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EP1131453A2 - PLANT AMINOACYL-tRNA SYNTHETASES - Google Patents

PLANT AMINOACYL-tRNA SYNTHETASES

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Publication number
EP1131453A2
EP1131453A2 EP99971858A EP99971858A EP1131453A2 EP 1131453 A2 EP1131453 A2 EP 1131453A2 EP 99971858 A EP99971858 A EP 99971858A EP 99971858 A EP99971858 A EP 99971858A EP 1131453 A2 EP1131453 A2 EP 1131453A2
Authority
EP
European Patent Office
Prior art keywords
isolated polynucleotide
polypeptide
isolated
trna synthetase
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99971858A
Other languages
German (de)
French (fr)
Inventor
Omolayo O. Famodu
Carl Simmons
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pioneer Hi Bred International Inc
EIDP Inc
Original Assignee
Pioneer Hi Bred International Inc
EI Du Pont de Nemours and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pioneer Hi Bred International Inc, EI Du Pont de Nemours and Co filed Critical Pioneer Hi Bred International Inc
Publication of EP1131453A2 publication Critical patent/EP1131453A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding aminoacyl-tRNA synthetases in plants and seeds.
  • Aminoacyl-tRNA Synthetases are enzymes that charge (acylate) tRNAs with amino acids. These charged aminoacyl tRNAs then participate in mRNA translation and protein synthesis.
  • the AARS show high specificity for charging a specific tRNA with the appropriate amino acid, for example valyl-tRNA with valine by valyl-tRNA synthetase or tryptophanyl-tRNA with tryptophan by tryptophanyl-tRNA synthetase.
  • AARS are ancient enzymes, having functioned in translation since early life evolution.
  • AARS aminoacyl-tRNA synthetases
  • AARS are structurally diverse, although AARSs for some amino acids are more closely related than for others.
  • AARSs are generally divided into two classes, class I and class II based on structural similarity and amino acid preferences (Eriani et al., (1990) Nature 547:203-206). Plants like all other cellular organisms have aminoacyl-tRNA synthetases.
  • Aminoacyl-tRNA synthetases play a critical role in protein translation by linking genetic nucleic acid information to protein synthesis. Aminoacyl-tRNA synthetases perform this role by "reading" the identity of the different tRNAs and acylating them with the correct cognate amino acid. A large volume of research over several decades has been focused on identifying inhibitors of this process. Inhibitors of aminoacyl-tRNA synthetases have been found to be cytotoxic due to their inhibition of protein synthesis.
  • Biochemical processes are often compartmentalized in regions of cells, such as mitochondria, plastids, and lysosomes. These organelles are key sites for many biochemical pathways. Bioengineering of these processes may require targeting protein products to specific organells.
  • One method to accomplish this involves the addition of an N-terminal prosequence (transit peptide) that directs protein entry into a specific organelle(s). Upon or shortly after transport into the organelle the transit peptide is usually proteolytically removed, and the mature protein is then able to function.
  • a few plant transit peptides have been shown empirically to be capable of directing fused proteins into specific organelles.
  • This invention describes a number of chloroplast-targeting and mitochondria- targeting transit peptides for (maize) aminoacyl-tRNA synthetases. These sequences will find utility in directing both aminoacyl-tRNA synthetase and other proteins into these organelles.
  • nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand protein synthesis in plants, provide genetic tools for the manipulation of gene expression, protein targeting to specific organells and provide possible targets for herbicides.
  • the present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 240 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn arginyl-tRNA synthetase polypeptide of SEQ ID NO:2, a rice arginyl-tRNA synthetase polypeptide of SEQ ID NO:4, a soybean arginyl-tRNA synthetase polypeptide of SEQ ID NO:6, a wheat arginyl-tRNA synthetase polypeptide of SEQ ID NO:8.
  • the present invention also relates to an isolated polynucleotide comprising the complement ofthe nucleotide sequences described above.
  • the present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 205 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 10, a rice glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 12, a soybean glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 14.
  • the present invention also relates to an isolated polynucleotide comprising the complement ofthe nucleotide sequences described above.
  • the present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 79 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a wheat glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 16.
  • the present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
  • the present invention further relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 243 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn histidyl-tRNA synthetase polypeptide of SEQ ID NO: 18, a soybean histidyl-tRNA synthetase polypeptide of SEQ ID NO:20, a wheat histidyl-tRNA synthetase polypeptide of SEQ ID NO:22.
  • the present invention also relates to an isolated polynucleotide comprising the complement ofthe nucleotide sequences described above.
  • the present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 30 amino acids that has at least 60% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:24, 26, 28, 30, 32, 34, 36 and 38.
  • the isolated polynucleotides ofthe claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.
  • the present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences.
  • the present invention relates to a chimeric gene comprising an isolated polynucleotide ofthe present invention operably linked to suitable regulatory sequences.
  • the present invention also relates to the identification of transit peptides associated with aminoacyl-tRNA synthetases ofthe instant invention and the use of those transit peptides to target aminoacyl-tRNA synthetases and other operably linked proteins to specific organelles within plant cells.
  • Transit peptide amino acid sequences are located just upstream ofthe mature aminoacyl-tRNA synthetase polypeptide sequences disclosed in the instant invention.
  • the present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide ofthe present invention.
  • the host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell.
  • the present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide ofthe present invention or a chimeric gene ofthe present invention.
  • the present invention relates to a process for producing an isolated host cell comprising a chimeric gene ofthe present invention or an isolated polynucleotide ofthe present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide ofthe present invention.
  • the present invention relates to an arginyl-tRNA synthetase polypeptide of at least 240 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, and 8.
  • the present invention relates to a glutamyl-tRNA synthetase polypeptide of at least 205 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 10, 12, 14 and 16.
  • the present invention relates to a glutamyl-tRNA synthetase polypeptide of at least
  • the present invention relates to a histidyl-tRNA synthetase polypeptide of at least 243 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 18, 20 and 22.
  • the present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histi
  • the present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide gene, preferably a plant arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences; and amplifying
  • the amplified nucleic acid fragment preferably will encode a portion of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase amino acid sequence.
  • the present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion ofthe amino acid sequence encoding an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide ofthe present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide ofthe present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
  • a further embodiment ofthe instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of an arginyl-tRNA synthetase, glutamyl- tRNA synthetase or histidyl-tRNA synthetase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression ofthe chimeric gene wherein expression ofthe chimeric gene results in production of arginyl-tRNA synthetase, glutamyl- tRNA synthetase or histidyl-tRNA synthetase in the transformed host cell; (c) optionally
  • the present invention relates to a composition comprising an isolated polynucleotide ofthe present invention.
