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WO2000040709A2 - PROCEDE PERMETTANT DE FAIRE CODER AUX PLANTES ET CELLULES DE PLANTES DE LA LYSINE OU DE LA METHIONINE OU DU TRYPTOPHANE OU DE LA THREONINE A DES CODONS D'ARNm DIFFERENTS DE CEUX SPECIFIANT NORMALEMENT CES ACIDES AMINES PARTICULIERS DURANT LA SYNTHESE PROTEIQUE - Google Patents

PROCEDE PERMETTANT DE FAIRE CODER AUX PLANTES ET CELLULES DE PLANTES DE LA LYSINE OU DE LA METHIONINE OU DU TRYPTOPHANE OU DE LA THREONINE A DES CODONS D'ARNm DIFFERENTS DE CEUX SPECIFIANT NORMALEMENT CES ACIDES AMINES PARTICULIERS DURANT LA SYNTHESE PROTEIQUE Download PDF

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WO2000040709A2
WO2000040709A2 PCT/IB1999/002125 IB9902125W WO0040709A2 WO 2000040709 A2 WO2000040709 A2 WO 2000040709A2 IB 9902125 W IB9902125 W IB 9902125W WO 0040709 A2 WO0040709 A2 WO 0040709A2
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plants
dna
plant
trna
plant cells
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PCT/IB1999/002125
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WO2000040709A3 (fr
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William R. Folk
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Folk William R
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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
    • C12N15/8242—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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8253—Methionine or cysteine
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • 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
    • C12N15/8242—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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • 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
    • C12N15/8242—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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8254—Tryptophan or lysine

Definitions

  • This invention relates to methods for altering protein synthesis in plant cells and plants so as to modify their properties, including the composition and sequences of proteins synthesized by the plant cells and plants.
  • DNA deoxyribonucleic acid
  • mRNAs messenger ribonucleic acids
  • tRNA transfer RNA
  • Amino acids are attached to tRNAs by their cognate aminoacyl tRNA synthetases and are then incorporated into proteins encoded by the mRNAs, such that the sequence of amino acids in proteins is determined by the sequence of mRNA codons corresponding to those amino acids.
  • the mRNA codons which normally specify lysine are AAA and AAG; the tRNA lys species which bind these codons have anticodon sequences UUU or CUU, respectively.
  • the mRNA codon which normally specifies methionine is AUG; the tRNA met species which binds this codon has anticodon sequence CAU.
  • the mRNA codon which normally specifies tryptophan is CCA; the tRNA* 1 ' species which binds this codon has anticodon sequence CCA.
  • the mRNA codons which normally specify threonine are ACU, ACC, ACA and ACG; the tRNA d ⁇ r species which bind these codons have anticodon sequences AGU, GGU, UGU or CGU, respectively. These codons are known by persons skilled in the art from the universal genetic code (Table 2). Table 2. Universal Genetic Code
  • GTT val v GCT ala A GAT asp D GGT gly G GTC val v GCC ala A GAC asp D GGA gly G GTA val v GCA ala A GAA glu E GGA gly G GTG val v GCG ala GAG glu E GGG gly G
  • Plants are the major nutritional source of proteins and amino acids for man and livestock, and proteins produced in plants have industrial and medical value. Modification of the sequences and compositions of these proteins can improve their value. In bacteria, use of altered tRNAs to modify the specificity of coding during protein synthesis has provided a most effective way to study the structure, function and interactions of proteins [Murgola, 1995. In D. Soil and U. RajBhandary (eds), tRNA: Structure, Biosynthesis, and Function, ASM Press, Washington, D.C.].
  • altered tRNAs to modify the specificity of coding during protein synthesis in plants can similarly facilitate analysis of the machinery by which proteins are made, will facilitate analysis of protein structure and function, and opens new pathways for the production of proteins having altered amino acid sequences and compositions.
  • the predominant seed proteins of cereals and some legumes are deficient in lysine.
  • a goal of agricultural science is to develop plant varieties encoding nutritionally balanced proteins.
  • One method which has been invented requires changing the DNA sequences encoding the zein seed storage proteins (U.S. Patents 4,885,357 and 4,886,878). The methods disclosed in this invention provides an alternative process to achieve this goal.
  • compositions for causing plant cells and plants to code lysine, methionine, tryptophan or threonine at mRNA codons other than those which normally code lysine (AAA, AAG), methionine (AUG), tryptophan (UGG) or threonine (ACU, ACC, ACA, ACG) during protein synthesis.
  • DNAs which will express in plant cells and plants, tRNAs that are charged with lysine, methionine, tryptophan or threonine and which pair with mRNA codons other than those normally specifying these amino acids are chemically synthesized or prepared from naturally occurring tRNA genes by mutagenesis.
  • DNAs are introduced into plant protoplasts or plant cells or plant tissues, together with DNAs encoding selectable traits such as antibiotic resistance or herbicide resistance, so as to produce stable transformed cell lines or plant tissues in which tRNAs expressed from the DNAs introduce lysine, methionine, tryptophan or threonine at mRNA codons other than those normally specifying these amino acids during protein synthesis.
  • Coding of lysine, methionine, tryptophan or threonine at alternative codons during protein synthesis in plant cells and plant tissues may be measured by the simultaneous or subsequent introduction into the plant cells or plant tissues or plants of reporter genes which encode proteins whose function requires coding of lysine, methionine, tryptophan or threonine at codons other than those normally specifying these amino acids.
