WO2004013092A2 - Beta-alanine n-methyl-transferase - Google Patents

Beta-alanine n-methyl-transferase Download PDF

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WO2004013092A2
WO2004013092A2 PCT/US2002/024936 US0224936W WO2004013092A2 WO 2004013092 A2 WO2004013092 A2 WO 2004013092A2 US 0224936 W US0224936 W US 0224936W WO 2004013092 A2 WO2004013092 A2 WO 2004013092A2
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nmtase
protein
seq
nucleic acid
purified
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PCT/US2002/024936
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WO2004013092A3 (fr
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Bala Rathinasabapathi
Suresh Badu Raman
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University Of Florida
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Publication of WO2004013092A3 publication Critical patent/WO2004013092A3/fr

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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
    • C12N15/8273—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 drought, cold, salt resistance
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

  • the invention relates generally to the fields of biology, botany, and agricultural sciences. More particularly, the invention relates to the cloning, purification, and characterization of an N-methyltransferase from Limonium latifolium, and to methods and compositions for modulating a plant's resistance to environmental stress.
  • quaternary ammonium compounds (QACs) in response to abiotic stresses such as drought and salinity.
  • QACs quaternary ammonium compounds
  • ⁇ -alanine betaine is a more suitable osmoprotectant than glycine betaine under saline hypoxic conditions since the first step in glycine betaine synthesis requires molecular oxygen. Id.
  • ⁇ -alanine betaine accumulation was proposed to be an evolutionary strategy to avoid metabolic limitations for choline (Hanson et al., Proc Natl Acad Sci USA 91:306-310, 1994) since ⁇ -alanine betaine is synthesized from the ubiquitous primary metabolite ⁇ -alanine.
  • ⁇ -alanine betaine is synthesized by S-adenosyl-L-methionine (AdoMet) dependent N-methylation of ⁇ -alanine via N-methyl ⁇ -alanine and NN-dimethyl ⁇ -alanine (Rathinasabapathi et al., Physiol Plant 109: 225-231, 2000; Figure 1).
  • NMTase AdoMet dependent N-methyltransferase activities were demonstrated in ⁇ -alanine betaine accumulating members of the Plumbaginaceae family (Rathinasabapathi et al., Physiol Plant 109: 225-231, 2000). Heretofore, however, the gene encoding the NMTase was not cloned, and the protein responsible for the NMTase activities was uncharacterized.
  • the invention relates to the purification and characterization of an NMTase from L. latifolium and the cloning of the gene that encodes the NMTase.
  • the NMTase was purified from L. latifolium leaf tissue using a seven-step protocol.
  • Biochemical characterization of the purified enzyme indicated that it had an isoelectric point (pi) of 5.1, and that it was a dimer of 43 kD subunits.
  • Functional studies indicated that the purified enzyme catalyzes all three of the N-methylations involved in the synthesis of ⁇ -alanine betaine.
  • Peptide sequencing studies indicated that the purified NMTase shared some sequence similarity to methyltransferases from other organisms.
  • Peptide sequences from purified ⁇ -alanine NMTase were used to design degenerate primers for RT-PCR that yielded a 500 bp cDNA clone.
  • a full-length 1414 bp cDNA representing NMTase A was cloned by screening a lambda gtlO L. latifolium cDNA library using the 500 bp cDNA clone as a probe.
  • Transgenic tobacco plants expressing L. latifolium ⁇ -alanine NMTase accumulated ⁇ -alanine betaine and exhibited a stress-tolerant phenotype.
  • a purified nucleic acid which includes a nucleotide sequence that encodes a protein that: (a) shares at least 90%> (e.g., 95%, 98%>, or 100%>) sequence identity with at least 50 (e.g., 100, 500, or more) contiguous amino acids of SEQ ID NO:29; and (b) has at least one functional activity of a native Limonium latifolium NMTase.
  • the nucleotide sequence can also be one that shares at least 65% (e.g., 75%, 85%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO:29.
  • a vector that includes a nucleic acid of the invention hi the vector, the nucleic acid can be operably linked to one or more expression control sequences (e.g., a promoter).
  • the invention features a purified protein that: (a) shares at least 90% (e.g., 95% ⁇ or 99%) sequence identity with at least 50 contiguous amino acids of SEQ ID NO:
  • the purified protein can also be one that shares at least 85% (e.g., 95% or 100%) sequence identity with SEQ LD NO:28.
  • the cell is a plant cell (e.g., one in a plant).
  • the invention further includes a purified antibody that specifically binds to the protein of the invention.
  • a method of modulating stress resistance in a plant cell or seed includes the steps of: (a) providing a plant cell or seed; and (b) introducing into the plant cell or seed a purified nucleic acid or purified protein of the invention.
  • Agene® is meant a nucleic acid molecule that codes for a particular protein, or in certain cases a functional or structural RNA molecule.
  • the NMTase gene encodes the NMTase protein.
  • nucleic acid or a “nucleic acid molecule” means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
  • a “purified” nucleic acid molecule is one that has been substantially separated or isolated away from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
  • the term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote.
  • purified nucleic acids include cDNAs, fragments of genomic nucleic acids, nucleic acids produced by polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules.
  • a "recombinant" nucleic acid molecule is one made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • NMTase gene ANMTase polynucleotide
  • ANMTase nucleic acid or simply “NMTase” is meant a native NMTase-encoding nucleic acid sequence, e.g., the native L. latifolium NMTase cDNA shown in Fig. 7A (SEQ ID NO: 28), genomic sequences from which NMTase cDNA can be transcribed, and/or allelic variants and homologs of the foregoing.
  • the terms encompass double-stranded DNA, single-stranded DNA, and RNA.
  • isolated and purified refer to a enzymatically active molecule substantially separated from other molecules that are present in a cell or organism in which the enzymatically active molecule naturally occurs.
  • a purified NMTase includes, e.g., a NMTase-containing cell extract that has been subjected to one or more number of chromatographic separations.
  • isolated and purified as used herein also refer to a molecule produced artificially (i.e., outside the organism in which the molecule naturally occurs) by molecular biological techniques (e.g., recombinant DNA technology) or chemical synthesis (e.g., peptide synthesis).
  • protein or “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
  • a “purified” polypeptide is one that has been substantially separated or isolated away from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
  • NMTase protein NMTase polypeptide
  • NMTase polypeptide an expression product of an NMTase gene such as the protein whose sequence is shown in Fig. 7B (SEQ LD NO:29); or a protein that shares at least 65% (but preferably 75, 80, 85, 90 , 95, 96, 97 ,98, or 99%) amino acid sequence identity with SEQ ID NO:29 and displays a functional activity of NMTase.
  • a “functional activity” of a protein is any activity associated with the physiological function of the protein.
  • NMTase For example, functional activities of NMTase include N-methyltransferase activity, the ability to interact with substrates of the enzyme (e.g., ⁇ -alanaine, N-methyl ⁇ -alanaine, and N,N-dimethyl ⁇ -alanaine), and the ability to impart stress resistance to a plant or plant cell.
  • substrates of the enzyme e.g., ⁇ -alanaine, N-methyl ⁇ -alanaine, and N,N-dimethyl ⁇ -alanaine
  • nucleic acid molecule or polypeptide When referring to a nucleic acid molecule or polypeptide, the term “native” refers to a naturally-occurring (e.g., a "wild-type") nucleic acid or polypeptide.
  • a “homolog” of a L. latifolium NMTase gene is a gene sequence encoding a NMTase polypeptide isolated from a plant other than L. latifolium.
