CA2325054A1 - Amp deaminase - Google Patents

Abstract

The invention relates to a DNA which codes for a polypeptide with AMP deaminase (EC 3.5.4.6, adenosine triphosphate aminohydrolase) activity. The invention also relates to the use of this nucleic acid for producing a test system.

Description

~~50/489Z4 CA 02325054 2000-09-27 AMP Deaminase The present invention relates to a DNA coding for a polypeptide with AMP deaminase (EC 3.5.4.6, adenosine triphosphate [sic]
aminohydrolase) activity. The invention also relates to the use of a nucleic acid coding for a protein having AMP deaminase activity of plant origin for producing a test system for identifying AMP deaminase inhibitors. The invention further relates to the use of the nucleic acid coding for plant AMP
deaminase for producing plants with increased resistance to AMP
deaminase inhibitors.
Plants are able to synthesize their cellular components from carbon dioxide, water and inorganic salts.
This process is possible only by utilization of biochemical reactions to synthesize organic substances. Plants also need to synthesize de novo the nucleotides as components of the nucleic acids.
It is to be assumed that efficient formation, utilization and distribution of nucleotides influence cell division and the growth of a plant. Since plants depend on a functioning nucleo-tide metabolism, this metabolism is a possible target for the use of herbicides. Some known agents (e. g. 5'-phosphohydantocidin, alanosin or hadacidin) inhibit adenylosuccinate synthetase (Stayton et al., 1983, Curr. Top. Cell. Regul., 22, 103-141;
Siehl et al., 1996, Plant Physiol., 110, 753-758). The reaction catalyzed by adenylosuccinate synthetase is depicted in Figure 1.
Hydantocidin acts as a prodrug in this case. The agent is metabolized in the plant by phosphorylation at the 5'OH group to give the herbicide (Siehl et al., 1996, Plant Physiol., 110, 753-758).
An important principle for regulating nucleotide metabolism is, besides the enzyme reactions of de novo purine biosynthesis (e. g.
adenylosuccinate synthetase), also recycling processes and breakdown mechanisms. However, AMP deaminase occupies a special position. Thus, it is also possible to deaminate AMP (adenosine monophospate) again to give IMP (inosine 5'-phosphate). The enzyme catalyzes the following reaction:
AMp + Hz0 <--> IMP + NH4 [sic]

a The protein has been partially purified from various plants, and its regulatory propert:Les have been investigated, for example from spinach leaves (Yoshino and Murakami, 198 [sicj, Z.
Pflanzenphysiologie, 99, 331-338), Jerusalem artichokes (Le Floc'h and Lafleuriel, 1983, Physiologie Vergetale [sic], 21 (1), 15-2), pea seeds (Turner and Turner, 1961, Biochem. J. 79, 143) and cell cultures of Catharantus roseus, lesser periwinkle (Yabuki et al. 1992, Phytochemistry, 31 (6), 1905-1909). It moreover appears that t:he enzyme plays a central part in regulation of the adenylate pool in a cell (Chapman and Atkinson, 1973, J. Biol. Chem., ~'.48, 8309; Solano and Coffee,1978; Yoshino et al., 1979).
It has been shown that the known natural substance coformycin has a herbicidal effect on plants, and that the,agent in its phosphorylated form (5'-phosphocoformycin) is the actual AMP
deaminase inhibitor (Frieden et al., 1980, Biochem. 5303; Merkler et al., 1990, Biochem. 29, 8358-8364; Dancer et al., 1997, Plant Physiol., 114 (1), 119-129). The patent WO 96/01326 describes a method for looking for herbicides in an AMP deaminase enzyme assay (see also: Pillmc>or Pestic. Sci., 1998, 52, 75-80).
Genes which code for AMP deaminases have been isolated from many organisms. In mammals there appear to be families of AMP
deaminase genes (Morisaki et al. 1990, J. Biol. Chem. 265 (20), 11482-11486). Further cading sequences have been isolated from Schizosaccharomyces pombe (accession P50998) and Saccharomyces cerevisiae (accession P15274). Only adenine deaminases are known from bacteria, and it has not been possible to isolate AMP
deaminases. Expressed ~~equence tags, called est sequences, can be found, by sequence comparisons, in rice (GenBank Acc: C26026) and Arabidopsis (T21250) hawing similarity with yeast AMP dea,minases.
Complete cDNA sequences of plant AMP deaminases have not been described as yet.
It is an object of the present invention to isolate a complete plant cDNA coding for the enzyme AMP deaminase and to carry out functional expression thereof in bacterial or eukaryotic cells to obtain the enzyme in a simple and low-cost manner to carry out inhibitor-enzyme binding studies.
It is a further object of the present invention to overexpress the AMP deaminase gene in plants to produce plants which are tolerant of AMP deamina.se inhibitors.

We have found that these objects have been achieved by isolating the gene coding for the plant enzyme AMP deaminase, and the functional expression thereof in bacterial or plant cells or plants.
The present invention firstly relates to a DNA sequence SEQ ID
N0:1 comprising the coding region of a plant AMP deaminase from Arabidopsis thaliana (see Figure 2).
The invention further relates to DNA sequences which are derived from this SEQ ID N0:1 or hybridized with the latter and which code for a protein which has the biological activity of an AMP
deaminase.
The invention also relates to expression cassettes whose sequence [sic] code for an AMP df~aminase from Arabidopsis thaliana or the functional equivalent thereof. The nucleic acid sequence can in this connection be, for example a DNA or a cDNA sequence.
The expression cassettes according to the invention additionally comprise regulatory nuc:Leic acid sequences which control the expression of the coding sequence in the host cell. In a preferred embodiment, an expression cassette according to the invention comprises upsi:ream, i.e. at the 5' end of the coding sequence, a promoter and downstream, i.e. at the 3' end, a polyadenylation signal, and, where appropriate, further regulatory elements which are operatively linked to the AMP
deaminase gene coding ss~quence lying between them. Operative linkage means sequentia:L arrangement of promoter, coding sequence, terminator and, where appropriate, further regulatory elements in such a way that each of the regulatory elements can carry out its function properly on expression of the coding sequence.
An expression cassette according to the invention is produced by fusing a suitable promoter to a suitable AMP deaminase DNA
sequence and a polyaden!~lation signal by conventional recom-bination and cloning techniques as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W.
Enquist, Experiments wiith Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols :in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

