WO1999050400A1 - Amp-deaminase - Google Patents
Amp-deaminase Download PDFInfo
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- WO1999050400A1 WO1999050400A1 PCT/EP1999/002016 EP9902016W WO9950400A1 WO 1999050400 A1 WO1999050400 A1 WO 1999050400A1 EP 9902016 W EP9902016 W EP 9902016W WO 9950400 A1 WO9950400 A1 WO 9950400A1
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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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
- the present invention relates to a DNA coding for a polypeptide with AMP deaminase (EC 3.5.4.6, adenosine triphosphate aminohydrolase) activity.
- the invention relates to the use of a nucleic acid coding for a protein with AMP deaminase activity of plant origin for the production of a test system for the identification of inhibitors of AMP deaminase.
- the invention further relates to the use of the nucleic acid coding for plant AMP deaminase for the production of plants with increased resistance to inhibitors of AMP deaminase.
- Plants are able to synthesize their cell components from carbon dioxide, water and inorganic salts.
- Hydantocidin acts as a prodrug.
- the active ingredient is metabolized in planta by phosphorylation on the 5'OH group to give the herbicide (Siehl et al., 1996, Plant Physiol., 110, 753-758).
- AMP deaminase occupies a special position.
- AMP adenosine monophosphate
- IMP inosine 5 'phosphate
- the protein was partially purified from various plants and regulatory properties were examined, e.g. from spinach leaves (Yoshino and Murakami, 198, Z. Plant Physiology, 99, pp. 331-338), Jerusalem artichokes (Jerusalem Artichokes (Le Floc'h and La fleuriel, 1983, Physiologie Vergetale, 21 (1), 15- 2), pea seeds (Turner and Turner, 1961, Biochem. J. 79, 143) and cell cultures of Catharantus roseus, small evergreen (Yabuki et al. 1992, Phytochemistry, 31 (6), 1905-1909).
- the enzyme appears to play a central role in the regulation of the adenylate pool of a cell (Chapman and Atkinson, 1973, J. Biol. Former, 248, 8309; Solano and Coffee, 1978; Yoshino et al., 1979).
- AMP deaminases Genes encoding AMP deaminases have been isolated from many organisms. Gene families of AMP deaminase appear to be present in mammals (Morisaki et al. 1990, J. Biol. Chem. 265 (20), 11482-11486). Further coding sequences were isolated from Schizosaccharomyces pombe (accession P50998) and Saccharomyces cerevisiae (accession P15274). Only adenine deaminases are known from bacteria; AMP deaminases could not be isolated. By means of sequence comparisons, so-called est sequences from rice (GenBank Acc: C26026) and Arabidopsis (T21250) with similarity to yeast AMP deaminases can be found. Complete cDNA sequences of plant AMP deaminases have not yet been described.
- the object of the present invention was to isolate a complete plant cDNA coding for the enzyme AMP deaminase and its functional expression in bacterial or eukaryotic cells for the simple and inexpensive extraction of the enzyme for the implementation of inhibitor-enzyme binding studies.
- Another object of the invention was the overexpression of the AMP-Deaminase gene in plants for the production of plants which are tolerant of inhibitors of AMP-Deaminase.
- the object was achieved by isolating the gene coding for the enzyme AMP deaminase vegetable ⁇ , and its functional expression in bacterial or plant cells or plants.
- a first object of the present invention is a DNA sequence SEQ ID NO: 1 containing 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 or hybridize with this SEQ ID NO: 1 and which code for a protein which has the biological activity of an AMP deaminase.
- the invention also relates to expression cassettes, the sequence of which encode an Arabidopsis thaliana AMP deaminase or its functional equivalent.
- the nucleic acid sequence can e.g. be a DNA or a cDNA sequence.
- 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, if appropriate, further regulatory elements which are operatively linked to the intermediate coding sequence for the AMP deaminase 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 AMP deaminase DNA sequence and a polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987). 4
- Sequences are particularly preferred which ensure targeting in the apoplasts, in plastids, the vacuole, the mitochondrium, the endoplasmic reticulum (ER) or, due to the lack of corresponding operative sequences, a retention in the compartment of formation, the cytosol ( Kermode, Crit. Rev. Plant S ⁇ i. 15, 4 (1996), 285-423).
- the plant expression cassette can be installed in the tobacco transformation vector pBinAR-Hyg (see example 5).
