WO2001025457A2 - Gmp-synthetase aus pflanzen - Google Patents
Gmp-synthetase aus pflanzen Download PDFInfo
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- WO2001025457A2 WO2001025457A2 PCT/EP2000/009245 EP0009245W WO0125457A2 WO 2001025457 A2 WO2001025457 A2 WO 2001025457A2 EP 0009245 W EP0009245 W EP 0009245W WO 0125457 A2 WO0125457 A2 WO 0125457A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
Definitions
- methylxanthines such as caffeine and theobromine, in particular in plant families of the Rubiaceae and Theaceae.
- IMP phosphoribosyl pyrophosphate
- PRPP phosphoribosyl pyrophosphate
- IMP is synthesized in a 10-step reaction sequence. In subsequent reactions, IMP can be converted to AMP by adenylosuccinate synthetase and adenylosuccinate lyase.
- IMP is first converted to XMP by IMP dehydrogenase, which is aminated to GMP by GMP synthetase, see Fig. 1.
- GMP-synthetase Genes coding for GMP-synthetase have been isolated from different organisms.
- GMP synthetase also plays a role in the balanced synthesis of guanosine nucleotides and adenosine nucleotides, since ATP is a substrate of GMP synthetase.
- the object of the present invention was to demonstrate that GMP synthetase is a suitable herbicidal target in plants, the isolation of a complete plant cDNA coding for the enzyme GMP synthetase and its functional expression in bacterial or eukaryotic cells, and the production of an efficient and simple GMP synthetase test system for performing inhibitor-enzyme binding studies.
- the object was achieved by isolating a gene coding for the plant enzyme GMP synthetase, the production of antisense constructs of the GMP synthetase, and the functional expression of the GMP synthetase in bacterial or eukaryotic cells.
- One object of the present invention relates to the isolation of a full-length cDNA coding for a functional glutamine-hydrolyzing GMP synthetase (EC 6.3.5.2.) From tobacco (Nicotiana tabacum).
- a first object of the present invention is a DNA sequence SEQ-ID NO: 1 containing the coding region of a plant GMP synthetase from tobacco, see Example 1.
- Another object of the invention is a DNA sequence SEQ-ID No. 3 containing a part of the coding region of a plant GMP synthetase from Physcomitrella patens, see Example 2.
- the invention furthermore relates to 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 code for a protein which has the biological activity of a GMP synthetase.
- Nicotiana tabacum cv. Samsun NN which carry an antisense construct of GMP synthetase, have been characterized in more detail.
- the plants show growth retardation to different degrees.
- the transgenic lines as well as the descendants as 1st and 2nd generation showed a reduced growth in soil.
- a reduced amount of GMP-7M RNA compared to the wild type could be detected in the Northern hybridization.
- a reduced amount of GMP synthetase in the transgenic lines could be detected in the Western blot experiment compared to wild type plants, see Example 7.
- a correlation between growth retardation and reduction of the GMP synthetase protein amount can be determined. This clear connection clearly shows GMP synthetase for the first time as a suitable target protein for herbicidal active ingredients.
- the expression cassette containing a DNA sequence SEQ-ID No. 1 are expressed, for example, in other bacteria, in yeasts, fungi, algae, plant cells, insect cells or mammalian cells, see Example 5.
- the GMP synthetase protein expressed with the aid of the expression cassette according to the invention is particularly suitable for the detection of inhibitors specific for GMP synthetase.
- the plant GMP synthetase can be used, for example, in an enzyme test in which the activity of the GMP synthetase is determined in the presence and absence of the active substance to be tested. By comparing the two activity determinations, a qualitative and quantitative statement about the Make inhibitory behavior of the active substance to be tested, see Example 8.
- test system With the help of the test system according to the invention, a large number of chemical compounds can be checked quickly and easily for herbicidal properties.
- the method makes it possible to selectively reproducibly select those with great potency from a large number of substances, in order to subsequently carry out further in-depth tests known to the person skilled in the art.
