AU7658700A - Gmp synthetase derived from plants - Google Patents

Description

GMP synthetase from plants The present invention relates to the identification of plant GMP 5 synthetase (guanosine-monophosphate synthetase) as novel target for herbicidal agents. The present invention further relates to DNA sequences coding for a polypeptide 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 10 activity of plant origin for 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 15 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 20 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 their cellular components from 25 carbon dioxide, water and inorganic salts. This process 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 30 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 35 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 coenzyme A and nicotinamide and flavin coenzymes, which are 40 involved in many cellular conversions. Guanosine 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 for the biosynthesis of 45 methylxanthines such as caffeine and theobromine, especially in the Rubiaceae and Theaceae families of plants.

2 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 5 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. 10 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 15 is fixed in the form of glutamine and aspartate in the root nodules of legumes is firstly converted via the de novo 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. Biophys. 220 (1983), 20 179-187; Shelp et al., Arch. Biochem. Biophys. 224 (1983), 429-441). However, more recent investigations have shown that enzyme activitites of the purine biosynthetic pathway are also to be found in mitochondria in the root nodules of Vigna ungiculata [sic] (Atkins et al., Plant Physiology 113 (1997), 127-135; Smith 25 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 30 regulation. The enzyme PRPP amidotransferase (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). 35 GMP-Synthetase also plays a part in relation to the balanced synthesis of guanosine nucleotides and adenosine nucleotides because ATP is a substrate of GMP synthetase. 40 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 novo purine biosynthesis have already been described. An example which may be mentioned is 45 5'-phosphohydantocidin which inhibits an enzyme of plant purine metabolism, adenylosuccinate synthetase (ASS) (Siehl et al., Plant Physiol. 110 (1996), 753-758). Inhibitors for enzymes of 3 this metabolic pathway also exist from animals and microorganisms. Folate analogs inhibit various folate-dependent reactions, inter alia the enzyme GAR transformylase and have antiproliferative, antiinflammatory and immunosuppressant 5 effects. Mycophenolate (MPA), as an inhibitor of IMP dehydrogenase, has antimicrobial, antiviral and immunosuppressant effects. (Kitchin et al., Journal of the American Academy of Dermatology 37 (1997), 445-449). 10 Demonstration of the suitability of an enzyme as herbicide target can be shown, for 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 15 action of herbicidal agents. This has been shown by way of example for acetolactate synthase on transgenic potato plants (Hbfgen et al., Plant Physiology 107 (1995), 469-477). It is an object of the present invention to prove that GMP 20 synthetase in plants is a suitable herbicidal target, to isolate a complete plant cDNA coding 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. 25 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 synthetase in bacterial or eukaryotic cells. 30 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 (Nicotiana tabacum). 35 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 invention is a DNA sequence SEQ-ID No. 3 40 comprising a portion of the coding region of a plant GMP synthetase from Physcomitrella patens, see Example 2. Further aspects of the invention are DNA sequences which are derived from SEQ-ID NO: 1 or SEQ-ID No: 3 or hybridize with one 45 of these sequences and code for a protein which has the biological activity of a GMP synthetase.

4 Tobacco plants of the line Nicotiana tabacum cv. Samsun NN harboring an antisense construct of GMP synthetase have been characterized in detail. The plants show growth retardation to differing extents. The transgenic lines, and the progeny as 1st 5 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, compared with wild-type plants, of GMP synthetase 10 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. 15 In order to be able to find efficient inhibitors of plant GMP synthetase, it is necessary 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 20 synthetase from tobacco is cloned 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 25 other bacteria, in yeasts, fungi, algae, plant cells, insect cells or mammalian cells, see Example 5. The GMP synthetase protein expressed with the aid of the expression cassette according to the invention is particularly 30 suitable for finding inhibitors specific for GMP synthetase. For this purpose, the plant GMP synthetase can be employed, for example, in an enzyme assay in which the activity of the GMP synthetase is measured in the presence and absence of the agent 35 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. 40 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, more intensive 45 tests which are familiar to the skilled worker on these substances.