  • the present invention relates to a composition comprising a polypeptide ofthe present invention.
  • the present invention relates to an isolated polynucleotide comprising the nucleotide sequence comprising at least one of 30 contiguous nucleotides of nucleic acid sequences selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such sequences.
  • the present invention relates to an expression cassette comprising an isolated polynucleotide ofthe present invention operably linked to a promoter.
  • the present invention relates to a method for positive selection of a transformed cell comprising: (a) transforming a plant cell with an expression cassette ofthe present invention;
  • the present invention relates to a method for positive selection of a transformed cell comprising:
  • Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing.
  • Table 1 also identifies the cDNA clones as individual ESTs ("EST"), the sequences of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS").
  • Nucleotide sequences, SEQ ID NOs: l, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 and amino acid sequences SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 were determined by further sequence analysis of cDNA clones encoding the amino acid sequences set forth in SEQ ID NOs:24, 26, 28, 30, 32, 34, 36 and 38.
  • Nucleotide SEQ ID NOs:23, 25, 27, 29, 31, 33, 35 and 37 and amino acid SEQ ID NOs:24, 26, 28, 30, 32, 34, 36 and 38 were presented in a U.S. Provisional Application No. 60/107,789, filed November 10, 1998.
  • Glutamyl-tRNA Contig Composed of: 13 14 Synthetase sdc5c.pk0002.el l sgslc.pk001.kl2 sgslc.pk004.e20
  • Histidyl-tRNA Synthetase Contig composed of: 19 20 sdp4c.pk007.c7 ssm.pk0012.d9
  • a "polynucleotide” is a nucleotide sequence such as a nucleic acid fragment.
  • a polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.
  • An isolated polynucleotide ofthe present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides, ofthe nucleic acid sequence ofthe SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such sequences.
  • sequence refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.
  • substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties ofthe polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co- suppression technology.
  • Substantially similar also refers to modifications ofthe nucleic acid fragments ofthe instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties ofthe resulting transcript vis-a-vis the ability to mediate gene silencing or alteration ofthe functional properties ofthe resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.
  • Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications ofthe nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level ofthe polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell.
  • a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell.
  • the level ofthe polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level ofthe polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
  • antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100%) sequence identity with the gene to be suppressed.
  • alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties ofthe encoded polypeptide are well known in the art.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product.
  • Nucleotide changes which result in alteration ofthe N-terminal and C-terminal portions ofthe polypeptide molecule would also not be expected to alter the activity ofthe polypeptide.
  • an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a polypeptide in a plant cell.
  • a method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide (such as aminoacyl-tRNA synthetase) in a host cell may comprise the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.
  • a polypeptide such as aminoacyl-tRNA synthetase
  • a host cell eukaryotic, such as plant or yeast, prokaryotic such as bacterial, or viral
  • substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
  • One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5%) SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5%) SDS at 50°C for 30 min.
  • a more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature ofthe final two 30 min washes in 0JX SSC, 0.5% SDS was increased to 60°C.
  • Another preferred set of highly stringent conditions uses two final washes in 0JX SSC, 0.1% SDS at 65°C.
  • nucleic acid fragments ofthe instant invention may also be characterized by the percent identity ofthe amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.
  • Suitable nucleic acid fragments encode polypeptides that are at least 70% identical, preferably at least 80% identical to the amino acid sequences reported herein.
  • Preferred nucleic acid fragments encode amino acid sequences that are at least 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least 90% identical to the amino acid sequences reported herein.
  • nucleic acid fragments that encode amino acid sequences that are at least 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS.
  • a "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification ofthe protein or gene that the amino acid or nucleotide sequence comprises.
  • Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
  • a sequence often or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
  • gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
  • oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
  • a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
  • the instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantial portion ofthe disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
  • Codon degeneracy refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion ofthe amino acid sequences set forth herein.
  • the skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage ofthe host cell.
  • Synthetic nucleic acid fragments can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. "Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines.
  • nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias ofthe host cell.
  • the skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
  • Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • “Native gene” refers to a gene as found in nature with its own regulatory sequences.
  • Chimeric gene refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • Coding sequence refers to a nucleotide sequence that codes for a specific amino acid sequence.
  • Regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation ofthe associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments.
  • promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 75: 1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
  • the "translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the fully processed mRNA upstream ofthe translation start sequence.
  • the translation leader sequence may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).
  • the "3' non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end ofthe mRNA precursor.
  • the use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 7:671-680.
  • RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy ofthe DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing ofthe primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into polypeptide by the cell.
  • cDNA refers to a double-stranded DNA that is complementary to and derived from mRNA.
  • Sense RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell.
  • Antisense RNA refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Patent No. 5,107,065, incorporated herein by reference).
  • the complementarity of an antisense RNA may be with any part ofthe specific nucleotide sequence, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
  • operably linked refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control ofthe promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression ofthe target protein.
  • Overexpression refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
  • Co-suppression refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231,020, incorporated herein by reference).
  • altered levels refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
  • Measure protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
  • Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
  • chloroplast transit peptide is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made.
  • Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast transit peptide.
  • a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53).
  • a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
  • an endoplasmic reticulum retention signal may be added.
  • any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).
  • Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Patent No. 4,945,050, inco ⁇ orated herein by reference).
  • nucleic acid fragments encoding at least a portion of several aminoacyl-tRNA synthetases have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art.
  • the nucleic acid fragments ofthe instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species.
  • sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
  • genes encoding other arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptides could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art.
  • Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis).
  • sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • specific primers can be designed and used to amplify a part or all ofthe instant sequences.
  • the resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
  • two short segments ofthe instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
  • the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence ofthe other primer takes advantage ofthe presence ofthe polyadenylic acid tracts to the 3' end ofthe mRNA precursor encoding plant genes.
  • the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl.
  • a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.
  • the present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a polypeptide of a gene (such as arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase) preferably a substantial portion of a plant polypeptide of a gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the
  • Synthetic peptides representing portions ofthe instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) dv. Immunol. 3-5:1-34; Maniatis).
  • nucleic acid fragments ofthe instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of in those cells.
  • Overexpression ofthe proteins ofthe instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development.
  • the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided.
  • the instant chimeric gene may also comprise one or more introns in order to facilitate gene expression. Plasmid vectors comprising the instant chimeric gene can then be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants.