- Another object of the invention is a method for identifying substances with herbicidal activity which inhibit GMP synthetase activity in plants, consisting of
- transgenic plants, plant tissues or plant cells which contain an additional DNA sequence coding for an enzyme with GMP synthetase activity and are capable of overexpressing an enzymatically active GMP synthetase;
- Another object of the invention is a method for the identification of inhibitors of plant GMP synthetases, with potentially herbicidal activity by the gene of a plant GMP synthetase is cloned, overexpressed in a suitable expression cassette - for example in insect cells -, the cells are opened and the cell extract is used directly or after enrichment or isolation of the enzyme GMP synthetases in a test system for measuring the enzyme activity in the presence of low molecular weight chemical compounds ,
- the invention further relates to compounds with herbicidal activity which can be identified using the test system described above.
- Another object of the invention is a method for eliminating undesirable plant growth, characterized in that the plants to be removed are treated with a compound which specifically binds to plant GMP synthetase and inhibits their function.
- Inhibitors of GMP synthetase with herbicidal activity can be used as defoliants, desiccants, haulm killers and in particular as weed killers. Weeds in the broadest sense are understood to mean all plants that grow up in places where they are undesirable. Whether the active ingredients found with the aid of the test system according to the invention act as total or selective herbicides depends, among other things, on the amount used.
- Inhibitors of GMP synthetase with herbicidal activity can be used, for example, against the following weeds:
- the invention also relates to expression cassettes, the sequence of which codes for a GMP synthetase from tobacco or its functional equivalent.
- the nucleic acid sequence can be, for example, a DNA or a cDNA sequence.
- the invention also relates to an expression cassette containing a DNA sequence SEQ-ID No. 3 coding for part of the plant GMP synthetase from Physcomitrella patens.
- an expression cassette according to the invention also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell.
- an expression cassette according to the invention comprises upstream, i.e. at the 5 'end of the coding sequence, a promoter and downstream, i.e. at the 3 'end, a polyadenylation signal and optionally further regulatory elements which are operatively linked to the intervening coding sequence for the GMP synthetase gene.
- An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence.
- An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable GMP synthetase DNA sequence and a polyadenylation signal using conventional recombination and cloning techniques, as described, for example, in J. Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Curent Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
- the invention also relates to functionally equivalent DNA sequences which code for a GMP synthetase and which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO: 1 or SEQ-ID No. 3 have from 40 to 100%.
- the preferred subject of the invention are functionally equivalent DNA sequences which code for a GMP synthetase and which have a sequence homology based on the total length of the DNA sequence with the DNA sequence SEQ-ID NO: 1 or SEQ-ID No. 3 have from 60 to 100%.
- a particularly preferred object of the invention are functionally equivalent DNA sequences which code for a GMP synthetase and which, based on the total length of the DNA sequence, have a sequence homology with the DNA sequence SEQ-ID NO: 1 or SEQ-ID No. 3 have from 80 to 100%.
- Functionally equivalent sequences which code for a GMP synthetase are, according to the invention, those sequences which, despite a different nucleotide sequence, still have the desired functions.
- Functional equivalents thus include naturally occurring variants of the sequences described here as well as artificial, e.g. nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a plant.
- a functional equivalent is also understood to mean, in particular, natural or artificial mutations of an originally isolated sequence coding for a GMP synthetase, which furthermore shows the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
- the present invention also includes those nucleotide sequences which are obtained by modifying this nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also the insertion of further restriction enzyme interfaces.
- Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.
- the expression cassette according to the invention can also be used to transform bacteria, cyanobacteria, yeasts, filamentous fungi and algae with the aim of producing sufficient quantities of the enzyme GMP synthetase.
- Another object of the invention is a protein from tobacco characterized by the amino acid sequence SEQ-ID NO: 2 or derivatives or parts of this protein with GMP synthetase activity.
- the invention also relates to vegetable proteins with GMP synthetase activity with an amino acid sequence homology to the tobacco GMP synthetase of 20-100% identity. Vegetable proteins with GMP synthetase activity with an amino acid sequence homology to the tobacco GMP synthetase of 50-100% identity are preferred.
- Vegetable proteins with GMP synthetase activity with an amino acid sequence homology to the tobacco GMP synthetase of 80-100% identity are particularly preferred.