5 A further aspect of the invention is a method for identifying substances having a herbicidal action, which inhibit the GMP synthetase activity in plants, consisting of 5 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; 10 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 15 transgenic and untransformed 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 20 and untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance; where suppression of the growth or survivability of the untransformed plants, plant cells, plant tissues or plant parts 25 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 enzymic activity in plants. 30 A further aspect of the invention is a method for identifying inhibitors of plant GMP synthetases, with potential herbidical action, by cloning the gene of a plant GMP synthetase, bringing about overexpression in a suitable expression cassette - for 35 example in insect cells, opening the cells and employing the cell extract directly or after concentration 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. 40 A further aspect of the invention comprises compounds having a herbicidal action which can be identified using the assay system described above. 45 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 6 synthetase and inhibits the function thereof. Inhibitors of GMP synthetase with a herbicidal action can be used as defoliants, desiccants, haulm destroyers and, in particular, 5 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 alia on the application rate. 10 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, 15 Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, Taraxacum. 20 Monocotyledonous weeds of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria, 25 Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, Apera. Another aspect of the invention comprises expression cassettes whose sequence codes for a GMP synthetase from tobacco or 30 functional equivalent thereof. The nucleic acid sequence may in this connection be, for example, a DNA or a cDNA sequence. Another aspect of the invention is an expression cassette comprising a DNA sequence SEQ-ID No. 3 coding for a portion of 35 the plant GMP synthetase from Physcomitrella patens. The expression cassettes according to the invention additionally comprise regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. In a 40 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 polyadenylation signal downstream, i.e. at the 3' end, and, where appropriate, further regulatory elements which are operatively linked to the GMP synthetase gene 45 coding sequence lying between them. An operative linkage means tha sequential arrangement of promoter, coding sequence, terminator and, where appropriate, further regulatory elements in 7 such a way that each of the regulatory elements is able to perform its function as intended on expression of the coding sequence. 5 An expression cassette according 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 cloning techniques as described, for example, in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 10 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 15 Wiley-Interscience (1987). Another 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 20 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 25 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 30 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 80 to 100%. 35 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 40 sequences, for example obtained by chemical synthesis, which are adapted to the codon usage of a plant. A functional equivalent also means in particular natural or artificial mutations of an originally isolated sequence which 45 codes for a GMP synthetase and additionally shows the required function. Mutations comprise substitutions, additions, deletions, transpositions or insertions of one or more nucleotide residues.

8 Thus, for example, the present invention also includes nucleotide sequences obtained by modification of the nucleotide sequence. The aim of such a modification may be, for example, further localization of the coding sequence present therein or, for 5 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. 10 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. 15 A further aspect of the invention 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. 20 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 synthetase activity have an 25 amino acid sequence homology with the tobacco GMP synthetase of 50 - 100% identity. Particularly preferred plant proteins with GMP synthetase activity have an amino acid sequence homology with the tobacco 30 GMP synthetase of 80 - 100% identity. Another aspect of the invention comprises plant proteins with GMP synthetase activity having an amino acid sequence homology with the Physcomitrella patens GMP synthetase of 20 - 100% identity. 35 Preferred plant proteins with GMP synthetase activity have an amino acid sequence homology with the Physcomitrella patens GMP synthetase of 50 - 100% identity. 40 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 invention was overexpression of the GMP 45 synthetase gene in plants to produce plants which are tolerant of inhibitors of GMP synthetase.

9 Overexpression 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. 5 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 10 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 15 transformed with an expression 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. 20 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 babyfood products in order to achieve a nutrient solution corresponding to breast milk. In addition, an optimized 30 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 dietary feeding of patients with gout. Nucleotides also have flavor-forming and flavor-enhancing effects so that an altered nucleotide content has effects on the 35 taste properties of plants. A further aspect of the invention therefore comprises plants which have a modified content of guanosine nucleotides after expression of the DNA sequence SEQ-ID NO: 1 or SEQ-ID No: 3 in 40 the plant. A plant with a modified content of guanosine nucleotides is based, for example, by expression of an additional DNA sequence SEQ-ID No. 1 or 3 in the sense or antisense orientation in the 45 plant. A modified content of guanosine nucleotides means that it is possible to produce plants with an increased content of guanosine nucleotides in the case of the sense orientation and 10 plants with a reduced content of guanosine nucleotides in the case of the sense orientation (cosuppression) 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 owing to functional overexpression of the GMP synthetase gene in the plant compared with the plant which 10 has not been genetically manipulated for 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 15 plants. Particularly preferred sequences are those which ensure targeting in the apoplasts, in plastids, the vacuoles, the mitochondrion, the endoplasmic reticulum (ER) or, through the absence of 20 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 plant expression cassette can be incorporated 25 into the tobacco transformation 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 30 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 transcriptional effectors which, in their totality, lead to 35 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 40 possible to control expression of the exogenous GMP synthetase gene in the plant at a particular point in time. Promoters of this type, such as, for example, PRP1 promoter (Ward et al., Plant. Mol. Biol. (1993) 22, 361-366), a promoter inducible b y salicylic acid (WO 95/19443), a benzenesulfonamide-inducible 45 (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 (WO 93/21334) promoter are 11 described in the literature and can, inter alia, be used. Further particularly preferred promoters are those which ensure expression in tissues or plant parts in which the biosynthesis of 5 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). 10 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 Conrad, Bio/Technology (1995) 10, 1090-1094). 15 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. 20 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 codons preferred by plants. These codons preferred by plants can identified by 25 codons 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 30 frame. Adaptors 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 35 the content of guanosine nucleotides in the plant through overexpression 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. 40 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 45 genes of the plant to be transformed.

