 100%.
A particularly preferred aspect of the invention comprises functionally equivalent DNA sequences which code for a GMP
synthetase and which have, based on the complete length of the DNA sequence, a sequ~ce homology with the DNA sequence SEQ-ID
NO: 1 or SEQ-ID NO. 3 of from 80 to 100%.
Functionally equivalent sequences which code for a GMP synthetase are, according to the invention, sequences which, despite a different nucelotide sequence, still have the desired functions.
Functional equivalents thus comprise naturally occurring variants of the sequences described herein, and artificial nucleotide sequences, far example obtained by chemical synthesis, which are adapted to the codan usage of a plant.
A functional equivalent also means in particular natural or artificial mutations of an originally isolated sEquence which codes for a GMP synthet-ase and additionally shows the required function. Mutations comprise substitutions, additions, deletions, transpositions or insertions of one or more nucleotide residues.

0050~5~7~ CA 02385875 2002-03-27 ..

Thus, farr example, the pre ent invention also includes nucleotide sequences obtained by modificatian~of the nucleotide sequence.
The aim of such a modification may be, for example, further localization of the coding sequence present therein or, for example, insertion of further restriction enzyme cleavage sites.
Functional equivalents are also variants whose function has been attenuated or enhanced by comparison with initial gene or gene fragment.
The expression cassette according to the invention can also be employed for transforming bacteria, cyanobacteria, yeasts, filamentous fungi and algae with the aim of producing adequate amounts of the enyzme GMP synthetase.
A further aspect of the i~ntian is a protein from tobacco which has the amino acid sequence SEQ-ID NO: 2 or derivatives or portions of this protein with GMP synthetase activity.
Another aspect of the invention comprises plant proteins with GMP
synthetase activity having an amino acid sequence homology with the tobacco GMP synthetase of 20 - 100% identity.
Preferred plant proteins with GMP spnthetase activity have an amino acid sequence homology with the tobacco GMP synthetase of~
50 - 100% identity.
Particularly gref~ed plant proteins with GMP synthetase activity have an amino acid sequence homology with the tobacco GMP synthetase of 80 - 100% identity.
Another aspect of the i~ntian comprises plant proteins with GMP
synthetase activity having an amino acid sequence homology with the Physcomitrella patens GMP synthetase of 20 - 100% identity.
Preferred plant prc3teins with GMP synthetase activity have an amino acid sequence homology with the Physcomitrella patens GMP
synthetase of 50 - 100% identity.
Particularly preferred plant proteins with GMP synthetase activity have an amino acid sequence homology with the Physcomitrella patens GMP synthesase of 80 - 100% identity.
A further object of the im~ention was averexpres~ion of the GMP
synthetase gene in plants to produce plants which are tolerant of inhibitors of GMP synthetase.

Overexpressian of the gene sequence SEQ-ID NO: 1 coding for a GMP
synthetase in a plant achieves increased resistance to inhibitors of GMP synthetase. The transgenic plants produced in this way are likewise an aspect of the invention.
The efficiency of expression of the transgEnically expressed GMP
synthetase gene can be measured, for example, in vitro by shoot meristem propagation or by a germination test. In addition, a change in nature and level of the expression of the GMP
synthetase gene and the effect thereof on the resistance to inhibitors of GMP synthetase can be tested on test plants in glasshouse experiments.
An additional aspect of the invention comprises transgenic plants transformed with an expres-si.on cassette according to the invention comprising DNA sequence SEQ-ID No. 1, which has become tolerant of inhibitors of GMP synthetase due to additional expression of DNA sequence SEQ-ID No. 1, and to transgenic cells, tissues, parts and propagation material of such plants.
Particular preference is given in this connection to transgenic crop plants such as, for example, barley, wheat, rye, corn, soybean, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species, and legumes.
25.
An alteration in the nucleotide content in plants may be beneficial in various cases. For example, nucleotides are added to plant-based babyfaod products in order to achieve a nutrient solution corresponding to breast milk. In addition, an optimized nucleotide content would be sensible in the enteral feeding of patients. A reduced purinE nucleotide content in plants of foods relevance is relevant to the diet-cry feeding of patients with gout. Nucleotides also have f.lavar-forming and flavor-enhancing effects so that an altered nucleotide content has effects on the taste properties of plants.
A further aspect of the irroentian therefore comprises plants which have a modified content of guanos-ine nucleotides after expression of the DNA sequence SEQ-ID NO: 1 or SEQ-ID No: 3 in the plant.
A plant with a modified content of guanosine nucleotides is based, for example, by express-ian of an additional DNA sequence SEQ-ID No. 1 or 3 in the sense or antisense orientation in the plant. A modified cament of guanasine nucleotides means that it is possible to produce plants with an increased content of guanosine nucleotides in the case of the sense orientation and ~ 0~50/5~7~7 CA 02385875 2002-03-27 plants with a reduced content of guanosine nucleotides in the case of the sense orientation (casupgression) or antisense orientation.
5 Increasing the content of guanosine nucleotides means, for example, within the scope of the present invention the artificially applied capability of increased biosynthesis of guanosine nucleotides awing to functional overexpression of the GMP synthetase ge-ne in the plant compared with the plant which 10 has not been genetically manipulated far the duration of at least one plant generation.
A further aspecet of the invention is the use of plant GMP
synthetases to alter the concentrations of methylxanthines in plants.
Particularly preferred ser~uencES are those which ensure targeting in the apoplasts, in plastids, the vacuoles, the mitochondrion, the endoplasmic reticulum (ER) or, through the absence of appropriate operative sequences, ensure retention in the compartment of production, the cytosol (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).
For example, the pla t exgrsss-ion cassette can be incorporated into the tobacco t ion vector pBinAR, see Example 6.
Suitable in principle as promoter for the expression cassette according to the invention is every promoter able to control expression of foreign genes in plants. It is particularly preferred to use a plant promoter or a promoter derived from a plant virus. The CaMV 35S promoter from Blumenkohl mosaic virus (Franck et al., Cell 21 (1980), 285-294) is particuliarly preferred. This promoter contains various recognition sequences for transcriptianal effectors which, in their totality, lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).
The expression cassette according to the invention may also comprise a chemically inducible promoter through which it is possible to control expression of the e~cogenous GMP spnthetase gene in the plant at a particular point in time. Promoters of this type, such as, far example, PRP1 promoter (Ward et al., Plant. Mol. Biol. (1993) 22, 361-366), a promoter indueible b y salicylic acid (WO 95/19443), a benzenesulfonamide-inducible (EP 388186), a tetracycline-inducible (Gatz et al., Plant J.
(1992) 2, 397-404), an abscisic acid-inducible (EP0335528) or an ethanol- or cyclohexanone-inducible (w0 93/21334) promoter are ~05~~5~7-77 CA 02385875 2002-03-27 described in the literature and can, inter olio, be used.
Further particularly preferred promoters are those which ensure expression in tissues or plant parts in which the biosynthesis of purines or their precursors takes place. Particular mention may be made of promoters which ensure leaf-specific expression.
Mention should also be made of the promoter of the potato cytosolic FBPase or the potato ST-LSI promoter (Stockhaus et al., EMBO J., (1989) 8, 2445-2451).
It is possible with the aid of a seed-specific promoter to express a foreign protein stably up to a content of 0.67% of the total soluble seed protein in the seed of transgenic tobacco plants (Fiedler and Canrad, Bio/Technology (1995) 10, 1090-1094).
The expression cassette according to the invention can therefore comprise, for example, a seed-specific promoter (preferably the phaseolin promoter, the USP or LEB4 promoter), the LEB4 signal peptide, the gene to be expressed and an ER retEntion signal.
The inserted nucleotide sequence coding for a GMP synthetase can be prepared synthetically or be obtained naturally or comprise a mixture of synthetic and natural DNA components. In general, synthetic nucleotide sequences are produced with cvdans preferred by plants. These codvns preferred by plants can identified by colons which have the highest protein frequency and are expressed in most plant species of interest. To prepare an expression cassette it is possible to manipulate various DNA fragments in order to obtain a nucleotide sequence which expediently reads in the correct direction and is equipped with a correct reading frame. Adaptr~rs or linkers can be attached to the framework to connect the DNA fragments together.
Artificial DNA sequences are also suitable as long as they confer, as described above, the required property of increasing the content of guanDSine nucleotides in the plant through overexgressivn of the GMP synthetase gene in crop plants. Such artificial DNA sequences can be found, for example, by translation back from proteins having GMP synthetase activity and constructed by molecular modeling, or by in vitro selection.
Particularly suitable coding DNA sequencES are those obtained by translation back from a polypeptide sequence in accordance with the codon usage specific for the host plant. The specific codon usage can easily be found by a skilled worker familiar with methods of plant genetics by computer analyses of other known genes of the plant to be transformed.