- the invention also relates to vegetable proteins with GMP synthetase activity with an amino acid sequence homology to the Physcomitrella patens GMP synthetase of 20-100% identity.
- Vegetable proteins with GMP synthetase activity with an amino acid sequence homology to the Physcomitrella patens GMP synthetase of 50-100% identity are preferred.
- Vegetable proteins with GMP synthetase activity with an amino acid sequence homology to the Physcomitrella patens GMP synthetase of 80-100% identity are particularly preferred.
- Another object of the invention was the overexpression of the GMP synthetase gene in plants for the production of plants which are tolerant of inhibitors of GMP synthetase.
- the effectiveness of the expression of the transgenically expressed GMP synthetase gene can be determined, for example, in vitro by proliferation or by a germination test.
- a change in the type and level of expression of the GMP synthetase gene and its effect on the resistance to inhibitors of GMP synthetase on test plants can be tested in greenhouse experiments.
- the invention also relates to transgenic plants, transformed with an expression cassette according to the invention, containing the DNA sequence SEQ-ID No. 1, which by additional expression of the DNA sequence SEQ-ID No. 1 have become tolerant of inhibitors of GMP synthetase, as well as transgenic cells, tissues, parts and propagation material of such plants.
- Transgenic crop plants such as e.g. Barley, wheat,
- a change in the nucleotide content in plants can be useful in various cases.
- Plant-based baby food products are added with nucleotides, for example, in order to achieve a nutritional composition that corresponds to breast milk.
- an optimized nucleotide content would be useful in the case of enteral feeding of patients.
- a reduced purine nucleotide content in nutritionally relevant plants is relevant for the dietary nutrition of patients with gout.
- Nucleotides also have a taste-enhancing and flavor-enhancing effect, so that a changed nucleotide content affects the taste properties of plants.
- the invention therefore furthermore relates to plants which, after expression of the DNA sequence SEQ-ID NO: 1 or SEQ-ID No: 3, have a modified content of guanosine nucleotides in the plant.
- a plant with a modified guanosine nucleotide content is, for example, expressed by expressing an additional DNA sequence SEQ-ID No. 1 or 3 produced in sense or antisense orientation in the plant.
- Modified content of guanosine nucleotides means that both plants with an increased content of guanosine nucleotides with sense orientation and also plants with a reduced content of guanosine nucleotides with sense orientation (cosuppression) or antisense orientation can be produced.
- increasing the content of guanosine nucleotides means, for example, the artificially acquired ability of an increased biosynthetic capacity for guanosine nucleotides by functional overexpression of the GMP synthetase gene in the plant compared to the non-genetically modified plant for at least one plant generation.
- Another object of the invention is the use of plant GMP synthetases to change the concentrations of methylxanthines in plants.
- sequences which target in the apoplasts, plastids, the vacuole, the mitochondrium, the endoplasmic reticulum (ER) or, owing to the lack of corresponding operative sequences, remain in the compartment of the ent Stand, the cytosol, ensure Kermode, Crit. Rev. Plant Sei. 15, 4 (1996), 285-423).
- the plant expression cassette can be built into the Tobacco transformation vector pBinAR, see Example 6.
- any promoter is suitable as promoters of the expression cassette according to the invention which express the expression of
- a plant promoter or a plant virus-derived promoter is preferably used.
- the CaMV 35S promoter from the cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294) is particularly preferred. This promoter contains different ones
- the expression cassette according to the invention can also contain a chemically inducible promoter, by means of which the expression of the exogenous GMP synthetase gene in the plant can be controlled at a specific point in time.
- a chemically inducible promoter by means of which the expression of the exogenous GMP synthetase gene in the plant can be controlled at a specific point in time.
- promoters as e.g. the PRPl promoter (Ward et al., Plant. Mol. Biol. (1993) 22,
- promoters are particularly preferred which ensure expression in tissues or parts of plants in which
- Promoters that ensure leaf-specific expression should be mentioned in particular.
- the promoter of the cytosolic FBPase from potatoes or the ST-LSI promoter from potatoes should be mentioned (Stockhaus et al., EMBO J., (1989) 8, 2445-2451).