  • a “homolog” of a native NMTase polypeptide is an expression product of an NMTase homolog.
  • a "fragment" of an NMTase nucleic acid is a portion of an NMTase nucleic acid that s less than full-length and comprises at least a minimum length capable of hybridizing specifically with a native NMTase nucleic acid under stringent hybridization conditions.
  • the length of such a fragment is preferably at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 30 nucleotides of a native NMTase nucleic acid sequence.
  • a "fragment" of an NMTase polypeptide is a portion of an NMTase polypeptide that is less than full-length (e.g., a polypeptide consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or more amino acids of native NMTase), and preferably retains at least one functional activity of native NMTase.
  • low stringency conditions means in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 42°C, followed by washing in IX SSPE, 0.2% SDS, at 50°C;
  • moderate stringency conditions means in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 65°C;
  • high stringency conditions means in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by wasliing in 0. IX SSPE, and 0.1% SDS at 65°C.
  • sequence identity means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions.
  • sequence identity means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions.
  • the length of the compared sequences is at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides.
  • Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705).
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705
  • silent changes are those that substitute one or more base pairs in the nucleotide sequence, but do not change the amino acid sequence of the polypeptide encoded by the sequence.
  • Constant changes are those in which at least one codon in the protein-coding region of the nucleic acid has been changed such that at least one amino acid of the polypeptide encoded by the nucleic acid sequence is substituted with a another amino acid having similar characteristics.
  • Examples of conservative amino acid substitutions are ser for ala, thr, or cys; lys for arg; gin for asn, his, or lys; his for asn; glu for asp or lys; asn for his or gin; asp for glu; pro for gly; leu for ile, phe, met, or val; val for ile or leu; ile for leu, met, or val; arg for lys; met for phe; tyr for phe or trp; thr for ser; tip for tyr; and phe for tyr.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors.”
  • a first nucleic-acid sequence is "operably" linked with a second nucleic-acid sequence when the first nucleic-acid sequence is placed in a functional relationship with the second nucleic-acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • a cell, tissue, or organism into which has been introduced a foreign nucleic acid, such as a recombinant vector, is considered “transformed,” “transfected,” or “transgenic.
  • AA “transgenic” or “transformed” cell or organism also includes progeny of the cell or organism, including progeny produced from a breeding program employing such a "transgenic" cell or organism as a parent in a cross.
  • a plant transgenic for NMTase is one in which NMTase nucleic acid has been introduced.
  • antibody means an immunoglobulin or fragment of an immunoglobulin that retains a function of an intact immunoglobulin, e.g., antigen-binding or effector functions.
  • NMTase-specific antibody is meant an antibody that binds NMTase (e.g., a protein having the amino acid sequence of SEQ ID NO:29), and displays no substantial binding to other naturally occurring proteins other than those sharing the same antigenic determinants as NMTase.
  • the term includes polyclonal and monoclonal antibodies.
  • bind means that one molecule recognizes and adheres to a particular second molecule in a sample, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample.
  • a first molecule that "specifically binds" a second molecule has a binding affinity greater than about 10 5 to 10 6 liters/mole for that second molecule.
  • labeled with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end- labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • Figure 1 is a schematic overview of the synthetic pathway to ⁇ -alanine betaine. Each downward arrow represents an AdoMet dependent N-methylation step.
  • Figure 2 is a graph showing the NMTase activity and protein amounts in fractions separated by anion exchange chromatography using DEAE-Fractogel. The procedure is described in the methods section.
  • NMTase activities (nmol h "1 /fraction) against ⁇ -alanine (BA), N-methyl ⁇ -alanine (MM) and N, N-dimethyl ⁇ -alanine (DM) are indicated by squares, triangles and stars, respectively.
  • the predicted KCl gradient (20 to 300 mM) is shown in a dotted line.
  • Protein content (mg/fraction), estimated by the modified Lowry's method (Peterson et al., Anal Biochem 83:346-356, 1977) is shown in open circles.
  • BA ⁇ -alanine
  • MM N-methyl ⁇ -alanine
  • DM N,N-dimethyl ⁇ - alanine
  • Figure 4 is a graph showing the NMTase activity and protein concentrations in fractions separated by adenosine agarose affinity cliromatography. Protein elution profile by OD 280 of fractions is shown for unbound (UB), 200 mM KCl wash (KW) and substrate elution with 5 mM AdoMet (AE). Note that absorbance in the AdoMet elution is largely due to the AdoMet and not protein.
  • NMTase activities (nmol h "1 /fraction) with ⁇ -alanine (BA), N-methyl ⁇ -alanine (MM) and NN-dimethyl ⁇ -alanine (DM) measured in the unbound fraction, 200 mM KCl wash and AdoMet elution are shown in the inset.
  • Figure 5 is an autoradiograph of a gel from SDS-PAGE analysis of the purified L. latifolium NMTase and Photoaffinity labeling.
  • Lane A Precision SDS-Protein markers (Bio- Rad 161-0362).
  • Lane B SDS-Denatured protein (20 ng) from the adenosine agarose step (Table I), separated in a 12% acrylamide gel and stained with silver stain.
  • Lane C Partially purified (100-fold) NMTase fraction following photoaffinity labeling with S-Adenosyl-L- [ et/zy/- 3 H]Met, SDS-PAGE and autoradiography.
  • Lane D Partially purified (100-fold) NMTase fraction following photoaffinity labeling with S-Adenosyl-L-[metAy/- H]Met in the presence of AdoHCy, SDS-PAGE and autoradiography.
  • Figure 6 illustrates two graphs showing the results of a kinetic analysis of L. latifolium NMTase protein.
  • A Effect of varying Ado-Met concentration on the reaction velocity shown in a plot of s/v versus s. Ado-Met concentration was varied from 0 to 300 ⁇ M and ⁇ -alanine concentration was kept at 10 mM. Inset shows the direct plot.
  • B Effect of varying ⁇ -alanine on the reaction velocity shown in a plot of s/v versus s. ⁇ -alanine levels were varied between 0 and 10 mM. Ado-Met concentration was kept at 100 ⁇ M.
  • Figure 7 (A) illustrates a full length NMTase cDNA of L. Latifolium (SEQ ID NO: 1
  • the invention provides compositions and methods relating to NMTase, as well as methods and compositions for modulating stress resistance in a plant cell or plant seed, including, in particular, resistance to salinity and hypoxia.
  • stress resistance in a plant cell is modulated by introducing into the cell a nucleic acid encoding a functional ⁇ -alanine NMTase.
  • Stress resistance in a plant cell can also be modulated by introducing into the cell a purified ⁇ -alanine NMTase.
  • the cell into which has been introduced a nucleic acid encoding ⁇ -alanine NMTase, a purified NMTase or NMTase fragment thereof, is preferably a plant cell, e.g., one other than L. latifolium.
  • the plant cell can be one within a plant.
  • PCR-primer pairs can be derived from known sequences by known techniques such as using computer programs intended for that purpose (e.g., Primer, Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA.). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862 and Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers.
  • Immunological methods e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting are described, e.g., in Current Protocols in Immunology, ed. Coligan et al. (1991) John Wiley & Sons, New York; and Methods of Immunological Analysis, ed. Masseyeff et al. (1992) John Wiley & Sons, New York.
  • Nucleic Acids Encoding NMTase The invention provides a purified nucleic acid (polynucleotide) that encodes a polypeptide having the amino acid sequence of FIG. 7B (SEQ ID NO:29).
