CA2388851A1

CA2388851A1 - Formylglycinamidinribotide synthase from plants

Info

Publication number: CA2388851A1
Application number: CA002388851A
Authority: CA
Inventors: Jens Lerchl; Thomas Ehrhardt; Uwe Sonnewald; Ralf Boldt; Gotthard Kunze
Original assignee: Individual
Current assignee: BASF SE
Priority date: 1999-10-25
Filing date: 2000-10-17
Publication date: 2001-05-03
Also published as: EP1224292A2; WO2001031025A3; WO2001031025A2; AU1853501A

Abstract

The invention relates to DNA sequences coding for a polypeptide with formylglycinamidinribotide synthase (E.C.6.3.5.3) activity. The invention further relates to the use of said nucleic acids for the production of a tes t system.

Description

FORMYLGLYCINAMIDINRIBOTIDE SYNTHASE FROM PLANTS
The present invention relates to the identification of plant formylglycinamidine ribotide synthase (E. C. 6.3.5.3.) as novel target for herbicidal active ingredients. The present invention furthermore relates to DNA sequences SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 encoding a polypeptide with formylglycinamidine ribotide synthase activity. Moreover, the invention relates to the use of a nucleic acid encoding a protein with formylglycinamidine ribotide synthase activity which originates from plants for generating an assay system for identifying herbicidally active formylglycinamidine ribotide synthase inhibitors. The invention furthermore relates to the use of the nucleic acid SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 encoding plant formylglycinamidine ribotide synthase for the generation of plants with an increased resistance to formylglycinamidine ribotide synthase inhibitors. Moreover, the invention relates to a method of eliminating undesired vegetation by treating the plants to be eliminated with a compound which specifically binds to formylglycinamidine ribotide synthase encoded by a DNA
sequence SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 or a DNA
sequence hybridizing with this DNA sequence, and which inhibits its function.
Plants are capable of synthesizing their cell components from carbon dioxide, water and inorganic salts.
This process is only possible by exploiting biochemical reactions for synthesizing organic substances. Nucleotides are synthesized de novo in plants. Being components of the nucleic acids, they are particularly important. Covalently bound, nucleotides activate carbohydrates for polysaccharide biosynthesis. They furthermore activate head groups for lipid biosynthesis.
Nucleotides are involved in virtually all metabolic pathways.
Nucleoside triphosphates, especially ATP, drive most of the energy-requiring reactions of the cell. Adenine nucleotides are additionally also found as components in essential factors such as coenzyme A and in nicotinamide and flavin coenzymes, which are involved in a large number of cellular reactions. The coupled hydrolysis of guanosine-5'-triphosphate (GTP) defines a direction of reaction for various cellular processes such as protein translation, assembly of microtubuli, vesicular transport, signal transduction and cell division. Furthermore, nucleotides constitute the starting metabolites for the biosynthesis of _ . CA 02388851 2002-04-23 - methylxanthines such as coffein and theobromin in the plant family of the Rubiaceae and Theaceae.
Since plants depend on an effective nucleotide metabolism, it can be assumed that enzymes which are involved in nucleotide biosynthesis are suitable as target protein for herbicides. Thus, there have already been described active ingredients which inhibit de novo purine biosynthesis in plants. An example which may be mentioned is the natural substance hydanthocidin, which, after phosphorylation in planta inhibits adenylosuccinate synthetase (ASS); (Siehl et al., Plant Physiol. 110(.1996), 753-758).
Other inhibitors for enzymes of purine biosynthesis are also known for their pharmacological action in animals and microorganisms: folate analogs inhibit, inter alia, the enzyme GAR transformylase and have an antiproliferative, antiinflammatory and immunosuppressant action. Mycophenolic acid (MPA), an IMP dehydrogenase inhibitor in the GMP synthetic pathway, has an antimicrobial, antiviral and immunosuppressant action (Kitchin et al, Journal of the American Academy of Dermatology 37(1997), 445-449).
Bacterial PRPP amidotransferase can be inhibited by glutamine antagonists such as, for example, acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid).
Glutarnine antagonists are not specific to PRPP amidotransferase, but also inhibit the formylglycinamidine ribotide synthase (E. C.
6.3.5.3.) of mammals and have an antiproliferative action on tumor tissue (Elliot and Weber, Biochemical Pharmacology 34(1985), 243-248). In the fourth step of purine biosynthesis, formylglycinamidine ribotide synthase converts the carboxamide oxygen of the formylglycinamide ribonucleotide (FGAR) into an imino group. During the hydrolysis of ATP to ADP, the amide nitrogen is transferred from the glutamine to FGAR, :resulting in formylglycinamidine ribonucleotide (FGAM) and glutamate, see Figure 1. The efficacy of glutamine antagonists on plant formylglycinamidine ribotide synthase is still to be proven.
Genes which encode formylglycinamidine ribotide synthase have been isolated from a variety of organisms.
cDNAs which encode formylglycinamidine ribotide synthase have been isolated and characterized from various bacterial and animal organisms.

005x/50827 CA 02388851 2002-04-23 - The genes which encode most of the enzymes of the purine biosynthesis pathway in plants have already been isolated. In the case of formylglycinamidine ribotide synthase, only two partial EST (expressed sequence tag) sequences from Arabidopsis thaliana (GenBank Number AA042492, 1295 bp) and barley (GenBank Number HV,7000235, 384 bp) exist as yet.
The suitability of an enzyme as herbicide target can be proved by reducing the enzyme activity in transgenic plants, for example by means of antisense technology. If this leads to reduced growth, it can be concluded therefore that the enzyme, whose activity is reduced, is suitable as site of action for herbicidal active ingredients. For example, antisense inhibition of acetolactate synthase (ALS) in transgenic potato plants and the treatment of control plants with ALS-inhibiting herbicides leads to comparable phenotypes (Hofgen et al., Plant Physiology 107(1995), 469-477).
It is an object of the present invention to provide proof that formylglycinamidine ribotide synthase in plants is a suitable herbicidal target, to isolate a complete plant cDNA encoding the enzyme formylglycinamidine ribotide synthase and functionally express it in bacterial or eukaryotic cells, and to produce an efficient and simple formylglycinamidine ribotide synthase assay system for carrying out inhibitor-enzyme binding studies.
we have found that this object is achieved by first isolating cDNAs encoding plant formylglycinamidine ribotide synthase. The present invention therefore relates to the isolation of cDNAs encoding Arabidopsis thaliana, Nicotiana tabacum and Chilopsis linearis formylglycinamidine ribotide synthase.
The present invention furthermore relates to processes for identifying formylglycinamidine ribotide synthase inhibitors in plants by high-throughput methods. The invention therefore relates to the functional expression of plant formyl.glycinamidine ribotide synthase, in particular of Arabidopsis tha3iana and tobacco formylglycinamidine ribotide synthase in suitable expression systems and to the use of the resulting enzymes in an in vitro assay system for measuring formylglycinamidine ribotide synthase activity.
We have found that this object is achieved by the isolation of genes which encode the plant enzyme formylglycinamidine ribotide synthase, the generation of formylglycinamidine ribotide synthase antisense constructs, and the functional expression of ~~50/50827 CA 02388851 2002-04-23 formylglycinamidine ribotide synthase in bacterial or eukaryotic cells.
It is an object of the present invention to isolate full-length cDNAs encoding functional formylglycinamidine ribotide synthase (E.C.6.3.5.3.) from Arabidopsis thaliana and Nicotiana tabacum and a partial cDNA sequence from Chilopsis linearis.
A first subject-matter of the present invention is a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 containing the encoding region of a plant formylglycinamidine ribotide synthase from Nicotiana tabacum and Arabidopsis thaliana, see Examples 1 and 2.
Another subject-matter of the invention is DNA sequences which are derived from SEQ-ID No. 1 or SEQ-ID No. 3 or which hybridize with one of these sequences and which encode a protein which has the biological activity of a formylglycinamidine ribotide synthase.
Arabidopsis thaliana plants and tobacco plants of the line Nicotiana tabacum co. Samsun NN which carry a formylglycinamidine ribotide synthase sense or antisense construct have been characterized in greater detail. The plants show different degrees of retarded growth. The transgenic lines and the progeny of the lgt and 2nd generation showed a reduced growth in soil.
Using Northern hybridization, it was detected that the RNA
quantity of the formylglycinamidine ribotide synthase was reduced in plants with reduced growth compared with the wild type.
Furthermore, measurement of the enzyme activity detected that the amount of formylglycinamidine ribotide synthase activity was reduced in the transgenic lines compared with wild-type plants, see Examples 5 and 6. The expression level and the reduction in formylglycinamidine ribotide synthase activity correlate with the growth retardation. This clear connection identifies formylglycinamidine ribotide synthase for the first time unambiguously as suitable target protein for herbicidal active ingredients.
To be able to find efficient inhibitors of plant formylglycinamidine ribotide synthase, it is necessary to provide suitable assay systems with which inhibitor/enzyme binding studies can be carried out. To this end, for example, the cDNA
sequence of formylglycinamidine ribotide synthase or suitable fragments of the cDNA sequence of formylglycinamidine ribotide synthase from Arabidopsis thaliana is, or are, cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.

0~'rJ~/50827 CA 02388851 2002-04-23 Alternatively, however, it is possible to express the expression cassette containing a DNA subsequence of SEQ-ID No. 1 or SEQ-ID No. 3 for example in other bacteria, in yeasts, fungi, algae, plant cells, insect cells or mammalian cells.

The formylglycinamidine ribotide synthase protein which is expressed with the aid of an expression cassette is particularly suitable for finding inhibitors which are specific to formylglycinamidine ribotide synthase.
To this end, for example, the plant formylglycinamidine ribotide synthase can be employed in an enzyme assay in which the formylglycinamidine ribotide synthase activity is determined in the presence and absence of the active ingredient to be tested. A
comparison of the two activity determinations allows a qualitative and quantitative statement to be made on the inhibitory behavior of the active ingredient to be tested, see Example 8.
The assay system according to the invention allows a multiplicity of chemicals to be tested rapidly and simply for herbicidal properties. Using this method, substances with a potent action can be selected specifically and reproducibly from amongst a large number of substances, in order that further in-depth tests with which the skilled worker is familiar be carried out subsequently with these substances.
Another subject-matter of the invention is a method of identifying herbicidally active substances which inhibit the formylglycinamidine ribotide synthase activity in plants, consisting of the following steps:
a) the generation of transgenic plants, plant tissues or plant cells comprising an additional DNA sequence encoding an enzyme with formylglycinamidine ribotide synthase activity and capable of overexpressing enzymatically active formylglycinamidine ribotide synthase;
b) applying a substance to transgenic plants, plant cells, plant tissues or plant parts and to untransformed plants, plant cells, plant tissues or plant parts;
c) determining the growth or the viability of the transgenic and the untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance; and ., d) comparing the growth or the viability of the transgenic and the untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance;
where suppression of the growth or viability of the untransformed plants, plant cells, plant tissues or plant parts without simultaneous severe suppression of the growth or the viability of the transgenic plants, plant cells, plant tissues or plant parts confirms that the substance of b) is herbicidally active and inhibits the formylglycinamidine ribotide synthase enzyme activity in plants.
Another subject-matter of the invention is a method of identifying plant formylglycinamidine ribotide synthase inhibitors with a potentially herbicidal action by cloning the gene of a plant formylglycinamidine ribotide synthase, overexpressing it in a suitable expression cassette - for example in insect cells - disrupting the cells and employing the cell extract in an assay system for measuring the enzyme activity in the presence of low-molecular-weight chemicals, either directly or after concentration or isolation of the enzyme formylglycinamidine ribotide synthase.
Another subject-matter of the invention is compounds with a herbicidal action which can be identified with the above-described assay system.
Another subject-matter of the invention is a method of eliminating undesired vegetation, which comprises treating the plants to be eliminated with a compound which binds specifically to formylglycinamidine ribotide synthase encoding a DNA sequence SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 or a DNA sequence hybridizing with this DNA sequence, and which inhibits its function.
Herbicidally active formylglycinamidine ribotide synthase inhibitors can be employed as defoliants, desiccants, haulm killers and, in particular, as herbicides. Weeds in the widest sense are to be understood as meaning all plants which grow in locations where they are undesired. Whether the active ingredients found with the aid of the assay system according to the invention act as total or selective herbicides depends, inter alia, on the quantity applied.
Herbicidally active formylglycinamidine ribotide synthase inhibitors can be used, for example, against the following weeds:

~~SU/50827 CA 02388851 2002-04-23 Dicotyledonous weeds of the genera:
Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, 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.
Monocotyledonous weeds of the genera:
Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, Apera.
A subject-matter of the invention is also expression cassettes whose sequence encodes a formylglycinamidine ribotide synthase from Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis or their functional equivalent. The nucleic acid sequence can be, for example, a DNA or a cDNA sequence.
In addition, the expression cassettes according to the invention comprise regulatory nucleic acid sequences which govern the expression of the encoding sequence in the host cell. In accordance with, a preferred embodiment, an expression cassette according to the invention encompasses upstream, i.e. at the 5' end of the encoding sequence, a promoter, and downstream, i.e. at the 3' end, a polyadenylation signal and, if appropriate, other regulatory elements which are operatively linked to the encoding sequence for the formylglycinamidine ribotide synthase gene, which sequence lies between the promoter and the polyadenylation signal. Operative linkage is to be understood as meaning the sequential arrangement of promoter, encoding sequence, terminator and, if appropriate, other regulatory elements in such a manner that each of the regulatory elements can function as intended when the encoding sequence is expressed.
An expression cassette according to the invention is generated by fusing a suitable promoter with a suitable formylglycinamidine ribotide synthase DNA sequence and a polyadenylation signal using customary recombination and cloning techniques as they are described, for example, by T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and by T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1984) and by Ausubel, F.M. et al., Current Protocols in ~05~~~~$27 CA 02388851 2002-04-23 Molecular Biology, Gree"ne Publishing Assoc. and Wiley-Interscience (1987).
A subject-matter of the invention is also functionally equivalent DNA sequences which encode a formylglycinamidine ribotide synthase gene and which show a sequence homology with the DNA
sequence SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 of 40 to 100, based on the total length of the DNA sequence.
A preferred subject-matter of the invention is functionally equivalent DNA sequences which encode a formylglycinamidine ribotide synthase gene and which show a sequence homology with the DNA sequence SEQ-ID No. 1, SEQ-ID No. 3 or SEQ-ID No. 5 of 60 to 100, based on the total length of the DNA sequence.
A particularly preferred subject-matter of the invention is functionally equivalent DNA sequences which encode a formylglycinamidine ribotide synthase gene and which show a sequence homology with the DNA sequence SEQ-ID No. 1, SEQ-ID No.
3 or SEQ-ID No. 5 of 80 to 100, based on the total length of the DNA sequence.
Functionally equivalent sequences which encode a formylglycinamidine ribotide synthase gene are in accordance with the invention those sequences which retain the desired functions, despite a deviating nucleotide sequence. Functional equivalents thus encompass naturally occurring variants of the sequences described herein, but also artificial nucleotide sequences, for example those which have been obtained by chemical synthesis and which are adapted to suit the codon usage of a plant.
A functional equivalent is also to be understood as meaning in particular natural or artificial mutations of an originally isolated sequence which encodes a formylglycinamidine ribotide synthase and which continues to show the desired function.
Mutations encompass substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Thus, the present invention for example also extends to those nucleotide sequences which are obtained by modifying this nucleotide sequence. The target of such a modification can be, for example, the further delimitation of the encoding sequence contained therein or else, for example, the introduction of further restriction enzyme cleavage sites.