- a foreign protein can be stably expressed up to a proportion of 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio / Technology (1995) 10, 1090-1094).
- the expression cassette according to the invention can therefore, for example, be a seed-specific promoter (preferably the phaseolin Promoter, the USP or LEB4 promoter), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
- a seed-specific promoter preferably the phaseolin Promoter, the USP or LEB4 promoter
- the LEB4 signal peptide the gene to be expressed and an ER retention signal.
- the inserted nucleotide sequence coding for a GMP synthase can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components.
- synthetic nucleotide sequences with codons are generated which are preferred by plants. These codons preferred by plants can be determined from codons with the highest protein frequency, which are expressed in most interesting plant species.
- various DNA fragments can 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.
- adapters or linkers can be attached to the fragments.
- artificial DNA sequences are suitable as long as, as described above, for example, they impart the desired property of increasing the content of guanosine nucleotides in the plant by overexpressing the GMP synthetase gene in crop plants.
- Such artificial DNA sequences can be determined, for example, by back-translation of proteins constructed using molecular modeling, which have GMP synthetase activity, or by in vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence in accordance with the codon usage specific for the host plant are particularly suitable. The specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other, known genes of the plant to be transformed.
- Sequences which code for fusion proteins are to be mentioned as further suitable nucleic acid sequences according to the invention, part of the fusion protein being a plant GMP synthetase polypeptide or a functionally equivalent part thereof.
- the second part of the fusion protein can be, for example, a further polypeptide with enzymatic activity or an antigenic polypeptide sequence with the aid of which it is possible to detect GMP synthetase expression (for example myc-tag or his-tag).
- this is preferably a regulatory protein sequence, such as a signal or transit peptide, which directs the GMP synthetase protein to the desired site of action.
- the promoter and terminator regions according to the invention should expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
- the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
- the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
- the promoter according to the invention can be native or homologous as well as foreign or heterologous to the host plant.
- the expression cassette according to the invention contains in the 5 '-3' transcription direction the promoter according to the invention, any sequence and a region for the transcriptional termination. Different termination areas are interchangeable.
- Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (Oc opinion synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J 3 (1984) 835 ff) or functional equivalents.
- an expression cassette according to the invention is installed as an insert in a recombinant vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration.
- Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology” (CRC Press), Chap. 6/7, p.71-119.
- transformation The transfer of foreign genes into the genome of a plant is called transformation.
- the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene cannon, the electroporation, the incubation of dry embryos in DNA-containing solution, the microinjection and the 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 SD Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol.
- the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
- Agrobacteria transformed with an expression cassette according to the invention can also be used in a known manner to transform plants, in particular crop plants, such as cereals, maize, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa , Lettuce and the various tree, nut and wine species and legumes can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
- crop plants such as cereals, maize, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa , Lettuce and the various tree, nut and wine species and legumes can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
- the biosythesis site of pyrimidines is generally the leaf tissue, so that leaf-specific expression of the GMP synthetase gene is useful.
- the pyrimidine biosynthesis need not be restricted to the leaf tissue, but can also be carried out in a tissue-specific manner in all other parts of the plant, for example in fatty seeds.
- constitutive expression of the exogenous GMP synthetase gene is advantageous.
- inducible expression may also appear desirable.
- the expression cassettes according to the invention can be cloned into suitable vectors that enable their multiplication, for example in E. coli, to be cloned.
- suitable cloning vectors include pBR322, pUC series, Ml3mp series and pA-CYC184.
- Binary vectors which can replicate both in E. coli and in agrobacteria are particularly suitable.
- Another object of the invention relates to the use of an expression cassette according to the invention for the transformation of plants, plant cells, plant tissues or parts of plants.
- the aim of the use is preferably to increase the GMP synthetase content in the plant.
- the expression can take place specifically in the leaves, in the seeds or in other parts of the plant.
- Such transgenic plants, their reproductive material and their plant cells, tissue or parts are a further subject of the present invention.
- Cloning methods such as Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of Escherichia coli cells, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
- the sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to 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 in order to avoid polymerase errors in constructs to be expressed.