  • the chimeric gene described above may be further supplemented by altering the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 5-5:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel ( 1992) Plant Phys.100:1621 -1632) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.
  • a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences.
  • a chimeric gene designed to express antisense RNA for all or part ofthe instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression ofthe corresponding endogenous genes are reduced or eliminated.
  • tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
  • a preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.
  • the instant polypeptides may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art.
  • the antibodies are useful for detecting the polypeptides ofthe instant invention in situ in cells or in vitro in cell extracts.
  • Preferred heterologous host cells for production ofthe instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides.
  • This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression ofthe encoded aminoacyl-tRNA synthetase.
  • An example of a vector for high level expression ofthe instant polypeptides in a bacterial host is provided (Example 8).
  • the instant polypeptides can be used as a targets to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the polypeptides described herein catalyze various steps in aminoacyl-tRNA biosynthesis. Accordingly, inhibition ofthe activity of one or more ofthe enzymes described herein could lead to inhibition of plant growth. Thus, the instant polypeptides could be appropriate for new herbicide discovery and design. All or a substantial portion ofthe nucleic acid fragments ofthe instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes.
  • the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers.
  • Southern blots Maniatis
  • the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map.
  • the nucleic acid fragments ofthe instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation ofthe DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
  • Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
  • nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).
  • FISH direct fluorescence in situ hybridization
  • current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20)
  • improvements in sensitivity may allow performance of FISH mapping using shorter probes.
  • a variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med.
  • Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 55:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways.
  • short segments ofthe instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra).
  • the amplification of a specific DNA fragment with these primers indicates the insertion ofthe mutation tag element in or near the plant gene encoding the instant polypeptides.
  • the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor.
  • an arbitrary genomic site primer such as that for a restriction enzyme site-anchored synthetic adaptor.
  • EXAMPLE 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various corn, rice, soybean and wheat tissues were prepared. The characteristics ofthe libraries are described below. TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat
  • cDNA libraries may be prepared by any one of many methods available.
  • the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). The Uni-ZAPTM XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript.
  • the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into D ⁇ 10B cells according to the manufacturer's protocol (GIBCO BRL Products).
  • T4 DNA ligase New England Biolabs
  • plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences.
  • Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
  • Example 1 The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The
  • DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the ⁇ CBI.
  • BLASTX BLASTX algorithm
  • the P-value (probability) of observing a match of a cD ⁇ A sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative ofthe logarithm ofthe reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cD ⁇ A sequence and the BLAST "hit" represent homologous proteins.
  • EST EST
  • FIS cDNA clones
  • Contig contigs assembled from two or more ESTs
  • Contig* contigs assembled from an FIS and one or more ESTs
  • CGS FIS and PCR
  • Table 4 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4, 6 and 8 and the Arabidopsis thaliana sequence.
  • Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a arginyl-tRNA synthetase. These sequences represent the first corn, rice, soybean and wheat sequences encoding arginyl-tRNA synthetase.
  • EST EST
  • FIS cDNA clones
  • Contig contigs assembled from two or more ESTs
  • Contig* contigs assembled from an FIS and one or more ESTs
  • CGS FIS and PCR
  • Contig composed of: Contig 97.40 (gi 2500981 ) sdc5c.pk0002.el l sgslc.pk001.kl2 sgslc.pk004.e20 wlm96.pk055.g5 (EST) 18.30 (gi 2507428)
  • Table 6 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs: 10, 12, 14 and 16 and the Arabidopsis thaliana, Hordum vulgare, Nicotina tabacum and Saccharomyces cerevisiae sequences.
  • Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a glutamyl-tRNA synthetase. These sequences represent the first corn, rice, soybean and wheat sequences encoding glutamyl-tRNA synthetase.
  • EST EST
  • FIS cDNA clones
  • Contig contigs assembled from two or more ESTs
  • Contig* contigs assembled from an FIS and one or more ESTs
  • CGS FIS and PCR
  • Contig composed of: Contig 84.22 (gi 3659909) sdp4c.pk007.c7 ssm.pk0012.d9 wrl.pk0079.dl (FIS) 98.30 (gi 3659909)
  • Table 8 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs: 18, 20 and 22 and the Oryza sativa and Arabidopsis thaliana sequences.
  • Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a histidyl-tRNA synthetase. These sequences represent the first corn, soybean and wheat sequences encoding histidyl-tRNA synthetase.
  • a chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed.
  • the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be inco ⁇ orated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the digested vector pML103 as described below.
  • Amplification is then performed in a standard PCR.
  • the amplified DNA is then digested with restriction enzymes Ncol and Smal and fractionated on an agarose gel.
  • the appropriate band can be isolated from the gel and combined with a 4.9 kb Ncol-Smal fragment ofthe plasmid pML103.
  • Plasmid pML103 has been deposited under the terms ofthe Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Boulevard., Manassas, VA 201 10-2209), and bears accession number ATCC 97366.
  • the DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment ofthe maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end ofthe maize 10 kD zein gene in the vector pGem9Zf(+) (Promega).
  • Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL 1 -Blue (Epicurian Coli XL-1 BlueTM; Stratagene).
  • Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (SequenaseTM DNA Sequencing Kit; U.S. Biochemical).
  • the resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3' region.
  • the chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses ofthe inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27°C.
  • Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos.
  • the embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
  • the plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker.
  • This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT).
  • PAT phosphinothricin acetyl transferase
  • the enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin.
  • T e pat gene in p35S/Ac is under the control ofthe 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region ofthe nopaline synthase gene from the T-DNA ofthe Ti plasmid of Agrobacterium tumefaciens.
  • the particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells.
  • gold particles (1 ⁇ m in diameter) are coated with DNA using the following technique.
  • Ten ⁇ g of plasmid DNAs are added to 50 ⁇ L of a suspension of gold particles (60 mg per mL).
  • Calcium chloride 50 ⁇ L of a 2.5 M solution
  • spermidine free base (20 ⁇ L of a 1.0 M solution) are added to the particles.
  • the suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 ⁇ m) and the supernatant removed.
  • the particles are resuspended in 200 ⁇ L of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 ⁇ L of ethanol.
  • An aliquot (5 ⁇ L) ofthe DNA-coated gold particles can be placed in the center of a KaptonTM flying disc (Bio-Rad Labs).
  • the particles are then accelerated into the corn tissue with a BiolisticTM PDS-1000/He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
  • the embryogenic tissue is placed on filter paper over agarose- solidified N6 medium.
  • the tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter.
  • the petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen.