' ~~5~~5~7~77 CA 02385875 2002-03-27 Further suitable equivalent nucleic acid sequences according to the invention which may be mentioned are sequences coding for fusion proteins where one constituent of the fusion protein is a plant GMP synthetase polypeptide or a functionally equivalent portion thereof. The second part of the fusion protein can be, for example, another polypeptide with enzymatic activity or an antigenic polypeptide sequence with whose aid it is possible to detect GMP syrrthetase expression (e. g. myc tag or his tag).
However, this is preferably a regulatory protein sequence such as, for example, a signal or transit peptide which guides the GMP
synthetase protein to the desired site of action.
The promoter regions according to the invention and the terminator regions ought expediently to be provided in the direction of transcription of a linker or polylinker captaining one or more restriction sites far insertion of this sequence. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. The size of the linker within the regulatory region is generally less than 100 bp, frequently less than 60 bp, but at least 5 bp. The promoter according to the invention may be both native or homologous and foreign or heterologous to the host plant. The expx~essian cassette according to the invention cvmpris~es in the 5'-3' direction of transcription the promoter according to the invention, any suitable seque cue and a region for the transcriptional termination. Different ternrinatian regions can be exchanged far one another if desired.
It is furthermore possible to employ manipulations which provide suitable restriction cleavage sites or delete the excess DNA or restriction cleavage 51te8. Where the insertions, deletions or substitutions such as, far example, transitions and transversions are considered, it is possible to use in vitro mutagenesis, primer repair, restriction or ligation. In the case of suitable manipulations such as, far example, restriction, chewing-back or filling in of overhangs far blunt ends, it is possible to make complementary ends of the fragments available for the ligation.
Preferred polyadenylation signals are plant polyadenylation signals, preferably thane which essentially correspond to T-DNA
polyadenylation signals from Agrabacterium tumefaciens, in particular of gene 3 of the T-DNA (octopine synthase) of Ti plasmid pTiACHS (Gielen et al., EMBO J. 3 (1984) 835 ff) or functional equivalents.
To transform a host plant with a DNA coding for a GMP synthetase, an expression cassette according to the invention is incorporated ~
~~50~5~77~ CA 02385875 2002-03-27 as insert into a recombinant vector whose vector DNA contains additional functional regulatory signals, for example sequences for replication or integration. Suitable vectors are described inter olio in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chapters 6/7, pages 71-119.
The transfer of foreign genes into the genome of a plant is referred to as tra~rs-formatian. The methods used for this purpose are those described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene gun, electroporation, incubation of dry embryos in DNA-containing solution, microinjection and gene transfer mediated by agroba~terium. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Acade~aic Press (1993) 128-143 and in Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
Agrobacteria transformed with an expression cassEtte according to the invention can likewise be used in known manner for transforming plants, especially plants such as cereals, corn, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and various tree, nut and vine species, and legumes, for example by bathing wounded leaves or pieces of leaf in a solution of agrobacteria and then cultivating in suitable media.
The site of pyrimidine biasythe-sis is generally the leaf tissue, so that leaf-specific expression of the GMP synthetase gene is sensible. However, it is obvious that pyrimidine biosynthesis need not be confined to leaf tissue but may also take place in all other parts of the plant, for example in fat-containing seeds, tissue-specifically.
In addition, constitutive expre~s~sion of the exogenous GMP
synthetase gene is advantageous. However, on the other hand, inducible expression may also appear desirable.
Using the recombinant and cloning techniques quoted above, the expression cassettes according to the invention can be cloned into suitable vectors which make it possible to replicate them, 0~'3~~~5~77~7 CA 02385875 2002-03-27 for example into E. coli. Suitable cloning vectors are, inter olio, pBR322, pUC series, Ml3mp series and pACYC184. Binary vectors able to rEplicate both in E. coli and in agrobacteria are particularly suitable.
A further aspect of the invention relates to the use of an expression cassette according to the invention for transforming plants, plant cells, plant tissues or parts of plants. The aim of the use is preferably to increase the GMP synthetase content in the plant.
This may involve, depending on the chosen promoter, expression specifically in the leaves, in the seeds or other parts of the plant. Such transgenic plants, their propagation material and their plant cells, tissues or parts form a further aspect of the present invention.
The invention is illustrated by the Examples which now follow, but is not co fined to these:
Examples Methods of genetic manipulation on which the examples are based:
General cloning methods Cloning methods such as, for example, restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrBCellulose and nylon memtrranes, linkage of DNA fragments, transformation of Escherichia coli cells, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as des~ribEd by Sambraok et al.
(1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
Sequence analysis of recombinant DNA
Recombinant DNA molecules were sequenced using a laser fluorescence DNA sEquencer supplied by ABI by the method of Sanger (Sanger et al. (1977), Proc. Natl. Acad. Sci. USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
The chemicals used were purchased, unless otherwise mentioned, in analytical quality from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Servo (Heidelberg) and Sigma (Deisenhofen):
Solutions were made up using prepared, pprvgen-free water, ~
0050/5~7'T7 CA 02385875 2002-03-27 referred to as H20 in the subsequent text, from a Milli-Q water treatment system (Millipore, Eschbarn). Restriction endvnucleases, DNA-modifying enzymes and molecular biology kits were purchased from AGS (Heidelberg), Amersham (Braunschweig), 5 Biometra (Gottingen), Roche (Mannheim), Genomed (Bad Oeynhausen), New England Biolabs (Schwalbach/Taunus), Nvvagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Heidelberg). They were used in accordance with the manufacturers' instruction 10 unless mentioned otherwise.
The strains of bacteria used hereinafter (E. coli, XL-1 Blue) were purchased from Stratagene. E. coli AT 2465 was purchased from the coli genetic stuck center (Yale University, New Haven).
15 The agrobacterial strain used for the plant transformation (Agrvbacterium tumsfaciens, C58C1 with the plasmid pGV2260 or pGV3850kan) has been described by Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777). An alternative possibility is also to employ the agrabacterial strain LBA4404 (Clontech) or other suitable strains. Vectors which can be used far the cloning are pUCl9 (Vanish-Perrvn, Gene 33(1985), 103-119) pBluescript SK-(Stratagene), pGEM-T (Promega), pZerO (Invitragen), pBinl9 (Bevan et al., Nucl. Acids Res. 12(1984) 8711-8720) and pBinAR (Hofgen and Wil.lmitzer, Plant Science 66 (1990) 221-230).
Example 1 Isolation of a cDNA of the guaA gene, coding for a GMP synthetase from tobacco.
An expressed sEqueTlce tag (EST) from Arabidvpsis thaliana (EST
F14426) which, on a partial reading frame, encodes a polypeptide of 68 amino acids with 60% similarity with a GMP synthetase from Helicobacter pylori was subjected to partial 5'-terminal sequencing. The oligonucleatides 5'-aag got cca agc tct aag acc cta tcc-3' and 5'-tta got ctt tat tcc cat tcg atg g-3' from the 5'- and 3'-terminal sequences were used for amplif.icatian by a polymerase chain reaction (PCR) of a 1000 by cDNA fragment with EST F14426 as template in a Perkin Elmer DNA thermal cycler. The reaction mixture contained 0.1 ng/~ul cDNA from tobacco, 0.5 ~M of the appropriate oLigonucleatide , 200 N.M nucleotides (Pharmacia), mM KC1, 10 mM Tris-HC1 (pH 8.3 at 25°C, 1.5 mM MgCl2) and 0.02 U/~,1 Taq polymers a (Perkin Elmer).
45 The ampLificativn conditions were sEt as follows:
Annealing temperature: 52°C, 1 min ' ~0~~~50'~'~7 CA 02385875 2002-03-27 _ I6 Denaturat.ion temperature: 92°C, 1 min Elongation temperature: 72°C, 1.5 min Number of cycles: 30 The fragment was employed for screening a cDNA library from callus tissue of Nicotiana tabacum (variety Samsun NN) in the vector zAP Express. For this purpose, 2.5 x 105 lambda phages from the cDNA library were plated out on agar plates with E. coli xLl-Blue as bacterial strain. The phage DNA was transferred by standard methods (Sambrook et al. (1989); Cold Spring Harbor Laboratory Press: ISBN 0=87969-309-6) to nitrocellulose filters (Gelman Sciences) and fixed on the filters. The hybridization probe used was the PCR fragment described above which had been radiolabeled using a multiprime DNA labeling system (Amersham Buchler) in the presence of a-32P-dCTP (specific activity 3000 Ci/mmol) in accordance with the manufacturer's information.
The hybridization of the membranes took place after prehybridizativn at 60°C in 3 x SSPE, 0.1% sodium dodecyl sulfate (w/o), 0.02% polyvi:nylpyrrolidone (w/o), 0.02% Ficoll 400 (w/o) and 50 mg/ml calf thymus DNA for 12-16 hours (Sambrook et al.
(1989); Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
The filters were then washed in 2 x SSPE, 0.1% sodium dodecyl sulfate (w/o) at 60°C far 60 minutes. Positively hybridizing phages were visualized by autoradiography and purified and isolated by standard techniques.
It was possible to identify and purify 13 hybridizing signals.
After restriction analysis, the clones GMP-6 and GMP-7M were selected for double-stranded sequencing. Evaluation of the sequencing data showed that the clone GMP-7M with a length of 1973 by contained a complete reading frame of 1614 by which codes for a protein of 538 amino acids with a calculated molecular weight of 60.1 KDa (SEQ-ID No. 1). In front of the presumed start codon there is a stop codan in the same reading frame, which suggests that GMP-7M is a full-length cDNA. GMP-7M thus represents the first full-length plant cDNA of a GMP synthetase.
GMP-6 is a partial clone which is 217 nucleotides shorter than GMP-7M on the 5'. GMP-7M shows similarities with GMP synthetases from microorganisms and animals. Apart from the partial amino acid sequence encoded on EST F14426 there are no other sequences from plants with homology with GMP synthetases in the databases.
The greatest similarity (62%) is with a GMP synthetase from Helicobacter pylori. It is also evident that the similarities between the C termini of the GMP synthetases are greater than those in the region of the N termini. The N terminus of the GMP-7M amino acid sequewce corresponds with the N termini of GMP
synthetases from other organisms such as E. coli and Synechocystis sp. (Table 1). GMP-7M has no marked signal sequences (found by the program PSbRT, Nakai, K., Institute for Molecular and Cellular Biology, Osaka University, Japan), which might indicate a cytosolic localization of the protein.
Table 1 Sequence comparison of GMP synthetases from Nicotiana tabacum (guaA N.t = GMP-7M), Arabidopsis thaliana (guaA est_A.t, Genbank No. F14426), E.coli (guaA e.c, Genbank No. 146276), Synechocystis sp. (guaA syn, Genbank No. 1001583), Helicobacter pylori (guaA h.p, Genbank No. 3122166), Homo sapiens (guaA human, Genbank No. 1708072).