- H0 treated, pyrogen-free water
- DNA-modifying enzymes and molecular biology kits were developed by the companies AGS (Heidelberg), Amersham (Braunschweig), Biometra (Göttingen), Röche (Mannheim), Genomed (Bad Oeynhausen), New England Biolabs (Schwalbach / Taunus), Novagen ( Madison, Wisconsin, USA), Perkin-Elmer (further Stadt), Pharmacia (Freiburg) Qiagen (Hilden) and Stratagene (Heidelberg). Unless otherwise stated, they were used according to the manufacturer's instructions.
- E. coli, XL-1 Blue The bacterial strains used below (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 Agrobacterium strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850kan) was developed by Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777).
- the LBA4404 agrobacterial strain (Clontech) or other suitable strains can be used.
- the vectors pUC19 (Yanish-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 (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230).
- EST expressed sequence tag
- the oligonucleotides 5'-aag gat cca agc tct aag acc cta tcc-3 x and 5 v -tta gat ctt tat tcc cat tcg atg g-3 ⁇ were used for polymerase amplification Chain reaction (PCR) of a 1000 bp cDNA frag with EST F14426 used as a template in a DNA thermal cycler from Perkin Elmer.
- PCR polymerase amplification Chain reaction
- the reaction mixtures contained 0.1 ng / ⁇ l cDNA from tobacco, 0.5 ⁇ M of the corresponding oligonucleotides, 200
- the amplification conditions were set as follows:
- the fragment was used to screen a cDNA library from Nicotiana tabacum callus tissue (variety Samsun NN) in the vector ZAP Express.
- Nicotiana tabacum callus tissue variety Samsun NN
- the PCR fragment described above was used as the hybridization probe and was radioactively labeled with the aid of a “Multiprime DNA labeling System” (Amersham Buchler) in the presence of ⁇ - 32 P-dCTP (specific activity 3000 Ci / mmol) according to the manufacturer's instructions.
- the membranes were hybridized after prehybridization at 60 ° C. in 3 ⁇ 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 (Sambrook et al.
- GMP-6 represents a partial clone which is 5'-sided shortened by 217 nucleotides compared to GMP-7M.
- GMP-7M is similar to GMP synthetics from microorganisms and animals.
- the greatest similarity (62%) is to a GMP synthetase from Helicobacter pylori. It is also striking that the similarities between the C-termini of the GMP synthetases are greater than those in the area of the N-termini.
- the N-terminus of the GMP-7M amino acid sequence corresponds to the N-termini of GMP synthetases from other organisms, such as E. coli and Synechocystis sp. (Table 1) .
- GMP-7M has no pronounced signal sequences (determined by the program PSORT, Nakai, K., Institute for Molecular and Cellular Biology, Osaka University, Japan), which could indicate a cytosolic localization of the protein.
- Table 1 The N-terminus of the GMP-7M amino acid sequence corresponds to the N-termini of GMP synthetases from other organisms, such as E. coli and Synechocystis sp.
- GMP-7M GMP-7M
- Arabidopsis thaliana gua_A_est_A. T, Genbank No. F14426
- E. coli guaA_e.c, Genbank No. 146276
- Synechocystis sp. guaA_syn, Genbank No. 1001583
- Helieobacter pyori guaA_h.p, Genbank No. 3122166
- Homo sapiens guaA_human, Genbank No. 1708072.
- Double-stranded cDNA was generated from mRNA of different ages Protonemata from Physcomitrella patens and for the production of a
- 093_dll has a length of 1232 nucleotides and codes on a continuous reading frame for 382 amino acids.
- the comparison with GMP-7M shows that 093_dll is a partial cDNA.
- the homology to GMP-7M is 66.7% at the nucleotide level and 74.6% at the amino acid level.
- the GMP-7M cDNA was used as a template for a PCR with the oligonucleotides 5 x -CCTAGCCATGGAACCTCAAAC-3 and 5 x -TATAGGATCCTACTTTG-GTCACC-3 x .
- the reaction mixtures contained approx. 0.1 ng GMP-7M DNA, 0.5 ⁇ of the corresponding oligonucleotides, 200
- the amplification conditions were set as follows:
- Annealing temperature 50 ° C, 30 sec denaturation temperature: 92 ° C, 30 sec elongation temperature: 72 ° C, 3 min number of cycles: 25
- the fragment of approx. 1670 bp obtained was ligated into the vector pTrc99A (Pharmacia) via the Ncol and BamHI sites inserted by the oligonucleotides.