  • the air in the chamber is then evacuated to a vacuum of 28 inches of Hg.
  • the macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
  • tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some ofthe plates containing the glufosinate- supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.
  • Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 5:833-839).
  • a seed-specific expression cassette composed ofthe promoter and transcription terminator from the gene encoding the ⁇ subunit ofthe seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 251:9228-9238) can be used for expression ofthe instant polypeptides in transformed soybean.
  • the phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin.
  • Nco I which includes the ATG translation initiation codon
  • Sma I which includes the ATG translation initiation codon
  • Kpn I The entire cassette is flanked by Hind III sites.
  • the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites can be inco ⁇ orated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.
  • PCR polymerase chain reaction
  • Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides.
  • somatic embryos cotyledons
  • Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
  • Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) N ⁇ twre (London) 327:70-73, U.S. Patent No. 4,945,050).
  • a DuPont BiolisticTM PDS1000/HE instrument helium retrofit
  • a selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed ofthe 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 373:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3' region ofthe nopaline synthase gene from the T-DNA ofthe Ti plasmid of Agrobacterium tumefaciens.
  • the seed expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptides and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site ofthe vector carrying the marker gene.
  • Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette.
  • approximately 5-10 plates of tissue are normally bombarded.
  • Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury.
  • the tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
  • Five to seven days post bombardment the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly.
  • green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
  • the cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430.
  • This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system.
  • Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector.
  • Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis.
  • Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTGTM low melting agarose gel (FMC). Buffer and agarose contain 10 ⁇ g/ml ethidium bromide for visualization ofthe DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 ⁇ L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA).
  • the fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above.
  • the vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above.
  • the prepared vector pBT430 and fragment can then be ligated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL).
  • Transformants can be selected on agar plates containing LB media and 100 ⁇ g/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.
  • a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol. 759:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1 , IPTG (isopropylthio- ⁇ -galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°.
  • IPTG isopropylthio- ⁇ -galactoside, the inducer
  • Cells are then harvested by centrifugation and re-suspended in 50 ⁇ L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0J mM phenyl methylsulfonyl fluoride.
  • a small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator.
  • the mixture is centrifuged and the protein concentration ofthe supernatant determined.
  • One ⁇ g of protein from the soluble fraction ofthe culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.
  • EXAMPLE 9 Evaluating Compounds for Their ability to Inhibit the Activity of Aminoacyl-tRNA Synthetases
  • the polypeptides described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 8, or expression in eukaryotic cell culture, inplanta, and using viral expression systems in suitably infected organisms or cell lines.
  • the instant polypeptides may be expressed either as mature forms ofthe proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags.
  • Common fusion protein partners include glutathione S-transferase ("GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide (“(His) ⁇ ").
  • GST glutathione S-transferase
  • Trx thioredoxin
  • (His) ⁇ C- and/or N-terminal hexahistidine polypeptide
  • the fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme.
  • proteases include thrombin, enterokinase and factor Xa.
  • any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.
  • Purification ofthe instant polypeptides may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor.
  • the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme.
  • the instant polypeptides may be expressed as a fusion protein coupled to the C-terminus of thioredoxin.
  • a (His)g peptide may be engineered into the N-terminus ofthe fused thioredoxin moiety to afford additional opportunities for affinity purification.
  • Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B.
  • a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include ⁇ -mercaptoethanol or other reduced thiol.
  • the eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired.
  • Proteolytic cleavage ofthe thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBondTM affinity resin or other resin.
  • Crude, partially purified or purified enzyme, either alone or as a fusion protein may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation ofthe instant polypeptides disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity. For example, assays for aminoacyl-tRNA synthetase activity are presented by (Lloyd et al., (1995) Nucleic Acid Research 23 (75j:2882-2892).

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Abstract

This invention relates to an isolated nucleic acid fragment encoding an aminoacyl-tRNA synthetase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the aminoacyl-tRNA synthetase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the aminoacyl-tRNA synthetase in a transformed host cell.

Description

TITLE PLANT AMINO ACYL-tRNA SYNTHETASES This application claims the benefit of U.S. Provisional Application No. 60/107,789, filed November 10, 1998. FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding aminoacyl-tRNA synthetases in plants and seeds.
BACKGROUND OF THE INVENTION Aminoacyl-tRNA Synthetases (AARS) are enzymes that charge (acylate) tRNAs with amino acids. These charged aminoacyl tRNAs then participate in mRNA translation and protein synthesis. The AARS show high specificity for charging a specific tRNA with the appropriate amino acid, for example valyl-tRNA with valine by valyl-tRNA synthetase or tryptophanyl-tRNA with tryptophan by tryptophanyl-tRNA synthetase. In general, per organism there are at least one AARS for each of the twenty amino acids. There are exceptions however. AARS are ancient enzymes, having functioned in translation since early life evolution. Some have speculated that the earliest aminoacyl-tRNA synthetases were mRNAs, not proteins, with the proteinaceous AARS described here emerging later (Neidhardt et al., (1975) Annu. Rev. Microbiol. 29:215-250). AARS are structurally diverse, although AARSs for some amino acids are more closely related than for others. AARSs are generally divided into two classes, class I and class II based on structural similarity and amino acid preferences (Eriani et al., (1990) Nature 547:203-206). Plants like all other cellular organisms have aminoacyl-tRNA synthetases. However, a full description of the plant 'complement' of aminoacyl-tRNA synthetases has not yet been described. Full-length cDNA, genomic clones, and EST sequences for a variety of plant aminoacyl-tRNA synthetases are known. However, several anticipated aminoacyl-tRNA synthetases have not been discovered.
Because of the central role of protein synthesis in life, any agent that inhibits or disrupts this activity is likely to be toxic. Aminoacyl-tRNA synthetases play a critical role in protein translation by linking genetic nucleic acid information to protein synthesis. Aminoacyl-tRNA synthetases perform this role by "reading" the identity of the different tRNAs and acylating them with the correct cognate amino acid. A large volume of research over several decades has been focused on identifying inhibitors of this process. Inhibitors of aminoacyl-tRNA synthetases have been found to be cytotoxic due to their inhibition of protein synthesis. As such they therefore could be used as herbicides or in aminoacyl-tRNA synthetase selectable marker systems (Lloyd et al., (1995) Nucleic Acid Research 23(15):2882-2892). The genes disclosed herein can serve as the basis for testing whether the encoded aminoacyl-tRNA synthetases are sensitive to known inhibitors or other chemicals.