12 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 5 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 synthetase expression (e.g. myc tag or his tag). However, this is preferably a regulatory protein sequence such 10 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 15 direction of transcription of a linker or polylinker containing one or more restriction sites for 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 20 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 expression cassette according to the invention comprises in the 5'-3' direction of transcription the promoter according to the invention, any 25 suitable sequence and a region for the transcriptional termination. Different termination regions can be exchanged for one another if desired. It is furthermore possible to employ manipulations which provide 30 suitable restriction cleavage sites or delete the excess DNA or restriction cleavage sites. Where the insertions, deletions or substitutions such as, for example, transitions and transversions are considered, it is possible to use in..vitro mutagenesis, primer repair, restriction or ligation. In the case of suitable 35 manipulations such as, for example, restriction, chewing-back or filling in of overhangs for blunt ends, it is possible to make complementary ends of the fragments available for the ligation. Preferred polyadenylation signals are plant polyadenylation 40 signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular of gene 3 of the T-DNA (octopine synthase) of Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 ff) or functional equivalents. 45 To transform a host plant with a DNA coding for a GMP synthetase, an expression cassette according to the invention is incorporated 13 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 alia in "Methods in Plant Molecular Biology and 5 Biotechnology" (CRC Press), Chapters 6/7, pages 71-119. The transfer of foreign genes into the genome of a plant is referred to as transformation. The methods used for this purpose are those described for the transformation and regeneration of 10 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 15 mediated by agrobacterium. 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, Academic Press (1993) 128-143 and in Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 20 (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). 25 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 30 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 biosythesis is generally the leaf tissue, 35 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. 40 In addition, constitutive expression of the exogenous GMP synthetase gene is advantageous. However, on the other hand, inducible expression may also appear desirable. 45 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, 14 for example into E. coli. Suitable cloning vectors are, inter alia, pBR322, pUC series, M13mp series and pACYC184. Binary vectors able to replicate both in E. coli and in agrobacteria are particularly suitable. 5 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 10 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 15 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 confined to these: 20 Examples Methods of genetic manipulation on which the examples are based: 25 General cloning methods Cloning methods such as, for example, restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, 30 linkage of DNA fragments, transformation of Escherichia coli cells, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as described by Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). 