- the construct GMP-7Trc obtained was transformed into the E.coli strain AT2465 (genetic marker: thi-1, guaA21, relAl, ⁇ , spoTl) and on M9 minimal media (Sambrook et al. (1989) Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) with and without 100 ⁇ g / ml guanosine plated.
- the minimal media contained 0.4% glucose, 0.2% casa noacids, 100 ug / ml thiamine, 100 ug / ml inosine, 100 ug / ml biotin, 100 ug / ml histidine, 100 ug / ml arginine, 100 ug / ml 2 'deoxyuridine, 100 ⁇ M IPTG and 25 ⁇ g / ml ampicillin.
- the cloning vector pTrc99A was transformed into AT2465.
- GMP-7M cDNA from tobacco in the expression vector pTrc 99 A (see Table 2), which strongly indicates that the GMP-7M cDNA encoded for an active GMP synthetase.
- the enzyme encoded by GMP-7M is the first functional GMP synthetase isolated from plants.
- IPTG-induced day cultures were harvested by centrifugation and the cell pellets were digested and further processed according to the manufacturer's instructions for nickel affinity chromatography ("Qia-Express-Kit", Qiagen). In this way, the GMP synthetase could be purified to more than 95% purity.
- the protein was used according to conventional protocols to generate antisera in rabbits (carried out on behalf of the company Eurogentec, Herstal, Belgium).
- GMP-7I construct was used according to the manufacturer's instructions (GibcoBRL) to generate recombinant Baculovirus.
- this virus was used to infect Sf21 insect cells in order to produce active GMP synthetase, the activity of which after disruption of the cells in 50 mM Tris-HCl, pH 7, 6, 10 mM KC1, 1 mM EDTA, 10 mM PMSF and desalination of the extract on a Sephadex G-25 column (Pharmacia, Sweden) could be measured.
- Leaf disks of sterile plants were incubated in a Petri dish with a 1:50 agrobacterial dilution for 5-10 minutes. This was followed by a 2-day incubation in the dark at 25 ° C. on 2MS medium with 0.8% Bacto agar. The cultivation was continued after 2 days with 16 hours of light / 8 hours of darkness and on a weekly basis 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 continued.
- agrobacterial dilution for 5-10 minutes. This was followed by a 2-day incubation in the dark at 25 ° C. on 2MS medium with 0.8% Bacto agar. The cultivation was continued after 2 days with 16 hours of light / 8 hours of darkness and on a weekly basis on MS medium with 500 mg / 1 claforan (cef
- 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, transferred to soil after rooting and after cultivation for two weeks in a climatic chamber in a 16 hour light / 8 hour dark rhythm at 60% humidity for foreign gene expression or altered metabolite levels and phenotypic growth characteristics examined. Altered nucleotide contents can, for example, according to the method of Stitt et al. (FEBS Letters, 145 (1982), 217-222).
- the transgenic GMP synthase antisense plants and their subsequent generation were characterized by reduced growth compared to WT control plants and by fading of the sink leaves. These phenotypic changes occurred at an early stage of growth (see Fig. 3).
- a reduced amount of GMP-7M RNA compared to the wild type could be detected in the Northern hydride.
- 40 ⁇ g total RNA from sink leaves were used. Total RNA from plant tissues was, as in Logemann et al. (Anal. Biochem. 163 (1987), 21).
- 40 ⁇ g RNA were separated in a 1.5% agarose gel containing formaldehyde and transferred to nylon membranes (Hybond, Amersham).
- GMP synthetase in the transgenic lines could be detected in the Western blot experiment in comparison with wild type plants.
- total protein extracts were produced from sink leaves, separated by standard methods in SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Detection was carried out using an IgG-alkaline phosphatase conjugate and the BCIP / NBT system (Sigma).
- Example 8 Furthermore, the in vitro assay described in Example 8 showed a reduced GMP synthetase activity in transgenic lines with reduced growth.