Biochemical processes are often compartmentalized in regions of cells, such as mitochondria, plastids, and lysosomes. These organelles are key sites for many biochemical pathways. Bioengineering of these processes may require targeting protein products to specific organells. One method to accomplish this involves the addition of an N-terminal prosequence (transit peptide) that directs protein entry into a specific organelle(s). Upon or shortly after transport into the organelle the transit peptide is usually proteolytically removed, and the mature protein is then able to function. A few plant transit peptides have been shown empirically to be capable of directing fused proteins into specific organelles. However this ability appears to depend upon the structure of the protein being imported and to date it is impossible to predict whether a protein will be imported into an organelle with a given transit peptide. As such, it is advantageous to have a diversity of potential transit peptides from which the most efficient candidate can be chosen to target a protein of interest to an organelle. A number of plant transit peptides are known which direct mature proteins to mitochondria or chloroplast organells. These transit peptides are diverse in structure (length and amino acid sequence) and there is no strong consensus sequence identifying them. In addition, there is no obvious clear relationship between chloroplast targeting and mitochondrial targeting transit sequences. This invention describes a number of chloroplast-targeting and mitochondria- targeting transit peptides for (maize) aminoacyl-tRNA synthetases. These sequences will find utility in directing both aminoacyl-tRNA synthetase and other proteins into these organelles.
Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand protein synthesis in plants, provide genetic tools for the manipulation of gene expression, protein targeting to specific organells and provide possible targets for herbicides.
SUMMARY OF THE INVENTION The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 240 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn arginyl-tRNA synthetase polypeptide of SEQ ID NO:2, a rice arginyl-tRNA synthetase polypeptide of SEQ ID NO:4, a soybean arginyl-tRNA synthetase polypeptide of SEQ ID NO:6, a wheat arginyl-tRNA synthetase polypeptide of SEQ ID NO:8. The present invention also relates to an isolated polynucleotide comprising the complement ofthe nucleotide sequences described above.
The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 205 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 10, a rice glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 12, a soybean glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 14. The present invention also relates to an isolated polynucleotide comprising the complement ofthe nucleotide sequences described above.
The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 79 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a wheat glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 16. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention further relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 243 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn histidyl-tRNA synthetase polypeptide of SEQ ID NO: 18, a soybean histidyl-tRNA synthetase polypeptide of SEQ ID NO:20, a wheat histidyl-tRNA synthetase polypeptide of SEQ ID NO:22. The present invention also relates to an isolated polynucleotide comprising the complement ofthe nucleotide sequences described above. The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 30 amino acids that has at least 60% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:24, 26, 28, 30, 32, 34, 36 and 38.
It is preferred that the isolated polynucleotides ofthe claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide ofthe present invention operably linked to suitable regulatory sequences. The present invention also relates to the identification of transit peptides associated with aminoacyl-tRNA synthetases ofthe instant invention and the use of those transit peptides to target aminoacyl-tRNA synthetases and other operably linked proteins to specific organelles within plant cells. Transit peptide amino acid sequences are located just upstream ofthe mature aminoacyl-tRNA synthetase polypeptide sequences disclosed in the instant invention.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide ofthe present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide ofthe present invention or a chimeric gene ofthe present invention. The present invention relates to a process for producing an isolated host cell comprising a chimeric gene ofthe present invention or an isolated polynucleotide ofthe present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide ofthe present invention.
The present invention relates to an arginyl-tRNA synthetase polypeptide of at least 240 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, and 8.
The present invention relates to a glutamyl-tRNA synthetase polypeptide of at least 205 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 10, 12, 14 and 16. The present invention relates to a glutamyl-tRNA synthetase polypeptide of at least
79 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO: 16.
The present invention relates to a histidyl-tRNA synthetase polypeptide of at least 243 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 18, 20 and 22.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide in the host cell containing the isolated polynucleotide with the level of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide in a host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide gene, preferably a plant arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion ofthe amino acid sequence encoding an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide ofthe present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide ofthe present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone. A further embodiment ofthe instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of an arginyl-tRNA synthetase, glutamyl- tRNA synthetase or histidyl-tRNA synthetase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression ofthe chimeric gene wherein expression ofthe chimeric gene results in production of arginyl-tRNA synthetase, glutamyl- tRNA synthetase or histidyl-tRNA synthetase in the transformed host cell; (c) optionally purifying the arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase expressed by the transformed host cell; (d) treating the arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase with a compound to be tested; and (e) comparing the activity ofthe arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase that has been treated with a test compound to the activity of an untreated arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase, thereby selecting compounds with potential for inhibitory activity.
The present invention relates to a composition comprising an isolated polynucleotide ofthe present invention. The present invention relates to a composition comprising a polypeptide ofthe present invention.
The present invention relates to an isolated polynucleotide comprising the nucleotide sequence comprising at least one of 30 contiguous nucleotides of nucleic acid sequences selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such sequences.
The present invention relates to an expression cassette comprising an isolated polynucleotide ofthe present invention operably linked to a promoter.
The present invention relates to a method for positive selection of a transformed cell comprising: (a) transforming a plant cell with an expression cassette ofthe present invention;
(b) growing the transformed plant cell under conditions allowing expression of the polynucleotide in an amount sufficient to complement an amino-acyl t-RNA synthesis auxotroph in a plant cell to provide a positive selection means. The present invention relates to a method for positive selection of a transformed cell comprising:
(a) transforming a plant cell with a chimeric gene ofthe present invention; and
(b) growing the transformed plant cell, wherein the plant cell is a monocot or a dicot and includes corn, rice, soybean or wheat under conditions allowing expression ofthe polynucleotide in an amount sufficient to complement an amino-acyl t-RNA synthesis. BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs ("EST"), the sequences of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs ("Contig"), contigs assembled from an FIS and one or more ESTs ("Contig*"), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR ("CGS"). Nucleotide sequences, SEQ ID NOs: l, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 and amino acid sequences SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 were determined by further sequence analysis of cDNA clones encoding the amino acid sequences set forth in SEQ ID NOs:24, 26, 28, 30, 32, 34, 36 and 38. Nucleotide SEQ ID NOs:23, 25, 27, 29, 31, 33, 35 and 37 and amino acid SEQ ID NOs:24, 26, 28, 30, 32, 34, 36 and 38 were presented in a U.S. Provisional Application No. 60/107,789, filed November 10, 1998.