35 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, 40 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 45 analytical quality from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutions were made up using prepared, pyrogen-free water, 15 referred to as H 2 0 in the subsequent text, from a Milli-Q water treatment system (Millipore, Eschborn). Restriction endonucleases, DNA-modifying enzymes and molecular biology kits were purchased from AGS (Heidelberg), Amersham (Braunschweig), 5 Biometra (Gbttingen), Roche (Mannheim), Genomed (Bad Oeynhausen), New England Biolabs (Schwalbach/Taunus), Novagen (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 stock center (Yale University, New Haven). 15 The agrobacterial strain used for the plant transformation (Agrobacterium tumefaciens, 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 agrobacterial strain LBA4404 (Clontech) or other 20 suitable strains. Vectors which can be used for the cloning are pUC19 (Yanish-Perron, Gene 33(1985), 103-119) pBluescript SK (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBin19 (Bevan et al., Nucl. Acids Res. 12(1984) 8711-8720) and pBinAR (Hbfgen and Willmitzer, Plant Science 66 (1990) 221-230). 25 Example 1 Isolation of a cDNA of the guaA gene, coding for a GMP synthetase from tobacco. 30 An expressed sequence tag (EST) from Arabidopsis 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 35 sequencing. The oligonucleotides 5'-aag gat cca agc tct aag acc cta tcc-3' and 5'-tta gat ctt tat tcc cat tcg atg g-3' from the 5'- and 3'-terminal sequences were used for amplification by a polymerase chain reaction (PCR) of a 1000 bp cDNA fragment with EST F14426 as template in a Perkin Elmer DNA thermal cycler. The 40 reaction mixture contained 0.1 ng/[tl cDNA from tobacco, 0.5 [tM of the appropriate oligonucleotides, 200 [tM nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25'C, 1.5 mM MgCl 2 ) and 0.02 U/Vl Taq polymerase (Perkin Elmer). 45 The amplification conditions were set as follows: Annealing temperature: 52'C, 1 min 16 Denaturation temperature: 92'C, 1 min Elongation temperature: 72'C, 1.5 min Number of cycles: 30 5 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 XL1-Blue as bacterial strain. The phage DNA was transferred by 10 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 15 Buchler) in the presence of a- 3 2 P-dCTP (specific activity 3000 Ci/mmol) in accordance with the manufacturer's information. The hybridization of the membranes took place after prehybridization at 60 0 C in 3 x SSPE, 0.1% sodium dodecyl sulfate (w/v), 0.02% polyvinylpyrrolidone (w/v), 0.02% Ficoll 400 (w/v) 20 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/v) at 60 0 C for 60 minutes. Positively hybridizing phages were visualized by autoradiography and purified and 25 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 30 sequencing data showed that the clone GMP-7M with a length of 1973 bp contained a complete reading frame of 1614 bp 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 codon in the same reading frame, which 35 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 40 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 45 those in the region of the N termini. The N terminus of the GMP-7M amino acid sequence corresponds with the N termini of GMP synthetases from other organisms such as E. coli and 17 Synechocystis sp. (Table 1). GMP-7M has no marked signal sequences (found by the program PSORT, Nakai, K., Institute for Molecular and Cellular Biology, Osaka University, Japan), which might indicate a cytosolic localization of the protein. 