~~5~/48924 CA 02325054 2000-09-27 Particularly preferred sequences are those which ensure targeting in the apoplasts, in plastids, the vacuoles, the mitochondrion, the endoplasmic reticul.um (ER) or, owing to the absence of appropriate operative sequences, ensure retention in the compartment of production, the cytosol (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285--423).
For example, the plant expression cassette can be incorporated into the tobacco transformation vector pBinAR-Hyg (see Example 5).
Suitable in principle as promoter for the expression cassette according to the invention is every promoter able to control expression of foreign genes in plants. It is particularly preferred to use a plant promoter or a promoter derived from a plant virus. The CaMV 35S-promoter from cauliflower mosaic virus (Franck et al., Cell 27.(1980) 285-294) is particularly preferred.
This promoter contains various recognition sequences for trans-criptional effectors which, in their totality, lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989) 2195-2202).
The expression cassette' according to the invention can also comprise a chemically .i.nducible promoter through which it is possible to control expression of the exogenous AMP deaminase gene in the plant at a particular point in time. Promoters of this type, such as, fox- example, the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22(1993), 361-366), a promoter inducible by salicylic acid (WO 95/7.919443), a benzenesufonamide-inducible [sic] (EP 388186), a tetracycline-inducible (Gatz et al., (1992) Plant J. 2,397-404), an abscisic acid-inducible (EP335528) or an ethanol- or cyclohexanone-inducible (W09321334) promoter are described in the literature and can, inter alia, be used.
Further particularly preferred promoters are those which ensure expression in tissues or plant parts in which the biosynthesis of purines or their precux-sors takes place. Particular mention may be made of promoters which ensure leaf-specific expression.
Mention should be made of the promoter of the potato cytosolic FBPase or the potato ST-LSI promoter (Stockhaus et al., EMBO J. 8 (1989) 2445-245 [sic])"
It is possible with the' aid of a seed-specific promoter to express a foreign protein stably up to a content of 0.67 of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio/Technology 10(1995), 1090-1094).
The expression cassette: according to the invention can therefore comprise, for example, a seed-specific promoter (preferably the phaseolin promoter, thcs USP or LEB4 promoter), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.

5 Many legumes transport fixed nitrogen in the form of ureides from the nodules to the above-ground tissue (Schubert, 1986, Ann. Rev.
Plant Phys. 37, 539-574). Ureides are formed via purine biosynthesis. It is possible with the aid of a node-(nodule-) specific promoter for i3MP deaminase expression and thus ureide biosynthesis in legumes to be modified specifically. Gene expression cassettes according to the invention may accordingly comprise the promoter of the phosphoribosyl-pyrophosphate amidotransferase from Glycine max (see also Genbank Accession Number U87999) or another node-specific promoter as described in EP 249676.
The inserted nucleotide: sequence coding for an AMP deaminase can be prepared synthetica:Lly or be obtained naturally or comprise a mixture of synthetic and natural DNA components. In general, synthetic nucleotide sequences are produced with codons preferred by plants. These codon:~ preferred by plants can be determined from codons which have the highest protein frequency and are expressed in most plant species of interest. To prepare an expression cassette it is possible to manipulate various DNA
fragments in order to obtain a nucleotide sequence which expediently reads in the correct direction and is equipped with a correct reading frame. Adapters or linkers can be attached to the fragments to connect the DNA fragments together.
The sequence homology between yeast and plant AMP deaminases at the DNA level is, based on a selected highly homologous region, 57$, for example in the 1345-2715 base region of the sequence M30449 (Genbank Accession Number), established using the BLAST
program (Altschul et a:L.,(1990), J. Mol. Bio1.:215:403-419; Gish and States,(1993), Nature Genet. ~:266-272). In the fragment described, regions of ;ZO-30 nucleotides are so homologous that there is a sufficient probability of success for an oligonucleotide-based screening for AMP deaminase from other plants. Methods of hybridization with short oligonucleotides to detect DNA sequences which have good homology only in small regions with respect to the comparison sequence are described in Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN
0-87969-309-6).

~~5~/48924 CA 02325054 2000-09-27 The invention also relates to functionally equivalent DNA
sequences which code for an AMP deaminase gene [sic) and which, based on the total length of the gene, have a sequence homology of from 40 to 100 with the DNA sequence SEQ ID NO: 1.
The invention preferably relates to functionally equivalent DNA
sequences which code for an AMP deaminase gene [sic] and which, based on the total length of the gene, have a sequence homology of from 60 to 100 with the DNA sequence SEQ ID NO: 1.
The invention particularly preferably relates to functionally equivalent DNA sequences which code for an AMP deaminase gene [sic) and which, based on the total length of the gene, have a sequence homology of from 80 to 100 with the DNA sequence SEQ ID
N0: 1.
Functionally equivalent sequences coding for an AMP deaminase gene [sic] are, according to the invention, those sequences which, despite a differing nucleotide sequence, still have the required functions. Functional equivalents thus comprise naturally occurring variants of the sequences described herein, and artificial artificial [sic] nucleotide sequences, for example obtained by chemical synthesis, adapted to the codon usage of a plant.
A functional equivalent also means in particular natural or artificial mutations of an originally isolated sequence coding for an AMP deaminase, which still show the required function.
Mutations comprise substitutions, additions, deletions, transpositions or insertions of one or more nucleotide residues.
This means, for example, that the present invention also comprises those nucleotide sequences which are obtained by modifying this nucleotide sequence. The aim of such a modifi-cation may be, for example, further localization of the coding sequence present therein or else, for example, insertion of further restriction enzyme cleavage sites.
Functional equivalents are also those variants whose function is attenuated or enhanced by comparison with the initial gene or gene fragment.
Artificial DNA sequences are also suitable as long as they confer, as described above, the required property of increasing the IMP content in the plant by overexpression of the AMP
deaminase gene in crop plants. Such artificial DNA sequences can be found, for example, by translation back from proteins having AMP deaminase activity and constructed by molecular modeling, or by in vitro selection. Particularly suitable coding DNA sequences are those obtained by translation back from a polypeptide sequence in accordance with the codon usage specific for the host plant. The specific codon usage can easily be found by a skilled worker familiar with methods of plant genetics by computer analyses of other known genes of the plant to be transformed.
Further suitable equivalent nucleic acid sequences according to the invention which may be mentioned are sequences coding for fusion proteins where one constituent of the fusion protein is a plant AMP deaminase 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 whose aid it is possible to detect AMP deaminase expression (e. g. myc tag or his tag).
However, this is preferably a regulatory protein sequence such as, for example, a signal or transit peptide which guides the AMP
deaminase protein to the required site of action.
The promoter regions according to the invention and the terminator regions ought expediently to 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, usually 1 to 8, preferably 2 to 6, restriction sites. The size of the linker within the regulatory regions is generally less than 100 bp, frequently less than 60 bp, but at least 5 bp. The promoter according to the invention may be both native or homologous and foreign or heterologous to the hast plant. The expression cassette according to the invention comprises in the 5'-3' direction of trans-cription the promoter according to the invention, any suitable sequence and a region for transcription termination. Different termination regions can be exchanged for one another as desired.
It is furthermore possible to employ manipulations which provide suitable restriction cleavage sites or delete the excess DNA or restriction cleavage sites. Where insertions, deletions or substitutions such as, for example, transitions and transversions are considered, it is possible to use in vitro mutagenesis, primer repair, restriction or ligation. In the case of suitable manipulations such as, for example, restriction, chewing-back or filling in of overhangs for blunt ends, complementary ends of the fragments can be made available for the ligation.
Particularly important for the result according to the invention is the attachment of th.e specific ER retention signal SEKDEL
~(Schouten, A. et al. Plant Mol. Biol. 30 (1996), 781 - 792), which triples or quadruples the average level of expression. It is also possible to employ other retention signals which naturally occur in plant and animal proteins located in the ER
for constructing the cassette.
Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA
polyadenylation signals from Agrobacterium tumefaciens, especially of gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al., EMBO J. 3 (1984) 835 ff.) or functional equivalents.
To transform a host plant with a DNA coding for an AMP deaminase, an expression cassette according to the invention is incorporated as insert into a recombinant vector whose vector DNA contains additional functional regulatory signals, for example sequences for replication or integration. Suitable vectors are described inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chapter 6/7, pages 71-119.
The transfer of foreign genes into the genome of a plant is referred to as transformation. The methods used for this purpose are those described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene gun, electroporation, incubation of dry embryos in DNA-containing solution, microinjection and gene transfer mediated by agrobacterium. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, 'Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefacie.ns, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
Agrobacteria transformed with an expression cassette according to the invention can likewise be used in a known manner for transforming plants, especially crop plants such as cereals, corn, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species, and legumes, for example by bathing wounded leaves or pieces of leaves in a solution of agrobacteria and then cultivating in suitable media.