The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
TABLE 1
Aminoacyl-tRNA Synthet∑ ises
SEQ ID NO:
Protein Clone Designation (Nucleotide (Amino Acid)
Arginyl-tRNA Synthetase cpclc.pk001.dl l (FIS) 1 2
Arginyl-tRNA Synthetase rl0n.pk086.pl 6 (FIS) 3 4
Arginyl-tRNA Synthetase ssm.pk0026.bl0 (FIS) 5 6
Arginyl-tRNA Synthetase wlkl.pk0007.f5 (FIS) 7 8
Glutamyl-tRNA p0010.cbpcpl0r (CGS) 9 10 Synthetase
Glutamyl-tRNA rlr2.pk0032.f2 (CGS) 11 12 Synthetase
Glutamyl-tRNA Contig Composed of: 13 14 Synthetase sdc5c.pk0002.el l sgslc.pk001.kl2 sgslc.pk004.e20
Glutamyl-tRNA wlm96.pk055.g5 (EST) 15 16 Synthetase
Histidyl-tRNA Synthetase p0102.cerbb73r (CGS) 17 18
Histidyl-tRNA Synthetase Contig composed of: 19 20 sdp4c.pk007.c7 ssm.pk0012.d9
Histidyl-tRNA Synthetase wrl.pk0079.dl (FIS) 21 22
Arginyl-tRNA Synthetase cpclc.pk001.dl l (EST) 23 24
Arginyl-tRNA Synthetase rl0n.pk086.pl 6 (EST) 25 26
Arginyl-tRNA Synthetase ssm.pk0026.bl0 (EST) 27 28
Arginyl-tRNA Synthetase wlkl.pk0007.f5 (EST) 29 30
Glutamyl-tRNA rlr2.pk0032.f2 (EST) 31 32
Synthetase
Glutamyl-tRNA sgslc.pk004.e20 (EST) 33 34 Synthetase
Histidyl-tRNA Synthetase ssm.pk0012.d9 (EST) 35 36 Histidyl-tRNA Synthetase wrl .pk0079.dl (EST) 37 38 The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 73:3021-3030 (1985) and in the Biochemical J. 219 (No. 2 :345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
DETAILED DESCRIPTION OF THE INVENTION In the context of this disclosure, a number of terms shall be utilized. As used herein, a "polynucleotide" is a nucleotide sequence such as a nucleic acid fragment. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA. An isolated polynucleotide ofthe present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides, ofthe nucleic acid sequence ofthe SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such sequences.
As used herein, "contig" refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence. As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties ofthe polypeptide encoded by the nucleotide sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co- suppression technology. "Substantially similar" also refers to modifications ofthe nucleic acid fragments ofthe instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties ofthe resulting transcript vis-a-vis the ability to mediate gene silencing or alteration ofthe functional properties ofthe resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications ofthe nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level ofthe polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level ofthe polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level ofthe polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100%) sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties ofthe encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration ofthe N-terminal and C-terminal portions ofthe polypeptide molecule would also not be expected to alter the activity ofthe polypeptide. Each ofthe proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity ofthe encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a polypeptide in a plant cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide (such as aminoacyl-tRNA synthetase) in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial, or viral) may comprise the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.
Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5%) SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5%) SDS at 50°C for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature ofthe final two 30 min washes in 0JX SSC, 0.5% SDS was increased to 60°C. Another preferred set of highly stringent conditions uses two final washes in 0JX SSC, 0.1% SDS at 65°C.
Substantially similar nucleic acid fragments ofthe instant invention may also be characterized by the percent identity ofthe amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides ofthe present invention) encode polypeptides that are at least 70% identical, preferably at least 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOWS and DIAGONALS SAVED=5.
A "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification ofthe protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence often or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantial portion ofthe disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
"Codon degeneracy" refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion ofthe amino acid sequences set forth herein. The skilled artisan is well aware ofthe "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage ofthe host cell. "Synthetic nucleic acid fragments" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. "Chemically synthesized", as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias ofthe host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available. "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.
"Coding sequence" refers to a nucleotide sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation ofthe associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
"Promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 75: 1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
The "translation leader sequence" refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream ofthe translation start sequence. The translation leader sequence may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236). The "3' non-coding sequences" refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end ofthe mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 7:671-680.
"RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy ofthe DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing ofthe primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into polypeptide by the cell. "cDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "Sense" RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Patent No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part ofthe specific nucleotide sequence, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The term "operably linked" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control ofthe promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression ofthe target protein. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231,020, incorporated herein by reference).
"Altered levels" refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms. "Mature" protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals. A "chloroplast transit peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. "Chloroplast transit sequence" refers to a nucleotide sequence that encodes a chloroplast transit peptide. A "signal peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).
"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or "gene gun" transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Patent No. 4,945,050, incoφorated herein by reference). Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis"). Nucleic acid fragments encoding at least a portion of several aminoacyl-tRNA synthetases have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments ofthe instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction). For example, genes encoding other arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase polypeptides, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all ofthe instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
In addition, two short segments ofthe instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence ofthe other primer takes advantage ofthe presence ofthe polyadenylic acid tracts to the 3' end ofthe mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 55:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 5^:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 7:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide. The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a polypeptide of a gene (such as arginyl-tRNA synthetase, glutamyl-tRNA synthetase or histidyl-tRNA synthetase) preferably a substantial portion of a plant polypeptide of a gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a polypeptide.
Availability ofthe instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions ofthe instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) dv. Immunol. 3-5:1-34; Maniatis). The nucleic acid fragments ofthe instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of in those cells.
Overexpression ofthe proteins ofthe instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. For reasons of convenience, the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression. Plasmid vectors comprising the instant chimeric gene can then be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware ofthe genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) E 73O J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 275:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.
For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by altering the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 5-5:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel ( 1992) Plant Phys.100:1621 -1632) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.
It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part ofthe instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression ofthe corresponding endogenous genes are reduced or eliminated.
Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Patent Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of specific phenotype to the reproductive tissues ofthe plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds, and is not an inherent part ofthe invention. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.
The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides ofthe instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production ofthe instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression ofthe encoded aminoacyl-tRNA synthetase. An example of a vector for high level expression ofthe instant polypeptides in a bacterial host is provided (Example 8).