5 Table 1 Sequence comparison of GMP synthetases from Nicotiana tabacum (guaAN.t = GMP-7M), Arabidopsis thaliana (guaAestA.t, Genbank 10 No. F14426), E.coli (guaA e.c, Genbank No. 146276), Synechocystis sp. (guaAsyn, Genbank No. 1001583), Helicobacter pylori (guaAh.p, Genbank No. 3122166), Homo sapiens (guaA human, Genbank No. 1708072). 15 1 50 guaAN.t ---------- ------MEPQ TQAKKSNLVL ILDYGSQYTH LITRRIRSLS guaA_estA.t -- ~~~~~~~- ~----~ guaA-e.c -~--------- --------- m tenihkhril ildfgsqytq lvarrvrelg guaAsyn mttqipvppv vsdqalpdri sdrlkgqiiv ildfgsqyse liarrirete guaAh.p ~---------- mil vldfgsqytq liarrlrerg 20 guaA_human -malcngdsk lenaggdlkd ghhhyegavv ildagaqygk vidrrvrelf 51 100 guaAN.t IFSLTINGTS SLDSIKELDP RVIILSGGPH SVHADGAPCF PPGFIEYVES guaA_estA.t --- ~--~~ ----- ~---- guaAe.c vycelwawdv teaqirdfnp sgiilsggpe stteenspra pq... .yvfe guaAsyn vysevlsyrt taqqlreikp kgiilsggpn svydqgapec dp.... eifq guaA_h.p iyteivpffe sieniqkkap kglilsggpa svyakdaykp sg... .kifd 25 guaA_human vqseifplet pafaikeqgf raiiisggpn svyaedapwf dpa.... ift 101 150 guaAN.t RGIHVLGICY GLQLIVQKLG GVVKIGEKHE YGRMEIEVGK NVV.... GGL guaA_estA.t ------ ~~- ----- ~ guaA_e.c agvpvfgvcy gmqtmamqlg ghveasnere fgyaqvevvn dsalvrgied guaAsyn lgvpvlgvcy gmqlmvkqlg grverakrge ygkaslhidd ptdlltnven 30 guaAh.p lnvpilgicy gmqylvdffg gvvvganeqe fgkavleitq nsvifegv.. guaA_human igkpvlgicy gmqmmnkvfg gtvhkksvre dgvfnisvdn tcslfrglqk 151 200 guaAN.t FGNTEIGDKQ VVWMSHGDEA VKLPEGFEVV ARSSQGAVAA IENRERRFYG guaA_est_A.t ~~~~~~-~~~ -- ~~~~guaAe.c altadgkpll dvwmshgdkv taipsdfitv astescpfai maneekrfyg guaAsyn dst....... .mwmshgdsc vdlptgfeil ahtdntpcaa iadhqkalfg guaA_h.p ...... kiks lvwmshmdkv ielpkgfttl akspnsphca iengk..ifg guaA_human ........ ee vvllthgdsv dkvadgfkvv arsgni.vag ianeskklyg 201 250 guaAN.t LQYHPEVTHS TEGMRTLRHF LFDVCGVTAG WKMEDVLEEE IKVIKGMVGP guaA_estA.t ~~~~~~~- ~------- guaA_e.c vqfhpevtht rqgmrmlerf vrdicqceal wtpakiidda varireqvg. 40 guaAsyn vqfhpevvhs vggialirnf vyhichcept wttaafiees irevrsqvg. guaAh.p lqfhpevvqs eeggkilenf allvcgcekt wgmqhfaqre iarlkekia. guaA_human aqfhpevglt engkvilknf lydiagcsgt ftvqnrelec ireikervgt 251 300 guaAN.t EDHVICALSG GVDSTVAAKL VHKAIG.DRL HCVFVDNGLL RYKERERVME guaAestA.t -~~~~~- ~---~ 45 guaA_e.c ddkvilglsg gvdssvtaml lhraig.knl tcvfvdngll rlneaeqvld guaA-syn drrvllalsg gvdsstlafl lhraig.dnl tcmfidqgfm rkgeperlve guaA_h.p nakvlcavsg gvdstvvatl lhraik.dnl iavfvdhgll rknekervqa guaA_human s.kvlvllsg gvdstvctal lnralnqeqv iavhidngfm rkresqsvee 18 301 350 guaAN.t LFEK ...... -.......... ... RLHLPVT CVDATEEFLS KLKGVTEPEM guaA_estA.t ~~-~~ -~~~-~ guaA-e.c mfgd...... .......... ...hfglniv hvpaedrfls alagendpea guaA-syn lfdh...... .......... ... qfhipvq yvnardrflk qlegvtdpee 5 guaA h.p mfkd...... .......... ....lkipln tidakevfls klkgvsepel guaAhuman alkklgiqvk vinaahsfyn gtttlpisde drtprkrisk tlnmttspee 351 400 guaAN.t KRKIIGKEFI NIFDLFAHDV EEKVGKKPSY LVQGTLYPDV IESC.. .PPP guaA_est_A.t ---- ~--- -- ~~~~~~~~ guaA-e.c krkiigrvfv evfd..eeal k... ledvkw laqgtiypdv iesaas.... 