. 9 Functional equivalents are also those variants whose function is reduced or increased compared with the starting gene, or gene fragment.
In addition, the expression cassette according to the invention can also be employed for the transformation of bacteria, cyanobacteria, yeasts, filamentous fungi and algae and eukaryotic cells (for example insect cells), with the purpose of producing sufficient amounts of the enzyme formylglycinamidine ribotide synthase.
Another subject-matter of the invention is a protein from Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis with the amino acid sequence SEQ-ID No. 2, SEQ-ID No. 4 or SEQ-ID No. 6, or derivatives or parts of this protein with formylglycinamidine ribotide synthase activity.
A subject-matter of the invention is also plant proteins with formylglycinamidine ribotide synthase activity with an amino acid sequence homology to the Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis formylglycinamidine ribotide synthase with the SEQ-ID No. 2, SEQ-ID No. 4 or SEQ-ID No. 6 of 20 - 100 identity.
Preferred are plant proteins with formylglycinamidine ribotide synthase activity with an amino acid sequence homology to the Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis formylglycinamidine ribotide synthase with the sequence SEQ-ID
No. 2, SEQ-ID No. 4 or sequence SEQ-ID No. 6 of 50 - 100 identity.
Particularly preferred are plant proteins with formylglycinamidine ribotide synthase activity with an amino acid sequence homology to the Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis formylglycinamidine ribotide synthase with the sequence SEQ-ID No. 2, SEQ-ID No. 4 or sequence SEQ-ID No. 6 of 80 - 100 identity.
It was another object of the invention to overexpress the formylglycinamidine ribotide synthase gene in plants in order to generate plants which tolerate formylglycinamidine ribotide synthase inhibitors.
Overexpression, in a plant, of the gene sequence SEQ-ID No. 1 or SEQ-ID No. 3, which encodes a formylglycinamidine ribotide synthase, results in an increased resistance to , 0050/50827 formylglycinamidine ribotide synthase inhibitors. The transgenic plants generated thus are also subject-matter of the invention.
Expressional efficacy of the recombinantly expressed formylglycinamidine ribvtide synthase gene can be determined, for example, in vitro by shoot-meristem propagation or by a germination test. Moreover, the expression of the formylglycinamidine ribotide synthase gene which has been altered in terms of type and level, and its effects an the resistance to formylglycinamidine ribotide synthase inhibitors can be tested in greenhouse experiments using test plants.
Subject-matter of the invention are also transgenic plants, transformed with an expression cassette containing the DNA
sequence SEQ-ID No. 1 or SEQ-ID No. 3, which have been made tolerant to formylglycinamidine ribotide synthase inhibitors by additionally expressing the DNA sequence SEQ-ID No. 1 or SEQ-ID
No. 3, and transgenic cells, tissues, parts and propagation material of such plants. Especially preferred in this context are transgenic crop plants such as, for example, barley, wheat, rye, maize, soya, rice, cotton, sugar beet, canola, sunflowers, flax, hemp, potatoes, tobacco, tomatoes, oilseed rape, alfalfa, lettuce and the various tree, nut and grapevine species, and also legumes.
Particularly preferred are sequences which ensure targeting into the apoplast, into plastids, into the vacuole, into the mitochondrium, into the endoplasmatic reticulum (ER), or which, owing to the absence of suitable operative sequences, ensure that the product remains in the compartment where it is formed, in the cytosol, (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).
For example, the plant expression cassette can be introduced into the plant transformation vector pBinAR, see Example 5.
A suitable promoter of the expression cassette according to the invention is, in principle, any promoter which is capable of governing the expression of foreign genes in plants. It is preferred to use, in particular, a plant gromoter or a promoter derived from a plant virus. Particularly preferred is the cauliflower mosaic virus CaMV 35S promoter (Franck et al., Cell 21(1980), 285-294). This promoter contains different recognition sequences for transcriptional effectors which, in their totality, lead to permanent and constitutive expression of the gene which has been introduced (Benfey et al., EMBO J., 8 (1989), 2195-2202).

The expression cassette according to the invention may also comprise a chemically inducible promoter which allows expression - of the exogenous formylglycinamidine ribotide synthase gene in the plant to be governed at a particular point in time. Such promoters which are described in the literature and which can be used are, inter alia, for example the PRP1 promoter (Ward et al., Plant.Mol. Biol. (1993) 22, 361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 388186), a tetracyclin-inducible promoter (Gatz et al., Plant J. (1992) 2, 397-404), an abscisic acid-inducible promoter (EP0335528) or an ethanol- or cyclohexanone-inducible promoter (WO 93/21334).
Particularly preferred promoters are furthermore those which ensure expression in tissues or parts of the plant in which the biosynthesis of purines or their precursors takes place.
Promoters which ensure leaf-specific expression must be mentioned in particular. Promoters which must be mentioned are the potato cytosolic FBPase or the potato ST-LSI promoter (Stockhaus et al., EMBO J., 8 (1989) 2445-245).
A foreign protein can be expressed stably in the seeds of transgenic tobacco plants to an extent of 0.67% of the total soluble seed protein with the aid of a seed-specific promoter (Fiedler and Conrad, Bio/Technology 10 (1995), 1090-1094). The expression cassette according to the invention can therefore contain, for example, a seed-specific promoter (preferably the phaseolin promoter, the USP promoter or the LEB4 promoter), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
The inserted nucleotide sequence encoding a formylglycinamidine ribotide synthase can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA
components. In general, synthetic nucleotide sequences are generated with codons which are preferred by plants. These codons which are preferred by plants can be determined from codons with the highest protein frequency expressed in the plant species of the highest interest. when preparing an expression cassette, a variety of DNA fragments may be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
Adaptors or linkers can be added to the fragments in order to link the DNA fragments to each other.

Other suitable DNA sequences are artificial DNA sequences as long as they mediate the desired property by increasing the purine nucleotide content in the plant by overexpressing the formylglycinamidine ribotide synthase gene. Such artificial DNA
sequences can be determined for example by backtranslating of proteins which have formylglycinamidine ribotide synthase activity and which have been constructed by means of molecular modeling, or they can be determined by in vitro selection.
Especially suitable are encoding DNA sequences which have been obtained by backtranslating a polypeptide sequence in accordance with the host-plant-specific codon usage. The specific codon usage can be determined readily by the skilled worker familiar with methods of plant genetics by means of computer evaluations of other, known genes of the plant to be transformed.
Other suitable equivalent nucleic acid sequences according to the invention which must be mentioned are sequences which encode fusion proteins, component of the fusion protein being a plant formylglycinamidine ribotide synthase polypeptide or a functionally equivalent part thereof. The second part of the fusion protein can be, for example, another polypeptide with enzymatic activity or an antigenic polypeptide sequence, with the aid of which detection of formylglycinamidine ribotide synthase expression is possible (for example myc-tag or his-tag). However, it is preferably a regulatory protein sequence such as, for example, a signal or transit peptide, which leads the formylglycinamidine ribotide synthase protein to the desired site of action.
The promoter and terminator regions according to the invention should expediently be provided, in the direction of transcription, with a linker or polylinker containing one or more restriction sites for insertion of this sequence. As a rule, the linker has 1 to 10, in most cases 1 to 8, preferably 2 to 6, restriction sites. In general, the linker within the regulatory regions has a size less than 100 bp, frequently less than 60 bp, but at least 5 bp. The promoter according to the invention may be native, or homologous, or else 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 sequence and a region of transcriptional termination. Various termination regions can be exchanged for each other as desired.
Manipulations which provide suitable restriction cleavage sites or which eliminate the excess DNA or restriction cleavage sites may also be employed. In vitro mutagenesis, primer repair, ', 0050/50827 CA 02388851 2002-04-23 restriction or ligation may be used in cases where insertions, deletions or substitutions such as, for example, transitions and transversions, are suitable. Complementary ends of the fragments may be provided for ligation in the case of suitable manipulations such as, for example, restriction, chewing-back or filling overhangs far blunt ends.
Preferred polyadenylation signals are plant polyadenylation signals, preferably those which correspond essentially to Agrobacterium tumefaciens T-DNA polyadenylation signals, in particular those of the gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al., EMBO J., 3 (1984), 835), or functional equivalents.
To transform a host plant with a DNA encoding formylglycinamidine ribotide synthase, an expression cassette according to the invention is incorporated, as insertion, 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 Biotechnology" (CRC Press, Chapters 6/7, 71-119).
The transfer of foreign genes into the genome of a plant is termed transformation. It exploits the above-described methods for transforming and regenerating plants from plant tissues or plant cells for transient or stable transformation. Suitable methods are the protoplast transformation by polyethylene-glycol-induced DNA uptake, the biolistic method using the gene gun, electroporation, incubation of dry embryos in DNA-containing solution, microinjection and agrobacterium-mediated gene transfer. The abovementioned methods are described by, for example, 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 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 the transformation of 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 equally be used in a known manner for transforming crop plants, in particular crop plants such as cereals, maize, soya, rice, cotton, sugar beet, canola, sunflowers, flax, hemp, potatoes, tobacco, tomatoes, oilseed rape, alfalfa, lettuce and the various tree, nut and grapevine ~ i ~05~/50827 CA 02388851 2002-04-23 species, or else legumes, for example by bathing wounded leaves or leaf sections in an agrobacterial suspension and subsequently growing them in suitable media.
The purine biosynthesis site is generally the leaf tissue, so that leaf-specific expression of the formylglycinamidine ribotide synthase gene is meaningful. However, it is obvious that the purine biosynthesis need not be limited to the leaf tissue, but may also take place in all other remaining parts of the plant in a tissue-specific fashion, for example in fatty seeds.
In addition, constitutive expression of the exogenous formylglycinamidine ribotide synthase gene is advantageous. On the other hand, inducible expression may also be desirable.
Using the recombination and cloning techniques cited above, the expression cassettes according to the invention can be cloned into suitable vectors which allow them to be multiplied, for example in E. coli. Suitable cloning vectors are, inter alia, pBR332, pUC series, Ml3mp series and pACYC184. Especially suitable are binary vectors which are capable of replication both in E. coli and in agrobacteria.
Another subject-matter 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 preferred purpose of the use is to increase the formylglycinamidine ribotide synthase content in the plant.
Depending on the choice of the promoter, expression may take place specifically in the leaves, in the seeds or in other parts of the plant. Such transgenic plants and their propagation material and their plant cells, tissue or parts are another subject of the present invention.
The invention will now be illustrated by the examples which follow, without being limited thereto.
Examples Recombinant methods on which the use examples are based:
General cloning methods Cloning methods such as restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA

fragments, transformation of Escherichia coli cells, growing bacteria and the 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).

Screening of cDNA libraries h-Phages of the relevant cDNA libraries were plated onto agar plates with E. coli XL1-Blue as bacterial strain. The phage DNA
10 was transferred to nitrocellulose filters (Gelman Sciences) by means of standard methods (Sambrook et al. (1989), Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) and fixed to the filters. The hybridization probes used were PCR fragments, or DNA
fragments obtained by restriction cleavage, which were 15 radiolabeled with the aid of a "Multiprime DNA labeling system"
(Amersham Buchler) in the presence of 32P-dCTP (specific activity 3000 Ci/mmol), following the manufacturer's instructions. The membranes were hybridized after prehybridization at 60°C in 3 x SSPE, 0.1~ sodium dodecyl sulfate (w/v), 0.02 polyvinylpyrrolidone (w/v), 0.02 Ficoll 400 (w/v) and 50 mg/ml calf thymus DNA for 12-16 hours. The filters were then washed for 60 minutes in 2 x SSPE, 0.1~ sodium dodecyl sulfate (w/v) at 60°C.
Positively hybridizing phages were visualized by autoradiography, singled out by means of standard techniques (Sambrook et al.
(1989); Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) and transferred into plasmids (Stratagene).
Sequence analysis of recombinant DNA
Recombinant DNA molecules were sequenced using an ABI laser fluorescence DNA sequences, following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA, 74(1977), 5463-5467).
Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
Analysis of total RNA from plant tissues Total RNA from plant tissues was isolated as described by Logemann et al.(Anal. Biochem. 163(1987), 21). For the analysis, in each case 20 ~,g of RNA were separated 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). The DNA
fragments employed as probe were radiolabeled with a Random Primed DNA Labeling Kit (Roche, Mannheim) and hybridized by standard methods, see Hybond instructions, Amersham.