  • the present invention relates to a method for causing plant cells or plant tissue or plants to code lysine or methionine or tryptophan or threonine at mRNA codons other than those normally specifying those particular amino acids during protein synthesis, which comprises: i) Preparing a DNA to express a tRNA which is charged with lysine and which binds to mRNA codons other than AAA or AAG during protein synthesis; or a DNA to express a tRNA which is charged with methionine and which binds to mRNA codons other than AUG; or a DNA to express a tRNA which is charged with tryptophan and which binds to mRNA codons other than UGG; or a DNA to express a tRNA which is charged with threonine and which binds to codons other than ACU, ACC, ACA, ACG, ii) introducing one or more of the said DNAs into plant protoplasts, plant cells or
  • Methods are also described to regulate the coding by tRNAs of lysine, methionine, tryptophan or threonine at mRNA codons other than those normally specifying these amino acids temporally or spatially in plant cells or plant tissues or plants.
  • Another subject of the invention is a method as above described, and further including the step of regulating the synthesis of the tRNA so as to cause lysine, methionine, tryptophan or threonine coding at codons other than those which normally specify lysine (AAA, AAG), methionine (AUG), tryptophan (UGG) or threonine (ACU, ACC, ACA, ACG) to occur at particular times or in particular tissues.
  • Altered coding of lysine, methionine, tryptophan or threonine during protein synthesis can be regulated by controlling the synthesis of the tRNAs from the introduced DNAs or by regulating the charging of the tRNAs by lysyl tRNA synthetase, methionyl tRNA synthetase, tryptophanyl tRNA synthetase or threonyl tRNA synthetase, respectively.
  • a binding site for a DNA binding protein is included in the 5' flanking region of the DNA which encodes the tRNA.
  • Altering the expression or the binding of the DNA binding protein to the DNA which encodes the tRNA will regulate synthesis of the tRNAs which code lysine, methionine, tryptophan or threonine at mRNA codons other than those normally specifying these amino acids during protein synthesis within the plant cell, plant tissue or plant.
  • a DNA encoding a lysyl tRNA synthetase, methionyl tRNA synthetase, tryptophanyl tRNA synthetase or threonyl tRNA synthetase which respectively charges the tRNA which codes lysine, methionine, tryptophan or tlrreonine at mRNA codons other than those normally specifying these amino acids is introduced into the plant cell or plant together with the DNA which expresses the tRNA.
  • a further subject of the invention is then a method as above described, wherein said DNA contains a site bound by a protein capable of interfering with the synthesis of the tRNA or wherein the synthesis of the protein which interferes with the synthesis of the tRNA is determined by a promoter whose activity is regulated.
  • the binding to said DNA by the protein which interferes with synthesis of the tRNA may be determined by a chemical inducer.
  • From the transformed plant cells and plant tissues can be regenerated plants which code lysine, methionine, tryptophan or threonine at codons other than those normally specifying these amino acids during protein synthesis to produce proteins whose amino acid sequences, compositions and properties are altered.
  • Another subject of the present invention is a method for producing plant cells or plant tissues or plants in which the utilization of tRNAs during protein synthesis is enhanced which comprises : i) preparing a DNA which will express a functional aminoacyl tRNA synthetase, ii) introducing said DNA into plant protoplasts, plant cells, or plant tissues or plants, iii) selecting the plant cells and plant tissues or plants which stably maintain said DNA, iv) and regenerating, if necessary, plants which stably maintain said DNA.
  • the aminoacyl tRNA synthetase charges tRNAs with lysine or with methionine or with tryptophan or with threonine.
  • This aminoacyl tRNA synthetase is preferably from a plant, and advantageously contains the sequence SEQ ID n° 1 specified in the sequence listing.
  • It may also be from a bacterium, a yeast, or an animal.
  • the synthesis of the aminoacyl tRNA synthetase is regulated by promoters which are tissue specific or whose activity is altered by chemical inducers.
  • said tRNA is preferably from a plant and may for example be selected from plant tRNA Iys , plant tRNA met , plant tRNA t ⁇ p and plant tRNA thr .
  • Said tRNA may also be from a yeast, or from an animal.
  • the mRNA codon to which the tRNA binds during protein synthesis specifies polypeptide chain termination.
  • the mRNA codon to which the tRNA binds during protein synthesis specifies one of the following amino acids: glutamine, asparagine, arginine, glutamic acid, serine.
  • the proteins synthesized by protein synthesis in the plant cells, tissues or plants include seed storage proteins, and non-plant proteins, such as immunoglobulines.
  • the plant encompassed by the present invention may preferably be selected from corn, soybeans, wheat, rice, rape and pea.
  • the amino acid coded using mRNA codons other than those which normally code it is one amino acid selected from lysine, methionine, tryptophan and threonine. More particularly, the amino acid coded using mRNA codons other than those which normally code it is lysine.
  • the process of this invention employs DNAs which express tRNAs that are charged with lysine in plant cells and plants.
  • tRNAs include the cytoplasmic tRNA Lys species of plants (one is known at present) and the tRNA Lys species of bacteria (E. coli) which also is charged with lysine by the plant lysyl tRNA synthetase.
  • E. coli tRNA Lys species of bacteria
  • the invention may employ the tRNA ⁇ g ⁇ of plants, which also is charged with lysine by the plant lysyl tRNA synthetase and which codes lysine at codons other than AAA and AAG.
  • the sequences of the DNAs corresponding to these tRNAs are:
  • Plant tRNA 1 ⁇ (SEQ ID n° 2) : gcccgtctag ctcagttggt agagcgcaag gctcttaacc ttgtggtcgt gggttcgagc 60
  • Plant tR A£e A (SEQ ID n° 3) : ggattcgtgg cgcaatggta gcgcgtctga ctctagatca gaaggttgcg tgttcgattc SO
  • E. coli tRNA 1 ⁇ (SEQ ID n° 4) : gggtcgttag ctcagttggt agagcagttg acttttaatc aattggtcgc aggttcggaa 60 tcctgcacga cccacca 77 (The tRNA anticodon sequences are underlined).
  • tRNA Lys species from other organisms can also serve because the tRNA sequence elements used by the plant lysyl tRNA synthetase for binding and charging tRNA have been conserved throughout evolution and are present in all known tRNA Lys species.