  • a preferred nucleic acid molecule of the invention is the native NMTase polynucleotide shown in FIG. 7A (SEQ ID NO:28).
  • native NMTase polynucleotide was discovered in a L. latifolium cDNA library, nucleic acid molecules encoding a polypeptide of the present invention can be obtained from L. latifolium plants.
  • Nucleic acid molecules of the present invention may be in the form of RNA or in the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA).
  • the DNA may be double- stranded or single-stranded, and if single-stranded may be the coding (sense) strand or non- coding (anti-sense) strand.
  • the coding sequence which encodes native NMTase may be identical to the nucleotide sequence shown in FIG. 7A (SEQ ID NO:28). It may also be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the polynucleotide of SEQ LD NO:28.
  • nucleic acid molecules within the invention are variants of NMTase such as those that encode fragments, analogs and derivatives of native NMTase.
  • Such variants may be, e.g., a naturally occurring allelic variant of native NMTase, a homolog of native NMTase, or a non-naturally occurring variant of native NMTase.
  • These variants have a nucleotide sequence that differs from native NMTase in one or more bases.
  • the nucleotide sequence of such variants can feature a deletion, addition, or substitution of one or more nucleotides of a native NMTase.
  • Nucleic acid insertions are preferably of about 1 to 10 contiguous nucleotides, and deletions are preferably of about 1 to 30 contiguous nucleotides.
  • variant nucleic acid molecules encode polypeptides that substantially maintain an NMTase functional activity.
  • variant nucleic acid molecules encode polypeptides that lack or feature a significant reduction in an NMTase functional activity.
  • preferred variant nucleic acids feature silent or conservative nucleotide changes.
  • variant NMTase polypeptides displaying substantial changes in one or more functional activities of native NMTase can be generated by making nucleotide substitutions that cause less than conservative changes in the encoded polypeptide.
  • nucleotide substitutions are those that cause changes in (a) the structure of the polypeptide backbone; (b) the charge or hydrophobicity of the polypeptide; or (c) the bulk of an amino acid side chain.
  • Nucleotide substitutions generally expected to produce the greatest changes in protein properties are those that cause non-conservative changes in codons.
  • codon changes that are likely to cause major changes in protein structure are those that cause substitution of (a) a hydrophilic residue, e.g., serine or threonine, for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histadine, for (or by) an electronegative residue, e.g., glutamine or aspartine; or (d) a residue having a bulky side chain, e.g., phenylalanine, for (or by) one not having a side chain, e.g., glycine.
  • a hydrophilic residue e.g., serine or threonine
  • a hydrophobic residue e.g.,
  • Naturally occurring allelic variants of native NMTase within the invention are nucleic acids isolated from L. latifolium that have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with native NMTase, and encode polypeptides having as at least one functional activity in common with native NMTase.
  • 75% e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%
  • Homologs of native NMTase within the invention are nucleic acids isolated from other species that have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with native NMTase, and encode polypeptides having at least one functional activity in common with native NMTase.
  • Naturally occurring allelic variants of NMTase and homologs of NMTase can be isolated by screening L. latifolium and non- .
  • latifolium for a native NMTase functional activity (e.g., ⁇ -alanine methylation) using a library screen similarly to the method of identification of native NMTase described herein, other assays described herein, or other techniques known in the art.
  • the nucleotide sequence of such homologs and allelic variants can be determined by conventional DNA sequencing methods. Alternatively, public or non-proprietary nucleic acid databases can be searched to identify other nucleic acid molecules having a high percent (e.g., 70, 80, 90%) or more) sequence identity to native NMTase. Once identified, these sequences can be incorporated into expression constructs that can be used in various assays such as those described herein to screen for those molecules that encode proteins which share or lack one or more functional activities of native NMTase.
  • Non-naturally occurring NMTase variants are nucleic acids that do not occur in nature (e.g., are made by the hand of man), have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%o, 98%, and 99%>) sequence identity with native NMTase, and encode polypeptides having as at least one functional activity in common with native NMTase.
  • 75% e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%o, 98%, and 99%>
  • non- naturally occurring NMTase nucleic acids are those that encode a fragment of an NMTase protein, those that hybridize to native NMTase or a complement of native NMTase under stringent conditions, those that share at least 65% sequence identity with native NMTase or a complement of native NMTase, and those that encode an NMTase fusion protein.
  • Nucleic acids encoding fragments of NMTase within the invention are those that encode, e.g., 2, 5, 10, 25, 50, 100, 150, 200, 250, 300, or more amino acid residues of NMTase.
  • Shorter oligonucleotides e.g., those of 6, 12, 20, 30, 50, 100, 125, 150 or 200 base pairs in length
  • Shorter oligonucleotides that encode or hybridize with nucleic acids that encode fragments of NMTase can be used as probes, primers, or antisense molecules.
  • Longer polynucleotides e.g., those of 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or 1300 base pairs
  • Nucleic acids encoding fragments of NMTase can be made by enzymatic digestion (e.g., using a restriction enzyme) or chemical degradation of full length NMTase or variants of NMTase.
  • nucleic acids that hybridize under stringent conditions to the nucleic acid of SEQ LD NO:28 or the complement of SEQ ID NO:28 are also within the invention.
  • such nucleic acids can be those that hybridize to SEQ ID NO:28 or the complement of SEQ ID NO:28 under low stringency conditions, moderate stringency conditions, or high stringency conditions are within the invention.
  • Preferred such nucleotide acids are those having a nucleotide sequence that is the complement of all or a portion of SEQ LD NO:28.
  • variants of NMTase within the invention are polynucleotides that share at least 65 % (e.g., 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99%) sequence identity to SEQ ID NO:28 or the complement of SEQ ID NO:28.
  • Nucleic acids that hybridize under stringent conditions to or share at least 65%> sequence identity with SEQ ID NO:28 or the complement of SEQ ID NO:28 can be obtained by techniques known in the art such as by making mutations in native NMTase, by isolation from an organism expressing such a nucleic acid (e.g., a L. latifolium plant expressing a variant of native NMTase), or a non- L. latifolium plant expressing a homolog of native NMTase.
  • Nucleic acid molecules encoding NMTase fusion proteins are also within the invention.
  • Such nucleic acids can be made by preparing a construct (e.g., an expression vector) that expresses an NMTase fusion protein when introduced into a suitable host.
  • a construct e.g., an expression vector
  • such a construct can be made by ligating a first polynucleotide encoding an NMTase protein fused in frame with a second polynucleotide encoding another protein such that expression of the construct in a suitable expression system yields a fusion protein.
  • oligonucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. Such oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. Oligonucleotides within the invention may additionally include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci
  • the oligonucleotides may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • a peptide e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • NMTase polypeptides disclosed herein those skilled in the art can create nucleic acid molecules that have minor variations in their nucleotides, by, for example, standard nucleic acid mutagenesis techniques or by chemical synthesis. Variant NMTase nucleic acid molecules can be expressed to produce variant NMTase polypeptides.
  • Antisense, Ribozyme, Triplex, and RNA Interference Techniques Although a major application of the invention involves increasing a plant cell's or plant's stress tolerance by increasing the expression of NMTase in the plant cell or plant, in other applications it may be desired to decrease NMTase expression in a cell. Thus, another aspect of the invention relates to the use of purified antisense nucleic acids to inhibit expression of NMTase. Antisense nucleic acid molecules within the invention are those that specifically hybridize (e.g.
  • binding under cellular conditions to cellular mRNA and/or genomic DNA encoding an NMTase protein in a manner that inhibits expression of the NMTase protein, e.g., by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • Antisense constructs can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an NMTase protein.