Hybridization signals were visualized by autoradiography with the aid of Kodak X-GMAT AR films.
Unless otherwise specified, the chemicals used were analytical grade and obtained from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen).
Solutions were made with a refined, pyrogen-free water, termed H20 hereinbelow, from a Milli-Q water refining system (Millipore, Eschborn). Restriction endonucleases, DNA-modifying enzymes and molecular biologic kits were obtained from AGS (Heidelberg), Amersham (Braunschweig), Biometra (Gottingen), Roche (Mannheim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach/Taunus), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Heidelberg). Unless otherwise specified, they were used in accordance with the manufacturer's instructions.
The bacterial strains used hereinbelow (E. coli, XL-1 Blue) were obtained from Stratagene. E. coli AT 2465 was obtained from the coli genetic stock center (Yale University, New Haven). The agrobactexial strain used for transforming plants (Agrobacterium tumefaciens, C58C1 with plasmid pGV2260 or pGV3850kan) was described by Deblaere et al. (Nucl. Acids Res. 13 (1985), 4777).
Alternatively, it is also possible to use the agrobacterial strain LBA4404 (Glontech) or other suitable strains. Vectors which can be used for cloning are pUCl9 (Vanish-Perron, Gene 33(1985), 103-119), pBluescript SK- (Stratagene), pGEM-T
(Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids Res. 12(1984), 8711-8720) and pBinAR (Hofgen and Willmitzer, Plant Science 66 (1990), 221-230).
Example 1 Isolation of a full-length formylglycinamidine ribotide synthase cDNA from Arabidopsis thaliana To isolate formylglycinamidine-ribotide-synthase-encoding cDNAs, an Arabidopsis thaliana expressed sequence tag (est) which partially encodes formylglycinamidine ribotide synthase (GenBank Accession number AA042492) was obtained, sequenced and used as template for generating a hybridization probe by means of polymerase chain reaction (PCR) in order to isolate formylglycinamidine-ribotide-synthase-encoding cDNAs.
The reaction mixtures contained approx. 1 ng/~,1 template DNA, 0.5 ~,M of the oligonucleotides 5'-GCTGCTTCAATAAGGTTCAAC-3' and 5'-CGCTATTCCCAACGGCACACC-3', 200 ~M deoxy-nucleotides (Pharmacia), 50 mM KC1, 10 mM Tris-HC1 (pH 8.3 at 25°C, 1.5 mM
MgCl2) and 0.02 U/~1 Taq polymerase (Perkin Elmer).
The amplification conditions were set as follows:
Annealing temperature: 50°C, 1 min Denaturation temperature: 94°C, 1 min Elongation temperature: 72°C, 2 min Number of cycles: 30 The resulting 465 by fragment was used for a heterologous screening of 6.5*105 pfu of a UniZAP XR cDNA library (Stratagene) from Arabidopsis thaliana (whole plant) mRNA. After restriction and sequence analysis, it was possible to identify two distinguishable clones (purl-19 and purl-23) which encoded reading frames with homology to formylglycinamidine ribotide synthase from Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Drosophila melanogaster. The sequence comparisons show that both purl-19 (length: 1267 bp) and purl-23 (length:
917 bp) are partial clones encoding 3' regions of formylglycinamidine ribotide synthase. To isolate a full-length cDNA, it is necessary to employ a size-fractionated cDNA library for the screening since only further partial clones were isolated from other cDNA libraries. Therefore, a 964 by fragment obtained by cleavage of purl-19 with EcoRI and KpnI was used for screening 5*105 pfu of an Arabidopsis thaliana cDNA library in the vector lambda-ZAP-Express (Stratagene). The library was prepared with RNA from entire plants. A cDNA fraction > 2 kbp long was prepared in order to generate the cDNA library.
A positively hybridizing clone with the longest insert {purl-48.1) was identified by means of restriction analysis and subsequently sequenced, see SEQ-ID No. 1. The purl-48.1 cDNA has a length of 4570 base pairs and is identical in the overlapping region (nucleotide 3328- 4570) with the shorter purl-19 cDNA. A
continuous reading frame encodes, in purl-48.1, a polypeptide of 1407 amino acids and a calculated mass of 153 952 kDa, see SEQ-ID
No. 2. The amino acid sequence shows a similarity to formylglycinamidine ribotide synthase from other organisms, see Table 1. A relationship exists with human formylglycinamidine ribotide synthase (GenBank Number AB002359) with 51.4 identity, or with 59.7 similarity at the amino acid level. purl-48.1 shows an N-terminal extension of 53 to 88 amino acids compared with formylglycinamidine ribotide synthase from other organisms, which suggests an important transit peptide. The computer -aided evaluation of the sequence predicts a possible function for amino acids 1-53 as signal peptide for an import into the plastids.

1$
Localization of the enzyme in plastids tallies with its function in purine biosynthesis. The reading frame upstream of the start methionine contains a stop codon. purl-48.1 is thus the first full-length plant cDNA for a formylglycinamidine ribotide synthase.
Table 1 Sequence formylglycinamidine synthase comparison ribotide of 10sequences organisms Saccharomyces from various (E.coli, cerevisiae,Drasophilamelanogaster, Sapiens, Homo Arabidopsis thaliana). amino Only those acids which do not correspond to the consensus shown.
sequence are 15PurL_e.coli-__________-________________________-__-_-________ purL_yeast -_-________---____-_--____-_-__-____-_-__-_-______ purl Drome ----_-__-________-_____,_________-__--___--_______ purl human ----_____________________-___________-_-__--_-__--purL-ara-48-mntsqatraalflngsnrqamllqrssmsqlwgsvrmrtsrlslnrtkav Consensus --___-_-_-_-_____-_____-____-----_-__--__---_--__-purl e.coli---------------------------_ me.-lr---a-saf-ink-1 -purl-yeast ---------_--------_--------- tdy-lp-pka-sqf--dn-i -purl Drome -__-______-_____________________--_-__m-ilr-ydvqa-purL human ---aasasstnlihl-skghispakdt--qqrtpa-m----h--vr-sg-purL-ara-48-slrcsaqpnkpkaav-tgsfvtadelp--vekpaa-... ih-----liq 25Consensus ---------------S-----------SLM---I-EGSPVL-FYRVP-LH

purl e.colia...rfqaa-lpvhniya---hfad.-nap--dd-haq--r-1--gpa-.

purL~reast kdi--ytnstsvinelrsc-ihyvngiaq--seqdt-1--v-lt-dsa-d purl Drome sa-ee-sv--rlreedg.av-s-rm-r--h-e.ys-qaeh--aldel-v-purL human -g-a-ghtr-kl-gklp.-1qg-e------v-.wt-ea-p-aeetkk-m-purl-ara-48--s--a-.l-kav-tkisnqi-slt--qsf-ig.l-s-lkd..e-lsv-k-Consensus E-ANSE--LR--Q-----EYV-V-TELCYNLN--EAK-LESL-KY--LLW

purl e.coli.............. -ha-q....-k...-1-t--pgtis---sk-td-purL_yeast iand--arql-d-v-nn--ssa-.-edty-ir-v--sgtis---sk-tn-35purl Drome lvk---skgqs-s-qpa-q...st--sq----i---fn-s--y---c-n-purL human lfgc--ll.ddv--e-w-.....-p--nd------n-s --t---i-sv purl-ara-48-il.-etyepe--gtd-f-erkkqe-lhaviv------s-t-a------s-Consensus ---QPL----NLARAS-LP---L-GS--LLLEVGPRL-F-TPWSTNAV-I

40purl e.coliahn--- n----gva-yiea..-tltn-qwqgvtae.-----m-tvf .q-purL_yeast ahv-----k-q-i--glal-iktvp-f-----lnd-slkcvy----qql-purL_Drome fqnl-y.s--r-m-tst---v..t---gskapes-r-vpl-g----qcl-purL human crat--.gp-d-v-tt---rl..s-ah-psaeve--al-t-------qhf purl-ara-48-cra-- . t----s----l..fsk------qike-a-mv------cv---Consensus ---CGLQDEV-RLER-RRYL----FGEPLLEN--AIF-A-LHDRMTE--Y

purl e.colifa.lddaeql-ahhq-t--ts-d-lgqg.....-q--id--lr-----ae purL_yeast lt-ppntm-i--hee-k-lvh---t-kdtkqspkdi-s---t-------s purl Drome -e-nt-ka--d.eql--rqa-whf--.. ----a---ri-------fnd -purl human ph...-iq-- spesm--.pl -g-.in..i- g---1----- ---------s purl-ara-48- -qk...lv-- e-nvv--e-. ..ky--..-m -k--k---ei ---m---f-a Consensus T-E--P--SF FT---PEPV- NVPLVP--VL EEGR-ALEKA NQELGLALD-purl e.coli dei--lq-a- tk.-g---nd i--ym---a- ---c--ki-- a-w----eq-purL-yeast genre-liha- vetmk-d--d ----m---v- ---c--ki-- a-wt---i--purL Drome y-----h-1- ak--g----t -----c---- ---------r -rm----ve-purL human w---f--kr- .q--q---st --a--1---- ---------k -qlhv--q-1 purl-ara-48- q--q---rl- redik-d--n -----i---- ---------a -nm----kpm Consensus -DLDYYTD-F --EL-RNPT- VELFDFAQSN SEHSRHWFFN GD-VIDG-KQ

purl e.coli-----k--kn-f-ttpdhvlsayk--aavme-s--gryfadhe-g.r.y-purL_yeast qft-----rn-hklnpeytisays--aavldsenda-ffapns-t.kewt purl Drome ----ir--md--ahtn---tik-s-----mv--dhqtiv-ssvva-gavr purl human vh---es-ms---ssn----lk-c-----iq-k--r--r-ed--r-srfq purl-ara-48-d---m-ivks-w-anrn-s-ig-k-----ir--1-nq-r-11-gsvcll-Consensus PKSLFQMI--TQE---PNNV--F-DNSSA--GFEV-FL-P--PT-P---D

purl e.colifh--pahi-mkv----h---is-w---a--s--e---eg-----a-pk--purL_yeast stk-ripl-ikv----h----s-----a--s--e---eg-----s-tkc-purL Drome 1-svqsdli-------m----a--s--t--t---1--vqgv---gvpi--Purl human q--glrhvv-------f--g-c--s--t--t------vqc----ahvv--purL-ara-48-vsardldi--------f-c--a-y---e--a------th-----sfvv-s Consensus -QQE----LFTAETHN-PTAV-PFPGA-TG-GGRIRD--ATGRG-K--AG

purl e.colilv-fs-s--r---f-----.ed-gk-erivta-d-mt-gp1-gaafn-e-purL_yeast ls-fs-sd-1---n---- -nigk-yhi--a-d-m---p1-saafn-e-.

purl Drome -a-----a------k--y-p-d-k--atf-p--qvl--------------purL human -a---f--------nl-----s-q--g-f-r--eva--------------purL-ara-48--s-------nme-sya----ss-q--s-1----q-1-d------------Consensus T-GYCVGNLHIPGYEQPWEDL-F-YP-N-ASPL-I-IEASNGASDYGNKF

Purl,_e.coli-r-al---f--yee--nshn-.e-1-gy-----la----ni--d-vq--.

purL_yeast -r-c----f--ltt--lnhq-ke-i-gf-----ia--f--v-pqfal-nt purl Drome -----s-falsy--nsaadas--..d-yv--------1--mp-tmre-lp purl human ----la-fa-sl--q..-pd-- ..--wi----------sme-d--s-ea purl-ara-48----m-q--t--f-mr..-ps-d-..--wl------a---qidht--t--a Consensus GEPVINGY-RT-GLKV-L--GQRE-RE-HKPIMFSGGIGT-RA-HI-KG-purL,e.colie-nv-ak--v1---amn--1-------m.a---s------as---dn---purL_yeast --tp-sc-iv1--qsml--1--------.a--egs-----as----n---purL Drome -.ar-q--a-i---v----v--------eiq-sg--e---n------a--purL_human -.-p--ev--v---v----v--------qvq-dnts----g---------purL-ara-48-.-v---v--i---a----m-------m.v---n--e---n------a--Consensus PIE-GMLLVK-GGP-YRIG-GGGAASSV--SGQ-DADLDF-AVQRGDPEM

purl e.coli-rrcqe--dr-wq--da----f---v----ls-amp---sd-gr-g-fel purl-yeast -rrcqq--d---a--nn---q----v----ls-a-p---hdndl---f--purL Drome -n-ln--v---ld---q----a---------g-------e--f---v-fs purl -q-mn--------apkg---c-1--------g------sd-..---i-yt human - sq-ly--v---i-m--k---i----------c--v--iiy-..q--e---purl-ara-48-Consensus E-K--RVIRACVELGE-NPILSIHDQGAGGN-NVLKELV-PG-AGAKIDI

purl e.coli--ilsde-g--p----cn-s---yv-a-a---lplfd-1-----a-y--i purL_yeast -kvlsle-g--pm---cn-s---yv-g-spqdlsif--------a-f---purL Drome k-fql----yta--1-------nn-i-cn-----1--k--r---c-isf-purL-human srfql----1na----------sn---lrspn-df-thusa---c-acf-purL-ara-48--avvv--h---v----------qd-i--k-es--i-qs------lsm--i 5 Consensus RE---GDPTMS-LEIWGAEYQER-ALLV-ADARE-LEEICKRER-P-AVV

purl e.coli-ea-eelh-s-h-...............rhfdnq-i--p-dvl---t--m purL_yeast -ha-aeqk-ive-...............-1lkt--i---mpilf--p--m purl Drome -vv--d--v--l.ekp---dleqa-n-fnrsevs-f----ky---d---r human --i--dr-iv-v-drec-yrrngqgd-p-t-----v-----w------rk purL

10_ --in-g--c--i-sta-a-cskeg-....p---pav-----k---d---k purl-ara-48-Consensus GT-TG-GRLTL-D---APK-----L-A-P-PPPTP-DLELE-VLGKMPK-purl e.coli-r-v-tlkak.gdalare-itia--vk---h--t-ae-t--v-ig-----purL_yeast sretit--In-pean-seips-q--iq---n--s-g--s--i-ig-----15purL_Drome -y--k--qtp-ke-s--k-11-de--e---s-va-g------n----c-g purl-human eff---kppm-qp-a--p-1svhq--e------a-a---y--n------g purl-ara-48--fkfn-i-yarep-diap-it-m---k------s-s-----------c--Consensus T-DLQREA--L--L-LP-G--L-DAL-RVLRLP-V-SKRFLTTKVDRSVT

20purl e.coli-m--rd-m---w---v-nc---ta-ldsyy... --m----rapva---f purL_yeast --idrd-f---w---v---g--gt-lgetiis----m-m--k--na-isa purl Drome --i----------a----y-1-tv-hfsh...s-i-ts--t--1-g----purL human -------------t-------val-hee-...i-a-t-1------s----purL-ara-48--------t-----it------iaqtftd-...--g-c------i-g----Consensus GLVAQQQCVGPLQVPLADVAVT--S---L---TGEA-AIGEQPVK-LLDP

purl e.colia-s-----g-----iaatqig-ikri-1-a---a--gh---d-g--e-v-purL_yeast s-s-k-s---s-1-ifa-d-ks-nhi-1-a---sp-shq---sk--e-vq purl Drome a-m--mc-----s---fv-ise-a---c---------------rmf--c-purL human kva--___________f_1__-_r___c______-_____-____a__ce purl-ara-48-k-m--___g_______~,,~____a_s___a_____y____e___s_m__-ai 30Consensus -A-ARLAVAEALTNLV-AKVTDL-DVK-SGNWMWAAKLPGEGAALYDA-K

purl e.coli-v-e--c----lt-pv----m--ktr-qegneeremts-1------f-rv purL_yeast ---ld-c----v--pv----m--kmk-....ddke-t--1--n-t-f--v purl Drome e-.cqilee-hi-------------k....-g--tiks--t----t----purL human -m.vavma---v-v-----------r....-gt-t-r-----------v-35purl-ara-48-- .s-amie--i-------------h....ad--v-k---n------vt-Consensus ALG-EL-PALG-AIDGGKDSLSMAA-W---V-GE-V-APGSLVISAYAPC

purl e.colie-vrh-i--q-..s-...e-na---i--g..--nna--at------r---purL-yeast fnts--w--1-nrn-... sv-v-----akqetks--a---1--yn-v--40Purl Drome --vrlk------g-.-a-skts--win-e...nsa---------aya-q-purL human --ita-------h-e-r-...h--y-a--..p-qh----t----c-s---purL-ara-48---it------- ..1-. gi--h---a.. -kr-----------g-i---Consensus PD--KTVTPDLK-PTG-GDD--LLLVDLS--KG--RLGGSALAQVF-QLG