- any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette according to the invention.
- 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 recognition sequences for transcriptional effectors, which in their entirety 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 contain a chemically inducible promoter, by means of which the expression of the exogenous AMP deaminase gene in the plant can be controlled at a specific point in time.
- a chemically inducible promoter as e.g. the PRPl promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a promoter inducible by salicylic acid (WO 95/1919443), one inducible by benzenesufonamide
- promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of purines or their precursors takes place. 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. 8 (1989) 2445-245).
- the expression cassette according to the invention can therefore contain, for example, a seed-specific promoter (preferably the phaseolin promoter, the USP or LEB4 promoter), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
- 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.
- gene expression cassettes according to the invention can contain 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 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.
- sequence homology between AMP deaminase from yeast and from plant is 57% at the DNA level in relation to a selected, highly homologous sub-region, for example in the region of base 1345-2715 of sequence M30449 (Genbank Accession Number), created using the BLAST program ( Altschul et al., (1990), J. Mol. Biol.: 215: 403-419; Gish and States, (1993), Nature Genet. ⁇ : 266-272).
- regions of 20-30 nucleotides are so homologous that there is a sufficient probability of success for an oligonucleotide-based screening for AMP deaminase from other plants.
- the invention also relates to functionally equivalent DNA sequences which code for an AMP deaminase gene and which, based on the total length of the gene, have a sequence homology with the DNA sequence SEQ ID NO: 1 of 40 to 100%.
- Preferred objects of the invention are functionally equivalent DNA sequences which code for an AMP deaminase gene and which, based on the total length of the gene, have a sequence homology with the DNA sequence SEQ ID NO: 1 of 60 to 100%.
- a particularly preferred object of the invention are functionally equivalent DNA sequences which code for an AMP deaminase gene and which, based on the total length of the gene, have a sequence homology with the DNA sequence SEQ ID NO: 1 of 80 to 100%.
- Sequences which are functionally equivalent and which code for an AMP deaminase gene 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 herein as well as artificial, e.g. artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a plant.
- a functional equivalent is understood to mean, in particular, natural or artificial mutations of an originally isolated sequence coding for an AMP deaminase, which furthermore show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
- the present invention also encompasses those nucleotide sequences which are obtained by modification of this nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also be 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.
- artificial DNA sequences are suitable as long as, as described above, they impart the desired property of increasing the IMP content in the plant by overexpressing the AMP deaminase gene in crop plants.
- Such artificial DNA 7 sequences can comprise or be determined by in vitro selection, for example by back-translation means Mo- lecular £ modeling of proteins constructed that tivity AMP deaminase activated. Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to 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 equivalent nucleic acid sequences according to the invention, a component of the fusion protein being a vegetable AMP deaminase polypeptide or a functionally equivalent part thereof.
- the second part of the fusion protein can e.g. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence that can be used to detect AMP deaminase expression (e.g. myc-tag or his-tag).
- this is preferably a regulatory protein sequence, such as e.g. a signal or transit peptide that directs the AMP deaminase 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 both native or homologous and 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 AgroJacterium-m tumefaciens, in particular gene 3 of T-DNA (octopine 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 inserted as an insert into a recombinant vector, the vector DNA of which contains additional functional regulatory signals, for example sequences for replication or integration.
- additional functional regulatory signals for example sequences for replication or integration.
- Suitable vectors are described 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 the 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, published by S.D. Kung and R.
- the construct to be expressed is preferably cloned into a vector which is suitable for transforming AgroJba ⁇ ter u-m turne faciens, 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, 9
- Flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and - the various tree, nut and wine species and legumes are used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
- the biosynthesis site of purines is generally the leaf tissue, so that leaf-specific expression of the AMP deaminase gene is useful.
- the purine bio-synthesis need not be limited to the leaf tissue, but can also be tissue-specific in all other parts of the plant, for example in fatty seeds.
- constitutive expression of the exogenous AMP deaminase gene is advantageous.
- inducible expression may also appear desirable.
- the expression cassettes according to the invention can be cloned into suitable vectors which enable their multiplication, for example in E. coli.
- suitable cloning vectors include pBR332, pUC series, M13mp series and pACYC184.
- 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 AMP deaminase 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 as well 10 whose plant cells, tissues or parts are a further subject of the present invention.