  • the antisense construct can take the form of an oligonucleotide probe generated ex vivo which, when introduced into an NMTase expressing cell, causes inhibition of NMTase expression by hybridizing with an mRNA and/or genomic sequences coding for NMTase.
  • Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g.
  • nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see, e.g., U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659- 2668.
  • oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of an NMTase encoding nucleotide sequence, are preferred.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to NMTase mRNA.
  • the antisense oligonucleotides will bind to NMTase mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
  • oligonucleotides complementary to either the 5' or 3' untranslated, non-coding regions of an NMTase gene could be used in an antisense approach to inhibit translation of endogenous NMTase mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably less that about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length.
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al.(Nucl. Acids Res.
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451, 1988), etc.
  • antisense molecules should be delivered into cells that express NMTase in vivo.
  • a number of methods have been developed for delivering antisense DNA or RNA into cells.
  • antisense molecules can be introduced directly into the tissue site using bombardment-based methodology (see, e. g., Christou P, Plant Mol Biol 35:197, 1997) or by Agrobacterium-mediated transformation (see, e.g., Hiei et al., Plant Mol Biol 35:205, 1997).
  • modified antisense molecules designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be used.
  • a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter.
  • the use of such a construct to transform L. latifolium plants will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous NMTase transcripts and thereby prevent translation of NMTase mRNA.
  • NMTase activity can also be inhibited in a cell using other technologies including ribozymes, gene inactivation, and RNA interference.
  • Ribozyme molecules designed to catalytically cleave NMTase mRNA transcripts can also be used to prevent translation o ⁇ NMTase mRNA and expression of NMTase. See, e.g., PCT Publication No. WO 90/11364, published Oct. 4, 1990; Sarver et al., Science 247:1222-1225, 1990; U.S. Pat. No. 5,093,246; and Haseloff and Gerlach, Nature 334:585-591, 1988.
  • Endogenous NMTase gene expression can also be reduced by inactivating or "knocking out" the NMTase gene or its promoter using targeted homologous recombination. See, e.g., Kempin et al., Nature 389:802, 1997; Smithies et al., Nature 317:230-234, 1985; Thomas and Capecchi, Cell 51:503-512, 1987; and Thompson et al, Cell 5:313-321, 1989.
  • endogenous NMTase gene expression might be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the NMTase gene (i.e., the NMTase promoter and/or enhancers) to form triple helical structures that prevent transcription of the NMTase gene in target cells.
  • deoxyribonucleotide sequences complementary to the regulatory region of the NMTase gene i.e., the NMTase promoter and/or enhancers
  • dsRNA double-stranded RNA
  • dsRNA double-stranded RNA
  • Agrobacteriwn-mediated transformation hi this manner, such dsRNA is persistent and inherited.
  • expression of dsRNA can interfere with accumulation of endogenous mRNA encoding a target protein (e.g., NMTase).
  • the invention also includes oligonucleotide probes (i.e., isolated nucleic acid molecules conjugated with a detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme); and oligonucleotide primers (i.e., isolated nucleic acid molecules that can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase).
  • a detectable label or reporter molecule e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme
  • oligonucleotide primers i.e., isolated nucleic acid molecules that can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target
  • Probes and primers within the invention are generally 15 nucleotides or more in length, preferably 20 nucleotides or more, more preferably 25 nucleotides, and most preferably 30 nucleotides or more.
  • Preferred probes and primers are those that hybridize to the native NMTase sequence (SEQ ID NO: 28) under high stringency conditions, and those that hybridize to NMTase homologs under at least moderately stringent conditions.
  • probes and primers according to the present invention have complete sequence identity with the native L.
  • latifolium NMTase sequence although probes differing from the L. latifolium NMTase sequence and that retain the ability to hybridize to native NMTase sequences under stringent conditions may be designed by conventional methods. Primers and probes based on the native L. latifolium NMTase sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed NMTase sequences by conventional methods, e.g., by re-cloning and sequencing a L. latifolium NMTase cDNA.
  • Vectors for Expressing NMTase can be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
  • a construct preferably is a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • conventional compositions and methods for preparing and using vectors and host cells are employed, as discussed, e.g., in Sambrook et al., supra, or Ausubel et al., supra.
  • NMTase genes in plants is achieved by introducing into a plant a nucleic acid sequence containing an NMTase gene encoding an NMTase polypeptide.
  • a number of vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants are known. See, e.g., Pouwels et al. (1985) Cloning Vectors: A Laboratory Manual, Supp. 1987; Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology, Academic Press; and Gelvin et al. (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers.
  • plant expression vectors include (1) one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker.
  • plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- , or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • CaMV cauliflower mosaic virus
  • CaMV 35S promoter a caulimovirus promoter
  • These promoters confer high levels of expression in most plant tissues, and are generally not dependent on the particular encoded proteins to be expressed.
  • CaMV is a source for both the 35S and 19S promoters.
  • the CaMV 35S promoter is a strong promoter. See, e.g., Odel et al., Nature 313:810, 1985; Dekeyser et al, Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220:389, 1990.
  • Plant promoters that may be useful in the invention are known. See, e.g., An et al, Plant Physiol. 88:547, 1988; Fromm et al., Plant Cell 1:977, 1989; Callis et al. Plant Physiol. 88: 965, 1988; Kuhlemeier et al., Plant Cell 1: 471, 1989; Schaffner and Sheen, Plant Cell 3: 997, 1991; Simpson et al., EMBO J. 4: 2723, 1985; Marcotte et al., Plant Cell 1 :969, 1989; Siebertz et al., Plant Cell 1: 961, 1989; Roshal et al, EMBO J.
  • Plant expression vectors may also include RNA processing signals such as introns, which have been shown to be important for efficient RNA synthesis and accumulation. Callis et al., Genes and Dev. 1: 1183, 1987. The location of the RNA splice sequences can influence the level of transgene expression in plants, hi view of this fact, an intron may be positioned upstream or downstream of an NMTase polypeptide-encoding sequence in the transgene to modulate levels of gene expression.
  • Expression vectors within the invention may also include regulatory control regions which are generally present in the 3' regions of plant genes. See, e.g., Thornburg et al., Proc. Natl Acad. Sci USA 84: 744, 1987; An et al., Plant Cell 1: 115, 1989.
  • a 3' terminator region may be included in the expression vector to increase stability of the mRNA.
  • 3' terminators derived from octopine or nopaline synthase genes could be used.
  • Plant expression vector within the invention preferably contain a selectable marker gene used to identify the cells that have become transformed.
  • Suitable selectable marker genes for plant systems include genes encoding enzymes that produce antibiotic resistance
  • herbicide resistance e.g., phosphinothricin acetyltransferase which confers resistance to the herbicide Basta (Hoechst AG, Frankfurt, Germany).
  • a 3' terminator region may be included in the expression vector to increase stability of the mRNA.
  • 3' terminators derived from octopine or nopaline synthase genes could be used.
  • Plant expression vector within the invention preferably contain a selectable marker gene used to identify the cells that have become transformed.
  • Suitable selectable marker genes for plant systems include genes encoding enzymes that produce antibiotic resistance (e.g., those conferring resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) or herbicide resistance (e.g., phosphinothricin acetyltransferase which confers resistance to the herbicide Basta (Hoechst AG, Frankfurt, Germany).
  • antibiotic resistance e.g., those conferring resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin
  • herbicide resistance e.g., phosphinothricin acetyltransferase which confers resistance to the herbicide Basta (Hoechst AG, Frankfurt, Germany).