  • the particular lysyl tRNA synthetase which charges a particular tRNA may be expressed together with that tRNA in the plant cell, so as to ensure the tRNA is charged with lysine.
  • the method disclosed in this invention permits the facile determination of whether a particular tRNA Lys species will code lysine at codons other than AAA or AAG during protein synthesis in plant cells and plants.
  • sequences of tRNA ys species of other organisms and their cognate lysyl tRNA synthetases may be accessed through the EMBO/Gen Bank/DDBJ nucleotide sequence databases and from the compilation of tRNA sequences and sequences of tRNA genes published annually by Nucleic Acids Research (Oxford University Pres, Oxford, U.K.).
  • flanking sequences of the DNA encoding a tRNA modulate expression in plant cells of that tRNA (Ulmasov and Folk, 1995. The Plant Cell 7: 1723-1734). Consequently, in the construction of DNAs to stably express tRNAs in plant cells and plants, it is desirable to include flanking sequences which permit efficient tRNA expression.
  • flanking sequences which permit efficient tRNA expression.
  • the 5' and 3' flanking sequences of the A. thaliana tRNA ys gene sufficient to cause efficient expression of tRNA Lys species in monocot and dicot cells and plants are:
  • the 5' and 3' flanking sequences of other plant tRNA genes (the tR A ⁇ , tRNA 1 ⁇ tRNA ⁇ tRNA ⁇ 5 and tRNA Leu genes) which are known to be expressed efficiently are also expected to function. These sequences are available in the EMBO/GenBank/DDBJ Nucleotide Sequence Databases or by reference to the sequences and citations in Ulmasov and Folk, 1995, which are incorporated by reference.
  • nucleotides 179-195 in the above 5' flanking sequence, or at a comparable position in the 5' flanking sequences of the other specified tRNA genes a site bound by a DNA binding protein capable of interfering with transcription, as for example the sequence bound by the lac repressor (SEQ ID n° 7): attgtgagcg ctcaat 18 or the sequence bound by the tet repressor (SEQ ID n° 8) : actctatcac tgatagagt 19 Substitution of these sequences at this site has been shown to not impair synthesis of the tRNA in the absence of protein binding.
  • Sequences bound by other proteins should also function and variations in these sequences and slight adjustments in the site of substitutions may be used so long as protein binding is sufficient to block expression of the tRNA, and introduction of the binding site for the protein does not impair synthesis of the tRNA in the absence of the protein.
  • a convenient method to analyze in transient assays in plant cells the synthesis of tRNAs from such DNA constructs is provided in Ulmasov et al. 1997( Plant Mol. Biol. 35:417-424) and this method is incorporated by reference.
  • Binding of proteins such as the lac repressor or the tet repressor to their respective sites introduced into the 5' flanking sequence of tRNA genes expressed in plant cells reduces tRNA expression three to ten fold, and the degree of repression can be controled by regulating either the concentration of the protein in the plant cell or by interfering with its capacity to bind to the 5' flanking sequences by exposing plant cells or plants to inducers such as lactose or tetracycline or their analogs, which bind to the repressors and prevent them from binding to DNA.
  • the concentration of the repressors in the cell is determined by their synthesis, which is a function of the promoter used to express the repressor.
  • a subsequent section will relate to the types of promoters which should be used to drive the synthesis of the repressor.
  • a complete double stranded DNA constructed to express a tRNA in plant cells and plants should include the coding sequence of the desired tRNA bounded by the 5' and 3' flanking sequences specified above. As the size of this DNA approaches 400 bp, it is convenient to assemble it from smaller oligonucleotides (which can be purchased commercially), each having a length of 40-50 nucleotides, with 5' phosphoryl termini.
  • the oligonucleotides which make up the sequence of one strand of the DNA should span the 5' and 3' termini of the complementary oligonucleotides which make up the sequence of the other strand of DNA so as to permit the complete DNA to be prepared by annealing and ligating the oligonucleotides together.
  • the oligonucleotides at the termini of the DNA may be synthesized so as to include cohesive termini which will facilitate ligation to a vector for cloning in E. coli. This method has been used for the synthesis of numerous genes (see for example, Wosnick et al., 1987. Gene 60:115-127).
  • Optimal conditions used for annealing oligonucleotides depend upon the precise sequences and concentrations of the oligonucleotides.
  • Pertinent variables include using a buffer at neutral pH, (such as Tris-HCl) including 10-100 mM Na + or K + to neutralize the charges of DNA phosphates and use of equimolar concentrations of each oligonucleotide in the range of one micromolar so as to enable association in a convenient time.
  • a buffer at neutral pH such as Tris-HCl
  • equimolar concentrations of each oligonucleotide in the range of one micromolar so as to enable association in a convenient time.
  • the full length DNA is then introduced into a suitable vector (available from commercial sources) with complementary termini to those present in the DNA, for transformation into E. coli and amplification by standard procedures.
  • the DNA should be extracted from the E. coli and purified by buoyant density gradient centrifugation in CsCl or by chromatography on commercially available resins suitable for DNA purification.
  • DNA encoding the tRNA together with flanking sequences may be constructed by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • tRNA ys _it is convenient to use PCR primers complementary to the 5' and 3' flanking sequences and which also include terminal sequences cleaved by a restriction endonuclease.
  • the tRNA ⁇ ugene can be isolated from thaliana genomic DNA (EMBO/GenBank DDB5 Accession No.
  • Standard conditions for PCR reactions usually include 10 mM Tris HC1, pH 8.0, 50 mM KCl, 1.5 mM MgCl 2 , 0.01% gelatin, 2mM of each dNTP and 0.1% Triton X-100, 20 pmoles of primer and 2.5 ⁇ mls of Taq polymerase in a final volume of 0.1 ml. Temperatures and lengths of cycles depend upon the base composition of the DNA, and general rules of determining such are available in standard laboratory manuals such as Sambrook et al, 1987 and Ausubel et al, 1997.