- 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 amounts of the enzyme AMP-deaminase.
- Another object of the invention is a protein from Arabidopsis thaliana characterized by the amino acid sequence SEQ ID NO: 2 or derivatives or parts of this protein with AMP deaminase activity. Compared to Saccharomyces cerevisiae, the homology at the amino acid level is 43-47% identity (see Figure 4).
- the invention also relates to vegetable proteins with AMP deaminase activity with an amino acid sequence homology to the Arabidopsis thaliana AMP deaminase of 20-100% identity.
- Vegetable proteins with AMP deaminase activity with an amino acid sequence homology to the Arabidopsis thaliana AMP deaminase of 50-100% identity are preferred.
- Vegetable proteins with AMP deaminase activity with an amino acid sequence homology to the Arabidopsis thaliana AMP deaminase of 80-100% identity are particularly preferred.
- AMP deaminase is a suitable target for herbicides.
- the complete cDNA sequence of the AMP deaminase from Arabidopis 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 the detection of inhibitors specific for AMP deaminase.
- the AMP deaminase can be used, for example, in an enzyme test in which the activity of the AMP deaminase 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 can be made about the inhibitory behavior of the active substance to be tested (see Example 4).
- test system Using 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.
- the invention further relates to herbicides which can be identified using the test system described above.
- the effectiveness of the expression of the transgenically expressed AMP-Deaminase gene can be determined, for example, in vitro by an increase in shoot meristem or by a germination test.
- a change in the type and level of expression of the AMP deaminase gene and its effect on the resistance to inhibitors of AMP deaminase 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, and to transgenic cells, tissues, parts and propagation material of such plants.
- Transgenic crop plants such as e.g. Barley, wheat, rye, corn, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and wine species, as well as legumes.
- the invention further relates to plants which, after expression of the DNA SEQ ID NO: 1, have an increased IMP content in the plant.
- increasing the inosine-5 '-phosphate (IMP) content means for at least the artificially acquired ability of an increased IMP biosynthesis by functional overexpression of the AMP deaminase gene in the plant compared to the non-genetically modified plant a generation of plants.
- 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.
- RNA from plant tissues was isolated as in Logemann et al. ((1987) Anal. Biochem. 163, 21). For the analysis, 20 ⁇ g RNA were separated in a 1.5% agarose gel containing formaldehyde and transferred to nylon membranes (Hybond, Amersham). The detection of specific transcripts was carried out as described for Amasino ((1986) Anal. Biochem. 152,
- the cDNA fragments used as a probe were radioactively labeled with a random primed DNA labeling kit (Boehringer, Mannheim) and hybridized according to standard methods (see Hybond user instructions, Amersham). Hyridization signals were visualized by autoradiography using X-OMAT AR films from Kodak.
- the bacterial strains used below (E. coli, XL-I Blue) were obtained 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) was developed by Deblaere et al. (Nucl. Acids 13
- the agrobacterial strain LBA4404 (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).
- the Arabidopsis est clone coding for the AMP deaminase was obtained from the Arabidopsis Biological Resource Center (Ohio State University). It is a partial cDNA clone (T21250) that does not correspond to the full-length transcript of the AMP deaminase.
- a PCR amplification of the Arabidopsis AMP deaminase partial fragment was carried out in a DNA thermal cycler from Perkin Elmer.
- the reaction mixtures contained 8 ng / ⁇ l genomic DNA from Escherichia coli, 0.5 ⁇ M of the corresponding oligonucleotides, 200 ⁇ M nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C., 1.5 mM) MgCl 2 ) and 0.02 U / ⁇ l Taq polymerase (Perkin Elmer).
- the amplification conditions were set as follows:
- the resulting fragment comprises a small part of the est clone T21250, with the aid of which a heterologous screening of an Arabidopsis thaliana cDNA bank was carried out (Stratagene).
- 3.0 ⁇ 10 5 lambda phages from the cDNA library from Arabidopsis thaliana (Stratagene) were plated on agar plates with E. coli XLI-Blue as a bacterial strain.
- the phage DNA was analyzed using
- the membranes were hybridized after prehybridization ⁇ at 60 ° C in 3 x SSPE, 0.1% sodium dodecyl sulfate (w / v), 0.02% polyvinylpyrolidone (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. Positive hybridizing phages were visualized by autoradiography and purified and isolated using standard techniques.