10 guaA-syn krrlighefi qvfe..eesn r... lgpfdy laqgtlypdv iesadsnvdp 0 guaAh.p krkiigetfi evfe..keak khhlkgkief laqgtlypdv iesvsv.... guaAhuman krkiigdtfv ki. .anevig emnlkpeevf laqgtlrpdl iesasl .... 401 450 guaAN.t GSGRTHSHTI KSHHNVGGLP KDMKL. .KLI EPLKLLFKDE VRELGKILDI guaAest_A.t -- ~--- -~~ ~~~~~-~--~ ~ guaAe.c atgk..ahvi kshhnvgglp kemkm..glv eplkelfkde vrkiglelgl 15 guaA syn ktgervavki kshhnvgglp knlrf..klv eplrklfkde vrklgrsigl guaA h.p .. .kgpskvi kthhnvgglp ewmdf. .kli eplrelfkde vrllgkelgv guaAhuman .vasgkaeli kthhndteli rklreegkvi eplkdfhkde vrilgrelgl 451 500 guaAN.t SEDFLKRHPF PGPGLAVRIP GDVTAGNSLD ILRQVDEIFI QSIRDAKIYD guaAestA.t --- ~~~~~~~ -- -- ~ ~ 20 guaAe.c pydmlyrhpf pgpglgvrvl gevkk.eycd llrradaifi eelrkadlyd guaA-syn peeivrrhpf pgpglairii gevts.erln ilrdadfivr deiskrgiyh guaA-h.p sqdflmrhpf pgpglavril geise.skik rlqeadfifi eelkkanlyd guaAhuman peelvsrhpf pgpglairvi c.aeepyick dfpetnnilk ivadfsasvk 501 550 guaAN.t EIWQAFAVFL PVKTVGVQGD QRTHSHAVAL RA.VTSQDGM TADWYYFDFK guaAestA.t ~~---------- ------aqgd kgtiphvgcp pcrlqaqvgl tadwfifehk 25 guaAe.c kvsqaftvfl pvrsvgvmgd grkydwvvsl ra.vetidfm tahwahlpyd guaA-syn dywqafavll pirsvgvmgd krtyahpvvl rf.itsedgm tadwarvpyd guaA-h.p kvwqafcvll nvnsvgvmgd nrtyenaicl ra.vnasdgm tasfsflehs guaAhuman kphtllqrvk actteedqek lmqitslhsl nafllpiktv gvqgdcrsys 551 600 guaAN.t FLDDVSRKIC NSVRGVNRVL LDITSKPPST IEWE~------ --------- 30 guaAest_A.t flddvsrkic nsvqgvnrvv lditskppst iewe------ guaA-e.c flgrvsnrii nevngisrvv ydisgkppat iewe------ guaA-syn ileaisnriv nevkgvnrvv yditskppgt iewe ------ ~guaAh.p flekvsnrit nevsginrvv yditskppgt iewe---~guaAhuman yvcgisskde pdweslifla rliprmchnv nrvvyifgpp vkepptdvtp 601 650 35 guaAN.t ~-----~~~~ -- ~~------ guaA_est_A.t -~~~~~--~ -- ~~~~~- ~ ~ guaA-e.c -~~~---~ ----- ~~~~ guaA-syn ~ ~- ~~~- ~ ~- ~~ guaA-h.p -~---~~ ----- ~ guaAhuman tflttgvlst lrqadfeahn ilresgyagk isqmpviltp lhfdrdplqk 651 700 40 guaAN.t ~~~~~~~~~~ ~ - ~~- ~ guaA_est_A.t ~~~~~~~--~ ~~~ -- guaA-e.c ~ ~- ~~~~ guaA-syn - ~~~~~~- ~~ ~~ ~ -- ~ guaA-h.p --- ~~~~~ ------ ~ ~ ~ guaAhuman qpscqrsvvi rtfitsdfmt gipatpgnei pvevvlkmvt eikkipgisr 45 701 716 guaAN.t ~~~~-~~--- ~~~~-~ guaA-est_A.t -- ~~~~~~~~ ~~~~~ guaA_e.c ~~~~~~~~~~ -- ~~~ 19 guaA-syn -- ~~~--~- --- ~ guaA h.p ------- ~-- guaAhuman imydltskpp gttewe Example 2 5 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 Physcomitrella patens and used to produce a cDNA bank in the vector pBluescript SKII (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 from Aquifex aeolicus. The complete sequence of 093-d1l was determined, see SEQ-ID No. 3. 15 093_dll has a length of 1232 nucleotides and codes on a continuous reading frame for 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. 20 Example 3 Demonstration of the function of GMP-7M by complementation of E. coli 25 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 [tM of the appropriate 30 oligonucleotides, 200 [tM nucleotides (Pharmacia), 50 mM KC1, 10 mM Tris-HCl (pH 8.3 at 25'C, 1.5 mM MgCl 2 ) and 0.02 U/Rl Pfu polymerase (Stratagene). The amplification conditions were set as follow: 35 Annealing temperature: 50*C, 30 sec Denaturation temperature: 92*C, 30 sec Elongation temperature: 72*C, 3 min Number of cycles: 25 40 The resulting fragment of about 1670 bp 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, guaA21, relA1, X, spoTI) 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 Rg/ml guanosine.