The site of purine biosynthesis is generally the leaf tissue, so that leaf-specific expression of the AMP deaminase gene is sensible. However, it is obvious that purine biosynthesis need not be confined to leaf tissue but may also take place in all other parts of the plant, for example in fat-containing seeds, tissue-specifically.
Many legumes transport fixed nitrogen in the form of ureides from the nodules to the above-ground tissue (Schubert, 1986, Ann. Rev.
Plant Phys. 37, 539-574). Ureides are formed via purine biosynthesis. It is possible with the aid of a node-(nodule-) specific promoter for AMP deaminase expression and thus ureide biosynthesis in legumes to be modified specifically, for example using the promoter of the phosphoribosyl-pyrophosphate amidotransferase from Glycine max (see also Genbank Accession Number U87999) or using another node-specific promoter (described for example in EP 249676.
In addition, constitutive expression of the exogenous AMP
deaminase gene is advantageous. However, on the other hand, inducible expression may also appear desirable.
Using the recombination and cloning techniques quoted above, the expression cassettes according to the invention can be cloned into suitable vectors which make it possible to replicate them, for example in E. coli. Suitable cloning vectors are, inter alia, pBR332 [sic], the pUC series, the Ml3mp series and pACYC184.
Binary vectors able to replicate both in E. coli and in agrobacteria are particularly suitable.

The invention further relates to the use of an expression cassette according to the invention for transforming plants, plant cells, plant tissues or parts of plants. The aim of the use is preferably to increase the AMP deaminase content in the plant.
This may involve, depending on the chosen promoter, expression specifically in the leaves, in the seeds or other parts of the plant. The present invention further relates to such transgenic plants, their propagation material and their plant cells, tissue or parts.
The expression cassette according to the invention can in addition be employed for transforming bacteria, cyanobacteria, yeasts, filamentous fungi and algae with the aim of producing adequate amounts of the enzyme AMP deaminase.

The invention further relates to a protein from Arabidopsis thaliana having the amino acid sequence SEQ ID N0:2 or derivatives or parts of this protein with AMP deaminase activity.
Compared with Saccharomyces cerevisiae, the homology at the amino 5 acid level is 43 - 47~ identity (see Figure 4).
The invention also relates to plant proteins having AMP deaminase activity with an amino acid sequence homology with the Arabidopsis thaliana AMP deaminase of 20 - 100 identity.
Plant proteins having AMP deaminase activity with an amino acid sequence homology with the Arabidopsis thaliana AMP deaminase of 50 - 100 identity are preferred.
Plant proteins having AMP deaminase activity with an amino acid sequence homology with the Arabidopsis thaliana AMP deaminase of 80 - 100 identity are particularly preferred.
As already mentioned, the AMP deaminase is a suitable target for herbicides. In order to be able to find even more efficient AMP
deaminase inhibitors, it is necessary to provide suitable test systems with which inhibitor-enzyme binding studies can be carried out. For this purpose, for example, the complete cDNA
sequence of the AMP dea:minase from Arabidopsis thaliana is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli (see Example 3).
The AMP deaminase protein expressed with the aid of the expression cassette according to the invention is particularly suitable for finding inhibitors specific for AMP deaminase.
To this end, the AMP deaminase can be employed, for example, in an enzyme assay in which the activity of the AMP deaminase is measured in the presence and absence of the agent to be tested.
Comparison of the two activity determinations allows a qualitative and quantitative statement to be made about the inhibiting behavior of the agent to be tested (see Example 4).
It is possible with the aid of the test system according to the invention to check a large number of chemical compounds rapidly and straightforwardly for herbicidal properties. The method allows reproducible selection from a large number of substances specifically those with great potency in order for further tests in depth, which are familiar to the skilled worker, then to be carried out with these substances.