  • a useful strategy for selection of transformants for herbicide resistance is described in Vasil I K (1984
  • Transgenic plants within the invention can be made by regenerating plant cells transformed with a plant expression vector by standard plant tissue culture techniques. See, e.g., in Vasil supra; Green et al., supra; Weissbach and Weissbach, supra; and Gelvin et al., supra.
  • a vector carrying a selectable marker gene e.g., kanamycin resistance
  • a cloned NMTase gene under the control of its own promoter and terminator or, if desired, under the control of exogenous regulatory sequences such as the 35S CaMV promoter and the nopaline synthase terminator is transformed into Agrobacterium.
  • Transformation of leaf tissue with vector-containing Agrobacterium is carried out as described in Ishida et al., Nature Biotech. 14:745-750, 1996 and Horsch et al., Science 227:1229, 1985.
  • Putative transformants are selected after a few weeks (e.g., 3 to 5 weeks) on plant tissue culture media containing kanamycin (e.g. 100 ⁇ g/ml).
  • Kanamycin-resistant shoots are then placed on plant tissue culture media without hormones for root initiation. Kanamycin-resistant plants are then selected for greenhouse growth. If desired, seeds from self-fertilized transgenic plants can then be sowed in a soil-less media and grown in a greenhouse.
  • Kanamycin-resistant progeny are selected by sowing surfaced sterilized seeds on hormone-free kanamycin-containing media. Analysis for the integration of the transgene is accomplished by standard techniques (see, e.g., Ausubel et al. supra; Gelvin et al. supra). Transgenic plants expressing the selectable marker are then screened for transmission of the transgene DNA by standard immunoblot and DNA and RNA detection techniques. Each positive transgenic plant and its transgenic progeny are unique in comparison to other transgenic plants established with the same transgene. Integration of the transgene DNA into the plant genomic DNA is in most cases random and the site of integration can profoundly effect the levels, and the tissue and developmental patterns of transgene expression. Consequently, a number of transgenic lines are usually screened for each transgene to identify and select plants with the most appropriate expression profiles.
  • Transgenic lines are evaluated for levels of transgene expression. Expression at the RNA level is determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis are employed and include PCR amplification assays using oligonucleotide primers designed to amplify only transgene RNA templates and solution hybridization assays using transgene-specific probes (see, e.g., Ausubel et al., supra). The RNA-positive plants are then analyzed for protein expression by Western immunoblot analysis using NMTase polypeptide-specific antibodies (see below and Ausubel et al., supra).
  • the present invention provides a purified ⁇ -alanine NMTase polypeptide isolated from L. latifolium. As described in the Examples section below, a ⁇ -alanine NMTase was isolated from L. latifolium using a series of purification steps. This protein was characterized both physically and functionally. Isoelectric focusing analysis showed that the purified enzyme exhibited an isoelectric point of 5.15. SDS-PAGE analysis showed that the purified enzyme migrated at about 43 kD. Functionally, the purified enzyme was capable of methylating ⁇ -alanine, N-methyl ⁇ -alanine, and N,N-dimethyl ⁇ -alanine.
  • the invention also provides fragments of the enzyme. Fragments of the enzyme can be made by treating the whole ⁇ - alanine NMTase polypeptide with one or more proteases, or by subjecting it to one of the techniques described below in the examples section. Peptide sequencing revealed the amino acid sequence of 5 oligopeptides (SEQ ID NOs: 1-5, 9, 10, and 11) making up parts of the whole ⁇ -alanine NMTase polypeptide. Thus, the invention also provides purified polypeptides including one or more of these sequences. Fragments of the whole ⁇ -alanine NMTase polypeptide can be made by chemically synthesizing the oligopeptides by known techniques. '
  • the present invention provides a purified NMTase polypeptide encoded by a nucleic acid of the invention.
  • a preferred form of NMTase is a purified native NMTase polypeptide that has the deduced amino acid sequence shown in Fig. 7B (SEQ ID NO. 29) or which is encoded by the nucleic acid of SEQ ID NO:28 (Fig. 7A).
  • Variants of native NMTase such as fragments, analogs and derivatives of native NMTase are also within the invention.
  • Such variants include, e.g., a polypeptide encoded by a naturally occurring allelic variant of native NMTase, a polypeptide encoded by a homolog of native NMTase, and a polypeptide encoded by a non-naturally occurring variant of native NMTase.
  • NMTase variants have a peptide sequence that differs from native NMTase in one or more amino acids.
  • the peptide sequence of such variants can feature a deletion, addition, or substitution of one or more amino acids of a native NMTase polypeptide.
  • Amino acid insertions are preferably of about 1 to 4 contiguous amino acids, and deletions are preferably of about 1 to 10 contiguous amino acids.
  • variant NMTase polypeptides substantially maintain a native NMTase functional activity.
  • variant NMTase polypeptides lack or feature a significant reduction in an NMTase functional activity.
  • preferred NMTase variants can be made by. expressing nucleic acid molecules within the invention that feature silent or conservative changes.
  • Variant NMTase polypeptides with substantial changes in functional activity can be made by expressing nucleic acid molecules within the invention that feature less than conservative changes.
  • NMTase fragments corresponding to one or more particular motifs and/or domains or to arbitrary sizes, for example, at least 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, and 350 amino acids in length are within the scope of the present invention.
  • Isolated peptidyl portions of NMTase proteins can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • an NMTase polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.
  • the fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of native NMTase.
  • NMTase proteins are encoded by a nucleic acid that has at least 85% sequence identity (e.g., 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) with the nucleic acid sequence of SEQ ID NO:28.
  • an NMTase protein of the present invention is a Plumbaginaceae NMTase protein.
  • an NMTase protein has one or more functional activities of native NMTase.
  • NMTase variants can be generated through various techniques known in the art. For example, NMTase variants can be made by mutagenesis, such as by introducing discrete point mutation(s), or by truncation. Mutation can give rise to an NMTase variant having substantially the same, or merely a subset of the biological activity of native NMTase. Other variants of NMTase that can be generated include those that are resistant to proteolytic cleavage, as for example, due to mutations which alter ubiquitination or other enzymatic targeting associated with the protein.
  • Whether a change in the amino acid sequence of a peptide results in an NMTase variant having one or more functional activities of native NMTase can be readily determined by testing the variant for a native NMTase functional activity in one or more of the assays described herein.
  • NMTase variants can be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector.
  • the purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential NMTase sequences.
  • the synthesis of degenerate oligonucleotides is well known in the art. See e.g., Narang, SA (1983) Tetrahedron 39:3; Itakura et al.(1981) Recombinant DNA, Proc 3rd Cleveland Sympos.
  • a library of coding sequence fragments can be provided for an NMTase clone in order to generate a variegated population of NMTase fragments for screening and subsequent selection of fragments having one or more native NMTase functional activities.
  • a variety of techniques are known in the art for generating such libraries, including chemical synthesis.
  • a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of an NMTase coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which car include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with SI nuclease; and (v) ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NMTase variants.
  • the most widely used techniques for screening large gene libraries typically comprise cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Combinatorial mutagenesis has a potential to generate very large libraries of mutant proteins, e.g., in the order of 10 molecules. Combinatorial libraries of this size may be technically challenging to screen even with high throughput screening assays.
  • techniques such as recursive ensemble mutagenesis (REM) that allow one to avoid the very high proportion of non-functional proteins in a random library and simply enhance the frequency of functional proteins (thus decreasing the complexity required to achieve a useful sampling of sequence space) can be used.