purl e.colidk-a-vr---q---fy-ai---v-q-k..l--y--r---------a----45purl-yeast -ks-tvy-n-i---flesliq-hqqkediv--y--r------i-------purL Drome kdt-n-trsdv-gka-av--s--gdg..-iq----v-------cv----i purl human eh-----lpen-vra-si--g--kd-..-1cs---v-----vtc------purL-ara-48--dc------py--nv--gv---i-en..-vs----i-----v--a-----Consensus N-PPDLDDVA -LKG-FD-TQ ALLA-R--L- LAGHD-SDGG LLVTLLEMAF

purl e.coli --h--ida-i............atl--..dr--a--n----a-iq-r-a-purL_yeast -sr---eini............dg.--lesq-tn--n----a-fqi--kn purl Drome g-ls--r--1se -lak-knfdksveklnrpe-----a--c-w-v--ld--purL human ------q--vpv-rvdv..............-s---a--p-1----qep-purL-ara-48----k-inl-1asngis-..............fet--s----1---i-k-n Consensus AGNCGL-VD---P---L--------GD----LAVLF-EELG-VLEVSATD

purl e.coli r----sv-aqh-1-.dcvhy--q-vsgd-f-ita--q-...-f--s-tt-10purL_yeast -skf-ki-nen---k--isi--kpsfq-qeikii-st-ndviyans----purL Drome --r-rstyek---pny-1-vtegf-ld----1-ngkse...l-dqplrv-purL human -aq-lkry-d--lhclel-ht-e--pham-r-s---av... -e-pvg--purL-ara-48--d--mek--afd-ta-ii-n-td...splie---d-i-...h---kt-f-Consensus LEAVE--LR-AGVA-EY-G-VG-AG--SRVVVKVNG-T---VLSE-RSEL

purl e.coli -vw-a--tw-mqr-rd-----dq-hqaksnda----nv--s-dine-vaa purL_yeast eqt-sk--yemq--rd--kt----fasitddr----q-a-ty--ad-mki purl Drome ykk--r--ye-e---a------a-yns-ey-qa-..q-rgpq-v.q.-el purl human -al-----f--dr--ae-r-va--erg-re-mg-..s-c-pptfpk.-sv purl-ara-48--dm--d--f--e---rlas-v-m-keg-kf-he-..nw--s-i-ss.tnn 20Consensus R--WEETS-QL-KLQ-NPECAEEE---L--R-DPGL-YKL-FNP--DA--purL_e.coli -yiatg----------q---shv-----------dai--h-----t--t-purL_yeast gl-1-sq-----i---q----qm---wc-qq---nsv----t---e--fh purl Drome tlkr-s-pvr----------se---m-cll--n--_h--------q-tas purl human -r-pggps-r--i-----s-------d---1----------q--cs-a--25purl-ara-48-nymsqdvk-----i----s------s---ya----p----v----a-d-t Consensus P-E-S-ARPKVAVLREEGVNGDREMP.AAFHRAGFEVWDVTMSDLL-GRIG

purl e.coli -ed-ha-vac-----g----age---k-----d--rde-at--h.--q-1 purl yeast --d-i--aac-----g----aga---k-v-yheg-r---sk--ne-q---30Purl Drome vsqy---i-p-------t---------n--h-p-llp--aa.-kr-q-v-purL human --t---va-v-----------------avt-hp-agaelrr.-r------purL-ara-48--_q___iv_v____-____d_________r__ep_1___qe,_y______ Consensus LD-FRGL-F-GGFSYADVLGSAKGWAASILFN-RV-SQF--FF-KRPDTF

purl e.coli a--v-----m-sn-...........r-li-g--.1-----..r-t-d---35purL_yeast af-a-----flsr-...........kdii-gc-.n--s-a..r-v-eqy-purL Drome ---i-------t-i-f--....saks---ad.....-dva-1--k-q---purL human ---v------la-------dpnedaa-m--d-qpar-gll-r--1---y-purL-ara-48--__i_______a_____p_p,.....q--gsldtsq---_" _e-____ Consensus SLG-CNGCQLM-LLGWVGG-------EVGP-SE--WPRFVL-HN-SGRFE

purl e.coli a-fsl-evt-...... 1-q--v--qm-ia-s-----vev.--aah-purL_yeast a-vcm-q-s-ekdns-ee-vf-n--a--k--ia------kat-sksa-q-purL Drome c-w-t-k-......p-nr-i--gs-kdl--gc--------f--. -ek-i purl human s-w---rv......gpg-al--r----a---v-s-----yv--.ssp--q purl-ara-48-c-ft--t-......kd---i--k-----t-gv-a------ay-.p-egv-Consensus -R-ASV-3-Q-----SSPS-ML-GMEGSVLP-WVAHGEGR-AF-RD-ELL

purl e.coli -a---k--va -----nf-k- --t--a---- -----tavtt es--vti---purLTyeast ekf-kd--ec i----ny--- --rf-f---- -t------k- -n--v-----X050/5082? CA 02388851 2002-04-23 purl Drome s--q-eq-vt -q----v-kp --1--1---- --q----1-- s------1--purL human -qi-ar--a- -hwa-----p --q--1---- --g-v----- c------v--purL-ara-48- d-mlhsd-a- ---c------ --a--f-1-- --1---a--- ---------Consensus AHLES-GL-P LRYVDDDGNV TE-YP-NPNG SPNGIAGICS PDGRHLAMMP

purl e.coli ----v--tvs ns-h-..... -nw-edg--m -i-----kql g----purL-yeast ----vc-lea ns---eg.ky -ew--yg--i -1--s--r-v g----purL Drome ----css-y- wpyv-s--e- sptqse---q im-n--y--c vk-nq purl human ----av-p-- wa-r-pp--. ..tltt---- qlsi-----t 1-gsc purl-ara-48- ----c-1--- fp---t-w-- -ka.-p---- km-q---d-. 1--c Consensus HPER-FRMWQ --WYP-SFDV E--GG-SPWL R-FRNARNW- -ES-Example 2 Isolation of a cDNA encoding a tobacco formylglycinamidine ribotide synthase To isolate further plant formylglycinamidine ribotide synthase cDNAs, an EcoRI/XhoI fragment of clone purl-48.1 was used for screening a tobacco leaf tissue cDNA library (Nicotiana tabacum var. Samsun NN). 19 positive clones were identified and isolated.
Following restriction analysis and sequencing, clone purl-Ntl.l was identified. purl-Ntl.l encodes, on a partial reading frame of 3434 by - see SEQ-ID No. 3 -, a polypeptide 1017 amino acids in length, see SEQ-ID No. 4. In the overlapping region, the polypeptide showed similarity to Arabidopsis thaliana formylglycinamidine ribotide synthase (purl-48.1) (80.1%
identity).
Example 3 Isolation of a Chilopsis linearis cDNA encoding a formylglycinamidine ribotide synthase Double-stranded cDNA was generated from Chilopsis linearis mRNA
and used to generate a cDNA library in the vector pBluescript SKII (lambda ZAP II RI library construction kit, Stratagene).
Individual clones of this library were partially sequenced. The sequence on the 3'-side of clone 74 chi 005 e10 showed similarity to tobacco purl-Ntl.l and Arabidopsis thaliana purl-48.1. Clone 74 chi_005 e10 was sequenced completely. On a partial cDNA 478 by in length, it encodes a polypeptide of 97 amino acids. At the amino acid level, 74 chi_005 e10 has 82.4% identity with purl-48.1 and 75.2% identity with purl-Ntl.l.

Example 4 Proof of the function of purl-48.I by complementation of E. coli To prove the function of the cDNA encoded by purl-48.1, it was cloned into suitable expression vectors (for example the pQE, Qiagen or pET series, Novagen). To this end, fragments of various lengths of the purl-48.1 cDNA can be generated, for example by PCR, and ligated into the vectors which have been prepared by treatment with restriction endonucleases. The resulting expression constructs can be used to complement the E. coli strain CGSC#4537 (genotype: fhuA2 , lacYl, g1nV44(AS), gal-6, ~,-, nadB4, purL66, rpsL9, malT1(~.R), xylA7, mtlA2, DargHl; E.coli genetic stock center, Yale University, New Haven). To this end, the strain is transformed with the expression construct in question and plated onto M9 minimal media without adenine. By way of example, the minimal media should contain 0.4~ glucose, 0.2%
casamino acids, 100 ~,g/ml thiamine, 100 ~g/ml inosine, 100 ~,g/ml biotin, 100 ~,g/ml nicotinate, 100 ~M IPTG and 50-100 ~,g/ml of the antibiotic in question to which the expression vector mediates resistance. In the parallel experiment, the cloning vector can be transformed into CGSC#4537 without purl-48.1 insert as negative control. It emerged that only the bacteria which have been transformed with purl-48.1 expression constructs are capable of growth on minimal media without adenine. Accordingly, the enzyme encoded by purl-48.1 is functional and the first functional formylglycinamidine ribotide synthase which has been isolated from plants.
Furthermore, yeast (Saccharomyces cerevisiae) may also be used for complementation. To this end, a yeast mutant is generated whose Ade6 gene, which encodes the yeast formylglycinamidine ribotide synthase, is rendered unfunctional. A method which can be relied on for generating such a yeast mutant which is suitable for complementation is, for example, that of Guldener et al., Nucleic Acids Research 24(I996), 2519-2524, applied to a suitable starting strain such as, for example, SEY6210 (Robinson et al., Protoplasma 150(1989), 79-82). The yeast mutant generated thus should not show growth on minimal media without adenine. To prove the function of the purl-48.1-encoded cDNA, the latter is cloned into suitable yeast expression vectors (for example pYEB112, Riesmeier et al., EMBO J. 11(1992), 4705-4713 or pYES2, Invitrogen). To this end, purl-48.1 cDNAs of various lengths can be generated for example by PCR and ligated into vectors which have been prepared by treatment with restriction endonucleases.
The resulting expression constructs are used to complement the yeast mutant generated. To this end, the latter is transformed with the expression construct and plated onto minimal media (for example SDG media, Clontech, Matchmaker System) without adenine.
In the parallel experiment, the cloning vector is transformed into yeast mutants without purl-48.1-insert as negative control.
It emerges that only those yeasts which have been transformed with purl-48.1 expression constructs are capable of growth on minimal media without adenine. Thus, the enzyme encoded by purl-48.1 is the first functional formylglycinamidine ribotide synthase which has been isolated from plants.
Example 5 Generation of transgenic Arabidopsis thaliana plants To prove the suitability of formylglycinamidine ribotide synthase as target for herbicidal active ingredients; the expression of formylglycinamidine ribotide synthase was lowered in transgenic Arabidopsis plants by cosuppression or antisense inhibition.
To this end, binary vectors for plant transformation were first generated. To achieve inhibition by means of cosuppression in Arabidopsis thaliana, clone purl-19 was cleaved with BamHI and SalI, and the resulting 1127 kb fragment was ligated into vector pBinAR (Hofgen and Willmitzer, Plant Science 66(1990), 221-230) which had been cleaved with the same restriction enzymes. To achieve antisense inhibition in Arabidopsis thaliana, clone purl-19 was cleaved with BamHI and KpnI, and the resulting 964 by fragment was ligated into vector pBinAR which had been cleaved with the same restriction enzymes. The resulting binary constructs AtSpurL and AtASpurL (Figure 2) were transformed into Arabidopsis thaliana.
To this end, a slightly modified form of the vacuum infiltration method of Bechtold et al. (C. R. Acad.Sci. Paris, Life Sciences 316(1993), 1194-1199) was applied.
To this end, the binary vectors AtSpurL and AtASpurL were transformed into Agrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al., Nucl. Acids. Res. 13(1984), 4777-4788). 20 ml of an overnight culture in YEB medium with 50 ~,g/ml kanamycin (Sambrook et al. (1989), Cold Spring Harbor Laboratory Press:
ISBN 0-87969-309-6) were diluted in 500 ml of YEB/kanamycin and shaken overnight at 28°C. The bacteria were harvested by centrifugation and resuspended in 500 ml of infiltration medium (2.15 g/1 Murashige and Skoog Basal salt mixture (Sigma), 50 g/1 sucrose, 1 mg/1 nicotinic acid, 1 mg/1 pyridoxin, 10 mg/1 thiamine, 100 mg/1 myo-inositol, 1 mg/1 glycine, 44 ~M

N6-benzylaminopurine, pH 5.7). The aerial parts of flowering Arabidopsis plants were dipped into the bacterial suspension and left to infiltrate for 10-15 minutes at 10-15 mbar. The plants were then grown on in the greenhouse until the seeds had matured.
5 The seeds were sterilized and then placed on solid medium (2.15 g/1 Murashige and Skoog Basal salt mixture (Sigma), 0.1 g/1 myo-inositol, 0.5 g/1 MES, 10 g/1, 8 g/1 agar-agar, 1 mg/1 nicotinic acid, 1 mg/1 pyridoxin, 10 mg/1 thiamine, 100 mg/1 myo-inositol, 1 mg/1 glycine, pH 5.7) supplemented with 15 ~g/ml 10 hygromycin, grown under sterile conditions for 2-4 weeks and then transferred into soil.
Culture conditions:
15 Sterile culture: 21°C, 150 ~E*m-Z*s-1, 75% atmospheric humidity Greenhouse: daytime temperature 22°C / night temperature 18°C, 16 h photoperiod, 450 ~E*m-2*s-1, 68% atmospheric humidity.
20 Example 6 Generation of transgenic tobacco plants To inhibit formylglycinamidine ribotide synthase in tobacco by 25 cosuppression, clone purl-Ntl.l was cleaved with Xbal and SalI, and the resulting 3.6 kbp fragment was ligated into vector pBinAR
which had been cleaved with the same restriction enzymes. To subject formylglycinamidine ribotide synthase in tobacco to antisense inhibition, clone purl-Ntl.l was cleaved with Asp718 and XbaI, and the resulting 3.6 kbp fragment was ligated into vector pBinAR which had been cleaved with the same restriction enzymes. The resulting binary constructs NtSpurL and NtASpurL -see Figure 2 - were transformed into tobacco.
To this end, the plasmids NtSpurL and NtASpurL were transformed into Agrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al., Nucl. Acids. Res. 13(1984), 4777-4788). To transform tobacco plants (Nicotiana tabacum cv. Samsun NN), a 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony in Murashige-Skoog medium (Murashige and Skovg, Physiol.
Plant. 15(1962), 473) supplemented with 2% sucrose (2MS medium) was used. Leaf disks of sterile plants (in each case approx.
1 cm2) were incubated in a Petri dish for 5-10 minutes with a 1:50 agrobacterial dilution. This was followed by 2 days' incubation in the dark at 25°C on 2MS medium with 0.8% Bacto agar.
Cultivation was continued after 2 days and at 16 hours light/8 hours dark and continued in a weekly rhythm on MS medium i with 500 mg/1 claforan (cefotaxime-sodium), 50 mg/1 kanamycin, 1 mg/1 benzylaminopurine (BAP), 0.2 mg/1 naphthylacetic acid and 1.6 g/1 glucose. Growing shoots were transferred to MS medium supplemented with 2~ sucrose, 250 mg/1 claforan and 0.8$ Bacto agar. Regenerated shoots were obtained on 2MS medium with kanamycin and claforan, transferred into soil after rooting, and, after cultivation in a controlled-environment cabinet in a 16-hour light/8-hour dark rhythm at 60$ atmospheric humidity, analyzed.
Example 7 Analysis of transgenic plants Transgenic plants were examined for formylglycinamidine ribotide synthase expression and activity, and for altered metabolite contents and phenotypical growth characteristics. Altered nucleotide contents can be determined, for example, by the method of Stitt et al., FEBS Letters 145(1982), 217-222.
Lines of transgenic plants which had been transformed with the constructs AtSpurL, AtASpurL, NtSpurL or NtASpurL are characterized by a growth which is reduced by different degrees in comparison with untransformed control plants. In transgenic lines, with the above-described phenotype, a reduced amount of purl-48.1 or purl-Ntl.l was found by RNA analysis using the Northern blot technique. The formylglycinamidine ribotide synthase activity can be determined by a method as described in Example 8.
These data constitute a direct connection between reduced formylglycinamidine ribotide synthase expression and reduced growth of Arabidopsis thaliana and tobacco plants, respectively, and therefore identify formylglycinamidine ribotide synthase as suitable target protein for herbicidal active ingredients.
Example 8 Measurement of formylglycinamidine ribotide synthase activity Formylglycinamidine ribotide synthase activity is measured by modifying methods which have been described (Methods in Enzymology. 51(1978), 193-201). After adapting the method to high-throughput methods, one of the above-described in vitro assays can be used to search for formylglycinamidine ribotide synthase activity inhibitors. To this end, the formylglycinamidine ribotide synthase activity can be prepared from plant tissues. Alternatively, a complete or truncated plant formylglycinamidine ribotide synthase (preferably Arabidopsis thaliana formylglycinamidine ribotide synthase) can be produced in a suitable prokaryotic (for example E.coli) or eukaryotic (for example yeasts, insect cells) expression system. After disrupting the plant tissues or cells in suitable buffers and, if appropriate, further purification steps, for example by chromatographic methods, the formylglycinamidine ribotide synthase activity can be quantified. This makes it possible to correlate the growth phenotype in transgenic lines with the formylglycinamidine ribotide synthase activity.
This method allows known formylglycinamidine ribotide synthase inhibitors such as glutamine antagonists, and in particular novel plant formylglycinamidine ribotide synthase inhibitors, to be identified.