The invention further relates to herbicides identifiable using the test system described above.
Overexpression of the gene sequence Seq ID N0: 1 coding for an AMP deaminase in a plant achieves increased resistance to AMP
deaminase inhibitors. The invention likewise relates to the transgenic plants produced in this way.
The efficiency of expression of the transgenically expressed AMP
deaminase gene can be measured, for example, in vitro by shoot meristem propagation or by a germination test. In addition, a change in the nature anal level of the expression of the AMP
deaminase gene and the effect thereof on the resistance to AMP
deaminase inhibitors ca.n be tested on test plants in glasshouse experiments.
The invention additionally relates to transgenic plants transformed with an expression cassette according to the invention, and to trans.genic cells, tissues, parts and propagation material of such plants. Particular preference is given in this connection to transgenic crop plants such as, for example, barley, wheat, rye, corn, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species, and legumes.
The invention further relates to plants which, after expression of the DNA-SEQ ID N0:1 in the plant, have an increased IMP
content.
An increased inosine 5'-phosphate (IMP) content means for the purpose of the present invention the artificially acquired capability of increasedl IMP biosynthesis owing to functional overexpression of the F~MP deaminase gene in the plant compared with the plant which ha.s not been genetically manipulated for the duration of at least one plant generation.
The invention is illustrated by the examples which now follow, but is not confined to these:
Examples A. Methods of genetic manipulation on which the examples are based:
General cloning method~o ~05~/48924 CA 02325054 2000-09-27 Cloning methods such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of Escherichia coli cells, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as described by Sambrook et al.
(1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
Sequence analysis of recombinant DNA
Recombinant DNA molecules were sequenced using a laser fluorescence DNA sequencer supplied by ABI by the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
Analysis of complete RNA from plant tissues Complete RNA from plant: tissues was isolated as described by Logemann et x1.((1987) Anal. Biochem. 163, 21). For the analysis, in each case 20 mg of RNA were fractionated in a formaldehyde-containing 1.5~ agarose gel and transferred to nylon membranes (Hybond, Amersham). Specific transcripts were detected as described by Amasino ((1986) Anal. Biochem. 152, 304). The cDNA fragments employed as probe were radiolabeled using a random primed DNA labeling kit: (Boehringer, Mannheim) and hybrized [sic]
by standard methods (see Hybond information for users, Amersham).
Hyridization [sic] signals were visualized by autoradiography using X-GMAT AR films supplied by Kodak.
The bacterial strains (E. coli, XL-I [sic] Blue) used hereinafter were purchased from Stratagene or Pharmacia in the case of NP66.
The agrobacterium strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850kan) has been described by Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777). Alternatively, it is also possible to use the agro- bacterium strain LBA4404 (Clontech) or other suitable strains. Vectors which can be used for cloning are pUCl9 (Vanish-Perron, Gene 3x(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 ;>cience 66 (1990) 221-230).
Example 1 PCR-Amplification of the AMP deaminase gene using synthetic oligonucleotides.
The Arabidopsis est clone coding for the AMP deaminase was purchased from the Arabidopsis Biological Resource Center (Ohio State University). This is a partial cDNA clone (T21250) which does not correspond to the full-length transcript of the AMP
deaminase. PCR amplific:ation of the arabidopsis AMP deaminase fragment was carried out in a DNA thermal cycler supplied by Perkin Elmer. The oligonucleotides used, 5' primer CGAGATAGCTCGTAAC and 3'' primer AGCCCACTCATATTATT, were taken from the established sequence. The reaction mixtures contained 8 ng/~1 genomic DNA from Eschez:ichia coli, 0.5 ~M of the appropriate oligonucleotides, 200 yM nucleotides (Pharmacia), 50 mM KC1, 10 mM tris-HC1 (pH 8.3 at 25~C, 1.5 mM MgCl2) and 0.02 U/~l Taq polymerase (Perkin Elmer).
The amplification conditions were set as follows:
Annealing temperature: 52~C, 1 min Denaturing temperature:. 92~C, 1 min Elongation temperature:. 72~C, 1.5 min Number of cycles: 40 The resulting fragment comprises a small part of the est clone T21250, which was used to carry out a heterologous screening of an Arabidopsis thaliana cDNA Bank (Stratagene). 3.0 x 105 lambda phages of the Arabidopsis thaliana cDNA library (Stratagene) were plated out on agar plal:es with E. coli XLI-Blue [sic] as bacterial strain. The phage DNA was transferred by standard methods (Sambrook et a7L. (1989); Cold Spring Harbor Laboratory Press: ISBN 0=87969-309-6) to nitrocellulose filters (Gelman Sciences) and fixed on the filters. The hybridization probes [sic] used was the PCR fragment described above, which were [sic]
radiolabeled using a multiprime DNA labeling system (Amersham Buchler) in the present:e of a-32P-dCTP (specific activity 3000 Ci/mmol) in accordance with the manufacturer's instructions.
Hybridization of the membranes took place after prehybridization at 60~C in 3 x SSPE, 0.1~ sodium dodecyl sulfate (w/v), 0.02 polyvinylpyrolidone [sic] (w/v), 0.02 Ficoll 400 (w/v) and 50 mg/ml calf thymus DNA for 12-16 hours (Sambrook et al. (1989);
Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). The filters were then washed in 2 x SSPE, 0.1$ sodium dodecyl sulfate (w/v) at 60~C for 60 minutes. Positively hybridizing phages were visualized by autoradiography and purified and isolated by standard techniques.

~~5~/48924 CA 02325054 2000-09-27 Example 2 Sequence analysis of the cDNA clones coding for a protein having AMP deaminase activity..
64 cDNA clones coding for a polypeptide with great homologies with yeast AMP deaminase resulted (see Figure 4). The two longest clones are identical and show in the restriction digest an EcoRI
fragment 2880 base pairs long. This longest clone coding for the AMP deaminase from Arabidopsis thaliana is called AMP1. The plasmid is referred to as pBS-AMP1. The cDNA (see Figure 2) has an open reading frame of 2578 base pairs with a stop codon in position 2579-2581. The' amino acid sequence starts with the third base after the linker sequences (bold-printed ends of the sequence in Figure 2) in the third reading frame and can be translated into a polypeptide 860 amino acids long, or, from the first methionine start codon, into a polypeptide of 839 amino acids (see Figure 3). Another alternative is to use the methionine in position 46, so that a polypeptide of 824 amino acids would result.
Whereas the protein aci:ivity is located in the cytosol in yeast, a cytosolic and a potentially mitochondrial activity has been described in plants (Yoshino and Murakami, 198, Z.
Pflanzenphysiologie, 99, 331-338). On the basis of the reading frame of the present cDNA sequence, it can be inferred that it might possibly be the mitochondrial form or the cytosolic form, either of which can arise by use of different start codons. It is noteworthy that with the start of the reading frame from base 3 of the coding sequence a large number of hydrophilic serine and threonine residues alternating with aliphatic amino acid residues are present in front o:E the methionine codons, which indicates a transit sequence for mitochondria or plastids. It is generally assumed that purine biosynthesis takes place in plastids.
However, it has recent:Ly been shown that the enzymes of purine biosynthesis are also :Located in mitochondria (Atkins et al., 1997, Plant Physiol. 113, 127-135). On the basis of the reading frame of the present cIDNA sequence, it is obvious to assume that it might possibly be t:he mitochondrial, plastidial or cytosolic form, each of which results by use of different start codons. The two longest clones show a continuous open reading frame with two potential start codons at the N terminus. Compared with the yeast sequence, the second start codon is approximately at the position where the yeast sequence also starts (see Figure 4). Over the entire length of the protein, the plant sequence is about 59-63~
similar to the yeast sequence and about 43-47~ identical, depending on the choice of the comparison parameters (Lasergene Software, MegAlign Program). It is moreover noticeable that the N
terminus of the plant AMP deaminase shows only little homology in contrast to the C terminus of the yeast sequence.
5 Example 3 Production of overexpression vectors in E. coli The following oligonucl.eotide sequences were derived from the 10 established sequence and were provided with a BamHI restriction cleavage site and to protruding bases. The oligonucleotides are underlined and numbered in Figure 2. Potential methionine start codons are shown bold.
15 1. 5' Primer aaggatccATGTTACTCTCTCTTCTGAG, 2. 5' Primer aaggatccATGGAACCCAATATTTAC, 3. 5' Primer aaggatccATGCATTTCAAGGCAC, 4. 3' Primer aaggatcc'.CTATGGAACAACTTCATCAG.
The use of the three different 5' primers in combination with the 3' primer lead [sic] to PCR products of different lengths (fragment I, II, III), which correspond to the complete reading frame (fragment I, primers 1 + 4), the sequence from the first Met start codon (fragment II, primers 2 + 4) and from the second start codon (fragment I:II, [lacuna] 3 + 4).
The PCR reaction [sic] mixtures contained 8 ng/~1 pBS-AMP1 DNA, 0.5 ~M of the appropriate oligonucleotides, 200 ~M nucleotides (Pharmacia), 50 mM KC1, 10 mM tris-HC1 (pH 8.3 at 25~C, 1.5 mM
MgCl2) and 0.02 U/~1 Ta.q polymerase (Perkin Elmer). The amplification conditions were set as follows:
Annealing temperature: 52~C, 1 min Denaturing temperature:. 92~C, 1 min Elongation temperature:: 72~C, 2.5 min Number of cycles: 30 The PCR fragments were cloned into the overexpression vectors pETlSb, pETlla and pQE9 and employed for protein production by means of IPTG induction by standard methods (see handbook: The Quiaexpressionist [sic] (1992), Quiagen [sic], Hilden).