  • REM recursive ensemble mutagenesis
  • REM is an algorithm which enhances the frequency of functional mutants in a library when an appropriate selection or screening method is employed. Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Yourvan et al. (1992) Parallel Problem Solving from Nature, 2., hi Maenner and Manderick, eds., Elsevier Publishing Co., Amsterdam, pp. 401-410; Delgrave et al. (1993) Protein Engineering 6(3):327-331. A technique such as RACHITT (random chimeragenesis on transient templates) can also be used to generate a combinatorial library of mutant proteins.
  • RACHITT random chimeragenesis on transient templates
  • RACHITT is a method of in vitro recombination or "DNA shuffling" that yields a high recombinatorial frequency, a large spectrum of phenotypic diversity, and a low frequency of nonfunctional clones. Coco et al., Nature Biotech. 19:354-358, 2001.
  • the invention also provides for reduction of NMTase proteins to generate mimetics.
  • the mutagenic techniques described can also be used to map which determinants of NMTase participate in protein-protein interactions involved in, for example, binding of NMTase to proteins which may function upstream (including both activators and repressors of its activity) of NMTase or to proteins or nucleic acids which may function downstream of NMTase, and whether such molecules are positively or negatively regulated by NMTase.
  • NMTase-derived peptidomimetics which competitively inhibit binding of NMTase with that moiety.
  • peptidomimetic compounds can be generated which mimic those residues of native NMTase. Such mimetics may then be used to interfere with the normal function of an NMTase protein.
  • non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. (1988) in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands), azepine (e.g., see Huffman et al.(1988) in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands), substituted gamma lactam rings (Garvey et al. (1988) in Peptides: Chemistry and Biology, G. R.
  • NMTase polypeptides may also be chemically modified to create NMTase derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
  • Covalent derivatives of NMTase can be prepared by linking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C-terminus of the polypeptide.
  • the present invention further pertains to methods of producing the subject NMTase polypeptides.
  • a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the peptide to occur.
  • the cells may be harvested, lysed, and the protein isolated.
  • a recombinant NMTase polypeptide can be isolated from host cells using techniques known in the art for purifying proteins including ion-exchange cliromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide.
  • NMTase has been expressed in any cell or in a transgenic plant
  • an anti-NMTase antibody e.g., produced as described below
  • the matrix can be used for immuno-affinity chromatography to purify NMTase from cell lysates by standard methods. See e.g., Ausubel et al., supra.
  • NMTase can be further purified by other standard techniques, e.g., high performance liquid chromatography. See e.g., Fisher (1980) Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier.
  • NMTase is expressed as a fusion protein containing an affinity tag (e.g., GST) that facilitates its purification.
  • affinity tag e.g., GST
  • Anti- ⁇ -alanine NMTase Antibodies ⁇ -alanine NMTase polypeptides (or immunogenic fragments or analogs thereof) can be used to raise antibodies useful in the invention. Such polypeptides can be isolated as described herein. Fragments of ⁇ -alanine NMTase can be prepared by digesting the native protein with proteases or by synthesizing oligopeptides based on known amino acid sequence information.
  • ⁇ -alanine NMTase polypeptides can be coupled to a carrier protein, such as KLH, as described in Ausubel et al., supra, mixed with an adjuvant, and injected into a host mammal. Antibodies produced in that animal can then be purified by peptide antigen affinity chromatography.
  • various host animals can be immunized by injection with a ⁇ -alanine NMTase polypeptide or an antigenic fragment thereof. Commonly employed host animals include rabbits, mice, guinea pigs, and rats.
  • Narious adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • Other potentially useful adjuvants include BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules that are contained in the sera of the immunized animals.
  • Antibodies within the invention therefore include polyclonal antibodies and, in addition, monoclonal antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, and molecules produced using a Fab expression library.
  • Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen can be prepared using the ⁇ -alanine NMTase polypeptides described above and standard hybridoma technology. See e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur.
  • monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described in Kohler et al, Nature 256:495, 1975; and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • a hybridoma producing a mAb of the invention may be cultivated in vitro or in vivo. The ability to produce high titers of mAbs in vivo makes this a particularly useful method of production.
  • polyclonal or monoclonal antibodies can be tested for specific ⁇ - alanine NMTase recognition by Western blot or immunoprecipitation analysis by standard methods, for example, as described in Ausubel et al., supra.
  • Antibodies that specifically recognize and bind to ⁇ -alanine NMTase are useful in the invention.
  • such antibodies can be used in an immunoassay to monitor the level of ⁇ -alanine NMTase produced by a plant (e.g., to determine the amount or subcellular location of ⁇ -alanine NMTase). In some cases it may be desirable to minimize the potential problems of low affinity or specificity of antisera.
  • two or three fusions can be generated for each protein, and each fusion can be injected into at least two rabbits.
  • Antisera can be raised by injections in a series, preferably including at least three booster injections.
  • Antiserum is also checked for its ability to immunoprecipitate recombinant ⁇ -alanine NMTase polypeptides or control proteins, such as glucocorticoid receptor, CAT, or luciferase.
  • the antibodies of the invention can be used, for example, in the detection of ⁇ -alanine NMTase in a biological sample. Antibodies also can be used in a screening assay to measure the effect of a candidate compound on expression or localization of ⁇ -alanine NMTase. Additionally, such antibodies can be used to interfere with the interaction of ⁇ -alanine NMTase and other molecules that interact with ⁇ -alanine NMTase.
  • Single chain antibodies can be adapted to produce single chain antibodies against a ⁇ -alanine NMTase polypeptide, or a fragment thereof.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments that recognize and bind to specific epitopes can be generated by known techniques.
  • such fragments include but are not limited to F(ab') 2 fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab expression libraries can be constructed (Huse et al., Science 246:1275, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • the invention encompasses methods for detecting the presence of NMTase protein or NMTase nucleic acid in a biological sample as well as methods for measuring the level of NMTase protein or NMTase nucleic acid in a biological sample. Such methods are useful for examining plant intracellular signaling pathways associated with stress resistance.
  • An exemplary method for detecting the presence or absence of NMTase in a biological sample involves obtaining a biological sample from a test plant (or plant cell) and contacting the biological sample with a compound or an agent capable of detecting an NMTase polypeptide or a nucleic acid encoding an NMTase polypeptide (e.g., mRNA or genomic DNA).
  • a preferred agent for detecting a nucleic acid encoding an NMTase polypeptide is a labeled nucleic acid probe capable of hybridizing to the nucleic acid encoding the NMTase polypeptide.
  • the nucleic acid probe can be, for example, all or a portion of NMTase itself (e.g., a nucleic acid molecule having the sequence of SEQ ID NO:28) or all or a portion of a complement o ⁇ NMTase.
  • the probe can also be all or a portion of an NMTase variant, or all or a portion of a complement of an NMTase variant.
  • oligonucleotides at least 15, 30, 50, 100, 250, or 500 nucleotides in length that specifically hybridize under stringent conditions to native NMTase or a complement of native NMTase can be used as probes within the invention.
  • a preferred agent for detecting an NMTase polypeptide is an antibody capable of binding to an NMTase polypeptide, preferably an antibody with a detectable label.
  • Such antibodies can be polyclonal, or more preferably, monoclonal.
  • An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • Detection methods of the invention can be used to detect an mRNA encoding NMTase, a genomic DNA encoding NMTase, or an NMTase polypeptide in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of mRNAs encoding NMTase include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of a NMTase polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of genomic DNA encoding , NMTase include Southern hybridizations.