guaA N.t MEPQ TAKKSNLVLILDYGSQYTHLITRAIR5L5 A
t A
t gua _es .

A ihkh ildf t i1 gua m en gsqytq lvarrvrelg e.c r guaA syn mttqipvgpvvsdqalpdrisd=lkgqiivildfgsqyseliarrirete guaA h.p -mil vldfgsqytq.liarrlrerg 20guy hen -malcagdsklenaggdlkdghhhyegavvildagaqygkvidrrvrelf guaA N.t IFSLTINGTSSLDS2KELDPAVI~ILSGGPHSVHADGAPCFFPGFIEYVES

A
t A
t gua es .

guaA e.c vycelwawdvteaqirdfnpsgiilsgggestteensprapq....yvfe guaA syn vysevlsyrttaqqlreikpkgiilsggpnsvydqgapecdp....eifq guaA h.p iyteivgffes-ieniqkkapkglilsggpasvyakdaykpsg....kifd Z5guaA human vqsEifpletgafaikeqgfraiiisggpnsvyaedapwfdpa....ift guaA_N.t RGIHVLGI~CYGLQI~IVQKLGGVVKIGEKHEYGRMEIEVGKNW....GGL

A
t A
t gua es .

guaA e.c agvpvfgvcygmqtmamqlgghveasne~refgyaqvevvndsalvrgied guaA syn lgvpvlgvcygmqlmvkqlggrverakrgeygkaslhiddptdlltnven 30guaA h.p lnvpilgicygmqylvdffggvvvganeqefgkavleitqnsvifegv..

guaA human igkpvlgicygmqmmnkvfggtvhkksvredgvfnisvdntcslfrglqk guaA N.t FGNTEI~GDKQVVpPiSIiGDEAVKLFEGFEVVARSSQGAVAAIERREARFYG

uaA
e t A
t g --s .

guaA e.c altadgkplldvwmshgdkvtaipsdfitvastESCgfaimanEekrfyg guaA syn dst........m~mtshgdscvdlptgfeilahtdntp~aaiadhqkalfg 35guaA h.p ......kikslvwmshmdkvielpkgfttlakspnsphcaiengk..ifg guaA human ........eevvllthgdsvdkvadgfkwsrrsgni.vagianeskklyg guaA N.t LQYHPEVTHBTEGPIATLRHFLFDVCGVTAGWKMEDVLEEErRVIKGI~fiTGP

uaA e -t A
t g -s .

guaA e.c vqfhpevthtrqgmrmlerfvrdicqcealwtpakiiddavarireqvg.

40guaA_syn vqfhgewhsvggialirnfvyhichceptwttaafieesirevrsqvg.

guaA h.p lqfhgevvqseeggkilenfallvcgcEktwgmqhfaqreiarlkekia.

guaA human aqfhpevgltengkvilknflydiagcsgtftvqnrelecireikervgt guaA N.t EDHVICALSGGVDSTVAAKLVHKAIG.DItLHCVFVDNGLLRYREFtERVIKE

uaA
est A
t g _ .