Example 4 Enzyme assay of the plant AMP deaminase from E. coli resulting from overexpression cultures E.coli was disrupted by the pressure disruption method in a French Press under maximum pressure in a 20 ml pressure chamber or with the aid of a glass bead mill (IMA disintegrator). 10 ml of buffer (O.1M KHzP04; pH 7.5; 0.4M sucrose, 0.1 mM DTT) are used per 1 g of cell pellet. The pellet is disrupted by addition of 2.5 times the amount of glass beads (d=0.5 mm) in the glass bead mill at 4°C and 2500 rpm for 20 min. The disrupted material is .
centrifuged at 4°C and 100,000 g for 20 minutes. The enzyme activity was determined. in a photometric assay by measurement at 260 nm in a photometer (Uvikon 933, Kontron). The choice of the overexpression vectors also made it possible to purify the AMP
deaminase to homogeneity in one step via the histidine anchor by standard methods when D~TT was omitted from the disruption buffer (compare also the handbook: The Quiaexpressionist [sic], Quiagen [sic], Hilden).
The homogenate buffer was changed in the following medium by dialysis in 40 mM citrate, pH 6.5 (adjusted with 5 N NaOH), 0.05%
BSA (w/v), 100 mM KC1.
10-100 ~.1 portions of the enzyme fraction in the changed buffer were made up to 700 ~1 with buffer and, by addition of 100 ~1 of a 1 mM AMP solution, 0.5 mM ATP solution and 1 ~M diadenosine pentaphosphate solutior.~, the decrease in extinction was measured for 2-10 min using a reference cuvette with 700 ~.1 of reaction buffer and 100 ~1 of a protein homogenate from an untransformed E. coli culture. Identical amounts of total protein were employed for the measurements of: the reference relative to the measured value.
Example 5 Production of plant expression cassettes A 35S CaMV promoter wa:~ inserted as EcoRI-KpnI fragment (corresponding to nucleotides 6909-7437 of cauliflower mosaic virus (Franck et al. (1980) Cell 21, 285)) into the plasmid pBinl9 (Bevan et al. (7.980) Nucl. Acids Res. 12, 8711). The polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACHS (Gielen et al., (1984) EMBO J. 3, 835), nucleotides 11749-11939, was isolated as PvuII-HindIII fragment and, after addition of SphI linkers, cloned onto the PvuII cleavage site ~05~~48924 CA 02325054 2000-09-27 between the SpHI-HindI:CI (sic) cleavage site of the vector. The result was the plasmid pBinAR (HSfgen and Willmitzer (1990) Plant Science 66, 221-230). The PCR fragments I, II and III (see above) were cloned into the BamHI cleavage site of the pBinAR vector in both orientations and employed for transforming tobacco plants.
The resulting plasmids are referred to as pBinAMP-20, pBinaAMP-0, pBinAMP-0, pBinaAMP -0, pBinAMP+26, pBinaAMP+26 and correspond to the fragments I, II, III, described in Example 3, from the PCR
mixtures described abo~re.
Example 6 Production of transgenic tobacco plants The plasmids pBinAMP-20, pBinaAMP-20, pBinAMP-0, pBinaAMP-0, pBinAMP+26, pBinaAMP+2fi were transformed into Agrobacterium tumefaciens C58C1:pGV2:?60 (Deblaere et al, 1984, Nucl. Acids.
Res. 13, 4777-4788). Tobacco plants (Nicotiana tabacum cv. Samsun NN) were transformed using a 1:50 dilution of an overnight culture of a positively transformed agrobacterium colony in Murashige-Skoog medium {(1962) Physiol. Plant. 15, 473) with 2%
sucrose (2MS medium). Leaf disks from sterile plants (about 1 cm2 each) were incubated in a Petri dish with a 1:50 agrobacterium dilution for 5-10 minui~es. This was followed by incubation in the dark at 25°C on 2MS medium with 0.8% Bacto agar for 2 days. The cultivation was continued after 2 days with 16 hours light/8 hours dark and continued in a weekly rhythm on MS medium with 500 mg/1 Claforan (cefotaxime sodium), 50 mg/1 kanamycin, 1 mg/1 benzylaminopurine (BAP), 0.2 mg/1 naphthylacetic acid and 1.6 g/1 glucose. Growing shoots were transferred to MS medium with 2%
sucrose, 250 mg/1 Claforan and 0.8% Bacto agar.
Regenerated shoots are obtained on 2MS medium with kanamycin and Claforan and, after rooting, transferred to soil and, after cultivation for two wea_ks in a climate chamber with 16 hours light/8 hours dark rhythm at 60% humidity, investigated for foreign gene expression or changed metabolite contents and phenotypical growth features. Changed nucleotide contents can be determined, for example, by the method of Stitt et al. (1982, FEBS Letters, 145, 217-222).
Example 7 To demonstrate the tolerance of AMP deaminase-overexpressing transgenic tobacco plants to AMP deaminase inhibitors, the latter were treated with various amounts of coformycin or other AMP

1$
deaminase inhibitors. :Ct was possible to show in a glasshouse in all cases that the plants overexpressing an AMP deaminase show tolerance to the inhibitors employed by comparison with the control.

SEQUENZPfZOTOKOLL
<110> BASF AG
<120> AMP-Deaminase <130> 19814512.8 <140> 48924 <141> 1998-04-O1 <160> 2 <170> PatentIn Vers. 2.0 <210> 1 <211> 2880 <212> DNA
<213> Arabidopsis thaliana <220>
<221> CDS
<222> (78)..!2594) <400> 1 gaattcggca cgaggtctta ctctctcttc tgagttctga cctgagcaca cacacagaga 60 ttttgattgt gtcactc atg gaa ccc aat att tac caa ctt gcc ctc gcg 110 Met Glu Pro Asn Ile Tyr Gln Leu Ala Leu Ala get cta ttc gga get tcc ttc gtt get gtt tct ggg ttt ttc atg cat 158 Ala Leu Phe Gly Ala Ser Phe Val Ala Val Ser Gly Phe Phe Met His ttc aag gca ctg aat cta gtc ctt gag cgt ggt aag gag cgt aaa gag 206 Phe Lys Ala Leu Asn Leu Val Leu. Glu Arg Gly Lys Glu Arg Lys Glu aac cct gat gga gac gag cct caa. aat ccg acc ttg gtg agg cgg cgg 254 Asn Pro Asp Gly Asp Glu Pro Gln. Asn Pro Thr Leu Val Arg Arg Arg agc caa gtt aga agg aag gtt aat. gac caa tat ggt cgc agt cct get 302 Ser Gln Val Arg Arg Lys Val Asn Asp Gln Tyr Gly Arg Ser Pro Ala tct ctt cca gat gcc act cct ttt. acc gat ggt ggc ggc ggc ggc ggc 350 1.