Additionally, the instant polypeptides can be used as a targets to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the polypeptides described herein catalyze various steps in aminoacyl-tRNA biosynthesis. Accordingly, inhibition ofthe activity of one or more ofthe enzymes described herein could lead to inhibition of plant growth. Thus, the instant polypeptides could be appropriate for new herbicide discovery and design. All or a substantial portion ofthe nucleic acid fragments ofthe instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments ofthe instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments ofthe instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation ofthe DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:31-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes. A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 77:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 7^:325-332), allele- specific ligation (Landegren et al. (1988) Science 247:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents ofthe mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 55:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments ofthe instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion ofthe mutation tag element in or near the plant gene encoding the instant polypeptides. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptides can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function ofthe instant polypeptides disclosed herein. EXAMPLES
The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments ofthe invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
EXAMPLE 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various corn, rice, soybean and wheat tissues were prepared. The characteristics ofthe libraries are described below. TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat
Library Tissue Clone cpclc Corn pooled BMS treated with chemicals related to cGMP** cpclc.pk001.dll pOOlO Corn log phase suspension cells treated with A23187® to pOOlO.cbpcplOr induce mass apoptosis**** p0102 Corn early meiosis tassels* p0102.cerbb73r rlOn Rice 15 day old leaf* rl0n.pk086.pl6 rlr2 Rice leaf 15 days after germination, 2 hours after infection rlr2.pk0032.f2 of strain Magaporthe grisea 4360-R-62 (AVR2-YAMO) sdc5c sdc5c.pk0002.ell sdp4c sdρ4c.pk007.c7 sgslc Soybean seeds 4 hours after germination sgslc.pk001.kl2 sgslc.pk004.e20 ssm Soybean shoot meristem ssm.pk0012.d9 ssm.pk0026.bl0 wlkl Wheat seedlings 1 hour after inoculation with Erysiphe wlkl.pk0007.f5 graminisf sp tritici and treatment with herbicide*** wlm96 Wheat Seedlings 96 hours after inoculation with Erysiphe wlm96.pk055.g5 graminisf. sp tritici wrl Wheat root from 7 day old seedling wrl.pk0079.dl
*These libraries were normalized essentially as described in U.S. Patent No. 5,482,845, incoφorated herein by reference. **Corn tissues were individually treated with one of the following then pooled; Suramin, MAS7, dipyryridamole, zaprinast, 8-bromo-cGMPtrequinsin HCL, Compound 48/80 all of which are commercially available from Calbiochem-Noavbiochem Coφ.
*** Application of 6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone; synthesis and methods of using this compound are described in USSN 08/545,827, incoφorated herein by reference.
****AJ3187® is commercially available from several vendors including Calbiochem
cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). The Uni-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DΗ10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
EXAMPLE 2
Identification of cDNA Clones cDNA clones encoding aminoacyl-tRNA synthetases were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol.
275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3 -dimensional structure Brookhaven Protein Data
Bank, the last major release ofthe SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The
DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the ΝCBI. For convenience, the P-value (probability) of observing a match of a cDΝA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative ofthe logarithm ofthe reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDΝA sequence and the BLAST "hit" represent homologous proteins.
EXAMPLE 3
Characterization of cDΝA Clones Encoding Arginyl-tRΝA Synthetase
The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity ofthe polypeptides encoded by the cDΝAs to arginyl-tRΝA synthetase from Arabidopsis thaliana (ΝCBI Identifier No. gi 2632105). Shown in Table 3 are the BLAST results for individual ESTs ("EST"), the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs ("Contig"), contigs assembled from an FIS and one or more ESTs ("Contig*"), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR ("CGS"): TABLE 3
BLAST Results for Sequences Encoding Polypeptides Homologous to Arabidopsis thaliana Arginyl-tRNA Synthetase
BLAST pLog Score to Clone Status gi 2632105 cpclc.pk001.dl l (FIS) 146.00 rl0n.pk086.pl 6 (FIS) >254.00 ssm.pk0026.bl0 (FIS) 129.00 wlkl.pk0007.f5 (FIS) 102.00
The data in Table 4 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4, 6 and 8 and the Arabidopsis thaliana sequence.
TABLE 4
Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Arabidopsis thaliana Arginyl-tRNA Synthetase
Percent Identity to SEQ ID NO. gi 2632105
2 75%
4 66%
6 74% 8 73%
Sequence alignments and percent identity calculations were performed using the Megalign program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Shaφ (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOWS and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a arginyl-tRNA synthetase. These sequences represent the first corn, rice, soybean and wheat sequences encoding arginyl-tRNA synthetase.
EXAMPLE 4 Characterization of cDNA Clones Encoding Glutamyl-tRNA Synthetase The BLASTX search using the EST sequences from clones listed in Table 5 revealed similarity ofthe polypeptides encoded by the cDNAs to glutamyl-tRNA synthetase from Arabidopsis thaliana (NCBI Identifier No. gi 3435196), Hordum vulgar (NCBI Identifier No. gi 2500980), Nicotina tabacum (NCBI Identifier No. gi 2500981) and Saccharomyces cerevisiae (NCBI Identifier No. gi 2507428). Shown in Table 5 are the BLAST results for individual ESTs ("EST"), the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs ("Contig"), contigs assembled from an FIS and one or more ESTs ("Contig*"), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR ("CGS"):
TABLE 5
BLAST Results for Sequences Encoding Polypeptides Homologous to Arabidopsis thaliana, Hordum vulgare, Nicotina tabacum and Saccharomyces cerevisiae Glutamyl-tRNA Synthetase Clone Status BLAST pLog Score pOOlO.cbpcplOr (CGS) >254.00 (gi 3435196) rlr2.pk0032.f2 (CGS) >254.00 (gi 2500980)
Contig composed of: Contig 97.40 (gi 2500981 ) sdc5c.pk0002.el l sgslc.pk001.kl2 sgslc.pk004.e20 wlm96.pk055.g5 (EST) 18.30 (gi 2507428)
The data in Table 6 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs: 10, 12, 14 and 16 and the Arabidopsis thaliana, Hordum vulgare, Nicotina tabacum and Saccharomyces cerevisiae sequences.
TABLE 6
Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Arabidopsis thaliana, Hordum vulgare, Nicotina tabacum and Saccharomyces cerevisiae Glutamyl-tRNA Synthetase
SEQ ID NO. Percent Identity to
10 67% (gi 3435196)
12 81% (gi 2500980)
14 87% (gi 2500981)
16 54% (gi 2507428)
Sequence alignments and percent identity calculations were performed using the Megalign program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Shaφ (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY-10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a glutamyl-tRNA synthetase. These sequences represent the first corn, rice, soybean and wheat sequences encoding glutamyl-tRNA synthetase.