- the amino acid sequence begins with the third base after the linker sequences (bold ends of the sequence in Figure 2) in the third reading frame and can be translated into an 860 amino acid polypeptide, or from the first methionine start codon into a polypeptide of 839 amino acids (see Figure 3). Alternatively, the methionine in position 46 can be used, so that a polypeptide of 824 amino acids would result.
- oligonucleotide sequences were derived from the determined sequence and provided with a BamHI restriction site and two overhanging bases.
- the oligonucleotides are underlined and numbered in Figure 2. Potential methionine start codons are shown in bold.
- fragment I, II, III which follow the complete reading frame
- fragment I, primer 1 + 4 correspond to the first Met start codon (fragment II, primer 2 +4) or the second start codon (fragment III, 3 + 4).
- the PCR reaction mixtures contained 8 ng / ⁇ l pBS-AMPl DNA, 0.5 ⁇ M of the corresponding oligonucleotides, 200 ⁇ M nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C., 1, 5 mM MgCl 2 ) and 0.02 U / ⁇ l Taq polymerase (Perkin Elmer).
- the amplification conditions were set as follows:
- PCR fragments were cloned into the overexpression vectors pET15b, pETlla and pQE9 and used for protein production by means of IPTG-5 induction according to standard methods (see manual: The Quiaexpressionist (1992), Quuiagen, Hilden).
- E.coli was subjected to pressure digestion on the French Press under maximum pressure in a 20 ml pressure chamber or using a
- the homogenate was buffered in the following medium by dialysis in 40 mM 30 citrate, pH 6.5 (adjusted with 5 N NaOH), 0.05% BSA (w / v), 100 mM KCl.
- Each 10-100 ⁇ l of the rebuffered enzyme fraction was made up to 700 ⁇ l with buffer and by adding 100 ⁇ l of a 1 mM AMP solution
- 35 solution, 0.5 mM ATP solution and 1 ⁇ M diadenosine pentaphosphate solution measured the decrease in extinction over 2-10 min against a reference cuvette with 700 ⁇ l reaction buffer and 100 ⁇ l of a protein homogenate of untransformed E. coli culture. Equal amounts of total protein were used for the measurements of the reference against the measured value
- nucleotides 11749-11939 was isolated as a PvuII-HindiII fragment and after addition of Sphl -Line cloned to the PvuII interface between the SpHI-HindIII interface of the vector.
- the plasmid pBinAR was produced (Höfgen and Willmitzer (1990) Plant Science 66, 221-230).
- the PCR fragments I, II and III were cloned into the BamHI site of the vector pBinAR in both orientations and used to transform tobacco plants.
- the resulting plasmids have the names pBinAMP-20, pBin ⁇ AMP-0, pBinAMP-0, pBin ⁇ AMP-0, pBinAMP + 26, pBintxAMP + 26 and correspond to fragments I, II, III described in Example 3 from the PCR batches described above.
- the plasmids pBinAMP-20, pBin ⁇ AMP-20, pBinAMP-0, pBin ⁇ AMP-0, pBinAMP + 26, pBin ⁇ AMP + 26 were found in Agrobacterium tumefaciens
- C58Cl pGV2260 transformed (Deblaere et al, 1984, Nucl. Acids. Res. 13, 4777-4788).
- a 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony in Murashige-Skoog medium ((1962) Physiol. Plant. 15, 473) with 2% sucrose ( 2MS medium) is used.
- Leaf disks of sterile plants (each about 1 cm 2 ) 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.
- 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 contents and phenotypic 18th
- Modified nucleotide contents can *, for example, according to the method of Stitt et al. (1982, FEBS Letters, 145, 217-222).