  • PCR products are purified by standard methods, such as adsorption and elution from glass beads, and then ligated into a suitable vector and transformed into E. coli, from which the DNA may be purified by standard methods described in laboratory manuals such as Sambrook et al., 1987 and Ausubel et al., 1997.
  • the process of this invention employs expressing, in plant cells and plants, tRNA species whose anticodon sequences pair with mRNA codons other than AAA or AAG.
  • tRNA anticodon sequences other than UUU or CUU.
  • changing the plant tRNA ys anticodon sequence from UUU to CCU causes the tRNA Lys to pair with the mRNA codon AAG and to code Lys in place of Arg; a change from UUU or CUU to GUU or to AUU causes these tRNA Lys species to pair with the mRNA codons, AAC or AAU, respectively and to code Lys in place of Asn.
  • Reference to the genetic code in a standard text provides the full list of possible codons and in turn, the complementary anticodon sequences of the tRNAs which will pair with them.
  • mRNA codons AAU, AAC, CAA, CAG, CGU, CGC, CGA, CGG, AGA, AGG, UAG, UAA, UGA. It is the corresponding anticodon sequences in the DNAs which should be used in place of the TTT or CTT anticodon sequences which normally are present in the tRNA Lys gene.
  • the desired anticodon sequence can be specified in the DNA encoding the tRNA during its synthesis, either in the sequence of the oligonucleotide used to prepare the DNA, or by site-directed mutagenesis of the DNA following its preparation.
  • Site-directed mutagenesis is standard to the art as a number of procedures are clearly described in the literature and in laboratory manuals such as Sambrook et al, 1987 and Ausubel et al, 1997 and kits with all of the reagents are detailed protocols and commercially available.
  • the procedure of Nickoloff et al, (Methods in Molecular Biology 58: 455-468, 1996) has been found to be particularly convenient.
  • oligonucleotide (of approximately 18-20 nt) complementary to the anticodon stem and loop of the tRNA sequence in the DNA with the desired anticodon sequence positioned near the middle of the oligonucleotide.
  • the oligonucleotide can be purchased commercially, and is mixed with the double stranded vector DNA containing the tRNA gene, together with a second oligonucleotide which contains a selectable marker such as a restriction enzyme site.
  • the DNAs are denatured and annealed and the oligonucleotides extended by DNA polymerase, and then ligated with DNA polymerase. They are transformed into E.
  • DNAs having the desired change in the tRNA gene are identified by DNA sequencing or by hybridization to oligonucleotides having the desired sequence by procedures which are standard to the art and may be found in Sambrook et al, 1987 and Ausubel et al., 1997.
  • tRNAs which are not normally charged by the plant lysyl tRNA synthetase may acquire this property when their anticodon sequences are altered. This is the case for the plant tRNA Trp whose anticodon has been changed to CUA by mutagenesis of the DNA which encodes tRNA T ⁇ p using the procedure described above.
  • the resulting tRNA 1 ⁇ transiently expressed in plant cells codes lysine at the chain terminating codon UAG during protein synthesis in plant cells (Ulmasov et al., 1998, Nucleic Acids Research 26:5139-5141).
  • DNAs encoding this tRNA or any other which is charged by lysyl tRNA synthetase can be employed in plant cells or plants to code lysine at codons other than AAA or AAG in plant cells or plants.
  • the process of this invention may employ expressing in plant cells or plants:
  • the preparation and use of DNAs expressing these proteins follows:
  • Expression of a tRNA in a plant cell can be regulated by a repressor protein which binds to the 5' flanking sequences of the tRNA gene and which interferes with transcription.
  • a repressor protein which binds to the 5' flanking sequences of the tRNA gene and which interferes with transcription. This permits coding of lysine at codons other than AAA or AAG to be controlled during the life of the plant cell or plant and to be directed to specific tissues during the development of the plant. This may be useful to prevent possibly deleterious effects of coding of lysine at alternative codons during the synthesis of proteins which are important for the growth of the plant cell or plant.
  • the repressor protein should be expressed by a chimeric gene under the control of a promoter whose activity is regulated during development, or in specific tissues, or by chemical or physical inducers which can be applied at will.
  • the tissue, developmental stage or time at which it is desired for the tRNA to be expressed so as to code lysine at codons other than AAA or AAG determines the promoter which should be used to drive the transcription of the lac repressor or the tet repressor or other repressor protein.
  • useful promoters include (but are not limited to) the CaMV35S promoter (U.S.
  • Patent 5352605 and selected sub-elements for tissue specific expression, or the PR-l ⁇ promoter which is activated by foliar sprays of benzothiadiazole (BTH) or isonicotinic acid (Gorlach et al, 1996. Plant Cell 8:629-643) or the dark and light regulated chlorophyll A B Binding Protein Promoter (U.S. Patent 5656496).
  • BTH benzothiadiazole
  • isonicotinic acid Gorlach et al, 1996. Plant Cell 8:629-643
  • the dark and light regulated chlorophyll A B Binding Protein Promoter U.S. Patent 5656496.
  • the construction of chimeric genes which express in plant cells and plants, repressors under the control of these plant promoters together with appropriate regulatory sequences such as polyadenylation signals, an intron to determine splicing and a viral translational enhancer is now standard to the art (for example, see U.S.
  • the chimeric gene for the expression of the repressor protein may be physically linked to the gene for the expression of the tRNA, or these genes may be present on separate DNA molecules. Additional genes necessary for the expression of antibiotic resistance or other selectable markers used for the introduction, selection and maintenance of DNAs in transformed plant cells and plants should be added to these DNAs, as is standard to the art.
  • Appropriate promoters for the expression of the lysyl tRNA synthetase include those specified above, and as well, particularly those which direct expression of plant storage proteins such as the French bean ⁇ phaseolin gene promoter or the maize zein promoters or the rice glutelin promoters, so that the lysyl tRNA synthetase is expressed at the same time and in the same tissues as the genes for the seed storage proteins.