45gu~_e.c ddkvilglsggvdssvtamllhraig.knltcvfvdngllrlneaeqvld guaA syn drrvllalsggvdsatlafllhraig.dnltcmfidqgfmrkgegerlve guaA h.p nakvlcavsggvdstwatl lhraik.dnliavfvdhgllrknekervqa guaA human s.kvlvllsggvdstvctallnralnqeqviavhidngfmrkresqsvee ~~5~~5~7~77 CA 02385875 2002-03-27 1$

guaA N.t LFEK...................RLHLPVTCVDATEEFLSKLKGVTEFEM

t -- --- -t A. -guaA es guaA_e.c mfgd...................hfglnivhvpaedrflsalagendpea guaA_syn lfdh...................qfhipvqyvnardrflkqlegvtdgee guaA h.p mfkd....................lkiplntidakevflsklkgvsepel guaA human alkklgiqvkvinaahsfyngtttlpisdedrtprkrisktlmnttspee guaA N.t KRKI~IGKEFINTFDL-FAHDVEERVGKRPSYLVQGTLYFDVIESC...PPP

A -t A
t gua -es .

guaA e.c krkiigrvfvevfd..eealk...ledvkwlaqgtiypdviesaas....

guaA syn krrlighefiqvfe..eesnr...lgpfdylaqgtlygdviesadsnvdp 10guaA h.p krkiigetfievfe..keakkhhlkgkieflaqgtlypdviesvsv....

guaA human krkiigdtfvki..anevigemnlkpesvflaqgtlrpdliesasl....

guaA N.t GSGRTHSFiTIKSAHNVGGLPKDMKL..KLIEPLKLLFKDEVRELGKILDI

----guaA est -A.t guaA e.c atgk..ahvikshhawgglpkemkm..glveplkelfkdevrkiglelgl 15guaA syn ktgervavkikshhnvgglpknlrf..klveplrklfkdevrklgrsigl guaA h.p ...kgpskvikthhnvgglpewmdf..klieplrelfkdevrllgkelgv guaA human .vasgkaelikthhndtelirklreegkvieplkdfhkdevrilgrelgl guaA N.t SEDFI;RRHFFPGPGLAVR~IPGDVTAGNSLDILROVDEIFIQSIADAKIYD

~ - -t A
t gua. -_es .

20guaA e.c pydmlyrhpfpggglgvrvlgevkk.eycdllrradaifieelrkadlyd guaA syn peeivrrhgfpgpglairiigevts.erlnilrdadfivrdeiskrgiyh guaA h.p sqdflmrhpfpgpglavrilgeise.skikrlqeadfifieelkkanlyd guaA human peelvsrhpfpgpglairvic.aeepyickdfpetimilkivadfsasvk guaA N.t EIWOAFAVFLPVRTVGVQGDQRTHSHAVALRA.VTSBDGMTADW7C7CFDFK

t A d k i h .t aqg gt gcrlqaqvgltadwfifehk guy es p vgcp 25guaA e.c kvs~aftvflpvrsvgvmgdgrkydwvvslra.vetidfmtahwahlpyd guaA syn dywqafavllpirsvgvmgdkrtyahpwlrf.itsedgmtadwarvpyd guaA h.p kvwqaf~cvllnvnsvgvmgdnrtyenaiclra.onasdgmtasfsflehs guaA human kphtllqrvkactt-eedqeklmgitslhslnafllpiktvgvqgdcrsys A N FLDDVSRKICNSVRGVNAVLLBITSKPFS
t qua T IEWE
.

30gu~_est_A.t flddvsrkicnsvqgvnrvvlditskppstiewe --guaA e.c flgrvsnriinevngisrvPydisgkpgatiewe guaA syn ileaisnrivn~vkgvnrwyditskppgtiewe --guaA h.p flekvsaritnevsginrvnyditskppgtiewe -guaA human yvcgisskdepdwesliflarliprmchxronrvvyifggpvkepptdvtp N
t 35. -guy guaA est --A.t A

gua -e.c A

gua -syn A h ---gua --.p -guaA human tflttgvlstlrqadfeahnilresgyagkisqmpviltplhfdrdplqk t gua .
-A ---t A
t gua es .

A

gua e.c A_ gua - -syn A h gua .p guaA human qpscqrsvvirtfitsdfmtgigatpgnEipvewlkmvt eikkipgisr guaA N.t -P
t A
t gua .
es .

A

gua e.c ' ~~5~/5~'~') CA 02385875 2002-03-27 guaA syn guaA h.p guaA human imydltskpp gttewe Example 2 Isolation of a cDNA of the guaA gene, coding for a GMP synthetase from the moss Physcomitrella patens Double-stranded cDNA was generated from mRNA from protonemata of various ages of Physcvmitrella patens and used to produce a cDNA
bank in the vector pBluescript SKLI (lambda ZAP II RI Library construction kit, Stratagene). Single clones from this bank were partially sequenced. The sequence of the clone 093-dll showed clear homology with the GMP synthetase frBm Aquifex aeolicus. The complete sequence of 093-dll was determined, see SEQ-ID No. 3.
Og3 dll has a length of 1232 nucleotides and codes on a continuous reading frame far 382 amino acids. Comparison with GMP-7M reveals that 093 dll is a partial cDNA. The homology with GMP-7M is 66.7% at the nucleotide level and 74.6% at the amino acid level.
Example 3 Demonstration of the function of GMP-7M by complementation of E.
coli The GMP-7M cDNA was employed as tEmplate for a PCR with the oligonucleotides 5'-CCTAGCCATGGAACCTCAAAC-3' and 5'-TATAGGATCCTACTTTGGTCACC-3'. The reaction mixtures contained about 0.1 ng of GMP-7M DNA, 0.5 ~M of the appropriate oligonucleotides, 200 N.M nucleotides (Pharmacia), 50 mM KC1, 10 mM Tris-HC1 (pH 8.3 at 25°C, 1.5 mM MgCl2) and 0.02 U/~1 Pfu polymerase (Stratagene).
The amplification conditions were set as follow:
Annealing temperature: 50°C, 30 sec Denaturation temperature: 92°C, 30 sec Elongation temperature: 72°C, 3 min Number of cycles: 25 The resulting fragment of about 1670 by was ligated via the NcoI
and BamHI cleavage sites introduced by the oligonucleotides into the vector pTrc99A (Pharmacia). The resulting construct GMP-7Trc was transformed into the E.coli strain AT2465 (genetic markers:
thi-1, guaA2l, relAl, ~,, spoil) and plated out on M9 minimal medium (Sambrook et al. (1989) Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) with and without 100 ~,g/ml guanosine.

~Qr~~/r~~~~7 CA 02385875 2002-03-27 The minimal media contained 0.4% glucose, 0.2% casamino acids, 100 ~g/ml thiamine, 100 ~.g/ml inosine, 100 ~g/ml biotin, 100 ~,g/ml histidine, 100 ~g/ml arginine, 100 ~.g/ml 2'-deaxyuridine, 100 N.M
IPTG and 25 ~g/ml ampicillin. The cloning vectors pTrc99A was 5 transformed into AT2465 in a parallel experiment. It emerged that only the transformed bacteria which contained a GMP-7M cDNA from tobacco in the exgreswion vector pTrc99A were capable of growth on minimal media without guanosine (see Tab. 2}, which points strongly to the GMP-7M cDNA coding far an active GMP synthetase.
10 The enzyme encoded by GMP-7M thus rEpresents the first functional GMP synthetase isolated from plants.
Table 2 15 Growth of E. coli AT2465 trans-farmed with various plasmids after 2 days at 37°C
pTrc99A + GMP-7M pTrc99A