SEQUENCE LISTING
<110> BASF Aktiengesellschaft <120> Formylglycinamidine ribotide synthase <130> NAE 1237-99 <140>
<141>
<160> 6 <170> PatentIn Vers. 2.0 <210> 1 <211> 4570 <212> DNA
<213> Arabidopsis thaliana <220>
<221> CDS
<222> (146)..(4366) <400> 1 gaattcggca cgaggaccag acaagaactc tgtgtcgact gccgtactct cggagtcgga 60 gcttcggtcc cgttaagaca gtaacagaga aaaaccctaa accctctttt cactttgttt 120 gctgttccgg tcatatctcg cgacg atg aat acc tcc caa gca act cgt gcg 172 Met Asn Thr Ser Gln Ala Thr Arg Ala get ctg ttt cta aac ggt tcc aat aga caa gcg atg ctt ttg cag cgg 220 Ala Leu Phe Leu Asn Gly Ser Asn Arg Gln Ala Met Leu Leu Gln Arg agc tca atg agt caa ttg tgg ggt tct gtt aga atg aga act tca cgg 268 Ser Ser Met Ser Gln Leu Trp Gly Ser Val Arg Met Arg Thr Ser Arg ctg tct ctg aat cga aca aaa get gtt agc ttg aga tgt tct get caa 316 Leu Ser Leu Asn Arg Thr Lys Ala Val Ser Leu Arg Cys Ser Ala Gln cct aat aaa cct aaa gca get gtt tct act ggt tct ttt gtt act get 364 Pro Asn Lys Pro Lys Ala Ala Val Ser Thr Gly Ser Phe Val Thr Ala i1 ~ CA 02388851 2002-04-23 gat gag cta cca agc ttg gtt gag aaa cct get get gag gtt atc cat 412 Asp Glu Leu Pro Ser Leu Val Glu Lys Pro Ala Ala Glu Val Ile His ttt tat cgc gtg cct ttg att caa gaa agc gcg aac gcg gaa ctc ctc 460 Phe Tyr Arg Val Pro Leu Ile Gln Glu Ser Ala Asn Ala Glu Leu Leu aag get gtt caa acc aaa atc agc aac cag att gtc agt ttg act aca 508 Lys Ala Val Gln Thr Lys Ile Ser Asn Gln Ile Val Ser Leu Thr Thr gaa cag tct ttc aat att ggg ctt gaa tcc aag tta aaa gat gaa aaa 556 Glu G1n Ser Phe Asn Ile Gly Leu Glu Ser Lys Leu Lys Asp Glu Lys ctt tct gtt cta aag tgg att ctt caa gaa aca tat gag cca gag aat 604 Leu Ser Val Leu Lys Trp Ile Leu Gln Glu Thr Tyr Glu Pro Glu Asn tta ggt acg gat agt ttt ctt gag agg aag aag caa gaa gga ctc cat 652 Leu Gly Thr Asp Ser Phe Leu Glu Arg Lys Lys Gln Glu Gly Leu His get gtt att gtt gaa gtt ggc ccc aga ttg tct ttc aca aca gca tgg 700 Ala Val Ile Val Glu Val Gly Pro Arg Leu Ser Phe Thr Thr Ala Trp tcc acc aat gcg gtt tcg ata tgt aga gca tgt ggg tta gac gag gtg 748 Ser Thr Asn Ala Val Ser Ile Cys Arg Ala Cys Gly Leu Asp Glu Val act cgc ttg gaa agg tca agg agg tac ctt ttg ttc agc aag gag cca 796 Thr Arg Leu Glu Arg Ser.Arg Arg Tyr Leu Leu Phe Ser Lys Glu Pro ctt ttg gaa aat cag ata aaa gaa ttt get gca atg gtt cat gat aga 844 Leu Leu Glu Asn Gln Ile Lys Glu Phe Ala Ala Met Val His Asp Arg atg acc gag tgc gtg tac act caa aag ctg gtt tca ttc gag acg aat 892 Met Thr Glu Cys Val Tyr Thr Gln Lys Leu Val Ser Phe Glu Thr Asn gtg gtt cca gaa gaa gtg aag tat gtg cct gtg atg gag aag ggt cgg 940 Val Val Pro Glu Glu Val Lys Tyr Val Pro Val Met Glu Lys Gly Arg aag gcg ctg gaa gaa atc aac cag gag atg ggt tta gcc ttt gat gag 988 Lys Ala Leu Glu Glu Ile Asn Gln Glu Met Gly Leu Ala Phe Asp Glu caa gat ttg cag tat tat act aga ctt ttc aga gag gac att aag cgt 1036 Gln Asp Leu Gln Tyr Tyr Thr Arg Leu Phe Arg Glu Asp Ile Lys Arg gat cct acc aat gtg gag cta ttt gat atc get caa tcc aac agc gag 1084 Asp Pro Thr Asn Val Glu Leu Phe Asp Ile Ala Gln Ser Asn Ser Glu cac agt cga cac tgg ttc ttt get ggg aac atg gtt att gat gga aag 1132 His Ser Arg His Trp Phe Phe Ala Gly Asn Met Val Ile Asp Gly Lys cca atg gat aag tct ctt atg cag att gta aag agc act tgg gag gca 1180 Pro Met Asp Lys Ser Leu Met Gln Ile Val Lys Ser Thr Trp Glu Ala aat cga aat aat tct gtc att ggg ttt aag gat aac tct agt get att 1228 Asn Arg Asn Asn Ser Val Ile Gly Phe Lys Asp Asn Ser Ser Ala Ile aga ggc ttt ctg gtg aac cag cta cgg cct cta ctt cct ggt tct gtg 1276 Arg Gly Phe Leu Val Asn Gln Leu Arg Pro Leu Leu Pro Gly Ser Val tgc tta ctt gat gtc agt gca cgt gat ctc gac ata ttg ttc act get 1324 Cys Leu Leu Asp Val Ser Ala Arg Asp Leu Asp Ile Leu Phe Thr Ala gaa acc cat aat ttc cct tgt get gtg get cct tat cct ggt get gag 1372 Glu Thr His Asn Phe Pro Cys Ala Val Ala Pro Tyr Pro Gly Ala Glu aca ggt get gga ggt cga atc aga gat aca cat gca aca gga aga ggt 1420 Thr Gly Ala Gly Gly Arg Ile Arg Asp Thr His Ala Thr Gly Arg Gly tct ttt gtg gtt gca tcc act tct ggt tac tgt gtt ggt aac ctt aac 1468 Ser Phe Val Val Ala Ser Thr Ser Gly Tyr Cys Val Gly Asn Leu Asn atg gag gga tct tat get cca tgg gaa gac tcg tct ttc caa tac cca 1516 Met G1u Gly Ser Tyr Ala Pro Trp Glu Asp Ser Ser Phe Gln Tyr Pro tcg aac cta gca tca cct cta cag ata ctc atc gac get agt aat ggt 1564 Ser Asn Leu Ala Ser Pro Leu Gln Ile Leu Ile Asp Ala Ser Asn G1y gca tct gac tat ggt aac aaa ttt gga gag ccc atg att cag gga tat 1612 Ala Ser Asp Tyr Gly Asn Lys Phe Gly Glu Pro Met Ile Gln Gly Tyr act agg act ttt gga atg aga ctg cca agt ggg gat aga cga gaa tgg 1660 Thr Arg Thr Phe Gly Met Arg Leu Pro Ser Gly Asp Arg Arg Glu Trp ttg aag ccg att atg ttc agt gca ggg att gga cag att gat cat act 1708 Leu Lys Pro Ile Met Phe Ser Ala Gly Ile Gly Gln Ile Asp His Thr cat ata act aaa ggg gag cca gag gtt ggg atg ctt gtt gtt aag att 1756 His Ile Thr Lys Gly Glu Pro Glu Val Gly Met Leu Val Val Lys Ile ggt gga cct get tac cgc att ggc atg gga gga gga gca get tcg agt 1804 Gly Gly Pro Ala Tyr Arg Ile Gly Met Gly Gly Gly Ala Ala Ser Ser atg gtt agt ggc cag aat gat gca gag ctt gat ttc aat get gtg caa 1852 Met Val Ser Gly Gln Asn Asp Ala Glu Leu Asp Phe Asn Ala Val Gln cgt gga gat get gaa atg tct cag aag ttg tac cgt gtt gtt cgt get 1900 Arg Gly Asp Ala Glu Met Ser Gln Lys Leu Tyr Arg Val Val Arg Ala tgt att gag atg ggt gag aag aat cct att atc agc att cat gat caa 1948 Cys Ile Glu Met Gly Glu Lys Asn Pro Ile Ile Ser Ile His Asp Gln ggt get ggt gga aac tgc aat gtt gtc aag gaa att att tat cca cag 1996 Gly Ala Gly Gly Asn Cys Asn Val Val Lys Glu Ile Ile Tyr Pro Gln ggt gca gag att gac ata aga gcg gtt gtt gtg ggt gat cat act atg 2044 Gly Ala Glu Ile Asp Ile Arg Ala Val Vai Val Gly Asp His Thr Met tcg gtg ttg gag ata tgg gga gca gaa tat caa gag caa gat gcg att 2092 Ser Val Leu Glu Ile Trp Gly Ala Glu Tyr Gln GIu Gln Asp Ala Ile ttg gtg aaa get gag agc cgg gag att ttg caa tca atc tgt aag agg 2140 Leu Val Lys Ala Glu Ser Arg Glu Ile Leu Gln Ser Ile Cys Lys Arg gaa agg ctt tcg atg get gtg att gga aca att aat ggg ggt ggt cgc 2188 Glu Arg Leu Ser Met Ala Val Ile Gly Thr Ile Asn Gly Gly Gly Arg tgt act tta att gac agc aca get gca gcg aag tgc agt aaa gaa ggg 2236 Cys Thr Leu Ile Asp Ser Thr Ala Ala Ala Lys Cys Ser Lys Glu Gly cta cct cca cct cca cct get gtg gat ctt gaa ctc gag aag gtt ctt 2284 Leu Pro Pro Pro Pro Pro Ala Val Asp Leu Glu Leu Glu Lys Val Leu ggt gat atg cct aag aag acg ttt aag ttc aac cgc att get tat gca 2332 Gly Asp Met Pro Lys Lys Thr Phe Lys Phe Asn Arg Ile Ala Tyr Ala cgg gag cca ctt gat att get cct gga att aca ttg atg gat get ttg 2380 Arg Glu Pro Leu Asp Ile Ala Pro Gly Ile Thr Leu Met Asp Ala Leu aaa aga gtt ctc cga tta cca tca gtt tct tca aag cgg ttc ttg aca 2428 Lys Arg Val Leu Arg Leu Pro Ser Val Ser Ser Lys Arg Phe Leu Thr acc aaa gtg gat aga tgt gtg aca ggt ctt gtt get cag caa caa aca 2476 Thr Lys Val Asp Arg Cys Val Thr Gly Leu Val Ala Gln Gln Gln Thr gtt ggg cca ttg cag atc acg ctt get gat gtt gca gtt att gca cag 2524 Val Gly Pro Leu Gln Ile Thr Leu Ala Asp Val Ala Val Ile Ala Gln aca ttc aca gat cta aca ggt ggt gca tgt gcc att ggt gag caa ccg 2572 Thr Phe Thr Asp Leu Thr Gly Gly Ala Cys Ala Ile Gly Glu Gln Pro atc aaa ggc ttg ctt gat cca aaa gcc atg get agg cta get gtt gga 2620 Ile Lys Gly Leu Leu Asp Pro Lys Ala Met Ala Arg Leu Ala Val Gly gag get ttg aca aat ctg gtt tgg gca aag gtc act gca ctt tct gat 2668 Glu Ala Leu Thr Asn Leu Val Trp Ala Lys Val Thr Ala Leu Ser Asp gtt aaa get agt ggt aac tgg atg tat get gcc aag ctt gaa gga gaa 2716 Val Lys Ala Ser Gly Asn Trp Met Tyr Ala Ala Lys Leu Glu Gly Glu gga tca gca atg tat gat get gcg att get cta tct gaa gcg atg att 2764 Gly Ser Ala Met Tyr Asp Ala Ala Ile Ala Leu Ser Glu Ala Met Ile gaa ctt ggc att gca att gat ggt gga aaa gac agt ctt tca atg gca 2812 Glu Leu Gly Ile Ala Ile Asp Gly Gly Lys Asp Ser Leu Ser Met Ala get cat gcg gac ggt gag gtt gtt aaa get cca gga aat ctt gtg att 2860 Ala His Ala Asp Gly Glu Val Val Lys Ala Pro Gly Asn Leu Val Ile agt gcc tat gtt acc tgt cca gac ata aca aag aca gtg act ccg gat 2908 Ser Ala Tyr Val Thr Cys Pro Asp Ile Thr Lys Thr Val Thr Pro Asp cta aag ctc gga ggt gat gat ggt att ctc ttg cat gtt gat ttg gca 2956 Leu Lys Leu GIy Gly Asp Asp Gly Ile Leu Leu His Val Asp Leu Ala aag gga aag agg aga ttg ggt gga tct gca ctg get cag gtt ttt ggt 3004 Lys Gly Lys Arg Arg Leu Gly Gly Ser Ala Leu Ala Gln Val Phe Gly cag ata gga aat gac tgt cct gat ctt gat gat gtt cca tat ctg aaa 3052 Gln Ile Gly Asn Asp Cys Pro Asp Leu Asp Asp Val Pro Tyr Leu Lys aac gtt ttc gat ggc gtt caa get ctc att gca gaa aac ttg gta tct 3100 Asn Val Phe Asp Gly Val GIn Ala Leu Ile Ala Glu Asn Leu Val Ser get gga cac gac atc agt gat ggt gga ctg gta gta aca get ttg gaa 3148 Ala Gly His Asp Ile Ser Asp Gly Gly Leu Val Val Thr Ala Leu Glu atg get ttt get gga aac aaa ggt ata aat ctc gac ttg get tca aat 3196 Met Ala Phe Ala Gly Asn Lys Gly Ile Asn Leu Asp Leu Ala Ser Asn gga att agc ctt ttt gag act ttg ttt tct gaa gaa ctt ggt ctc gtg 3244 Gly Ile Ser Leu Phe Glu Thr Leu Phe Ser Glu Glu Leu Gly Leu Val ~ CA 02388851 2002-04-23 ctt gag att agt aag aca aac ctg gac get gtg atg gaa aaa ctc cgt 3292 Leu Glu Ile Ser Lys Thr Asn Leu Asp Ala Val Met Glu Lys Leu Arg get ttc gat gtt act get gag atc att gga aat gtc act gat tcg cct 3340 Ala Phe Asp Val Thr Ala Glu Ile Ile Gly Asn Val Thr Asp Ser Pro ctg ata gag gta aaa gtg gat ggg att act cat ttg agt gag aaa act 3388 Leu Ile Glu Val Lys Val Asp Gly Ile Thr His Leu Ser Glu Lys Thr tca ttc ctc aga gac atg tgg gaa gac acc agt ttc cag ttg gaa aag 3436 Ser Phe Leu Arg Asp Met Trp Glu Asp Thr Ser Phe Gln Leu Glu Lys ctg caa cga ttg gca tct tgt gta gag atg gag aaa gaa ggt ttg aag 3484 Leu Gln Arg Leu Ala Ser Cys Val Glu Met Glu Lys Glu Gly Leu Lys ttt agg cat gag cct aat tgg aaa ctc tca ttt att cca tcc tcg acc 3532 Phe Arg His Glu Pro Asn Trp Lys Leu Ser Phe Ile Pro Ser Ser Thr aac aat aat tat atg tct cag gat gtt aag cca aaa gta gca gtg atc 3580 Asn Asn Asn Tyr Met Ser Gln Asp Val Lys Pro Lys Val Ala Val Ile cga gaa gaa ggc agc aat gga gac aga gaa atg tca get gca ttt tac 3628 Arg Glu Glu Gly Ser Asn Gly Asp Arg Glu Met Ser Ala Ala Phe Tyr get gcc ggt ttt gaa cct tgg gac gtg aca gtg tct gat ctt cta get 3676 Ala Ala Gly Phe Glu Pro Trp Asp Val Thr Val Ser Asp Leu Leu Ala gga gac atc acc ctt gat cag ttc cgc ggt att gtg ttt gtg gga ggg 3724 Gly Asp Ile Thr Leu Asp Gln Phe Arg Gly Ile Val Phe Val Gly Gly ttc agt tat gcg gat gtt ctc gac tca gcc aaa gga tgg get get tca 3772 Phe Ser Tyr Ala Asp Val Leu Asp Ser Ala Lys Gly Trp Ala Ala Ser ata agg ttc aac gag cct gtg ttg agt caa ttt cag gag ttc tac aaa 3820 Ile Arg Phe Asn Glu Pro Val Leu Ser Gln Phe Gln Glu Phe Tyr Lys aga cca gac acg ttc agt ctt gga atc tgc aat gga tgt cag tta atg 3868 Arg Pro Asp Thr Phe Ser Leu Gly Ile Cys Asn Gly Cys Gln Leu Met get ctg tta gga tgg gtt cca ggt cct caa gtt ggc ggg tca ctt gac 3916 Ala Leu Leu Gly Trp Val Pro Gly Pro Gln Val Gly Gly Ser Leu Asp acc tcg cag ccg agg ttt gtt cac aac gaa tca gga agg ttt gag tgc 3964 Thr Ser Gln Pro Arg Phe Val His Asn Glu Ser Gly Arg Phe Glu Cys 1260 1265 ' 1270 agg ttc aca agc gtg acc ata aag gac tcg cca tcg ata atg ctg aaa 4012 Arg Phe Thr Ser Val Thr Ile Lys Asp Ser Pro Ser Ile Met Leu Lys gga atg gag gga agt act tta ggg gtt tgg gcg gcg cac gga gaa gga 4060 Gly Met Glu Gly Ser Thr Leu Gly Val Trp Ala Ala His Gly Glu Gly cgg get tat ttc ccg gac gaa gga gtc ttg gac cat atg ctt cac tca 4108 Arg Ala Tyr Phe Pro Asp Glu Gly Val Leu Asp His Met Leu His Ser gat ttg gca cca ttg aga tac tgt gat gat gat ggg aac gtg act gaa 4156 Asp Leu Ala Pro Leu Arg Tyr Cys Asp Asp Asp Gly Asn Val Thr Glu gcg tac cct ttt aac ctc aat ggt tca ccg ttg gga ata gcg get ata 4204 Ala Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu Gly Ile Ala Ala Ile tgt tca cct gat ggg aga cat ttg gcg atg atg cct cat cct gaa cgt 4252 Cys Ser Pro Asp Gly Arg His Leu Ala Met Met Pro His Pro Glu Arg tgt ttc ttg atg tgg cag ttt cca tgg tac cca aca agc tgg gac gtt 4300 Cys Phe Leu Met Trp Gln Phe Pro Trp Tyr Pro Thr Ser Trp Asp Val gag aaa get ggc ccg agc ccg tgg ttg aag atg ttc cag aat gca agg 4348 Glu Lys Ala Gly Pro Ser Pro Trp Leu Lys Met Phe Gln Asn Ala Arg gac tgg tta gag tcg tgt taagcgtgtt tttttagtat aatttttatc 4396 Asp Trp Leu Glu Ser Cys ~ CA 02388851 2002-04-23 tgcatcattt tgttgtgcat ttggtgtttt gcagagtctt tgaaagttgt cgacaaatca 4456 tgtgcttttt agtgagactt gatgtgtgct ttttgtcctt cacgttgtat tcaaactctg 4516 gaaattaagc atgagttact ctcttcgatc aaaaaaaaaa aaaaaaaact cgag 4570 <210> 2 <211> 1407 <212> PRT
<213> Arabidopsis thaliana <400> 2 Met Asn Thr Ser Gln Ala Thr Arg Ala Ala Leu Phe Leu Asn Gly Ser Asn Arg Gln Ala Met Leu Leu Gln Arg Ser Ser Met Ser Gln Leu Trp Gly Ser Val Arg Met Arg Thr Ser Arg Leu Ser Leu Asn Arg Thr Lys Ala Val Ser Leu Arg Cys Ser Ala Gln Pro Asn Lys Pro Lys Ala Ala Val Ser Thr Gly Ser Phe Val Thr Ala Asp Glu Leu Pro Ser Leu Val Glu Lys Pro Ala Ala Glu Val Ile His Phe Tyr Arg Val Pro Leu Ile Gln Glu Ser Ala Asn Ala Glu Leu Leu Lys Ala Val Gln Thr Lys Ile Ser Asn Gln Ile Val Ser Leu Thr Thr Glu Gln Ser Phe Asn Ile Gly Leu Glu Ser Lys Leu Lys Asp Glu Lys Leu Ser Val Leu Lys Trp Ile Leu Gln Glu Thr Tyr Glu Pro Glu Asn Leu Gly Thr Asp Ser Phe Leu Glu Arg Lys Lys Gln Glu Gly Leu His Ala Val Ile Val Glu Val Gly Pro Arg Leu Ser Phe Thr Thr Ala Trp Ser Thr Asn Ala Val Ser Ile ~ CA 02388851 2002-04-23 Cys Arg AIa Cys Gly Leu Asp Glu Val Thr Arg Leu Glu Arg Ser Arg Arg Tyr Leu Leu Phe Ser Lys Glu Pro Leu Leu Glu Asn Gln Ile Lys Glu Phe Ala Ala Met Val His Asp Arg Met Thr Glu Cys Val Tyr Thr Gln Lys Leu Val Ser Phe Glu Thr Asn Val Val Pro Glu Glu Val Lys Tyr Val Pro Val Met Glu Lys Gly Arg Lys Ala Leu Glu Glu Ile Asn Gln Glu Met Gly Leu Ala Phe Asp Glu Gln Asp Leu Gln Tyr Tyr Thr Arg Leu Phe Arg Glu Asp Ile Lys Arg Asp Pro Thr Asn Val Glu Leu Phe Asp Ile Ala Gln Ser Asn Ser Glu His Ser Arg His Trp Phe Phe Ala Gly Asn Met Val Ile Asp Gly Lys Pro Met Asp Lys Ser Leu Met Gln Ile Val Lys Ser Thr Trp Glu~.Ala Asn Arg Asn Asn Ser Val Ile Gly Phe Lys Asp Asn Ser Ser Ala Ile Arg Gly Phe Leu Val Asn Gln Leu Arg Pro Leu Leu Pro Gly Ser Val Cys Leu Leu Asp Val Ser Ala Arg Asp Leu Asp Ile Leu Phe Thr Ala Glu Thr His Asn Phe Pro Cys Ala Val Ala Pro Tyr Pro Gly Ala Glu Thr Gly Ala Gly Gly Arg Ile Arg Asp Thr His Ala Thr Gly Arg Gly Ser Phe Val Val Ala Ser Thr Ser Gly Tyr Cys Val Gly Asn Leu Asn Met Glu Gly Ser Tyr Ala Pro Trp Glu Asp Ser Ser Phe Gln Tyr Pro Ser Asn Leu Ala Ser Pro Leu G1n Ile Leu Ile Asp Ala Ser Asn Gly Ala Ser Asp Tyr Gly Asn Lys Phe Gly Glu Pro Met Ile Gln Gly Tyr Thr Arg Thr Phe Gly Met Arg Leu Pro Ser Gly Asp Arg Arg Glu Trp Leu Lys Pro Ile Met Phe Ser Ala G1y Ile Gly Gln Ile Asp His Thr His Ile Thr Lys Gly Glu Pro Glu Val GIy Met Leu Val Val Lys Ile Gly Gly Pro Ala Tyr Arg Ile Gly Met Gly G1y Gly Ala Ala Ser Ser Met Val Ser Gly Gln Asn Asp Ala Glu Leu Asp Phe Asn Ala Val Gln Arg Gly Asp Ala Glu Met Ser Gln Lys Leu Tyr