  • the specified promoter should be placed in an appropriate position to direct the synthesis of the coding sequences for the lysyl tRNA synthetase which is to be expressed.
  • lysyl tRNA synthetase Regulatory elements required for efficient expression in plant cells and plants, such as a polyadenylation signal, an intron to promote splicing and a viral translational enhancer should be included in the DNA encoding the lysyl tRNA synthetase, as is now standard to the art of expressing proteins efficiently in plant cells or plants.
  • the DNA for the expression of the lysyl tRNA synthetase may be physically linked to the DNA for the expression of the tRNA, or they may be present in separate molecules. Additional genes, including those expressing antibiotic resistance or other selectable traits used for the introduction, selection and maintenance of the DNAs in transformed plant cells and plants should be added to either DNA as is standard to the art. Expression of the A.
  • thaliana lysyl tRNA synthetase in plant cells will enhance utilization of plant tRNAs which code lysine at mRNA codons other than AAA or AAG.
  • the amino acid sequence for the A. thaliana lysyl tRNA synthetase is provided in the Sequence Listing as Seq. ID n° 1.
  • a lysyl tRNA synthetase of another organism is also expected to function with tRNAs from that organism, when expressed in plant cells. Coding of lysine at mRNA codons other than AAA or AAG may be sensitively detected and measured by the use of reporter genes whose products rely upon coding of lysine at codons other than AAA or AAG for their activity.
  • the firefly luciferase and the TEM-1 ⁇ - lactamase enzymes require a lysine in the substrate binding site or catalytic center, respectively, so genes for these enzymes with codons other than AAA or AAG in place of the codon for that critical lysine express proteins with greatly reduced, or undetectable enzymatic activity.
  • utilization in protein synthesis of tRNAs which code lysine in place of the AAA or AAG mRNA codons causes the synthesis of functional enzymes whose activity can be sensitively measured in plant cells and plants.
  • Measurement of the activity of these "reporter” enzymes is thereby a way to measure coding of lysine at codons other than AAA and AAG during protein synthesis within the plant cell or plant, and should be used to identify the plant cells or plants modified by the process of this invention.
  • LUC reporter gene having alternate codons in place of the AAG (Lys) codon 206 which is important for luciferase function is performed by site- directed mutagenesis of the LUC gene at codon 206 by standard procedures.
  • TEM-1 ⁇ lactamase reporter having a codon other than AAA (Lys) at position 73 in the consensus numbering system of Ambler et al., (Biochem. J. 276: 269-270, 1991) is performed by site-directed mutagenesis of the TEM-1 ⁇ lactamase gene using standard procedures.
  • the TEM-1 ⁇ lactamase coding sequence is made part of a chimeric gene for expression in plant cells using the CaMN35S promoter and a polyadenylation sequence using standard procedures, as is described for the construction of the LUC reporter gene (Ulmasov and Folk, 1995). Expression of luciferase may be measured as described by Ulmasov and Folk, 1995. Expression of ⁇ lactamase in plant cells may be measured with chromogenic substrates as described by Moore et al., 1997, Analytical Biochemistry 247:203- 209.
  • the process of this invention requires introducing D ⁇ As encoding tR ⁇ As which code lysine at codons other than AAA or AAG, as described in the previous sections, into plant protoplasts or plant cells or plant tissues together with genes encoding antibiotic resistance or herbicide resistance so the cells taking up the D ⁇ As are selected and the expression of the D ⁇ As stably maintained. Plants may be regenerated from the transformed plant cells or tissues using methods standard to the art.
  • D ⁇ As encoding the repressor proteins, a lysyl tR ⁇ A synthetase and the reporter gene(s) may be introduced together with the D ⁇ As encoding the tR ⁇ As so as to regulate tR ⁇ A synthesis and utilization and to measure lysine coding at alternative codons.
  • Procedures by which to introduce these D ⁇ As into particular plant cells or plant tissues and to regenerate plants from them are now standard to the art. A few representative procedures are provided below for specific families of plants, but it should be understood that as improvements and new procedures are developed, they may be substituted.
  • D ⁇ As prepared as described in the previous sections may need to be modified for introduction into plant cells or tissues by Agrobacterium-mediated transfer.
  • Procedures for the construction of Agrobacterium tumefaciens transformation vectors are well known in the art; see, for example, Hajdukiewicz et al., 1994, Plant Mol. Biol. 25:989-994 and McCormac et al., 1997, pBECKS. Molecular Biotechnology 8: 199-213.
  • D ⁇ As introduced by physical or chemical means, such as biolistic bombardment or PEG-mediated transformation do not require the specific sequences used by Agrobacterium to transfer D ⁇ A.
  • the DNAs encoding the tRNA and any proteins used to regulate or measure coding of Lys at codons other than AAA or AAG should contain or be cotransformed with DNAs containing traits suitable for the selection of transformed cells and transformed tissue that are appropriate for the plant species. Examples include the antibiotic resistance genes neomycin phosphotransferase II and hygromycin phosphotransferase or the phosphinothricin acetyltransferase or EPSP synthase genes conferring resistance to the herbicides basta or glyphosate, respectively. These DNAs should be homogenous and highly purified by CsCl gradient centrifugation or by chromatography through ion exchange or adsorption resins suitable for purifying DNA (available from commercial sources) by procedures standard to the art.
  • one appropriate method employs microprojectile bombardment of embryogenic maize suspension cultures with the aforementioned DNAs modified to include antibiotic resistance genes such as the bar gene or the neomycin phosphotransferase II (NPTII) gene having the appropriate regulatory signals to ensure their expression.
  • DNAs containing antibiotic resistance genes may be mixed together with the DNAs coding the tRNA and proteins and used to co-transform plant cells or tissues or plants.