  • in vivo techniques for detection of a NMTase polypeptide include introducing into a plant or plant cell labeled anti-NMTase antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a plant can be detected by standard imaging techniques.
  • N-methyl ⁇ -alanine and N,N-dimethyl ⁇ -alanine were synthesized as described previously
  • NN-dimethyl ⁇ -alanine sepharose 4B affinity resin was prepared by coupling the amino group of 1,6 diaminohexane in EAH- sepharose (Amersham-Pharmacia Biotech, Piscataway, NJ) to the carboxyl group of N,N- dimethyl ⁇ -alanine, using a carbodiimide procedure. Hoare and Datta (1990) Arch Biochem Biophys 277:122-129. Adenosine agarose affinity resin was prepared from 5'-AMP-agarose by the method of James et al. (1995) J. Biol. Chem. 270:22344-22350.
  • Magna Lift nylon (0.45 ⁇ , 137 mm) circles were from Osmonics, Inc. (Minnetonka, M ⁇ ).
  • dGTP ⁇ - 32 P, 800 Ci.mmor 1
  • Plasmid purification and gel extraction kits were from Qiagen (Valencia, CA).
  • RACE protocol kit was from BD Biosciences Clontech (Palo Alto, CA).
  • Molecular weight markers, Taq polymerase, d ⁇ TPs and restriction enzymes were from Promega (Madison, WI).
  • Oligonucleotide primers were synthesized from the custom primer synthesis unit of Life Technologies (Carlsbad, CA).
  • Leaves were sliced into about 1 cm wide strips, frozen in liquid nitrogen and ground to a powder in a mortar. The powder was transferred to a blender containing freshly prepared extraction medium, 400 mL per 100 g fresh weight leaves.
  • the extraction medium contained the following in 0.1 M Tris- HCl pH 8: 0.2 M sodium tefraborate, 2 mM DTT, 5 mM EDTA, 10% (v/v) glycerol, 4 % (w/v) insoluble PVPP, 6 % (w/v) Amberlite XAD-4, 10 ⁇ M leupeptin, 0.2 mM AEBSF, 1 ⁇ M pepstatin A, 1 ⁇ M Bestatin, 1 ⁇ M E-64 and 1 mM 1,10-phenanthroline.
  • the tissue was blended in the extraction buffer for 3 min at maximum speed, filtered through four layers of autoclaved cheesecloth and centrifuged at 20,000 g for 30 min in a refrigerated centrifuge (model J2-HS, Beckman Instruments, Fullerton, CA). The supernatant (crude extract) was saved for further purification (see below). An aliquot of the crude extract was desalted by passage through Sephadex G-25 columns (PD10, Amersham Pharmacia, Piscataway, NJ) prior to assays for total protein and NMTase activities. Enzyme Assay.
  • the NMTase activities with ⁇ -alanine, N-methyl ⁇ -alanine and N, N-dimethyl ⁇ -alanine were assayed using a radiometric method (Rathinasabapathi et al., Physiol Plant 109:225-231, 2000), with modifications as stated below.
  • the assay mixture contained 54 ⁇ L of enzyme preparation in a total volume of 100 ⁇ L containing 0.1 M Tris-HCl buffer pH 8.0, 2 mM DTT, 10 mM methyl acceptor, 100 ⁇ M AdoMet and 0.027 ⁇ M S-Adenosyl-L-[me /- 3 H]Met (200 nCi of radioactivity).
  • Protein precipitating between 10% (w/v) and 15% (w/v) PEG 8000 was dissolved in buffer A.
  • the MTase activities were stable in this fraction for at least two months when stored at -80°C.
  • 25 mL of the PEG-precipitated protein dissolved in buffer A was exposed to 50°C in a water bath for 15 min. The preparation then was centrifuged at 20,000 g for 20 min and the supernatant was collected.
  • protein (about 40 to 50 mg) from the heat treatment step was loaded onto a column (13.5 cm x 3 cm ) containing 50 mL DEAE-Fractogel EMD ion exchanger (EM Separations Technology, Gibbstown, NJ ).
  • the column was washed with 50 mL buffer A and then with 90 mL buffer A containing 20 mM KCl .
  • the bound proteins were then eluted from the column with 104-mL linear 20 mM to 300 mM KCl gradient in buffer A containing 0.1 mM AEBSF. Fractions (7.5 mL) were collected and assayed for NMTase activities and protein.
  • the bound proteins were eluted using buffer A containing 10 mM each of ⁇ -alanine and NN-dimethyl ⁇ -alanine and using buffer A containing 200 mM KCl. Substrate elution and the 200 mM KCl elution were pooled and concentrated to 1.3 mL before being loaded on to a continuous elecfrophoresis prep cell (Model 491, Bio-Rad, Hercules, CA). The prep cell used a native-gel column made up of 40 mL of 6% (w/v) acrylamide in 24 mM Tris-CAPS buffer, pH 9.3 (McLellan, 1982).
  • Elecfrophoresis was at 300 N for 2 h with 24 mM Tris-CAPS buffer, pH 9.3 and the proteins were eluted with buffer A. Fractions (3 mL each) were assayed for MTase activities and protein. Fractions with specific activities equal to and above that of the load were pooled, concentrated and loaded onto an adenosine agarose affinity gel (3 mL column). Non-specific proteins were washed off the column with buffer A containing 0.2 M KCl and the bound proteins were eluted with 5 mM AdoMet and 0.2 M KCl in buffer A. The eluate was concentrated prior to NMTase and protein assays.
  • Photoaffinity Labeling To identify the protein subunit(s) binding to AdoMet, photoaffinity labeling (Som and Friedman, J Biol Chem 265:4278-4283, 1990) was done on protein samples at various stages of purification from the ion exchange cliromatography stage onward using the method as described by Smith et al., Physiol Plant 108:286-294, 2000.
  • PEG precipitation step was employed primarily to concentrate the extracted protein in a stable form, achieving a 2-fold purification. In separate trials, heat treatment of the PEG fraction resulted in 2-fold improvement in specific activities.
  • DEAE-Fractogel anion exchange column chromatography improved specific activities to about 6-fold (Table I) as shown in Figure 2.
  • NMTase activities eluted from DEAE-fractogel column between 125 mM and 200 mM KCl, ahead of the majority of proteins ( Figure 2).
  • NMTase activity eluted as a single peak with an elution volume corresponding to a native molecular weight of 80 kD .
  • the use of protease inhibitors proved extremely valuable in this step. Without inhibitors, NMTase activity eluted in four peaks corresponding to 110, 80, 40 and 20 kD, the 80 kD NMTase being more than 50%) of the total recovered activity, and the total activity recovered was substantially reduced. Activity at 110 kD was probably due to protein aggregation.
  • NMTase activities eluted six to nine mL after the dye front eluted.
  • Adenosine agarose effected about a 1890-fold increase in specific activities (Figure 4).
  • the purified fraction methylated ⁇ -alanine, N-methyl ⁇ -alanine, and NN-dimethyl ⁇ -alanine (Table I).
  • the specific activities observed using N-methyl ⁇ -alanine and NN-dimethyl ⁇ -alanine as substrates were less than those observed using ⁇ -alanine (Table I).
  • the enzyme was labile in this fraction, especially for the activity against N, N-dimethyl ⁇ -alanine, with about 50% loss of activity over 12 h on ice.
  • V max /K m values were comparable for the three methyl acceptors (Table II).
  • AdoHCy was highly inhibitory to the NMTase: 50% > inhibition was achieved at 40 ⁇ M AdoHCy at 10 mM ⁇ -alanine and 100 ⁇ M Ado-Met.