20 Minimal medium without+

guanosine Minimal medium with + +

guanosine (100 ~.g/ml) Example 4 Overexgressian of the GMP synthet-ase from tobacco in E.coli and production of antibodies For overrexpres-sion in E.coli, BamHI cleavage sites were introduced by PCR with GMP-7M as template and the oligonucleotides GMPA: 5'-GCAATGGATCCTZAAACACAGGCG-3' and GMPB:
5'-AAAAGGATCCTACTTTGGTCACC-3' and made it possible to clone the fragment in the vector pETlSb (Novagen). A GMP-7M reading frame with hexahistidine anchor at the N terminus was produced in this way. After the carrec~t orientation had been checked by restriction digestion and golymerase errors had been excluded by sequencing, the resulting construct GMP-7E was transformed into E. coli BL21(DE3} (Stratagene). IPTG-induced one-day cultures were harvested by centrifugation, and the cell pellets were lyzed and treated further in accordance with the manufacturer's information far nickel affinity chromatography ("Qia-Express-Kit", Qiagen). It was possible in this way to purify the GMP synthetase to mare than 95% purity. The protein was used far producing antisera in rabbits by conventional ~05~~507'r7 CA 02385875 2002-03-27 protocols (carried out on cantract,by Eurogentec, Herstal, Belgium).
Example 5 Expression of the GMP spnthetase from tobacco in baculovirus-infected insECt cells In order to obtain sufficient active GMP synthetase far mass testing of chemicals, a 1.65 kb fragment was excized from GMP-7E
with BamHI and cloned into the transfer vector pFastBacHTa (GibcoBRL). The resulting construct GMP-7I was used to generate recombinant baculavirus in accordance with the manufacturer's information (GibcoBRL). This virus was used in accordance with the manufacturer's information (GibcoBRL) far infecting Sf21 insect cells irk order to praducE active GMP synthetase whose activity could be measured after disruption of the cells in 50 mM
Tris-HC1, pH 7.6, 10 mM RC1, 1 mM EDTA, 10 mM PMSF and desalting of the extract on a Sephadex G-25 column (Pharmacia, Sweden).
Example 6 Production of plant expression cas~sEttes The antisEnsE and cosuppress-ian tEChniquss were used with the aim of reducing the GMP spnthetase activity in trans~enic tobacco plants. For this purposE, plasmid constructs were produced in the vector pBinAR (HSfgen and Wil.lmitzer, Plant Science (1990) 66, 221-230). A fragment of 1599 by obtained from GMP-7M with BamHI
and Bg.II~ was ligated into the BamHI-cut vector pBinAR. The 1599 by fragment a codes the 5'-terminal part of the GMP synthetase cDNA. Clones obtained after traxreformation into E.coli XL1-blue were examined far the ari.entatian of the 1599 cassEtte by cutting with HindII1 as a check. The plasmids pGMP7AS (antisense construct) and pGMP7EX (sense construct) were identified in this way, see Figure 2.
Example 7 Generation and analysis of tra-~rsgenic plants The plasmids pGMP7AS and pGMP7EX - see Figure 2 - were transformed into Agrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al., Nucl. Acids. Res. 13(1984), 4777-4788). A 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony in Murashige-Skoog medium (Physiol. Plant.
15(1962), 473) with 2% sucrose (2MS medium) was used for 0050/5~7~7'~ CA 02385875 2002-03-27 transforming tobacco plants (Nicotiana tabacum co. Samsun NN).
Leaf disks from sterile plants (each about 1 cmz) were incubated in a Petri dish with a 1:50 agrabacteria dilution for 5-10 minutes. This was followed by incubation on 2MS medium with 0:8% Bacto agar in the dark at 25°C for 2 days. Cultivation was continued after 2 days with 16 hours light/8 hours dark, and continued in a weekly rhythm on MS medium with 500 mg/1 Claforan (cefotaxime sodium), 50 mg/1 kanamycin, 1 mg/1 benzylaminvpurine (BAP), 0.2 mg/1 naphthylacetic acid and 1.6 g/1 glucose. Growing shoots were transfexxed to MS medium with 2% sucrose, 250 mg/1 Claforan and 0.8% Bacto agar. RegEnerated shoots were obtained on 2MS medium with kanamycin and Claforan and, after rooting, were transferred into soil and, after cultivation for two weeks in a controlled-envirocabinet with a 16 hour light/8 hour dark rhythm at 60% humidity, investigated for foreign gene expression and altered metabolite cements and phenotypieal growth traits.
Altered nucleotide contents can be determined, far example, by the method of Stitt et al. (FEES Letters, 145 (1982), 217-222).
The transgenic GMP synthetase aritisense plants and their filial generation showed reduced growth, cr~mpared with WT plant controls, and bleaching of the sink leaves. These phenvtppical changes occurred in an early growth stage (see Fig. 3). In plants with reduced growth it was possible to detect in Northern hybridization a reduced amount of GMP-7M RNA, compared with the wild type. 40~,g portions of complete RNA from sink leaves were employed for this purp~s~e. Complete RNA was isolated from plant tissues as described by Logemann et al. (Anal. Biochem. 163 (1987), 21). For the analysis in each case 40 ~,g of RNA were fractionated in a formaldehyde-containing 1.5% agarose gel and transferred to nylon membranes (Hybond, Amersham). Specific transcripts were detected as described by Amasino (Anal. Biochem.
152(1986), 304). A specific c-DNA probe of the antisense strand was generated. This was done by cleaving the plasmid GMP-7M with BamHI and Bgl.LI and isolating a fragment comprising 1600 bp. The oligvnucleotide 5'-GAT ACG TCG TCA AGG AAC TTG-3' was used for the labeling reaction. The probe was hybridized by standard methods, see Hybvnd information for users, Amersham.
Hydridizativn [sic] signals were visualized by autaradiography using Kodak X-GMAT AR films. A clear correlation between expression of the growth phenotype and a reduction in the amount of GMP-7M RNA was shown (Fig. 3).
It was moreover possible in a WestErn blot experiment to detect a reduced amount, compared with wild-type plants, of GMP synthetase in the transgenic lines. This was done by preparing total protein extracts from sink leaves, separating in SDS polyacrylamide gel ~

electrophoresis by standard meth~ads and transferring to nitrocellulose membrane's. Detection took place with an IgG-alkaline phasphatase conjugate and the BCIP/NBT system (Sigma).
In addition, it was gos~sible by the in vitro assay described in Example 8 to establish that there was reduced GMP synthetase activity in transgenic lines with reduced growth.
The correlation between the level of expression and the GMP
synthetase activity and growth phenotype suggest that GMP
synthetase is a suitable target far herbicides.
Example 8 Assay systems for measuring GMP spnthetase activity The systems developed by Specter (Methods in Enzymology LI, 1978, 219-224) far animal errzpmes can be used to measure plant GMP
synthetase activity. In the first system, the AMP formation is made possible by coupling the reaction with AMP kinase, pyruvate kinase, lactate dehydrvgerrase and measurement at 340 em. The second system is based an direct detection of GMP (guanvsine monophasphate) by employing the radiolabeled substrate XMP
(xanthine monophosphate) and fractionation by thin-layer chromatography., Alternatively, the GMP synthetase activity can also be measured by a novel system, namely coupled detection of the produced glutamate. This system has the advantage of a smaller number of coupled reaction steps and provides greatEr signal strengths.
XMP + ATP + L-glutamine + HZO G~ GMP + AMP + L-glutamate + PPi L-glutamate + ApAD + H2p GluD' oxoglutaratE + APADH + NH4+
(GMP-S = GMP synthetase, GluDH = glutamate dehydrogenase, APAD =
3-acetylpyridine adenine dinucleutide) For this, the reaction mixture (see below) was incubated at 37°C
for 60 minutes, and the reaction was stopped by incubation at 95~C
for 5 minutes. The glutamate farmed was detected in the detection mixture (sEe below) by ghotometric measurement of .the increase in APADH at 363 em.

' ~050~507~ CA 02385875 2002-03-27 Reaction mixture:
100 ~L 750 mM Tris/HC1 buffer pH 7.8 100 ~,L 100 mM MgCl2 100 80 mM RCl ~L

100 ~L 20 mM XMP

100 ~L 200 mM L-glutamine 400 ~L Hy0 100 uL grBtein extract 1000 ~L
Detection mixture:
375 N.L 100 mM Tris-HC1 buffer pH 8.0 7 5 N.L 5 0 0 mM RC1 12 5 N,L H20 75 ~L 3 mM APAD
100 u~ of the reaction mixture 750 ~tL
Example 9 Search for inhibitors of GMP synthetase activity The in vitro assay described in Example 8 can be used with high throughput methods to search for inhibitors of GMP synthetase activity. The GMP synthetase activity far this can be prepared from plant tissues. It is possible and preferred for a plant GMP
synthetasE to be expressed in E.croli, in5e~t cells or another suitable expression system and then be concentrated or isolated.
It was possible in this way to ideritify known inhibitors such as 6-thio-XMP.