  • Selectable traits include those encoded by the hygromycin gene, or the NPT II gene or the bar gene under the control of suitable promoters and regulatory sequences, all of which can be obtained from laboratories practicing these methods.
  • suitable promoters and regulatory sequences all of which can be obtained from laboratories practicing these methods.
  • appropriate procedures now employ microprojectile bombardment or transformation mediated by Agrobacterium vectors with bar or hygromycin or glyphosate resistance genes as selectable markers (Ortiz et al., 1996, Plant Cell Reports 15:159-163; Cheng et al., 1997. Plant Physiol. 115:971-9801
  • methods for stable transformation include agrobacterium-mediated introduction of DNA into leaf discs, biolistic bombardment and PEG- mediated transformation of protoplasts, cells and embryogenic callus, all of which are standard to the art.
  • a convenient method is transformation by vacuum infiltration of seeds with Agrobacterium vectors (Bechtold et al, 1993 C.R. Acad. Sci., Paris, 316:1194-1199).
  • Plant cells or plants into which the DNA which expresses a tRNA that codes lysine at mRNA codons other than AAA or AAG has been introduced together with the selected antibiotic resistance trait should be characterized to ascertain the DNA encoding the tRNA is present and to that lysine coding at mRNA codons other than AAA or AAG occurs.
  • specific proteins made in the plant cell or plant which are abundant and easily purified, such as the small subunit of ribulose carboxylase or a seed storage protein may be purified and characterized for their composition and amino acid sequence by standard procedures.
  • Specific applications of the process include modification of the amino acid composition of plant storage proteins.
  • the alternative codons most likely to be of value will be those which are highly represented in those particular proteins, and these usually include asparagine and glutamine.
  • coding of Lys at these mRNA codons will be of value to increase the composition of lysine in these proteins.
  • the tRNA genes are most likely to function best when accompanied by the gene for the plant lysyl tRNA synthetase under the control of a seed-specific promoter, so that enhanced utilization of the tRNA occurs during seed storage protein synthesis.
  • An alternative application of the process in plant cells or plants is to generate diversity in protein structure in particular proteins which rely upon such diversity for their function. This is the case for the antibodies, which can be expressed in plant cells and plants. Antibodies with greater structural diversity react with more antigens and thereby have utility. Regulation of altered coding by tRNA lys in plant cells or plants into which genes encoding antibodies have been introduced will cause them to be synthesized with lysine at alternative codons. Regulation of such altered coding may be determined by altering the synthesis of the tRNA lys or by regulating the synthesis of a lysyl tRNA synthetase, as described above.
  • DNAs . Isolation of A. thaliana tRNA Lys :
  • DNA encoding the A thaliana tRNA ys (GenBank accession no. U67679) was isolated from genomic DNA by PCR, utilizing primers specific for the 5' and 3' flanking sequences. The conditions for PCR included 25 cycles of 30s at 94EC, 30s at 55EC and 60s at 72EC with 5' flanking sequence primer: (SEQ ID n 0 9) : gccaaggatg tataagtgtg tcggaact 28 and
  • 3' flanking sequence primer (SEQID n° 10) : tccttgggtt tgactaacta acggg 25
  • the PCR product was purified by gel electrophoresis and ligated to Srf-1 cleaved PCR-script vector (Stratagene, Inc, La Jolla, CA), yielding ptLys-1.
  • the DNA sequence specifying the tRNA ys anticodon was altered by oligonucleotide-directed mutagenesis from CTT to GTT (Asn) or CTC (Glu) by use of the Chameleon Double-Stranded
  • LUC reporter genes which detect Lys coding at AAC or GAG codons:
  • LUC firefly luciferase
  • HindlJJ to BamHJ fragments from these plasmids were subcloned into the Hind III-BamHI site of the pPZP211 plasmid (Hajdukiewicz et al., 1994) to generate the pLUC/Lys ' -(wt), pLUC/Asn 1 -206 and pLUC/Glu reporter D ⁇ As, respectively.
  • Nicotiana benthamiana cell suspension cultures were maintained at 25E C with weekly subculture in MS-1 medium composed of Murashige and Skoog salts, Gamborg vitamins, 1 mg/1 2,4-dichlorophenozyacetic acid (2,4-D) and 3% sucrose at pH5.8. Cells were incubated on plates with MS-1 medium plus 0.25 M mannitol and 0.25 M sorbitol (MS-ms medium) for 4 hours. Prior to bombardment, the plasmid D ⁇ As were precipitated onto 1 ⁇ m gold particles and sonicated lightly to completely suspend the particles.
  • Southern Blots also confirmed the presence in the plant cells of the D ⁇ As containing the tR ⁇ A genes and the LUC genes in the plant cells.
  • Genomic D ⁇ As from individual callus lines were digested with Hind III and electrophoresed through 0.8% agarose gels. D ⁇ A fragments were transferred to nylon membranes by capillary action and UN cross- linked. Prehybridization and hybridization were performed by standard methods as described in Sambrook et al., 1987 and Ausubel et al. 1997. Probes were prepared with the Multiprime D ⁇ A labeling system (Amersham Life Science), using as template a BamH I-Sacl 1.8 Kb restriction fragment of Uid-1 ( ⁇ glucuronidase) coding sequence. (Jefferson et al., 1987 EMBO J. 6:3901-3907). These data indicate the D ⁇ As encoding the tR ⁇ A lys gene is stably maintained by the plant cells.
  • calli containing the pLUC/Asnl-206 reporter gene together with the ptLys/Asnl DNA containing the tRNA ⁇ , gene exhibited a median luciferase activity of 34% of the wild type, or approximately sevenfold greater than that of calli transformed with pLUC/Asnl -206 alone (Table 1), indicating that the tRNA ⁇ uu expressed by ptLys/Asnl DNA causes coding of Lys at AAC codons during protein synthesis, restoring luciferase activity. The efficiency of such coding is approximately 30%.