Arg Val Val Arg Ala Cys Ile Glu Met Gly Glu Lys Asn Pro I1e Ile Ser Ile His Asp Gln Gly Ala Gly Gly Asn Cys Asn Val Val Lys Glu Ile Ile Tyr Pro Gln Gly Ala Glu Ile Asp Ile Arg Ala Val Val Val Gly Asp His Thr Met Ser Val Leu Glu Ile Trp Gly Ala Glu Tyr Gln Glu Gln Asp Ala Ile Leu Val Lys Ala Glu Ser Arg Glu Ile Leu Gln Ser Ile Cys Lys Arg Glu Arg Leu Ser Met Ala Val Ile Gly Thr Ile Asn Gly Gly Gly Arg Cys Thr Leu Ile Asp Ser Thr Ala Ala Ala Lys Cys Ser Lys Glu Gly Leu Pro Pro Pro Pro Pro Ala Val Asp Leu Glu Leu Glu Lys Val Leu Gly Asp Met Pro Lys Lys Thr Phe Lys Phe Asn Arg Ile Ala Tyr Ala Arg Glu Pro Leu Asp Ile Ala Pro Gly Ile Thr Leu Met Asp Ala Leu Lys Arg Val Leu Arg Leu Pro Ser Val Ser Ser Lys Arg Phe Leu Thr Thr Lys Val Asp Arg Cys Val Thr Gly Leu Val Ala Gln Gln Gln Thr Val Gly Pro Leu Gln Ile Thr Leu Ala Asp Val Ala Val I1e Ala Gln Thr Phe Thr Asp Leu Tlir Gly Gly Ala Cys Ala Ile Gly Glu Gln Pro Ile Lys Gly Leu Leu Asp Pro Lys Ala Met Ala Arg Leu Ala Va1 Gly Glu Ala Leu Thr Asn Leu Val Trp Ala Lys Val Thr Ala Leu Ser Asp Val Lys Ala Ser Gly Asn Trp Met Tyr Ala A1a Lys Leu Glu Gly Glu Gly Ser Ala Met Tyr Asg Ala Ala Ile Ala Leu Ser Glu Ala Met Ile Glu Leu Gly Ile Ala Ile Asp Gly Gly Lys Asp Ser Leu Ser Met Ala Ala His Ala Asp Gly Glu Val Val Lys Ala Pro Gly Asn Leu Val Ile Ser Ala Tyr Val Thr Cys Pro Asp Ile Thr Lys Thr Val Thr Pro Asp Leu Lys Leu Gly Gly Asp Asp G1y Ile Leu Leu His Val Asp Leu Ala Lys Gly Lys Arg Arg Leu Gly Gly Ser Ala Leu Ala Gln Val Phe Gly Gln Ile Gly Asn Asp Cys Pro fl ~ CA 02388851 2002-04-23 Asp Leu Asp Asp Val Pro Tyr Leu Lys Asn Val Phe Asp Gly Val Gln Ala Leu Ile Ala Glu Asn Leu Val Ser Ala Gly His Asp Ile Ser Asp Gly Gly Leu Val Val Thr Ala Leu Glu Met Ala Phe Ala Gly Asn Lys Gly Ile Asn Leu Asp Leu Ala Ser Asn Gly Ile Ser Leu Phe Glu Thr Leu Phe Ser Glu Glu Leu GIy Leu Val Leu Glu Ile Ser Lys Thr Asn Leu Asp Ala Val Met Glu Lys Leu Arg Ala Phe Asp Val Thr Ala Glu Ile Ile Gly Asn Val Thr Asp Ser Pro Leu Ile Glu Val Lys Val Asp Gly Ile Thr His Leu Ser Glu Lys Thr Ser Phe Leu Arg Asp Met Trp Glu Asp Thr Ser Phe Gln Leu Glu Lys Leu Gln Arg Leu Ala Ser Cys Val Glu Met Glu Lys Glu Gly Leu Lys Phe Arg His Glu Pro Asn Trp Lys Leu Ser Phe Ile Pro Ser Ser Thr Asn Asn Asn Tyr Met Ser Gln Asp Val Lys Pro Lys Val Ala Val Ile Arg Glu Glu Gly Ser Asn Gly Asp Arg Glu Met Ser Ala Ala Phe Tyr Ala Ala Gly Phe Glu Pro Trp Asp Val Thr Val Ser Asp Leu Leu Ala Gly Asp Ile Thr Leu Asp Gln Phe Arg Gly Ile Val Phe Val Gly Gly Phe Ser Tyr Ala Asp Val Leu Asp Ser Ala Lys Gly Trp Ala Ala Ser Ile Arg Phe Asn Glu Pro Val Leu Ser Gln Phe Gln Glu Phe Tyr Lys Arg Pro Asp Thr Phe Ser Leu Gly Ile Cys Asn Gly Cys Gln Leu Met Ala Leu Leu Gly Trp Val Pro Gly Pro Gln Val Gly Gly Ser Leu Asp Thr Ser Gln Pro Arg Phe Val His Asn Glu Ser Gly Arg Phe Glu Cys Arg Phe Thr Ser Val Thr Ile Lys Asp Ser Pro Ser Ile Met Leu Lys Gly Met Glu Gly Ser Thr Leu Gly Val Trp Ala Ala His Gly Glu Gly Arg Ala Tyr Phe Pro Asp Glu Gly Val Leu Asp His Met Leu His Ser Asp Leu Ala Pro Leu Arg Tyr Cys Asp Asp Asp Gly Asn Val Thr Glu Ala Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu Gly Ile Ala Ala Ile Cys Ser Pro Asp Gly Arg His Leu Ala Met Met Pro His Pro Glu Arg.Cys Phe Leu Met Trp Gln Phe Pro Trp Tyr Pro Thr Ser Trp Asp Val Glu Lys Ala Gly Pro Ser Pro Trp Leu Lys Met Phe Gln Asn Ala Arg Asp Trp Leu Glu Ser Cys <210> 3 <211> 3434 <212> DNA
<213> Nicotiana tabacum <220>
<221> CDS
<222> (2)..(3052) m <400> 3 g aat tcg cgg ccg cgc ggc cgc gaa ttc gcg gcc gca cct tat cct ggt 49 Asn Ser Arg Pro Arg Gly Arg Glu Phe Ala Ala Ala Pro Tyr Pro Gly 1 ~ 5 10 15 gcc gag aca ggt gca ggc ggc cgt atc cgg gat acc cat get act gga 97 Ala Glu Thr Gly AIa Gly Gly Arg Ile Arg Asp Thr His Ala Thr Gly agg ggt tct ttt gtt gtt gca tct aca gcc gga tat tgt gtt gga aat 145 Arg Gly Ser Phe Val Val Ala Ser Thr Ala Gly Tyr Cys Val Gly Asn ctt cat att gaa ggt tca tat get cct tgg gaa gat cct tct ttc aca 193 Leu His Ile Glu Gly Ser Tyr Ala Pro Trp Glu Asp Pro Ser Phe Thr tac-cca gca-aat ttg get tca ccg ctg cag atc ctt att gat get agt 241 Tyr Pro Ala Asn Leu Ala Ser Pro Leu Gln Ile Leu Ile Asp Ala Ser aat gga gca tcg gac tat ggg aac aaa ttc ggg gag cct ttg att cag 289 Asn Gly AIa Ser Asp Tyr Gly Asn Lys Phe Gly Glu Pro Leu Ile Gln ggt tat tgt cga acg ttt gga atg aga ctg cca agt ggc gag agg agg 337 Gly Tyr Cys Arg Thr Phe Gly Met Arg Leu Pro Ser Gly Glu Arg Arg gaa tgg ttg aag ccg atc atg ttt agt get ggc att ggg caa ata gat 385 Glu Trp Leu Lys Pro Ile Met Phe Ser Ala Gly Ile Gly Gln Ile Asp cac ctt cac tta tca aag gga gaa ccc gag att ggt atg ttg gta gtt 433 His Leu His Leu Ser Lys Gly Glu Pro Glu Ile Gly Met Leu VaI Val aag att gga gga cca gca tat cgt att gga atg gga ggt ggc get gca 481 Lys Ile Gly Gly Pro Ala Tyr Arg Ile Gly Met Gly Gly Gly Ala Ala tcc agc atg gtc agt gga cag aat gat gcc gag ctt gac ttc aac gcc 529 Ser Ser Met Val Ser Gly Gln Asn Asp Ala Glu Leu Asp Phe Asn Ala gtg cag cgt gga gat get gag atg gca cag aag ttg tat cgg gtt gtt 577 Val Gln Arg Gly Asp Ala Glu Met Ala Gln Lys Leu Tyr Arg Va1 Val . CA 02388851 2002-04-23 cgt get tgc att gag atg ggg gac aac aac ccg atc ata agc att cat 625 Arg Ala Cys Ile Glu Met Gly Asp Asn Asn Pro Ile Ile Ser Ile His gat cag ggt get ggt gga aac tgt aat gtc gtg aag gaa ata ata cat 673 Asp Gln Gly Ala Gly Gly Asn Cys Asn Val Val Lys Glu Ile Ile His cca cag ggc gcc aaa att gat ata agg gca att gta gtt ggc gat cac 72I
Pro Gln Gly Ala Lys Ile Asp Ile Arg Ala Ile Val Val Gly Asp His acg atg tct gtt ctg gaa att tgg ggt gca gaa tat cag gag caa gat 769 Thr Met Ser Val Leu Glu Ile Trp Gly Ala Glu Tyr Gln Glu Gln Asp gcc ata cta gtg aag cct gaa agt cgt gat ctt ttg caa gca atc tgt 817 Ala Ile Leu Val Lys Pro Glu Ser Arg Asp Leu Leu Gln Ala Ile Cys gcg agg gaa aga gtt tcc atg get gtt att gga aca att aat ggt gaa 865 Ala Arg Glu Arg Val Ser Met Ala Val Ile Gly Thr Ile Asn Gly Glu ggg cgt att gtc ctg gag gat agc gtg gca att gaa aaa acc agg tct 913 Gly Arg Ile Val Leu Glu Asp Ser Val Ala Ile Glu Lys Thr Arg Ser agt gga ttg cct cct cct cca cct gca gtg gat ctt gag ctt gag sag 961 Ser Gly Leu Pro Pro Pro Pro Pro Ala Val Asp Leu Glu Leu Glu Lys gtg ctt ggc gat atg cct aaa aag aca ttt gaa ttt cgt cgc atg aac 1009 Val Leu Gly Asp Met Pro Lys Lys Thr Phe Glu Phe Arg Arg Met Asn tat ctg cgt gaa cca ctt gat att get cct gca aca aca gtc tta gat 1057 Tyr Leu Arg Glu Pro Leu Asp Ile Ala Pro Ala Thr Thr Val Leu Asp tca ttg aag agg gtc ctg agg ctc cct tct gtt tgt tcg aaa agg ttc 1105 Ser Leu Lys Arg Val Leu Arg Leu Pro Ser Val Cys Ser Lys Arg Phe ttg acc act aaa gtt gac agg tgt gtc aca ggc ctt gtg gca cag cag 1153 Leu Thr Thr Lys Val Asp Arg Cys Val Thr Gly Leu Val Ala Gln Gln caa act gtg ggc ccc ctg cag att cct ctt get gat gtt get gtt ata 1201 Gln Thr val Gly Pro Leu Gln Ile Pro Leu Ala Asp Val Ala Val Ile get caa act tat aca gac tta act gga ggt gca tgc tca atc ggg gag 1249 Ala Gln Thr Tyr Thr Asp Leu Thr Gly Gly Ala Cys Ser Ile Gly Glu cag cca ata aaa ggt ctt ttg gat cca aaa gca atg gca cgg ctg get 1297 Gln Pro Ile Lys Gly Leu Leu Asp Pro Lys Ala Met Ala Arg Leu Ala gtc gga gaa gca ctc aca aat ctt gtt tgg gcg aaa att aca tct ctt 1345 Val Gly Glu Ala Leu Thr Asn Leu Val Trp Ala Lys Ile Thr Ser Leu tct gat gtt aaa gca agt ggg aat tgg atg tat get gca aag cta.gat 1393 Ser Asp Val Lys Ala Ser Gly Asn Trp Met Tyr Ala Ala Lys Leu Asp ggt gaa gga get gca atg tat gac get get gtt get ctt tct gaa get 1441 Gly Glu Gly Ala Ala Met Tyr Asp Ala Ala Val Ala Leu Ser Glu Ala atg att gaa ctt gga att gca att gat ggg ggg aaa gac agc ctt tcc 1489 Met Ile Glu Leu Gly Ile Ala Ile Asp Gly Gly Lys Asp Ser Leu Ser atg gca gcc cac tcg tct ggg gaa gtt gtt aaa gcc cca ggg aat cta 1537 Met Ala Ala His Ser Ser Gly Glu Val Val Lys Ala Pro Gly Asn Leu gtc atc agt acc tat gtg aca tgt cct gat ata acc aag aca gtt acg 1585 Val Ile 5er Thr Tyr Val Thr Cys Pro Asp Ile Thr Lys Thr Val Thr cca gac ttg aag ctt gga gat gat ggt gta ctg ctt cat att gac ttg 1633 Pro Asp Leu Lys Leu Gly Asp Asp Gly Val Leu Leu His Ile Asp Leu get aaa gga aaa cga cga ctt ggt gga tct get ctt gcc cag gtt ttt 1681 Ala Lys Gly Lys Arg Arg Leu Gly Gly Ser Ala Leu Ala Gln Val Phe gat caa att ggg gac gaa agt cct gat ctg gat gac gta tct tat ctt 1729 Asp Gln Ile GIy Asp Glu Sex Pro Asp Leu Asp Asp Val Ser Tyr Leu fl aag act gtt ttt aat gag gtt cag aat cta atc tct gat gag ctg ata 1777 Lys Thr Val Phe Asn Glu Val Gln Asn Leu Ile Ser Asp Glu Leu Ile tct get ggt cat gat atc agt gat ggg gga ctt tta gtg aat get ctg 1825 Ser Ala Gly His Asp Ile Ser Asp Gly Gly Leu Leu Val Asn Ala Leu gaa atg gca ttc gca ggg aac tgt ggc att cac ttg gat tta act tct 1873 Glu Met Ala Phe Ala Gly Asn Cys Gly Ile His Leu Asp Leu Thr Ser tta ggg agt agt gta ccc caa aca ctt ttt gca gag gag ctt ggc ctg 1921 Leu Gly Ser Ser Val Pro Gln Thr Leu Phe Ala Glu Glu Leu Gly Leu ctc att gaa gtt agc agg aag aac ttg gat tta gtt ctg gaa aag ctc 1969 Leu IIe Glu Val Ser Arg Lys Asn Leu Asp Leu Val Leu Glu Lys Leu tgc agt ggt get gtt tca get aat att att ggt caa gtt act tca tct 2017 Cys Ser Gly Ala Val Ser Ala Asn Ile Ile Gly Gln Val Thr Ser Ser cca ata gtt gaa ttg agg gtt gac ggg gtt act cat ttg aat gac aaa 2065 Pro Ile Val Glu Leu Arg Val Asp Gly Val Thr His Leu Asn Asp Lys act tct gtg ctc agg gat atg tgg gaa gaa acc agc ttt caa ttg gas ~ 2113 Thr Ser Val Leu Arg Asp Met Trp Glu Glu Thr Ser Phe Gln Leu Glu aag ctc caa aga ctg get tcg tgt gta gaa tta gaa aaa gaa gga ttg 2161 Lys Leu Gln Arg Leu Ala Ser Cys Val Glu Leu Glu Lys Glu Gly Leu 705 710 715 72p aag aat cgg cat gaa cca tcc tgg aaa cta tcc ttc aca cca aca ttt 2209 Lys Asn Arg His Glu Pro Ser Trp Lys Leu Ser Phe Thr Pro Thr Phe ?25 730 735 act gat gat aag tat atg act gcc gtt tca aag cca aag gtc gca att 2257 Thr Asp Asp Lys Tyr Met Thr Ala Val Ser Lys Pro Lys Val Ala Ile att cgc gag gaa ggc agc aat ggt gat aga gaa atg act gca get ttt 2305 Ile Arg Glu Glu Gly Ser Asn Gly Asp Arg Glu Met Thr Ala A1a Phe r tat get get gga ttt gag cca tgg gat gtt gca atg tca gac ctt ctc 2353 Tyr Ala Ala Gly Phe Glu Pro Trp Asp Val Ala Met Ser Asp Leu Leu aat gga gtc atc atg ctt gat gaa ttt aga gga att gtg ttt gtt gga 2401 Asn Gly Val Ile Met Leu Asp Glu Phe Arg Gly Ile Val Phe Val Gly ggt ttt agt tat get gac gtg ctt gat tct gca aaa ggc tgg gca gcg 2449 Gly Phe Ser Tyr Ala Asp Val Leu Asp Ser Ala Lys Gly Trp Ala Ala tcc att cgc ttt aat caa cct ctt tta aac caa ttt cag gca ttt tat 2497 Ser Ile Arg Phe Asn Gln Pro Leu Leu Asn Gln Phe G1n Ala Phe Tyr aac cgt cca gac act ttc agc ctt gga gtt tgc aat ggg tgc caa ctt 2545 Asn Arg Pro Asp Thr Phe Ser Leu Gly Val Cys Asn Gly Cys Gln Leu atg get ctg ttg ggt tgg gtt ccg ggg ccc caa gtg gga ggt gtt ttc 2593 Met Ala Leu Leu Gly Trp Val Pro Gly Pro Gln Val Gly Gly Val Phe ggt gcc ggc ggg gac cca tca cag cct agg ttt gta cat aat gag tct 2641 G.ly Ala Gly Gly Asp Pro Ser Gln Pro Arg Phe Val His Asn Glu Ser gga aga ttt gaa tgc cgc ttc acg agt gtg aca ata gaa gaa tca ccg 2689 Gly Arg Phe Glu Cys Arg Phe Thr Ser Val Thr Ile Glu Glu Ser Pro gcc ata atg ttc aaa ggt atg gaa ggt agt aca ctg ggc gtt tgg get 2737 Ala Ile Met Phe Lys Gly Met Glu Gly Ser Thr Leu Gly Val Trp Ala get cat ggt gaa gga aga get tat ttc cct gat gat agt gtt ttc aat 2785 Ala His Gly Glu Gly Arg Ala Tyr Phe Pro Asp Asp Ser Val Phe Asn cat att gtt ggc tcc aac ttg gca cca gtg aaa tat tgc gat gat gat 2833 His Ile Val Gly Ser Asn Leu Ala Pro Val Lys Tyr Cys Asp Asp Asp ggc aga cca aca gat ata tat cct ttc aat ctt aat ggt tct ccc ttg 2881 Gly Arg Pro Thr Asp Ile Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu ' CA 02388851 2002-04-23 ggt gtg gcg gca att tgt tct cca gat ggg cga cat ctt gcg ata atg 2929 Gly Val Ala Ala Ile Cys Ser Pro Asp Gly Arg His Leu Ala Ile Met cct cat cca gaa cgc tgt ttc ttg atg tgg cag ttc cca tgg tat cct 2977 Pro His Pro Glu Arg Cys Phe Leu Met Trp Gln Phe Pro Trp Tyr Pro aaa aat tgg gat gtt gaa aag aaa ggt cca agt ccc tgg ttg cgc atg 3025 Lys Asn Trp Asp Val Glu Lys Lys Gly Pro Ser Pro Trp Leu Arg Met ttc caa aat gcc aga gaa tgg tgc tca tgagattttc tgaaaattct 3072 Phe Gln Asn Ala Arg Glu Trp Cys Ser gaagtcatga cattgtttga cagacaaata ttattagagg ggcaaagctg agaggcaggt 3132 gctgttcttt atcacataga ggttaaagtg caaagattgg acagaagcga cacagtttaa 3192 tagtcagtgc tgaaggtgca gagattaaga tcagtgcaga gcttgagatc tctgcaattt 3252 tgtttctgat accatggcac aaagcagatg aatttttcaa tttaactggc tgattgtcgt 3312 ttctttcgat actgatggac tttttcattg cctcttagct gtttcgcttg tttcttgtat 3372 accataattg gtttttctta tgtcttacat atctgatcaa ctgattttgc ggccgcgaat 3432 tc <210> 4 <211> 1017 <212> PRT
<213> Nicotiana tabacum <400> 4 Asn Ser Arg Pro Arg Gly Arg Glu Phe Ala Ala Ala Pro Tyr Pro Gly Ala Glu Thr Gly Ala Gly Gly Arg Ile Arg Asp Thr His Ala Thr Gly Arg Gly Ser Phe Val Val Ala Ser Thr Ala Gly Tyr Cys Val Gly Asn Leu His Ile Glu Gly Ser Tyr Ala Pro Trp G1u Asp Pro Ser Phe Thr i _ 50 55 60 Tyr Pro Ala Asn Leu Ala Ser Pro Leu Gln Ile Leu Ile Asp Ala Ser Asn Gly Ala Ser Asp Tyr Gly Asn Lys Phe Gly Glu Pro Leu Ile Gln Gly Tyr Cys Arg Thr Phe Gly Met Arg Leu Pro Ser Gly Glu Arg Arg Glu Trp Leu Lys Pro Ile Met Phe Ser Ala Gly Ile Gly Gln Ile Asp His Leu His Leu Ser Lys Gly Glu Pro Glu Ile Gly Met Leu Val Val Lys Ile Gly Gly Pro Ala Tyr Arg Ile Gly Met Gly Gly Gly Ala Ala Ser Ser Met Val Ser Gly Gln Asn Asp Ala Glu Leu Asp Phe Asn Ala Val Gln Arg Gly Asp Ala Glu Met Ala Gln Lys Leu Tyr Arg Val Val Arg Ala Cys Ile Glu Met Gly Asp Asn Asn Pro Ile Ile Ser Ile His Asp Gln Gly Ala Gly Gly Asn Cys Asn Val Val Lys Glu Ile Ile His 21o z15 2zo Pro Gln Gly Ala Lys Ile Asp Ile Arg Ala Ile Val Val Gly Asp His Thr Met Ser Val Leu Glu Ile Trp Gly Ala Glu Tyr Gln Glu Gln Asp Ala Ile Leu Val Lys Pro Glu Ser Arg Asp Leu Leu Gln Ala Ile Cys Ala Arg Glu Arg Val Ser Met Ala Val Ile Gly Thr Ile Asn Gly Glu Gly Arg Ile Val Leu Glu Asp Ser Val Ala Ile Glu Lys Thr Arg Ser Ser Gly Leu Pro Pro Pro Pro Pro Ala Val Asp Leu Glu Leu Glu Lys ~ CA 02388851 2002-04-23 Val Leu Gly Asp Met Pro Lys Lys Thr Phe Glu Phe Arg Arg Met Asn Tyr Leu Arg Glu Pro Leu Asp Ile Ala Pro Ala Thr Thr Val Leu Asp Ser Leu Lys Arg Val Leu Arg Leu Pro Ser Val Cys Ser Lys Arg Phe Leu Thr Thr Lys Val Asp Arg Cys Val Thr Gly Leu Val Ala Gln Gln Gln Thr Val Gly Pro Leu Gln Ile Pro Leu Ala Asp Val Ala Val Ile Ala Gln Thr Tyr Thr Asp Leu Thr Gly Gly Ala Cys Ser Ile Gly Glu Gln Pro Ile Lys Gly Leu Leu Asp Pro Lys Ala Met Ala Arg Leu Ala Val Gly Glu Ala Leu Thr Asn Leu Val Trp Ala Lys Ile Thr Ser Leu Ser Asp Val Lys Ala Ser Gly Asn Trp Met Tyr Ala Ala Lys Leu Asp Gly Glu Gly Ala Ala Met Tyr Asp Ala Ala Val Ala Leu Ser Glu Ala Met Ile Glu Leu Gly Ile Ala Ile Asp Gly Gly Lys Asp Ser Leu Ser Met Ala Ala His Ser Ser Gly Glu Val Val Lys Ala Pro Gly Asn Leu Val Ile Ser Thr Tyr Val Thr Cys Pro Asp Ile Thr Lys Thr Val Thr Pro Asp Leu Lys Leu Gly Asp Asp Gly Val Leu Leu His Ile Asp Leu Ala Lys Gly Lys Arg Arg Leu Gly Gly Ser Ala Leu Ala Gln Val Phe Asp Gln Ile Gly Asp Glu Ser Pro Asp Leu Asp Asp Val Ser Tyr Leu f.