- the correlative relationship between the level of expression and the activity of the GMP synthetase and the growth phenotype suggests the suitability of the GMP synthetase as a target for herbicides.
- the systems developed according to Spector for animal enzymes can be used to measure the plant GMP synthetase activity.
- AMP formation is made possible by coupling the reaction with AMP kinase, pyruvate kinase, lactate dehydrogenase and measurement at 340 nm.
- the second system is based on the direct detection of GMP (guanosine monophosphate) by using the radioactively labeled substrate XMP (xanthine monophosphate) and separation in thin layer chromatography.
- the GMP synthetase activity can also be measured using a new system, namely the coupled detection of the glutamate formed.
- This system offers the advantage of a smaller number of coupled reaction steps and delivers higher signal strengths.
- GMP-S GMP synthetase
- GluDH glutamate dehydrogenase
- APAD 3-acetylpyridine adenine dinucleotide
- reaction mixture (see below) was incubated at 37 ° C for 60 minutes and the reaction was stopped by incubating at 95 ° C for 5 minutes.
- detection of the glutamate formed was carried out in the detection batch (see below) by photometric measurement of the APADH increase at 363 nm.
- the GMP synthetase activity can be prepared from plant tissues.
- a plant GMP synthetase can be found in E. coli, insect cells or another
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002385875A CA2385875A1 (en) | 1999-10-01 | 2000-09-21 | Gmp synthetase derived from plants |
JP2001528609A JP2003511036A (ja) | 1999-10-01 | 2000-09-21 | 植物から得られるgmpシンテターゼ |
EP00966064A EP1222293A2 (de) | 1999-10-01 | 2000-09-21 | Gmp-synthetase aus pflanzen |
AU76587/00A AU7658700A (en) | 1999-10-01 | 2000-09-21 | Gmp synthetase derived from plants |
IL14864400A IL148644A0 (en) | 1999-10-01 | 2000-09-21 | Gnp synthetase derived from plants |
BR0014455-0A BR0014455A (pt) | 1999-10-01 | 2000-09-21 | Sequência de dna, proteìna, uso de uma sequência de dna, métodos para encontrar substâncias, para identificar substâncias com ação herbicida e para eliminar crescimento de plantas indesejadas, sistema de teste, e, inibidor |
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DE19947490.7 | 1999-10-01 | ||
DE19947490 | 1999-10-01 |
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WO2001025457A2 true WO2001025457A2 (de) | 2001-04-12 |
WO2001025457A3 WO2001025457A3 (de) | 2001-12-20 |
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PCT/EP2000/009245 WO2001025457A2 (de) | 1999-10-01 | 2000-09-21 | Gmp-synthetase aus pflanzen |
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EP (1) | EP1222293A2 (de) |
JP (1) | JP2003511036A (de) |
CN (1) | CN1390261A (de) |
AU (1) | AU7658700A (de) |
BR (1) | BR0014455A (de) |
CA (1) | CA2385875A1 (de) |
IL (1) | IL148644A0 (de) |
WO (1) | WO2001025457A2 (de) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2199304A1 (de) * | 2004-12-17 | 2010-06-23 | Metanomics GmbH | Verfahren zur Steuerung der Chemikalienherstellung |
US8541208B1 (en) | 2004-07-02 | 2013-09-24 | Metanomics Gmbh | Process for the production of fine chemicals |
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PE20180674A1 (es) * | 2015-06-10 | 2018-04-19 | Univ Danmarks Tekniske | Uso de octacetidos sintasa para producir acido kermesico y acido flavokermesico |
MX2022010627A (es) * | 2021-09-23 | 2023-05-03 | Cj Cheiljedang Corp | Novedosa variante de gmp sintasa que hidroliza glutamina y procedimiento para la produccion de nucleotidos de purina mediante el uso de la misma. |