Ser Leu Pro Asp Ala Thr Pro Phe Thr Asp Gly G1y Gly Gly Gly Gly ggt gat aca cga cgg agc aac ggt: cac gtt tat gtc gat gaa att cct 398 Gly Asp Thr Arg Arg Ser Asn Gly His Val Tyr Val Asp Glu Ile Pro cct ggt ctc cct agg ctt cat acct cca tct gaa ggg aga get tct gta 446 Pro Gly Leu Pro Arg Leu His Thr Pro Ser Glu Gly Arg Ala Ser Val cat gga get agt agt atc agg aaa act gga agc ttt gtt aga cca ata 494 His Gly Ala Ser Ser Ile Arg Lys Thr Gly Ser Phe Val Arg Pro Ile tct ccg aaa tcc cct gtt get agt: get agt get ttt gag agt gtg gaa 542 Ser Pro Lys Ser Pro Val Ala Ser Ala Ser Ala Phe G1u Ser Val Glu gaa tca gat gat gat gat aat ttc~ act aat agt gag ggt tta gat get 590 Glu Ser Asp Asp Asp Asp Asn Leu. Thr Asn Ser Glu Gly Leu Asp Ala tcc tac ttg caa get aat ggt gac aat gag atg cct gca gat get aat 638 Ser Tyr Leu Gln Ala Asn Gly Asp Asn Glu Met Pro Ala Asp Ala Asn gaa gaa caa ata tct atg get gct. tca agt atg att cga tcc cat agt 686 Glu Glu Gln Ile Ser Met Ala Ala. Ser Ser Met Ile Arg Ser His Ser gtg tct ggt gac tta cat gga gtt. cag ctg agt cct att get get gac 734 Val Ser Gly Asp Leu His Gly Val Gln Leu Ser Pro Ile Ala Ala Asp att ctt cgt aag gag cca gag caa. gag acc ttt gtc cgt ctt aat gtt 782 Ile Leu Arg Lys Glu Pro Glu Gln Glu Thr Phe Val Arg Leu Asn Val cct ctt gag gtg cca acg tcc gat. gaa gtt gaa gcc tat aaa tgt ctg 830 Pro Leu Glu Val Pro Thr Ser Asp Glu Val Glu Ala Tyr Lys Cys Leu caa gaa tgt ctt gaa ctg cgg aac~ agg tat gtc ttc caa gaa aca gtt 878 Gln Glu Cys Leu Glu Leu Arg Lys; Arg Tyr Val Phe Gln-Glu Thr Val gca cca tgg gaa aaa gaa gtc ata tct gat cct agt act cca aag cct 926 L.

Ala Pro Trp Glu Lys Glu Val Ile Ser Asp Pro Ser Thr Pro Lys Pro aat aca gag cca ttt gca cac tat cct cag gga aaa tct gat cat tgt 974 Asn Thr Glu Pro Phe Ala His Tyr Pro Gln Gly Lys Ser Asp His Cys ttt gag atg caa gat ggg gtt gtc cac gtg ttt gca aat aaa gat gca 1022 Phe Glu Met Gln Asp Gly Val Val His Val Phe Ala Asn Lys Asp Ala aaa gaa gat ctc ttc ccg gta get gat gcc aca gcg ttt ttc act gac 1070 Lys Glu Asp Leu Phe Pro Val Ala Asp Ala Thr Ala Phe Phe Thr Asp ttg cat cac gta ctc aaa gtc ata get gca gga aac atc cgg act ttg 1118 Leu His His Val Leu Lys Val Ile A1a Ala Gly Asn Ile Arg Thr Leu tgc cac cgt cga cta gtg ctc cta gaa cag aaa ttt aat ctc cat ttg 1166 Cys His Arg Arg Leu Val Leu Leu Glu Gln Lys Phe Asn Leu His Leu atg ctt aat gcg gat aaa gaa ttt ctt get caa aaa agt gca cca cat 1214 Met Leu Asn Ala Asp Lys Glu Phe Leu Ala Gln Lys Ser Ala Pro His cgt gat ttt tat aac gtt agg aaa gtc gac act cat gtg cat cat tca 1262 Arg Asp Phe Tyr Asn Val Arg Lys Val Asp Thr His Val His His Ser get tgc atg aac cag aaa cac ctt tta agg ttt att aag tca aag ctc 1310 Ala Cys Met Asn Gln Lys His Leu Leu Arg Phe Ile Lys Ser Lys Leu cgg aaa gaa ccc gat gag gtt gta ata ttc cga gat gga aca tat ttg 1358 Arg Lys Glu Pro Asp Glu Val Val Ile Phe Arg Asp Gly Thr Tyr Leu acc ttg aga gaa gtt ttt gag agc ctg gat ctg act gga tat gac ctg 1406 Thr Leu Arg Glu Val Phe Glu Ser Leu Asp Leu Thr Gly Tyr Asp Leu aac gtc gac ctt ttg gat gtt cat gca gac aaa agt acc ttt cat cgt 1454 Asn Val Asp Leu Leu Asp Val His Ala Asp Lys Ser Thr Phe His Arg ttt gat aag ttc aac cta aag tat aac cct tgt ggt caa agt agg ctt 1502 Phe Asp Lys Phe Asn Leu Lys Tyr Asn Pro Cys Gly Gln Ser Arg Leu agg gag att ttc ctt aaa cag gat: aat ctc atc caa ggt cga ttt ctt 1550 Arg Glu Ile Phe Leu Lys Gln Asp Asn Leu Ile Gln Gly Arg Phe Leu 480 485 ~ 490 ggt gag ata aca aag eaa gtc ttc; tet gac ctt gaa get agt aaa tat 1598 Gly Glu Ile Thr Lys Gln Val Phe Ser Asp Leu Glu Ala Ser Lys Tyr eag atg get gaa tac aga ata tet. ata tat gge aga aaa atg age gag 1646 Gln Met Ala Glu Tyr Arg Ile Ser Ile Tyr Gly Arg Lys Met Ser Glu tgg gac caa ctc get agt tgg att gtg aac aat gat cta tac agt gag 1694 Trp Asp Gln Leu Ala Ser Trp Ilea Val Asn Asn Asp Leu Tyr Ser G1u aat gtt gtc tgg tta att cag ctc cca cgc ttg tac aac att tac aag 1742 Asn Val Val Trp Leu Ile Gln Leu. Pro Arg Leu Tyr Asn Ile Tyr Lys gac atg ggt att gtg aca tcg ttc cag aat atc ctg gac aat ata ttc 1790 Asp Met Gly Ile Val Thr Ser Phe Gln Asn Ile Leu Asp Asn Ile Phe att cct ctg ttt gaa gcc acg gta. gat cct gat tcc cat cct cag ctc 1838 Ile Pro Leu Phe Glu Ala Thr Val Asp Pro Asp Ser His Pro Gln Leu cat gtt ttt ttg aag cag gtt gtt gga ttt gat ttg gtt gat gat gaa 1886 His Val Phe Leu Lys Gln Val Val Gly Phe Asp Leu Val Asp Asp Glu agc aaa cct gaa aga cgt cce aca. aaa cac atg cce act eca get caa 1934 Ser Lys Pro Glu Arg Arg Pro Thr Lys His Met Pro Thr Pro Ala Gln tgg act aac gca ttc aat cct gca. ttt tcg tat tat gtc tac tat tgt 1982 Trp Thr Asn Ala Phe Asn Pro Ala. Phe 5er Tyr Tyr Val Tyr Tyr Cys tat get aac ctc tat gtg tta aat aag ctt cga gag tca aag ggc atg 2030 Tyr Ala Asn Leu Tyr Val Leu Asn. Lys Leu Arg Glu Ser Lys Gly Met act act ate acg eta cga cca cat. tct gga gag get ggt gac att gac 2078 9:

Thr Thr Ile Thr Leu Arg Pro His Ser Gly Glu Ala Gly Asp Ile Asp cac ttg get get acg ttt cta aca tgc cat agc atc gca cat gga atc 2126 His Leu Ala Ala Thr Phe Leu Thr Cys His Ser Ile Ala His Gly Ile 670 67'_i 680 aat ctg cga aag tct cct gtg ctt: cag tat ctg tac tac ctc gcc cag 2174 Asn Leu Arg Lys Ser Pro Val Leu Gln Tyr Leu Tyr Tyr Leu Ala Gln att ggt ctg gcc atg tca cca ctc~ agc aac aac tct ttg ttt cta gat 2222 Ile Gly Leu Ala Met Ser Pro Leu Ser Asn Asn Ser Leu Phe Leu Asp tac cac cgg aac ccg ttt cct gtg ttt ttc tta aga ggt ctc aat gtt 2270 Tyr His Arg Asn Pro Phe Pro.Val Phe Phe Leu Arg Gly Leu Asn Val tct ctg tct act gat gac ccc ctt cag att cac tta act aaa gaa cct 2318 Ser Leu Ser Thr Asp Asp Pro Leu. Gln Ile His Leu Thr Lys Glu Pro ctc gtg gaa gag tat agc ata get gca tca gtt tgg aag ctg agt gcg 2366 Leu Val Glu Glu Tyr Ser Ile A1a Ala Ser Val Trp Lys Leu Ser Ala tgt gac ctg tgc gag ata get cgt aac tca gtg tac cag tca ggt ttc 2414 Cys Asp Leu Cys Glu Ile Ala Arg Asn Ser Val Tyr Gln Ser Gly Phe tca cac gcc ctg aag tcg cac tgg att gga aaa gat tac tac aaa aga 2462 Ser His Ala Leu Lys Ser His Trp Ile Gly Lys Asp Tyr Tyr Lys Arg gga cct gat gga aac gac att cac aaa aca aac gtg cca cac ata agg 2510 Gly Pro Asp Gly Asn Asp Ile His Lys Thr Asn Val Pro His Ile Arg gtg gag ttc cgt gac acg atc tgg aaa gag gag atg caa cag gtt tat 2558 Val Glu Phe Arg Asp Thr Ile Trp Lys Glu Glu Met Gln Gln Val Tyr ctg ggc aag get gtt atc tct gat gaa gtt gtt cca taaaaaccac 2604 Leu Gly Lys Ala Val Ile Ser Asp Glu Val Val Pro aatcagaaat ggcaagacgt aagaatccaa catattgcag gggaacaaag agagcatttt 2664 gagaagtata cgaaagcagg aacctagtag atagggtaat aatatgagtg gctctgtgcc 2724 ctgagaaagc gattaggctg tgccaaaatc ttattgtttt ataaagcttt ttagataatg 2784 agatacaaag agaccagttg agaaccggtt ttaatataat ggatttcagt ttttggatta 2844 aaaaaaaaaa aaaaaaaaaa acctcgtgcc gaattc 2880 <210> 2 <211> 839 <212> PRT
<213> Arabidopsis thaliana <400> 2 Met Glu Pro Asn Ile Tyr Gln Leu Ala Leu Ala Ala Leu Phe Gly Ala Ser Phe Val Ala Val Ser Gly Phe Phe Met His Phe Lys Ala Leu Asn Leu Val Leu Glu Arg Gly Lys Glu Arg Lys Glu Asn Pro Asp Gly Asp Glu Pro Gln Asn Pro Thr Leu Val Arg Arg Arg Ser Gln Val Arg Arg Lys Val Asn Asp Gln Tyr Gly Arg Ser Pro Ala Ser Leu Pro Asp Ala Thr Pro Phe Thr Asp Gly Gly Gly Gly Gly Gly Gly Asp Thr Arg Arg Ser Asn Gly His Val Tyr Val Asp Glu Ile Pro Pro Gly Leu Pro Arg Leu His Thr Pro Ser Glu Gly Arg Ala Ser Val His Gly Ala Ser Ser Ile Arg Lys Thr Gly Ser Phe Val Arg Pro Ile Ser Pro Lys Ser Pro Val Ala Ser Ala Ser Ala Phe Glu Ser Val Glu Glu Ser Asp Asp Asp Asp Asn Leu Thr Asn Ser Glu Gly Leu Asp Ala Ser Tyr Leu Gln Ala 165 170 ~ 175 Asn Gly Asp Asn Glu Met Pro Ala. Asp Ala Asn Glu Glu Gln Ile Ser Met Ala Ala Ser Ser Met Ile Argr Ser His Ser Val Ser Gly Asp Leu His Gly Val Gln Leu Ser Pro Ilea Ala Ala Asp Ile Leu Arg Lys Glu Pro Glu Gln Glu Thr Phe Val Arg~ Leu Asn Val Pro Leu Glu Val Pro Thr Ser Asp Glu Val Glu Ala Tyr Lys Cys Leu Gln Glu Cys Leu Glu Leu Arg Lys Arg Tyr Val Phe Gln. Glu Thr Val Ala Pro Trp Glu Lys Glu Val Ile Ser Asp Pro Ser Thr Pro Lys Pro Asn Thr Glu Pro Phe Ala His Tyr Pro Gln Gly Lys Ser Asp His Cys Phe Glu Met Gln Asp Gly Val Val His Val Phe Ala Asn. Lys Asp Ala Lys Glu Asp Leu Phe Pro Val Ala Asp Ala Thr Ala Phe Phe Thr Asp Leu His His Val Leu Lys Val Ile Ala Ala Gly Asn Ile Arg Thr Leu Cys His Arg Arg Leu Val Leu Leu Glu Gln Lys Phe Asn. Leu His Leu Met Leu Asn Ala Asp Lys Glu Phe Leu Ala Gln Lys Ser Ala Pro His Arg Asp Phe Tyr Asn Val Arg Lys Val Asp Thr His Val. His His Ser Ala Cys Met Asn Gln Lys His Leu Leu Arg Phe I1e Lysc Ser Lys Leu Arg Lys Glu Pro Asp Glu Val Val Ile Phe Arg Asp Gly Thr Tyr Leu Thr Leu Arg Glu Val Phe Glu Ser Leu Asp Leu Thr Gly Tyr Asp Leu Asn Val Asp Leu Leu Asp Val His Ala Asp Lys Ser Thr Phe His Arg Phe Asp Lys Phe Asn Leu Lys Tyr Asn Pro Cys Gly Gln Ser Arg Leu Arg Glu Ile Phe Leu Lys Gln Asp Asn Leu Ile Gln Gly Arg Phe Leu Gly Glu Ile Thr Lys Gln Val Phe Ser Asp Leu Glu Ala Ser Lys Tyr Gln Met Ala Glu Tyr Arg Ile Ser Ile Tyr Gly Arg Lys Met Ser Glu Trp Asp Gln Leu Ala Ser Trp Ile Val Asn Asn Asp Leu Tyr Ser Glu Asn Val Val Trp Leu Ile Gln Leu Pro Arg Leu Tyr Asn Ile Tyr Lys Asp Met Gly Ile Val Thr Ser Phe Gln Asn Ile Leu Asp Asn Ile Phe Ile Pro Leu Phe Glu Ala Thr Val Asp Pro Asp Ser His Pro Gln Leu His Val Phe Leu Lys Gln Val Val Gly Phe Asp Leu Val Asp Asp Glu Ser Lys Pro Glu Arg Arg Pro Thr Lys His Met Pro Thr Pro Ala Gln Trp Thr Asn Ala Phe Asn Pro Ala Phe Ser Tyr Tyr Val Tyr Tyr Cys Tyr Ala Asn Leu Tyr Val Leu Asn Lys Leu Arg Glu Ser Lys Gly Met Thr Thr Ile Thr Leu Arg Pro His Ser Gly Glu Ala Gly Asp Ile Asp His Leu Ala Ala Thr Phe Leu Thr Cys His Ser Ile Ala His Gly Ile Asn Leu Arg Lys Ser Pro Val Leu Gln Tyr Leu Tyr Tyr Leu A1a Gln Ile Gly Leu Ala Met Ser Pro Leu Ser Asn Asn Ser Leu Phe Leu Asp Tyr His Arg Asn Pro Phe Pro Val Phe Phe Leu Arg Gly Leu Asn Val Ser Leu Ser Thr Asp Asp Pro Leu Gln Ile His Leu Thr Lys Glu Pro Leu Val Glu Glu Tyr Ser Ile Ala Ala Ser Val Trp Lys Leu Ser Ala Cys Asp Leu Cys Glu Ile Ala Arg Asn Ser Val Tyr Gln Ser Gly Phe Ser His Ala Leu Lys Ser His Trp Ile Gly Lys Asp Tyr Tyr Lys Arg Gly Pro Asp Gly Asn Asp Ile His Lys Thr Asn Val Pro His Ile Arg Val Glu Phe Arg Asp Thr Ile Trp Lys Glu Glu Met Gln Gln Val Tyr Leu Gly Lys Ala Val Ile Ser Asp Glu Va1 Val Pro