Lys Thr Val Phe Asn Glu Val Gln Asn Leu Ile 5er Asp Glu Leu Ile Ser Ala Gly His Asp Ile Ser Asp Gly Gly Leu Leu Val Asn Ala Leu Glu Met Ala Phe Ala Gly Asn Cys Gly Ile His Leu Asp Leu Thr Ser Leu Gly Ser Ser Val Pro Gln Thr Leu Phe Ala Glu Glu Leu Gly Leu Leu Ile Glu Val Ser Arg Lys Asn Leu Asp Leu Val Leu Glu Lys Leu Cys Ser Gly Ala Val Ser Ala Asn Ile Ile Gly Gln Val Thr Ser Ser 660 665 6?0 Pro Ile Val Glu Leu Arg Val Asp Gly Val Thr His Leu Asn Asp Lys Thr Ser Val Leu Arg Asp Met Trp Glu Glu Thr Ser Phe Gln Leu Glu Lys Leu Gln Arg Leu Ala Ser Cys Val Glu Leu Glu Lys Glu Gly Leu Lys Asn Arg His Glu Pro Ser Trp Lys Leu Ser Phe Thr Pro Thr Phe Thr Asp Asp Lys Tyr Met Thr Ala Val Ser Lys Pro Lys Val Ala Ile Ile Arg Glu Glu Gly Ser Asn Gly Asp Arg Glu Met Thr Ala Ala Phe Tyr Ala Ala Gly Phe Glu Pro Trp Asp Val Ala Met Ser Asp Leu Leu Asn Gly Val Ile Met Leu Asp Glu Phe Arg Gly Ile Val Phe Val Gly Gly Phe Ser Tyr Ala Asp Val Leu Asp Ser Ala Lys Gly Trp Ala Ala Ser Ile Arg Phe Asn Gln Pro Leu Leu Asn Gln Phe Gln Ala Phe Tyr s, .. a Asn Arg Pro Asp Thr Phe Ser Leu Gly Val Cys Asn Gly Cys Gln Leu Met Ala Leu Leu Gly Trp Val Pro Gly Pro Gln Val Gly Gly Val Phe Gly Ala Gly Gly Asp Pro Ser Gln Pro Arg Phe Val His Asn Glu Ser Gly Arg Phe Glu Cys Arg Phe Thr Ser Val Thr Ile Glu Glu Ser Pro Ala Ile Met Phe Lys Gly Met Glu Gly Ser Thr Leu Gly Val Trp Ala Ala His Gly Glu Gly Arg Ala Tyr Phe Pro Asp Asp Ser Val Phe Asn His Ile Val Gly Ser Asn Leu Ala Pro Val Lys Tyr Cys Asp Asp Asp Gly Arg Pro Thr Asp Ile Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu Gly Val Ala Ala Ile Cys Ser Pro Asp Gly Arg His Leu AIa Ile Met Pro His Pro Glu Arg Cys Phe Leu Met Trp Gln Phe Prv Trp Tyr Pro Lys Asn Trp Asp Val Glu Lys Lys Gly Pro Ser Pro Trp Leu Arg Met Phe Gln Asn Ala Arg Glu Trp Cys Ser <210> 5 <211> 47B
<212> DNA
<213> Chilopsis linearis <220>
<221> CDS
<222> (3)..(293) i <400> 5 ga att cgg cac gag gat gtt ttt ggc agt ata ctg gag tcg aac ttg 47 Ile Arg His Glu Asp Val Phe Gly Ser Ile Leu Glu Ser Asn Leu get cct gtg aga tat tgt gat gat gtt ggg aat cct aca gaa gtc tat 95 Ala Pro Val Arg Tyr Cys Asp Asp Val Gly Asn Pro Thr Glu Val Tyr cca ttc aac ctt aat ggt tct cct tta ggt gtt gca get att tgt tcc 143 Pro Phe Asn Leu Asn Gly Ser Pro Leu Gly Val Ala Ala Ile Cys Ser cca gat ggg agg cat ctt gcc atg atg cct cat cca gag cgt tgc ttc 191 Pro Asp Gly Arg His Leu Ala Met Met Pro His Pro GIu Arg Cys Phe ttg atg tgg cag tat ccg tgg tat ccc aag aac tgg aag gtg gag aag 239 Leu Met Trp Gln Tyr Pro Trp Tyr Pro Lys Asn Trp Lys Val Glu Lys aaa ggt cct agt ccg tgg tta cgc atg ttt cag aat get agg gag tgg 287 Lys Gly Pro Ser Pro Trp Leu Arg Met Phe Gln Asn Ala Arg Glu Trp tgt tca tgaggaaatg aaggcctaga agaataagaa ggataatact tggagaggac 343 Cys Ser nactgaaaag ggatgttagt agctttttat gatttgaatt ggttttctga acttttgtag 403 aaagccctga gctaattttg gtgtttagtt gagattttca gaaaaaaaaa aaaaaaaaaa 463 aaaaaaaaac tcgag 478 <210> 6 <211> 97 <212> PRT
<213> Chilopsis linearis <400> 6 Ile Arg His Glu Asp Val Phe Gly Ser Ile Leu Glu Ser Asn Leu Ala Pro Val Arg Tyr Cys Asp Asp Val Gly Asn Pro Thr Glu Val Tyr Pro Phe Asn Leu Asn Gly Ser Pro Leu Gly Val Ala Ala Ile Cys Ser Pro Asp Gly Arg His Leu Ala Met Met Pro His Pro Glu Arg Cys Phe Leu Met Trp Gln Tyr Pro Trp Tyr Pro Lys Asn Trp Lys Val Glu Lys Lys Gly Pro Ser Pro Trp Leu Arg Met Phe Gln Asn Ala Arg Glu Trp Cys Ser