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU37021/99A AU3702199A (en) | 1998-04-01 | 1999-03-25 | Amp deaminase |
EP99919138A EP1070120A1 (de) | 1998-04-01 | 1999-03-25 | Amp-deaminase |
CA002325054A CA2325054A1 (en) | 1998-04-01 | 1999-03-25 | Amp deaminase |
JP2000541288A JP2002527039A (ja) | 1998-04-01 | 1999-03-25 | Ampデアミナーゼ |
Applications Claiming Priority (2)
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DE19814512 | 1998-04-01 | ||
DE19814512.8 | 1998-04-01 |
Publications (1)
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WO1999050400A1 true WO1999050400A1 (de) | 1999-10-07 |
Family
ID=7863184
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1999/002016 WO1999050400A1 (de) | 1998-04-01 | 1999-03-25 | Amp-deaminase |
Country Status (5)
Country | Link |
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EP (1) | EP1070120A1 (de) |
JP (1) | JP2002527039A (de) |
AU (1) | AU3702199A (de) |
CA (1) | CA2325054A1 (de) |
WO (1) | WO1999050400A1 (de) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001031025A2 (de) * | 1999-10-25 | 2001-05-03 | Basf Aktiengesellschaft | Formylglycinamidinribotid-synthase aus pflanzen |
WO2002006319A2 (de) * | 2000-07-17 | 2002-01-24 | Bayer Cropscience Gmbh | Nucleinsäuremolekül, das für eine pflanzliche amp-deaminase codiert |
JP2005532790A (ja) * | 2002-03-20 | 2005-11-04 | ビーエーエスエフ アクチェンゲゼルシャフト | 除草剤の標的としてのセリンヒドロキシメチルトランスフェラーゼ |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103805629B (zh) * | 2014-01-28 | 2016-10-05 | 江南大学 | 鼠灰链霉菌amp脱氨酶基因的毕赤酵母真核表达方法 |
-
1999
- 1999-03-25 EP EP99919138A patent/EP1070120A1/de not_active Withdrawn
- 1999-03-25 AU AU37021/99A patent/AU3702199A/en not_active Abandoned
- 1999-03-25 CA CA002325054A patent/CA2325054A1/en not_active Abandoned
- 1999-03-25 JP JP2000541288A patent/JP2002527039A/ja not_active Withdrawn
- 1999-03-25 WO PCT/EP1999/002016 patent/WO1999050400A1/de not_active Application Discontinuation
Non-Patent Citations (5)
Title |
---|
DANCER, J.E. ET AL.: "Adenosine-5'-phosphate deaminase", PLANT PHYSIOL., vol. 114, 1997, pages 119 - 129, XP002110870 * |
MEYER, S.L. ET AL.: "Characterization of AMD, the AMP Deaminase gene in yeast.", BIOCHEMISTRY, vol. 28, 1989, pages 8734 - 8743, XP002110869 * |
NEWMAN, T. ET AL.: "3258 Lmbda-PRL2 Arabidopsis thaliana cDNA clone 93F5T7 - EST", EMBL - EMEST8 DATABASE: ACCESSION NUMBER T21250, 27 June 1994 (1994-06-27), XP002110868 * |
ROUNSLEY S.D. ET AL.: "Arabidopsis thaliana chromosome II BAC F16M14 genomic sequence", EMBL DATABASE: ACCESSION NUMBER AC003028, 31 October 1997 (1997-10-31), XP002110867 * |
YABUKI, N. & ASHIHARA, H.: "AMP deaminase and the control of adenylate catabolism ...", PHYTOCHEMISTRY, vol. 31, no. 6, 1992, pages 1905 - 1909, XP002110871 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001031025A2 (de) * | 1999-10-25 | 2001-05-03 | Basf Aktiengesellschaft | Formylglycinamidinribotid-synthase aus pflanzen |
WO2001031025A3 (de) * | 1999-10-25 | 2001-11-29 | Basf Ag | Formylglycinamidinribotid-synthase aus pflanzen |
WO2002006319A2 (de) * | 2000-07-17 | 2002-01-24 | Bayer Cropscience Gmbh | Nucleinsäuremolekül, das für eine pflanzliche amp-deaminase codiert |
WO2002006319A3 (de) * | 2000-07-17 | 2002-05-30 | Aventis Cropscience Gmbh | Nucleinsäuremolekül, das für eine pflanzliche amp-deaminase codiert |
JP2005532790A (ja) * | 2002-03-20 | 2005-11-04 | ビーエーエスエフ アクチェンゲゼルシャフト | 除草剤の標的としてのセリンヒドロキシメチルトランスフェラーゼ |
Also Published As
Publication number | Publication date |
---|---|
JP2002527039A (ja) | 2002-08-27 |
EP1070120A1 (de) | 2001-01-24 |
AU3702199A (en) | 1999-10-18 |
CA2325054A1 (en) | 1999-10-07 |
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