SEQUENCE LISTING
<110> BASF Aktienge-sellsuhaft <120> GMP syrtthetase from plax~ts <130> DE 19947490.7 <140> 0050-50777 <141> 1999-10-O1 <160> 4 <170> Patentln Vers. 2.0 <210> 1 <211> 1973 <212> DNA
<213> Nicotiana tabacum <220>
<221> CDS
<222> (65)..(1678) <400> 1 gaattcggca cqa~attt-ct ctutatuttt cttcctucua cccaucacuu aucctuucct 60 agca atg gaa cut caa aca cag gcg aag aaa tua aac ctu gta cta atc 109 Met Glu Pro Gln Thr Gln Ala Lys Lys Ser Asn Leu Val Leu Ile cta gac tac ggt tut cag tac aut cau cta atc acc cgc cga atc cga 157 Leu Asp Tyr Gly Ser Gln Tyr Thr His Leu Ile Thr Arg Arg Ile Arg agc cta tca att ttc tua ctu suc att aac ggc acc tct tcg tta gac 205 Ser Leu Ser Ile Phe Ser Leu Thr Ile Asn Gly Thr Ser Ser Leu Asp tcc ata aaa gaa ctc gac cca cgt gtu att atu ctu tcg ggt gga ccu 253 Ser Ile Lys Glu Leu Asp Pro Arg Val Ile Ile Leu Ser Gly Gly Pro cac agc gtc cac get gac ggc gca ccg tgt ttu cca cct ggg ttc atc 301 His Ser Val His Ala Asp Gly Ala Pro Cys Phe Pro Pro Gly Phe Ile gaa tac gtc gag tua cgt ggg att cac gtg tt-g ggt ata tgt tat ggg 349 ' CA 02385875 2002-03-27 Glu Tyr Val Glu Ser Arg Gly Lle His Val Leu Gly Ile Cys Tyr Gly ctg cag ttg att gtt cag aaa ctt ggc ggg gtt gtg aaa att gga gag 397 Leu Gln Leu Lle Val Gln Lys Leu Gly Gly Val Val Lys Lle Gly Glu aaa cat gag tat ggg aga atg gaa att gag gtt gga aag sat gtt gtt 445 Lys His Glu Tyr Gly Arg Met Glu Lle Glu Val Gly Lys Asn Val Val ggg ggg ttg ttt ggg sat acg gaa att ggt gat aaa cag gtg gtt tgg 493 Gly Gly Leu Phe Gly Asn Thr Glu Ile Gly Asp Lys Gln Val Val Trp atg agc cac ggt gat gag get gtg aaa ttg ccg gaa ggg ttt gag gtt 541 Met Ser His Gly Asp Glu Ala Val Lys Leu Prey Glu Gly Phe Glu Val gtg gcg agg agt agt cag ggt get gtt get get att gag aat egg gaa 589 Val Ala Arg Ser Ser Gln Gly Ala Val Ala Ala Lle Glu Asn Arg Glu cgg agg ttt tat ggg ctg cag tat cat ccc gag gta acg cac tcg act 637 Arg Arg Phe Tyr Gly Leu Gln Tyr His Pro Glu Val Thr His Ser Thr gaa ggg atg aga aca tta aga cac ttt ctg ttt gat gta tgt ggc gtt 685 Glu Gly Met Axg Thr Leu Arg His Phe Leu Phe Asp Val Cys Gly Val aca get ggc tgg aag atg gaa gat gtt etg gag gaa gaa ata aaa gtt 733 Thr Ala Gly Trp Lys Met Glu Asp Val Leu Glu Glu Glu Ile Lys Val atc aaa ggt atg gtt gga cct gaa gat cac gtg att tgt get tta tct 781 Ile Lys Gly Met Val Gly Pro Glu Asp His Val Ile Cys Ala Leu Ser ggt ggt gtt gat tcc aca gtt gca get aaa ttg gta cac aag get atc 829 Gly Gly Val Asp Ser Thr Val Ala Ala Lys Leu Val His Lys Ala Ile ggg gac agg ctt cac tgt gtt ttt gtt gat aat ggt cta tta agg tat 877 Gly Asp Arg Leu His Cys Val Phe Val Asp Asn Gly Leu Leu Arg Tyr aag gag aga gaa agg gtg atg gaa ctc ttt gag aag cgc ctt cat ttg 925 " CA 02385875 2002-03-27 Lys Glu Arg Glu Arg Val Met Glu Leu Phe Glu Lys Arg Leu His Leu cct gtt acc tgt gt~ gat get aca gaa gaa ttt ctc agc aaa cta aaa 973 Pro Val Thr Cys Val Asp Ala Thr Glu Glu Phe Leu Ser Lys Leu Lys ggc gta aca gaa cct gaa atg aag agg aaa ata att ggg aag gag ttc 1021 Gly Val Thr Glu Pro Glu Met Lys Arg Lys Ile Ile Gly Lys Glu Phe atc aac ata ttt gat ctt ttt gcc cat gat gtg gag gaa aaa gta ggg 1069 Ile Asn Ile Phe Asp Leu Phe Ala His Asp Val Glu Glu Lys Val Gly aaa aaa cct agt tac cta gtc caa gga acc ttg tat cct gat gta ata 1117 Lys Lys Pro Ser Tyr Leu Val Gln Gly Thr Leu Tyr Pro Asp Val Ile gag tct tgt cct cca cct gga a-gt gga aga aca cat tct cat aca atc 1165 Glu Ser Cys Pra Pro Pro Gly Ser Gly Arg Thr His Ser His Thr Ile aag agc cat cat aat gtt gga ggt ctt cca aag gac atg aag ctg aag 1213 Lys Ser His His Asn Val Gly Gly Leu~Pro Lys Asp Met Lys Leu Lys ctc atc gag caa ctg aaa ctt cta ttc aag gat gag gtt cgt gaa ttg 1261 Leu Ile Glu Pro Leu Lys Leu Leu Phe Lys Asp Glu Val Arg Glu Leu gga aag att ttg gat ata tct gag gac ttt ctt aaa cgc cau ccg ttc 1309 Gly Lys Ile Leu Asp Ile Ser Glu Asp Phe Leu Lys Arg His Pro Phe cct ggg ccc gga ctc get gtg cga att cca ggt gat gtc aca gca ggg 1357 Pro Gly Pro Gly Leu Ala Val Arg Ile PrD Gly Asp Val Thr Ala Gly aat tcc ttg gat att ctt cgt cag gtt gat gaa atc ttc att c$a tca 1405 Asn Ser Leu Asp I1e Leu Arg Gln Val Asp Glu Ile Phe Ile Gln Ser atc aga gat get aa-a atc tat gat gaa ata tgg caa get ttt get gtc 1453 Ile Arg Asp Ala Lys Ile Tyr Asp Glu Lle Trp Gln Ala Phe Ala Val ttc tta cca gtg aaa act gtt gga gta caa gga gac caa aga acc cat 1501 ' CA 02385875 2002-03-27 0~50/50T77 Phe Leu Pro Val Lys Thr Val Gly Val Gln Gly Asp Gln Arg Thr His tcc cac get gtt gca ctt aga gca gtc aca agt caa gat gga atg act 1549 Ser His Ala Val Ala Leu Arg Ala Val Thr Ser Gln Asp Gly Met Thr gca gac tgg tac tac ttt gat tta aag ttc ctt gac gac gta tca aga 1597 Ala Asp Trp Tyr Tyr Phe Asp Phe Lys Phe Leu Asp Asp Val Ser Arg aag atc tgc aat agt gtt cgt ggt gta a-at cga gtt ctg ctg gat att 1645 Lys Ile Cys Asn Ser Val Arg Gly Val Asn Arg Val Leu Leu Asp Ile aca tca aag cct cca tca aca atc gaa tgg gaa taatttgtta taaagaatgc 1698 Thr Ser Lys Pro Pro Ser Thr Ile Glu Trp Glu tatatttggt gaccaaargta ggattctttt gtgattttt-g gtgcataa~a aaaaggaaga 1758 aaatcataat agaaatttag gtccttttgt t-atgt-ggtag aactggtt~t tgggtaatta 1818 tgtgcaatgc tct~aacaat tttgtat~tt tatgggtatg atgat-a~caa attttactca 1878 gatcttggta gtacattttt cttatccaag tatagta-aca tgtggccagg catcaaaagc 1938 ctattccact caaaaaaaaa ara~aaaaaac t~gag 1973 <210> 2 <211> 538 <212> PRT
<213> Nicatiana tabacum <400> 2 Met Glu Pro Gln Thr Gln Ala Lys Lys Ser Asn Leu Val Leu Ile Leu Asp Tyr Gly Ser Gln Tyr Thr His Leu Ile Thr Arg Arg Ile Arg Ser 20 ~ 25 30 Leu Ser Ile Phe Ser Leu Thr Lle Asn Gly Thr Ser Ser Leu Asp Ser Ile Lys Glu Leu Asp Pra Arg Val Lle Ile Leu Ser Gly Gly Pro His ' CA 02385875 2002-03-27 Ob50/50T77 Ser Val His Ala Asp Gly Ala Pro Cys Phe Pro Pro Gly Phe Ile Glu Tyr Val Glu Ser Arg Gly Ile His Val Leu Gly Ile Cys Tyr Gly Leu Gln Leu Ile Val Gln Lys Leu Gly Gly Val Val Lys I1e Gly Glu Lys His Glu Tyr Gly Arg Met Glu Zle Glu Val Gly Lys Asn Val Val Gly Gly Leu Phe Gly Asn Thr Glu Lle Gly Asp Lys Gln Val Val Trp Met Ser His Gly Asp Glu Ala Val Lys Leu Pro Glu Gly Phe Glu Val Val Ala Arg Ser Ser Gln Gly Ala Val Ala Ala I1e Glu Asn Arg Glu Arg Arg Phe Tyr Gly Leu Gln Tyr His Pro Glu Val Thr His Ser Thr Glu Gly Met Arg Thr Leu Arg His Phe Leu Phe Asp Val Cys Gly Val Thr Ala Gly Trp Lys Met Glu Asp Val Leu Glu Glu Glu I1e Lys Val Ile Lys Gly Met