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WO1995027789A1 (en) * | 1994-04-08 | 1995-10-19 | Syntex (Usa) Inc. | Cloning and expression of human gmp synthetase, its use in screening for inhibitors of human gmp synthetase and inhibitors of human gmp synthetase |
WO1996019576A1 (en) * | 1994-12-22 | 1996-06-27 | Novartis Ag | Plant adenylosuccinate synthetase and dna coding therefor |
WO1998010074A2 (de) * | 1996-09-04 | 1998-03-12 | Basf Aktiengesellschaft | Adenylosuccinat synthetase |
US5780254A (en) * | 1995-05-04 | 1998-07-14 | Sandoz Ltd | Method for detection of herbicides |
US5780253A (en) * | 1995-05-04 | 1998-07-14 | Sandoz Ltd. | Screening method for detection of herbicides |
WO1999027119A1 (en) * | 1997-11-26 | 1999-06-03 | Novartis Ag | Method and compositions useful for the activation of silent transgenes |
EP0927761A2 (de) * | 1997-12-23 | 1999-07-07 | Basf Aktiengesellschaft | Gene der Purinbiosyntese aus Ashbya gossypii und deren Verwendung in der mikrobiellen Riboflavinsynthese |
-
2000
- 2000-09-21 EP EP00966064A patent/EP1222293A2/de not_active Withdrawn
- 2000-09-21 AU AU76587/00A patent/AU7658700A/en not_active Abandoned
- 2000-09-21 JP JP2001528609A patent/JP2003511036A/ja not_active Withdrawn
- 2000-09-21 WO PCT/EP2000/009245 patent/WO2001025457A2/de not_active Application Discontinuation
- 2000-09-21 CA CA002385875A patent/CA2385875A1/en not_active Abandoned
- 2000-09-21 BR BR0014455-0A patent/BR0014455A/pt not_active IP Right Cessation
- 2000-09-21 CN CN00813718.8A patent/CN1390261A/zh active Pending
- 2000-09-21 IL IL14864400A patent/IL148644A0/xx unknown
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WO1995027789A1 (en) * | 1994-04-08 | 1995-10-19 | Syntex (Usa) Inc. | Cloning and expression of human gmp synthetase, its use in screening for inhibitors of human gmp synthetase and inhibitors of human gmp synthetase |
WO1996019576A1 (en) * | 1994-12-22 | 1996-06-27 | Novartis Ag | Plant adenylosuccinate synthetase and dna coding therefor |
US5780254A (en) * | 1995-05-04 | 1998-07-14 | Sandoz Ltd | Method for detection of herbicides |
US5780253A (en) * | 1995-05-04 | 1998-07-14 | Sandoz Ltd. | Screening method for detection of herbicides |
WO1998010074A2 (de) * | 1996-09-04 | 1998-03-12 | Basf Aktiengesellschaft | Adenylosuccinat synthetase |
WO1999027119A1 (en) * | 1997-11-26 | 1999-06-03 | Novartis Ag | Method and compositions useful for the activation of silent transgenes |
EP0927761A2 (de) * | 1997-12-23 | 1999-07-07 | Basf Aktiengesellschaft | Gene der Purinbiosyntese aus Ashbya gossypii und deren Verwendung in der mikrobiellen Riboflavinsynthese |
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DATABASE EMBL [Online] ACCESSION NO: AW127061, 24. Oktober 1999 (1999-10-24) QUATRANO, R., ET AL.: " ga20f03.y1 Moss EST library PPU Physcomitrella patens cDNA clone PEP_SOURCE_ID:PPU021506 5' similar to TR:O66601 O66601 GMP SYNTHASE. ;,mRNA sequence." XP002167642 * |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8541208B1 (en) | 2004-07-02 | 2013-09-24 | Metanomics Gmbh | Process for the production of fine chemicals |
EP2199304A1 (de) * | 2004-12-17 | 2010-06-23 | Metanomics GmbH | Verfahren zur Steuerung der Chemikalienherstellung |
Also Published As
Publication number | Publication date |
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EP1222293A2 (de) | 2002-07-17 |
CA2385875A1 (en) | 2001-04-12 |
JP2003511036A (ja) | 2003-03-25 |
IL148644A0 (en) | 2002-09-12 |
AU7658700A (en) | 2001-05-10 |
BR0014455A (pt) | 2002-06-11 |
CN1390261A (zh) | 2003-01-08 |
WO2001025457A3 (de) | 2001-12-20 |
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