Claims (23)

We claim:

1. A DNA sequence comprising the coding region of a plant AMP
deaminase, wherein this DNA sequence has the nucleotide sequence SEQ ID NO:1.

2. A DNA sequence which hybridizes with the DNA sequence SEQ ID
N0: 1 as claimed in claim 1 or parts thereof or derivatives which are derived from this sequence by insertion, deletion or substitution, and codes for a protein which has the biological activity of an AMP deaminase.

3. An expression cassette comprising regulatory nucleic acid sequences and a DNA sequence as claimed in claim 1 or 2.

4. A protein having AMP deaminase activity and comprising an amino acid sequence which represents a partial sequence of at least 100 amino acids from SEQ ID NO: 2.

5. A protein as claimed in claim 4, which comprises as amino acid sequence the partial sequence 50-750 from SEQ ID NO: 2.

6. A protein as claimed in claim 5, which comprises as amino acid sequence the sequence depicted in SEQ ID NO: 2.

7. A bacterium comprising a DNA sequence as claimed in claim 1 or 2 or parts thereof or derivatives derived from these sequences by insertion, deletion or substitution.

8. A yeast comprising a DNA sequence as claimed in claim 1 or 2 or parts thereof or derivatives derived from these sequences by insertion, deletion or substitution.

9. A plant cell comprising an expression cassette as claimed in claim 3.

10. A plant comprising an expression cassette as claimed in claim 3.

11. The use of DNA sequences as claimed in claim 1 or 2 or parts or derivatives derived from these sequences by insertion, deletion or substitution for introduction into pro- or eukaryotic cells, these sequences being linked where appropriate to control elements which ensure transcription and translation in the cells and leading to the expression of a translatable mRNA which brings about the synthesis of an AMP

deaminase.

12. The use of the expression cassette as claimed in claim 3 for transforming plants.

13. The use of the expression cassette as claimed in claim 3 for producing a test system for identifying AMP deaminase inhibitors.

14. The use of a plant comprising an expression cassette as claimed in claim 3 for preparing AMP deaminase.

15. The use of the expression cassette as claimed in claim 3 for producing plants with increased resistance to AMP deaminase inhibitors by enhanced expression of a DNA sequence as claimed in claim 1 or 2.

16. The use of the expression cassette as claimed in claim 3 for producing plants with an increased content of IMP.

17. A test system based on the expression of an expression cassette as claimed in claim 3 for identifying AMP deaminase inhibitors.

18. A herbicidal agent which can be identified using a test system as claimed in claim 16 [sic].

19. A process for transforming a plant, which comprises introducing an expression cassette as claimed in claim 3 into a plant cell, into callus tissue, a whole plant or protoplasts from plant cells.

20. A process for producing plants with increased resistance to AMP deaminase inhibitors by enhanced expression of a DNA
sequence as claimed in claim 1 or 2.

21. A process for producing plants with an increased content of IMP by enhanced expression of a DNA sequence as claimed in claim 1 or 2.

22. A plant with increased resistance to AMP deaminase inhibitors, comprising an expression cassette as claimed in claim 3.

23. A plant with an increased content of IMP, comprising an expression cassette as claimed in claim 3.