  • Calli containing the pLUC/Glu-206 reporter gene expressed 0.7% the median luciferase activity, respectively, of calli containing pLUC/Lys (wt) DNA, because of the presence of Glu at residue 206 in place of the critical lysine (Table 1). This level of luciferase activity is in agreement with that observed with this LUC gene in bacteria and in plant cell transient assays (Chen et al., 1998).
  • Coding of lysine at GUU or CUC codons did not affect the viability or growth of the plant cells.
  • the efficiency of transformation of the calli with DNAs containing the ptLys/Asn and ptLys/Glu DNAs was not significantly different than the efficiency of transformation of calli with DNAs lacking the tRNA sequences, indicating that expression of the tRNAs which code lysine at alternative codons does not impair the viability of cells.
  • DNA encoding the A. thaliana tRNA Lys was isolated from genomic A. thaliana
  • Rice (oryzae sativa L, cv Taipei 309) callus was initiated from developing embryos, subcultured weekly in a phytagel-solidified Ms-1 medium composed of Murashige and Skoog salts, Gamborg vitamins, 2 mg/1 2-4D and 3% sucrose at pH 5.8 and maintained at 28E in the dark. Rice callus was incubated in MS-1 medium plus 0.25 M mannitol and 0.25 M sorbitol for 4 hours prior to biolistic bombardment. The D ⁇ A containing the reporter was mixed with the D ⁇ A containing the tR ⁇ A gene in a 2:1 ratio and precipitated onto gold particles as described by Sanford et al., 1993. The particles were lightly sonicated and then the callus was bombarded twice using a Biolistics acceleration device (PDS-1000/lte, BioRad).
  • PDS-1000/lte, BioRad Biolistics acceleration device
  • callus was moved to Ms-1 medium for three to seven days and then subjected to selection with 1 mg/1 bialaphos for two weeks, followed by subculturing biweekly on Ms-1 medium with 3 mg/1 bialaphos (Ms-3b).
  • Bialaphos resistant, transformed callus was isolated seven to nine weeks post-bombardment and maintained as individual callus lines on Ms-3b medium. Regeneration of transformed plants was initiated by placing the transformed callus on 1% phytagel-solidified Ms-1 medium with 5 mg/1 of kinetin for 3 days.
  • the slightly desiccated callus was placed in a lighted growth room and grown on Ms-1 medium containing 0.2% phytagel, 1 mg/1 of bialaphos and 5 mg/1 of kinetin or zeatin for an additional 20 days. Regenerated plantlets/shoots were transferred to the Ms-1 medium without hormones and once the plants grew strong roots, they were transferred to artificial soil and grown to maturity at 28EC/22EC day /night and 10-hour light periods.
  • PCR was carried out with primers complementary to the 3' and 5' flanking sequences of the tRNA gene, and with primers to the Ubi promoter and LUC coding sequence, respectively.
  • the PCR conditions included 30 cycles of 94EC for 1 min, 60EC for 2 min. 72EC for 2 min.
  • the PCR products were blotted onto nylon filters and hybridized with ⁇ - 32 P- dATP-labeled oligonucleotide probe complementary to the sequences and the wild type or mutated LUC genes or to the anticodon of the tRNA gene (Ausubel et al., 1997).
  • Hybridization was carried out at 50EC in a SSPE solution (Sambrook et al, 1987).
  • the filters were washed at 65EC to 75EC for 20 min in 0.5M tetramethylammonium chloride buffered with Tris-HCl, 50mM, pH 8.0 and .002M EDTA with 0.1% SDS at 59C, and the patterns were visualized by autoradiography.
  • the bar resistant callus transformed with the 3 different DNA preparations (LUCwt, LUC/am and LUC/am plus ptLys-am/Gus) were tested for lysine coding at the UAG codon by measuring luciferase activity from the Luc/am reporter gene.
  • Samples from 43 independent callus lines were examined.
  • Callus with the Luc/am-206 gene alone exhibited 0.04% of the luciferase activity of callus with the LUCwt DNA, while callus containing the LUC/am-206 DNA together with the ptLys-am/Gus DNA encoding tRNA L c y u A exhibited 11% of the wild type luciferase activity, on average a 250 fold increase.
  • Transformants containing the LUCwt DNA exhibited proteins that reacted with the LUC antibody which ranged from 0.3% to 2% of the total soluble protein, compared with standard luciferase protein.
  • the transformed callus and plants containing the LUC/am DNA exhibited no proteins which reacted with antibody directed against luciferase.
  • Transformants containing both the LUC/am and pt Lys-am/GUS DNAs exhibited approximately 0.1% of total soluble protein that reacted with the antibody directed against luciferase ptLys-am/GUS.
  • PCR products were generated with primers complementary to either tRNA flanking sequences or the Ubi promoter and to luciferase sequences from transformed callus lines and plants. The PCR products hybridized with radiolabeled oligonucleotides specific for the sequences of wild type and mutant LUC and the tRNA gene.
  • PCR products from callus lines and plants containing LUC/am and tRNAam constructs showed clear bands of hybridization when probed with oligonucleotides complementary to the region of the LUC and DNA with the chain termination codon, and the anticodon region in ptRNAam, but no hybridizing bands were detected when probed with the oligonucleotide coding wild type sequence.
  • LUC/am and ptLys-am/GUS constructs maintained their original mutated codons after integration into the rice genomes of the transformed callus and plants and therefore the luciferase activity detected in these transformants is due to expression of the tRNA ys species which causes coding of lysine at the UAG chain termination codon in LUC/am and restores luciferase activity and protein.
  • DNAs expressing tRNA L g s UA which encode the A. thaliana lysyl tRN synthetase and luciferase and the tet repressor.
  • DNA encoding the A. thaliana tRNA Lys was isolated and mutagenized to encode tRNA L s UA as described in the previous sections.