  • SDS sodium dodecyl sulfate
  • Active ⁇ -alanine ⁇ -methyltransferase protein was purified from leaves of L. latifolium using the procedure described previously (Rathinasabapathi et al., Plant Physiol. 126:1241- 1249, 2001).
  • the purified protein (20 ⁇ g) was separated on an SDS-PAGE gel and stained with Coomassie Blue.
  • the 43 kDa band was eluted from the gel and digested with LysC.
  • Peptide sequencing was done by Edman degradation (Tempst et al., Elecfrophoresis 11:537- 553, 1990). Based on the peptide sequences degenerate primers were designed for RT-PCR. Each primer included one or two inosines.
  • primers 2F and 3R amplified a 500 bp cD ⁇ A.
  • Primer combinations 2F and 1R and IF and 3R produced shorter products of 400 bp and 100 bp respectively.
  • the 500 bp PCR product was named clone 23 and sequenced. The D ⁇ A sequence of clone 23 is shown herein as SEQ ID ⁇ O:20.
  • RT-PCR was performed using a RT-PCR kit (Thermoscript RT-PCR system, hiVitrogen, Carlsbad, CA) according to manufacturer's instructions. RT-PCR reactions were in a volume of 50 ⁇ L in thin walled amplification tubes. The reactions contained 10 ⁇ L of first strand reaction, 200 ⁇ M of each of the four dNTPs, 2 mM of magnesium chloride, 4 ⁇ M each of the sense and antisense primers, 5 units of Taq DNA polymerase in 10 mM Tris-HCl pH 9, 50 mM KCl and 0.1% Triton X-100.
  • SEQ LD NO:21 is a PCR product obtained using SEQ ID NO:23 as a primer.
  • cDNA library construction Poly A+ RNA was isolated from L . latifolium leaves from plants salinized with 200 mM NaCl. First and second strand cDNAs were made using M-MLV reverse transcriptase (Sambrook et al., supra).
  • cDNAs size-selected for 1000 bp were cloned into the EcoRI site of lambda vector gtlO (Clontech, Palo Alto, CA).
  • the primary library had a titer of 1.5 x 10 plaque forming units per mL.
  • Library screening. Clone 23 was labeled with 32 PdGTP (800 Ci per mmol,
  • DNA sequencing and analysis DNA sequencing was in both strands using the fluorescent chain terminating dideoxynucleotides method. DNA sequences were analyzed by several software packages including BLAST (Patnaik and Blumenfeld, Anal. Biochem. 289:1-9, 2001).
  • Partial cDNA clones represented two groups, NMTase A (SEQ ID NOs:25-27) and NMTase B (SEQ ID NO: 24) differing only in their 3' untranslated region.
  • the open reading frame had two ATGs within the first 30 bp.
  • ⁇ -alanine NMTase had 53%o identity and 72% homology to alfalfa O-diphenol-O-methyltransferase (E.G. 2.1.1.6). Sequence relatedness to O-methyltransferases suggests the possibility of ⁇ -alanine NMTase having phylogenetic relations to O-methyltransferases. There is no recognizable signal sequence based upon sequence analyses, suggesting that b-alanine methylation occurs in the cytoplasm. Emanuelsson et al., J Mol Biol 300: 1005-1016, 2000. RNA blots. Total RNA from L. latifolium and L.
  • sequences were obtained from the purified NMTase protein from L. latifolium (Sequences A-H below). Amino acids in parentheses are alternate possibilities arising from ambiguities in sequencing. Sequence A
  • Sequence A shared some homology with the peptide sequences of several other methyltransferases (see below), but Sequence B did not. Sequence A showed homology to: a. Caffeic acid O-methyltransferase-like protein of Arabidopsis thaliana emb
  • EYRQLGLLA (SEQ ID NO:7)162; and c. O-diphenol-O-methyltransferase of Medicago sativa subsp. varia.
  • Example 4 Expression in Yeast
  • the open reading frame for the ⁇ -alanine NMTase was amplified using primers 5' to 3' gcggatccaatggcgaaccactcctcagctg (SEQ ID NO:30) and 5' to 3' ctcgagtcacttctggaactctaccacgg (SEQ ID NO:31) andZ.
  • latifolium cDNA was the template. These primers were designed with an add-on containing Bell and Xhol restriction sites.
  • the amplified 1143 bp product was subcloned into pCR2.1 TOPO vector.
  • a BamHI/XhoI fragment containing the cDNA was directionally cloned in frame in the yeast expression vector pYES NT-C (InVitrogen, Carlsbad, CA). The plasmid was introduced into
  • Saccharomyces cerevisiae InVSc strain by a LiCl procedure (Ausubel et al., 1995).
  • the recombinant protein, as a hexa-histidine fusion protein on its N-terminus was purified using the nickel affinity column as per manufacturer's instructions.
  • the purified protein was subjected to enterokinase cleavage and assayed for N-methyltransferase activities using the radiometric method described previously (Rathinasabapathi et al., Plant Physiol. 126:1241- 1249, 2001).
  • Example 5 Expression in Tobacco A full-length cDNA for ⁇ -alanine N-methyltransferase was subcloned into the EcoRI site of pMON979-R5 vector under the control of a modified Cauliflower mosaic virus 35S promoter.
  • the recombinant vector pR5-NMT was transferred into Agrobacterium tumefaciens strain ABI via triparental mating (Walkerpeach and Velten, Plant Molecular Biology Manual Bl:l-19, 1994). Transformation of tobacco by Agrobacterium was achieved by leaf disk procedure (Horsch et al., Science 227:1229-1231, 1985). Putative fransformants were identified based on their kanamycin resistance. Transgenic plants were analyzed for the expression of ⁇ -alanine NMTase using PCR,
  • RNA blots were then transferred to a greenhouse and seeds were collected following selfing.
  • vector alone control and ⁇ -alanine N-methyltransferase fransformants were grown in vermicuhte irrigated daily with a half-strength Hoagland solution. After a two-week period, NaCl was included at 50 mM increments for every three days until reaching 200 mM NaCl. The plants were harvested and leaves, stem and roots were dissected and weighed. Growth data were analyzed using analysis of variance comparing fresh weight and dry weight increases during the experimental period for the vector alone control and ⁇ -alanine NMTase transformants.
  • Table I Purification of an AdoMet dependent NMTase from 550 g fresh weight leaves of L. latifolium. Fold-purification was calculated based on specific activities measured with ⁇ - alanine.

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Abstract

L'invention porte sur l'isolation de β-alanine N-méthyl-transférase (NMTase) à partir de limonium latifolium. L'enzyme purifiée catalyse la N-méthylation de la β-alanine bétaïne, et possède un point iso-électrique d'environ 5,15 et une masse moléculaire apparente d'environ 43 kilodaltons. L'invention concerne également le clonage par criblage d'une banque d'ADNc de L. latifolum, et le séquençage, d'un ADNc complet codant pour cette NMTase. L'expression de cet ADNc dans des plantes transgéniques autres que L. latifolium entraîne une modulation de la résistance au stress dans les plantes.
PCT/US2002/024936 2002-08-06 2002-08-06 Beta-alanine n-methyl-transferase WO2004013092A2 (fr)

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Title
RAMAN S.B. ET AL.: 'beta-alanine N-methyltransferase of Limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectant beta-alanine betaine.' PLANT PHYSIOLOGY vol. 132, no. 3, July 2003, pages 1642 - 1651 *

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