20 The minimal media contained 0.4% glucose, 0.2% casamino acids, 100 Vg/ml thiamine, 100 Vg/ml inosine, 100 Vg/ml biotin, 100 Vg/ml histidine, 100 Vg/ml arginine, 100 pg/ml 2'-deoxyuridine, 100 [tM IPTG and 25 Vg/ml ampicillin. The cloning vectore 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 expression vector pTrc99A were capable of growth on minimal media without guanosine (see Tab. 2), which points strongly to the GMP-7M cDNA coding for 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 transformed with various plasmids after 2 days at 37*C pTrc99A + GMP-7M pTrc99A 20 Minimal medium without + guanosine Minimal medium with + + guanosine (100 pg/ml) 25 Example 4 Overexpression of the GMP synthetase from tobacco in E.coli and production of antibodies 30 For overexpression in E.coli, BamHI cleavage sites were introduced by PCR with GMP-7M as template and the oligonucleotides GMPA: 5'-GCAATGGATCCTCAAACACAGGCG-3' and GMPB: 5'-AAAAGGATCCTACTTTGGTCACC-3' and made it possible to clone the fragment in the vector pET15b (Novagen). A GMP-7M reading frame 35 with hexahistidine anchor at the N terminus was produced in this way. After the correct orientation had been checked by restriction digestion and polymerase errors had been excluded by sequencing, the resulting construct GMP-7E was transformed into E. coli BL21(DE3) (Stratagene). IPTG-induced one-day cultures 40 were harvested by centrifugation, and the cell pellets were lyzed and treated further in accordance with the manufacturer's information for nickel affinity chromatography ("Qia-Express-Kit", Qiagen). It was possible in this way to purify the GMP synthetase to more than 95% purity. The protein 45 was used for producing antisera in rabbits by conventional 21 protocols (carried out on contract by Eurogentec, Herstal, Belgium). Example 5 5 Expression of the GMP synthetase from tobacco in baculovirus-infected insect cells In order to obtain sufficient active GMP synthetase for mass 10 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-71 was used to generate recombinant baculovirus in accordance with the manufacturer's information (GibcoBRL). This virus was used in accordance with 15 the manufacturer's information (GibcoBRL) for infecting Sf21 insect cells in order to produce active GMP synthetase whose activity could be measured after disruption of the cells in 50 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM EDTA, 10 mM PMSF and desalting of the extract on a Sephadex G-25 column (Pharmacia, Sweden). 20 Example 6 Production of plant expression cassettes 25 The antisense and cosuppression techniques were used with the aim of reducing the GMP synthetase activity in transgenic tobacco plants. For this purpose, plasmid constructs were produced in the vector pBinAR (Hbfgen and Willmitzer, Plant Science (1990) 66, 221-230). A fragment of 1599 bp obtained from GMP-7M with BamHI 30 and BglII was ligated into the BamHI-cut vector pBinAR. The 1599 bp fragment encodes the 5'-terminal part of the GMP synthetase cDNA. Clones obtained after transformation into E.coli XL1-blue were examined for the orientation of the 1599 cassette by cutting with HindIII as a check. The plasmids pGMP7AS (antisense 35 construct) and pGMP7EX (sense construct) were identified in this way, see Figure 2. Example 7 40 Generation and analysis of transgenic plants The plasmids pGMP7AS and pGMP7EX - see Figure 2 - were transformed into Agrobacterium tumefaciens C58Cl:pGV2260 (Deblaere et al., Nucl. Acids. Res. 13(1984), 4777-4788). A 1:50 45 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 22 transforming tobacco plants (Nicotiana tabacum cv. Samsun NN). Leaf disks from sterile plants (each about 1 cm 2 ) were incubated in a Petri dish with a 1:50 agrobacteria dilution for 5-10 minutes. This was followed by incubation on 2MS medium with 5 0.8% Bacto agar in the dark at 25 0 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/i Claforan (cefotaxime sodium), 50 mg/i kanamycin, 1 mg/i benzylaminopurine (BAP), 0.2 mg/i naphthylacetic acid and 1.6 g/l glucose. Growing 10 shoots were transferred to MS medium with 2% sucrose, 250 mg/l 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-environment cabinet with a 16 hour light/8 hour dark 15 rhythm at 60% humidity, investigated for foreign gene expression and altered metabolite contents and phenotypical growth traits. Altered nucleotide contents can be determined, for example, by the method of Stitt et al. (FEBS Letters, 145 (1982), 217-222). 20 The transgenic GMP synthetase antisense plants and their filial generation showed reduced growth, compared with WT plant controls, and bleaching of the sink leaves. These phenotypical changes occurred in an early growth stage (see Fig. 3). In plants with reduced growth it was possible to detect in Northern 25 hybridization a reduced amount of GMP-7M RNA, compared with the wild type. 40p[g portions of complete RNA from sink leaves were employed for this purpose. 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 Rg of RNA were 30 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 35 BamHI and BglII and isolating a fragment comprising 1600 bp. The oligonucleotide 5'-GAT ACG TCG TCA AGG AAC TTG-3' was used for the labeling reaction. The probe was hybridized by standard methods, see Hybond information for users, Amersham. Hydridization [sic] signals were visualized by autoradiography 40 using Kodak X-OMAT 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 45 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 23 electrophoresis by standard methods and transferring to nitrocellulose membranes. Detection took place with an IgG-alkaline phosphatase conjugate and the BCIP/NBT system (Sigma). 5 In addition, it was possible by the in vitro assay described in Example 8 to establish that there was reduced GMP synthetase activity in transgenic lines with reduced growth. 10 The correlation between the level of expression and the GMP synthetase activity and growth phenotype suggest that GMP synthetase is a suitable target for herbicides. Example 8 15 Assay systems for measuring GMP synthetase activity The systems developed by Spector (Methods in Enzymology LI, 1978, 219-224) for animal enzymes can be used to measure plant GMP 20 synthetase activity. In the first system, the AMP formation is made possible by coupling the reaction with AMP kinase, pyruvate kinase, lactate dehydrogenase and measurement at 340 nm. The second system is based on direct detection of GMP (guanosine monophosphate) by employing the radiolabeled substrate XMP 25 (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 30 glutamate. This system has the advantage of a smaller number of coupled reaction steps and provides greater signal strengths. XMP + ATP + L-glutamine + H 2 0 GMP-S GMP + AMP + L-glutamate + PPi 35 GluDH L-glutamate + APAD + H 2 0 --- oxoglutarate + APADH + NH 4 + (GMP-S = GMP synthetase, GluDH = glutamate dehydrogenase, APAD = 40 3-acetylpyridine adenine dinucleotide) For this, the reaction mixture (see below) was incubated at 37 0 C for 60 minutes, and the reaction was stopped by incubation at 95 0 C for 5 minutes. The glutamate formed was detected in the detection 45 mixture (see below) by photometric measurement of the increase in APADH at 363 nm.