Val Gly Pro Glu Asp His Val I1e Cys Ala Leu Ser Gly Gly Val Asp Ser Thr Val Ala Ala Lys Leu Val His Lys Ala Ile Gly Asp Arg Leu His Cys Val Phe Val Asp Asn Gly Leu Leu Arg Tyr Lys Glu Arg Glu Arg Val Met Glu Leu Phe Glu Lys Arg Leu His Leu Pro Val Thr Cys VaI Asp Ala Thr Glu Glu Phe Leu Ser Lys Leu Lys Gly Val Thr Glu Pro Glu Met Lys Arg Lys Ile Ile Gly Lys Glu Phe Ile Or050/507-77 Asn Ile Phe Asp Leu Phe Ala His Asp Val Glu Glu Lys Val Gly Lys Lys Pro Ser Tyr Leu Val Gln Gly Thr Leu Tyr Pro Asp Val Ile Glu Ser Cys Pro Pro Pro Gly Ser Gly Arg Thr His Ser His Thr Ile Lys Ser His His Asn Val Gly Gly Leu Pro Lys Asp Met Lys Leu Lys Leu Ile Glu Pro Leu Lys Leu Leu Phe Lys Asp Glu Val Arg Glu Leu Gly Lys Ile Leu Asp Ile Ser Glu Asp Phe Leu Lys Arg His Pro Phe Pro Gly Pro Gly Leu Ala Val Arg Lle Pro Gly Asp Val Thr Ala Gly Asn Ser Leu Asp I-le Leu Arg Gln Val Asp Glu Ile Phe Ile Gln Ser Ile Arg.Asp Ala Lys Ile Tyr Asp Glu Ile Trp Gln Ala Phe Ala Val Phe Leu Pro Val Lys Thr Val Gly Val Gln Gly Asp Gln Arg Thr His Ser His Ala Val Ala Leu Arg Ala Val Thr Ser Gln Asp Gly Met Thr Ala Asp Trp Tyr Tyr Phe Asp Phe Lys Phe Leu Asp Asp Val Ser Arg Lys Ile Cys Asn Ser Val Arg Gly Val Asn Arg Val Leu Leu Asp Ile Thr Ser Lys Pro Pro Ser Thr Ile Glu Trp Glu <210> 3 <211> 1232 <212> DNA
<213> Physcomitrella patens Ob50/50T7-7 <220>
<221> CDS
<222> (3)..(1148) <400> 3 ga att cgg cac gag gcc act agt ate cag ggt aat att gcc get att 47 Ile Arg His Glu Ala Thr Ser Thr Gln Gly Asn Ile Ala Ala Ile gaa aat gtg gat tcc aga atc tac gcc ctc caa tac'cat ccc gag gtt 95 Glu Asn Val Asp Ser Arg Lle Tyr Ala Leu Gln Tyr His Pro Glu Val acg cac tca gag aaa ggg aca gag act ttg aga cac ttt ttc ctg sat 143 Thr His Ser Glu Lys Gly Thr Glu Thr Leu Arg His Phe Phe Leu Asn gtc tgc ggc atg aag get gac tgg cag atg cad aat gtg ttg gag gaa 191 Val Cys Gly Met Lys Ala Asp Trp Gln Met Gln Asn Val Leu Glu Glu gag att aaa aag gtc act gcg arc gtc ggc cca gat gat cat gtt att 239 Glu Ile Lys Lys Val Thr Ala Thr Val Gly Pro Asp Asp His Val Ile tgt gca ctc tcc ggg ggc gtg gac tca aca gta gca get act ctg gtg 287 Cys Ala Leu Ser Gly Gly Val Asp Ser Thr Val Ala Ala Thr Leu Val cac cgt get att gga gat cgc ctt cat tgt gtg ttt gta gat aat ggc 335 His Arg Ala Ile Gly Asp Arg Leu His Cys Val Phe Val Asp Asn Gly ctt tgc aga tac aag gaa aga gaa aga gtg atg gcc acs ttt gtg aaa 383 Leu Cys Arg Tyr Lys Glu Arg Glu Arg Val Met Ala Thr Phe Val Lys gac ctt cat ctg cca gtc act tgt gtg gat gcc act gag cag ttt ctc 431 Asp Leu His Leu Pro Val Thr Cys Val Asp Ala Thr Glu Gln Phe Leu agc aaa ttg aag ggc gtg gta gat cca gag aga aag agg aag atc atc 479 Ser Lys Leu Lys Gly Val Val Asp Pro Glu Arg Lys Arg Lys Ile Ile gga gca gag ttt att gca gtc ttt gat gaa ttt tcg cac aga ttg gag 527 Gly Ala Glu Phe Ile Ala Val Phe Asp Glu Phe Ser His Arg Leu Glu 0b50/50777 aga gag att gga aag atg cct get ttc ctt gtg cag gga aca ctt tat 575 Arg Glu Ile Gly Lys Met Pro Ala Phe Leu Val Gln Gly Thr Leu Tyr cca gat gtc att gag tag tgt cct cct cca ggg agc ggg aag tcg cat 623 Pro Asp Val Ile Glu Ser Cys Pro Pro Pro Gly Ser Gly Lys Ser His tcc cac aca atc aaa agt cat cac aac gtc ggt ggc ttg ccc gag aac 671 Ser His Thr Ile Lys Ser His His Asn Val Gly Gly Leu Pro Glu Asn atg aaa ttg aag ttg gtt gag cct ctc aag tgg ctc ttc aaa gac gag 719 Met Lys Leu Lys Leu Val Glu Pro Leu Lys Trp Leu Phe Lys Asp Glu gta cgc gaa atg ggt gca ttg ttg gat gta cct gtt tcc ttt ttg aag 767 Val Arg Glu Met Gly Ala Leu Leu Asp Val Pra Val Ser Phe Leu Lys cgc cat cct ttc cct gga cct gga ttg gcc gtg cga att ctt ggg gat 815 Arg His Pra Phe Pro Gly Pra Gly Leu Ala Val Arg Ile Leu Gly Asp gta act cag gac ggc gca ctc gac act atc cgc ttg gtt gat gag atc 863 Val Thr Gln Asp Gly Ala Leu Asp Thr Ile Arg Leu Val Asp Glu Ile ttt gtg aac agc att cga gag gca ggt ctt talc gat aag atc tgg cag 911 Phe Val Asn Ser Ile Arg Glu Ala Gly Leu Tyr Asp Lys Lle Trp Gln gca ttt get gtt tat ctg cca gta a-ag a-ct gtt ggc gtt caa gga gac 959 Ala Phe Ala Val Tyr Leu Pro Val Lys Thr Val Gly Val Gln Gly Asp aaa cgg aca cat tca cac get gtt get cta cgt gca att aca agt gaa 1007 Lys Arg Thr His Ser His Ala Val Ala Leu Arg Ala Lle Thr Ser Glu gac gga atg act get gac t~gg ttt cat ttt gat gga aag ttt ctt gcc 1055 Asp Gly Met Thr Ala Asp Trp Phe His Phe Asp Gly Lys Phe Leu Ala gag gta tca tct aaa atc tgc aac agc gta agg ggt atu aat agg gtg 1103 Glu Val Ser Ser Lys Ile Cys Asn Ser Val Arg Gly Lle Asn Arg Val ' CA 02385875 2002-03-27 gta tac gac att acg trt aaa cct cca tca act gtt gag tgg gaa 1148 Val Tyr Asp Ile Thr Ser Lys Pro Pro Ser Thr Val Glu Trp Glu tagacgtcag tact tggaagtact gtt~gttatg acgattcact gcaatactta 1208 acaaactatt ttatacttca aaaa 1232 <210> 4 <211> 382 <212> PRT
<213> Physcomitrella patens <400> 4 Ile Arg His Glu Ala Thr Ser Thr Gln Gly Asn I1e Ala Ala Ile Glu Asn Val Asp Ser Arg Ile Tyr Ala Leu Gln Tyr His Pro Glu Val Thr His Ser Glu Lys Gly Thr Glu Thr Leu Arg His Phe Phe Leu Asn Val Cys Gly Met Lys Ala Asp Trp Gln Met Gln Asn Val Leu Glu Glu Glu Ile Lys Lys Vai Thr Ala Thr Val Gly Pro Asp Asp His Val Ile Cys Ala Leu Ser Gly Gly Val Asp Ser Thr Val Ala Ala Thr Leu Val His Arg Ala Ile Gly Asp Arg Leu His Cps Val Phe Val Asp Asn Gly Leu Cys Arg Tyr Lys Glu Arg Glu Arg Val Met Ala Thr Phe Val Lys Asp Leu His Leu Pro Val Thr Cys Val Asp Ala Thr Glu Gln Phe Leu Ser Lys Leu Lys Gly Val Val Asp Pro Glu Arg Lys Arg Lys Ile Ile Gly Ala Glu Phe Ile Ala Val Phe Asp Glu Phe Ser His Arg Leu Glu Arg Glu Ile Gly Lys Met Pro Ala Phe Leu Val Gln Gly Thr Leu Tyr Pro Asp Val Ile Glu Ser Cys Pro Pro Pra Gly Ser Gly Lys Ser His Ser His Thr Ile Lys Ser His His Asn Val Gly Gly Leu Pro Glu Asn Met Lys Leu Lys Leu Val Glu Pro Leu Lys Trp Leu Phe Lys Asp Glu Val Arg Glu Met Gly Ala Leu Leu Asp Val Pro Val Ser Phe Leu Lys Arg His Pro Phe Pro Gly Pro Gly Leu Ala Val Arg Ile Leu Gly Asp Val Thr Gln Asp Gly Ala Leu Asp Thr Ile Arg Leu Val Asp Glu Ile Phe Val Asn Ser Ile Arg Glu Ala Gly Leu Tyr Asp Lys Lle Trp Gln Ala Phe Ala Val Tyr Leu Pro Val Lys Thr Val Gly Val Gln Gly Asp Lys Arg Thr His Ser His Ala Val Ala Leu Arg Ala ile Thr Ser Glu Asp Gly Met Thr Ala Asp Trp Phe His Phe Asp Gly Lys Phe Leu Ala Glu Val Ser Ser Lys Ile Cys Asn Ser Val Arg Gly Lle Asn Arg Val Val Tyr Asp Ile Thr Ser Lys Pro Pro Ser Thr Val Glu Trp Glu