Claims

We claim:

1. A DNA sequence containing the encoding region of a plant formylglycinamidine ribotide synthase as novel target for herbicidal active ingredients, wherein this DNA sequence has the nucleotide sequence SEQ-ID No.1, SEQ-ID No.3 or SEQ-ID
No.5.

2. A DNA sequence hybridizing with the DNA sequence SEQ-ID No.
1, SEQ-ID No.3 or SEQ-ID No.5 as claimed in claim 1 or parts thereof or derivatives, derived from this sequence by insertion, deletion or substitution and encoding a protein which has the biological activity of a formylglycinamidine ribotide synthase.

3. A protein with formylglycinamidine ribotide synthase activity, which contains, as amino acid sequence, the sequence shown in SEQ-ID No.2.

4. A protein with formylglycinamidine ribotide synthase activity containing an amino acid sequence which constitutes a subsequence of at least 100 amino acids from SEQ-ID No.2.

5. 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 the expression of a translatable mRNA which causes the synthesis of a plant formylglycinamidine ribotide synthase.

6. The use of a DNA sequence as claimed in claim 1 or 2 for generating an assay system for identifying herbicidally active plant formylglycinamidine ribotide synthase inhibitors.

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

8. A method of identifying herbicidally active substances which inhibit the formylglycinamidine ribotide synthase activity in plants, consisting of the following steps:

a) the generation of transgenic plants, plant tissues or plant cells comprising an additional DNA sequence encoding an enzyme with formylglycinamidine ribotide synthase activity and capable of overexpressing enzymatically active formylglycinamidine ribotide synthase;

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

c) determining the growth or the viability of the transgenic and the untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance; and d) comparing the growth or the viability of the transgenic and the untransformed plants, plant cells, plant tissues or plant parts after application of the chemical substance;

where suppression of the growth or viability of the untransformed plants, plant cells, plant tissues or plant parts without simultaneous severe suppression of the growth or the viability of the transgenic plants, plant cells, plant tissues or plant parts confirms that the substance of b) is herbicidally active and inhibits the formylglycinamidine ribotide synthase enzyme activity in plants.

9. An assay system based on the expression of a DNA sequence SEQ-ID No.1, SEQ-ID No.3 or SEQ-ID No.5 as claimed in claim 1 or 2 for identifying herbicidally active plant formylglycinamidine ribotide synthase inhibitors.

10. An assay system as claimed in claim 9 for identifying inhibitors of plant formylglycinamidine ribotide synthase, which comprises incubating the enzyme with a test substrate to be tested and, after a suitable reaction time, determining the enzymatic activity of the enzyme in comparison with the activity of the uninhibited enzyme.

11. A plant formylglycinamidine ribotide synthase inhibitor.

12. A plant formylglycinamidine ribotide synthase inhibitor, identified using an assay system as claimed in claim 9 or 10.

13. An inhibitor identified as claimed in claim 9 or 10 for use as herbicide.

14. A method of eliminating undesired vegetation, which comprises treating the plants to be eliminated with a compound which binds specifically to formylglycinamidine ribotide synthase encoding a DNA sequence as claimed in claim 1 or 2, and which inhibits its function.