24 Reaction mixture: 100 VL 750 mM Tris/HCl buffer pH 7.8 100 VL 100 mM MgCl 2 5 100 sL 80 mM KC1 100 VL 20 mM XMP 100 VL 200 mM L-glutamine 400 [tL

H

2 0 100 [lL protein extract 10 1000 VL Detection mixture: 375 [tL 100 mM Tris-HCl buffer pH 8.0 15 75 pL 500 mM KCl 125 ptL

H

2 0 75 [tL 3 mM APAD 100 VtL of the reaction mixture 750 [L 20 Example 9 Search for inhibitors of GMP synthetase activity 25 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 for this can be prepared from plant tissues. It is possible and preferred for a plant GMP synthetase to be expressed in E.coli, insect cells or another 30 suitable expression system and then be concentrated or isolated. It was possible in this way to identify known inhibitors such as 6-thio-XMP. 35 40 45

Claims (10)

1. 2. A DNA sequence which hybridizes with the DNA sequence SEQ-ID 10 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. 15 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.
2. 4. A protein as claimed in claim 3, which comprises as amino 20 acid sequence the part-sequence 50 - 300 from SEQ-ID No: 2 or SEQ-ID No: 4.
3. 5. A protein as claimed in claim 4, which comprises as amino 25 acid sequence the sequence depicted in SEQ-ID No: 2 or SEQ-ID No: 4.
4. 6. The use of a DNA sequence as claimed in claim 1 or 2 for introduction into pro- or eukaryotic cells, this sequence 30 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. 35 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.
5. 8. A method for finding substances which inhibit the activity of 40 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. 45
6. 9. A method as claimed in claim 8, wherein the measurement of the plant GMP synthetase is carried out in a high throughput 26 screening (HTS).
7. 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 10 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; i5 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 20 d) comparison of the growth or survivability of the transgenic and untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance; 25 where suppression of the growth or survivability of the untransformed plants, plant cells, plant tissues or plant parts without, however, greatly suppressing the growth or the survivability of the transgenic plants, plant cells, plant 30 tissues or plant parts demonstrates that the substance from b) shows herbicidal activity and inhibits the enzymic activity in plants. 35 11. An assay system based on 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. 40 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 45 uninhibited enzyme. 27
8. 13. An inhibitor of plant GMP synthetase.
9. 14. An inhibitor of plant GMP synthetase identified using an assay system as claimed in claim 11 or 12. 5
10. 15. An inhibitor as claimed in either of claims 13 or 14 for use as herbicide. 10 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. 15 20 25 30 35 40 45