Claims (16)

We claim:

1. A DNA sequence comprising the coding region of a plant GMP
synthetase, wherein this DNA sequence has the nucleotide sequence SEQ-ID No: 1 or SEQ-ID No: 3.

2. A DNA sequence which hybridizes with the DNA sequence SEQ-ID
No: 1 or SEQ-ID No: 3 as claimed in claim 1 or parts thereof or derivatives derived from these sequences by insertion, deletion or substitution, and codes for a protein which has the biological activity of a GMP synthetase, this DNA
sequence having a homology of at least 60% with SEQ ID NO: 1.

3. A protein having GMP synthetase activity and comprising an amino acid sequence which represents a portion of at least 100 amino acids of the sequence SEQ-ID No: 2 or 4.

4. A protein as claimed in claim 3, which comprises as amine acid sequence the part-sequence 50-300 from SEQ-ID No: 2 or SEQ-ID No: 4.

5. A protein as claimed in claim 4, which comprises as amino acid sequence the sequence depicted in SEQ-ID No: 2 or SEQ-ID
No: 4.

6. The use of a DNA sequence as claimed in claim 1 or 2 for introduction into pro- or eukaryotic cells, this sequence optionally being linked to control elements which ensure transcription and translation in the cells, and leading to expression of a translatable mRNA which brings about the synthesis of a plant GMP synthetase.

7. The use of a DNA sequence as claimed in claim 1 or 2 for producing an assay system for identifying inhibitors of plant GMP synthetase with a herbicidal action.

8. A method for finding substances which inhibit the activity of plant GMP synthetase, which comprises in a first step using a DNA sequence as claimed in claim 1 or 2 preparing GMP
synthetase, and in a second step measuring the activity of the plant GMP synthetase in the presence of a test substance.

9. A method as claimed in claim 8, wherein the measurement of the plant GMP synthetase is carried out in a high throughput screening (HTS).

10. A method for identifying substances with a herbicidal action, which inhibit the GMP synthetase activity in plants, consisting of a) preparation of transgenic plants, plant tissues, or plant cells which comprise an additional DNA sequence coding for an enzyme having GMP synthetase activity and are able to overexpress an enzymatically active GMP synthetase;

b) application of a substance to transgenic plants, plant cells, plant tissues or plant parts and to untransformed plants, plant cells, plant tissues or plant parts;

c) determination of the growth or survivability of the transgenic and untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance; and d) comparison of the growth ar survivability of the transgenic and untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance;

where suppression of the growth ar survivability of the untransformed plants, plant cells, plant tissues ar plant parts without, however, greatly suppressing the growth or the survivability of the transgenic plants, plant cells, plant tissues or plant parts demonstrates that the substance from b) shows herbicidal activity and inhibits the enzymic activity in plants.

11. An assay system based an the expression of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 as claimed in claim 1 or 2 for identifying inhibitors of plant GMP synthetase with a herbicidal action.

12. An assay system as claimed in claim 11 for identifying inhibitors of plant GMP synthetase, wherein the enzyme is incubated with a test substrate to be investigated and, after a suitable reaction time, the enzymatic activity of the enzyme is measured by comparison with the activity of the uninhibited enzyme.

13. An inhibitor of plant GMP synthetase.

14. An inhibitor of plant GMP synthetase identified using an assay system as claimed in claim 11 or 12.

15. An inhibitor as claimed in either of claims 13 or 14 for use as herbicide.

16. A method for eliminating unwanted plant growth, which comprises treating the plants to be eliminated with a compound which specifically binds to GMP synthetase encoded by a DNA sequence as claimed in claim 1 or 2, and inhibits the function thereof.