  • tet 0 1 operator sequence SEQ ID n° 16
  • actctatcac tgatagagt 19 by ligation of a double stranded oligonucleotide of which one strand has this sequence.
  • DNA encoding the A. thaliana tRNA A was prepared as described previously by site-directed mutagenesis of a plasmid encoding A. thaliana tRNA T ⁇ . (Ulmasov and Folk,
  • DNA encoding the A. thaliana lysyl tRNA synthetase was isolated by identifying sequences in the EMBL/GenBank/DDBJ database with homology to the S. cerevisae lysyl tRNA synthetase. These sequences were used to prepare PCR primers with which to isolate the coding sequence the A. thaliana lysyl tRNA synthetase from an A. thaliana cDNA library (Sequence ID No:001). This gene, when expressed in E. coli, produces an active lysyl tRNA synthetase which charges A. thaliana tRNA Lys .in vitro. It was placed under the control of the CaMV35S promoter and upstream of the octopine synthase polyadenylation signal for expression in plant cells.
  • Sequences encoding the Luc/am-206 reporter gene which requires Lys coding at UAG codon and sequences encoding the tetR repressor were placed downstream of the CaMN35S promoter and upstream of the octopine synthase polyadenylation signal for expression in plant cells as described in Chen et al., 1998 and Ulmasov et al, 1997.
  • Carrot (Daucus carota) protoplasts were prepared from carrot cells grown under ambient light and temperature in a medium containing 4.3 gn/L Murashige and Skoog salts,
  • Protoplasts were pelleted and washed with a solution containing 154 mM NaCl, 5 mM KCl, 125 mM CaCl 2 and 5 mM glucose, pH 6.0 and stored for 0.5 to 4 hours at 5NC. Immediately prior to transformation, the protoplasts were sedimented and resuspended in a solution containing 5mM MES, 20 mM CaCl 2 , 0.5M Mannitol, pH 5.7. Plasmid DNA was mixed with 10 6 protoplasts in 0.3 ml and 0.3 ml of 40% solution of polyethylene glycol (PEG) was added and incubated for 5 min at room temperature. Samples were diluted with hormone-free growth medium and incubated for 24-48 hours at room temperature prior to analysis.
  • PEG polyethylene glycol
  • This result was obtained in six independent experiments, indicating that charging and subsequent utilization of the tRNA L cu A is modulated by expression of high levels of lysyl tRNA synthetase. The most likely explanation for this is that the tRNA lys is not fully charged by lysine within the cell, and expression of high levels of lysyl tRNA synthetase increases charging and concomitant utilization during protein synthesis.
  • a tRNA T c P UA which, as a result of changing of its anticodon from CGG to CUA is charged by purified A. thaliana lysyl tRNA synthetase.
  • Expression of tRNA ⁇ A in plant cells causes lysine coding at UAG chain terminating codons, as evidenced by the restoration of luciferase activity encoded by LUCam-206 by DNA encoding tRNA T ⁇ jA . Utilization of tRNA ⁇ to code lysine at UAG codons was increased significantly by expression of the gene for A. thaliana lysyl tRNA synthetase, as with tRNA 1 ⁇ ,. These observations indicate that it is possible to regulate coding of lysine at alternative codons by expressing A. thaliana lysyl tRNA synthetase.
  • Type I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.). Planta 196: 7-14. Wilde, R.J., Shufflebottom, D., Cooke, S., Jasinska, I., Merryweather, A., Beri, R., Brammar, W.J., Bevan, M., and Schuch, W. 1992. Control of gene expression in tobacco cells using a bacterial operator-repressor system. EMBO J. 11_: 1251-1259.

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Abstract

La présente invention concerne un procédé permettant de faire coder aux cellules de plantes, ou aux tissus de plantes, de la lysine, ou de la méthionine, ou du tryptophane, ou de la thréonine, à des codons d'ARNm différents de ceux spécifiant normalement ces acides aminés particuliers durant la synthèse protéique. Ce procédé consiste: i) à préparer un ADN à exprimer un ARNt chargé en lysine qui se lie, lors de la synthèse protéique, à des codons d'ARNm autres que AAA ou AAG, ou à préparer un ADN qui exprime un ARNt chargé en méthionine qui se lie à des codons d'ARNm autres que AUG, ou à préparer un ADN qui exprime un ARNt chargé en tryptophane qui se lie à des codons d'ARNm autres que UGG, ou à préparer un ADN qui exprime un ARNt chargé en thréonine qui se lie à des codons d'ARNm autres que ACU, ACC, ACA, ACG, ii) et à introduire un ou plusieurs desdits ADN dans des protoplastes de plantes, dans des cellules de plantes, ou dans des plantes.
PCT/IB1999/002125 1998-12-24 1999-12-24 PROCEDE PERMETTANT DE FAIRE CODER AUX PLANTES ET CELLULES DE PLANTES DE LA LYSINE OU DE LA METHIONINE OU DU TRYPTOPHANE OU DE LA THREONINE A DES CODONS D'ARNm DIFFERENTS DE CEUX SPECIFIANT NORMALEMENT CES ACIDES AMINES PARTICULIERS DURANT LA SYNTHESE PROTEIQUE WO2000040709A2 (fr)

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AU41354/00A AU4135400A (en) 1998-12-24 1999-12-24 Process for causing plant cells and plants to code lysine or methionine or tryptophan or threonine at mrna codons other than those normally specifying these particular amino acids during protein synthesis

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WO2018092072A1 (fr) * 2016-11-16 2018-05-24 Cellectis Méthodes de modification de la teneur en acides aminés de plantes par décalages du cadre de lecture

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WO2018092072A1 (fr) * 2016-11-16 2018-05-24 Cellectis Méthodes de modification de la teneur en acides aminés de plantes par décalages du cadre de lecture
US11312972B2 (en) 2016-11-16 2022-04-26 Cellectis Methods for altering amino acid content in plants through frameshift mutations

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