EXAMPLE 5 Characterization ofcDNA Clones Encoding Histidyl-tRNA Synthetase The BLASTX search using the EST sequences from clones listed in Table 7 revealed similarity ofthe polypeptides encoded by the cDNAs to histidyl-tRNA synthetase from Oryza sativa (NCBI Identifier No. gi 3915070) and Arabidopsis thaliana (NCBI Identifier No. gi 3659909). Shown in Table 7 are the BLAST results for individual ESTs ("EST"), the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs ("Contig"), contigs assembled from an FIS and one or more ESTs ("Contig*"), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR ("CGS"):
TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous to Oryza sativa and Arabidopsis thaliana Histidyl-tRNA Synthetase Clone Status BLAST pLog Score
P0102.cerbb73r (CGS) >254.00 (gi 3915070)
Contig composed of: Contig 84.22 (gi 3659909) sdp4c.pk007.c7 ssm.pk0012.d9 wrl.pk0079.dl (FIS) 98.30 (gi 3659909)
The data in Table 8 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs: 18, 20 and 22 and the Oryza sativa and Arabidopsis thaliana sequences.
TABLE 8
Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Oryza sativa and Arabidopsis thaliana Histidyl-tRNA Synthetase
SEQ ID NO. Percent Identity to
18 87%
20 67% 22 67%
Sequence alignments and percent identity calculations were performed using the Megalign program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Shaφ (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a histidyl-tRNA synthetase. These sequences represent the first corn, soybean and wheat sequences encoding histidyl-tRNA synthetase.
EXAMPLE 6 Expression of Chimeric Genes in Monocot Cells A chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incoφorated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes Ncol and Smal and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb Ncol-Smal fragment ofthe plasmid pML103. Plasmid pML103 has been deposited under the terms ofthe Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, VA 201 10-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment ofthe maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end ofthe maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL 1 -Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3' region.
The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses ofthe inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27°C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. T e pat gene in p35S/Ac is under the control ofthe 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region ofthe nopaline synthase gene from the T-DNA ofthe Ti plasmid of Agrobacterium tumefaciens.
The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 φm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) ofthe DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
For bombardment, the embryogenic tissue is placed on filter paper over agarose- solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some ofthe plates containing the glufosinate- supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.
Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 5:833-839).
EXAMPLE 7 Expression of Chimeric Genes in Dicot Cells A seed-specific expression cassette composed ofthe promoter and transcription terminator from the gene encoding the β subunit ofthe seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 251:9228-9238) can be used for expression ofthe instant polypeptides in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.
The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incoφorated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.
Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons,
3-5 mm in length dissected from surface sterilized, immature seeds ofthe soybean cultivar A2872, can be cultured in the light or dark at 26°C on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.
Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nαtwre (London) 327:70-73, U.S. Patent No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed ofthe 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 373:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3' region ofthe nopaline synthase gene from the T-DNA ofthe Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptides and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site ofthe vector carrying the marker gene.
To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL ofthe DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above. Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
EXAMPLE 8 Expression of Chimeric Genes in Microbial Cells
The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430.
Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer and agarose contain 10 μg/ml ethidium bromide for visualization ofthe DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.
For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol. 759:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1 , IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0J mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration ofthe supernatant determined. One μg of protein from the soluble fraction ofthe culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight. EXAMPLE 9 Evaluating Compounds for Their Ability to Inhibit the Activity of Aminoacyl-tRNA Synthetases The polypeptides described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 8, or expression in eukaryotic cell culture, inplanta, and using viral expression systems in suitably infected organisms or cell lines. The instant polypeptides may be expressed either as mature forms ofthe proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags. Common fusion protein partners include glutathione S-transferase ("GST"), thioredoxin ("Trx"), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide ("(His)^"). The fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.
Purification ofthe instant polypeptides, if desired, may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor. When the instant polypeptides are expressed as fusion proteins, the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme. For example, the instant polypeptides may be expressed as a fusion protein coupled to the C-terminus of thioredoxin. In addition, a (His)g peptide may be engineered into the N-terminus ofthe fused thioredoxin moiety to afford additional opportunities for affinity purification. Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B. In an alternate embodiment, a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include β-mercaptoethanol or other reduced thiol. The eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired. Proteolytic cleavage ofthe thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBond™ affinity resin or other resin. Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation ofthe instant polypeptides disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity. For example, assays for aminoacyl-tRNA synthetase activity are presented by (Lloyd et al., (1995) Nucleic Acid Research 23 (75j:2882-2892).

Claims

CLAIMS What is claimed is:
1. An isolated polynucleotide comprising a nucleotide sequence encoding a first polypeptide of at least 240 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of arginyl-tRNA synthetase polypeptides of SEQ ID NOs:2, 4, 6 and 8, or an isolated polynucleotide comprising the complement ofthe nucleotide sequence.
2. The isolated polynucleotide of Claim 1 , wherein the isolated nucleotide sequence consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOsJ, 3, 5, and 7 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, and 8.
3. The isolated polynucleotide of Claim 1 wherein the nucleotide sequence is DNA.
4. The isolated polynucleotide of Claim 1 wherein the nucleotide sequence is RNA.
5. A chimeric gene comprising the isolated polynucleotide of Claim 1 operably linked to suitable regulatory sequences.
6. An isolated host cell comprising the chimeric gene of Claim 5.
7. An isolated host cell comprising an isolated polynucleotide of Claim 1.
8. The isolated host cell of Claim 7 wherein the isolated host is selected from the group consisting of yeast, bacteria, plant, and virus.
9. A virus comprising the isolated polynucleotide of Claim 1.
10. A polypeptide of at least 240 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NO:2, 4, 6 and 8.
11. An isolated polynucleotide comprising a nucleotide sequence encoding a first polypeptide of at least 205 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of glutamyl-tRNA synthetase polypeptides of SEQ ID NOs: 10, 12 and 14, or an isolated polynucleotide comprising the complement ofthe nucleotide sequence.
12. The isolated polynucleotide of Claim 1 1 , wherein the isolated nucleotide sequence consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:9, 11 and 13 that codes for the polypeptide selected from the group consisting of SEQ ID NOs: 10, 12 and 14.
13. The isolated polynucleotide of Claim 11 wherein the isolated polynucleotide is
DNA.
14. The isolated polynucleotide of Claim 11 wherein the isolated polynucleotide is RNA.
15. A chimeric gene comprising the isolated polynucleotide of Claim 11 operably linked to suitable regulatory sequences.
16. An isolated host cell comprising the chimeric gene of Claim 15.
17. An isolated host cell comprising an isolated polynucleotide of Claim 11.
18. The isolated host cell of Claim 17 wherein the isolated host selected from the group consisting of yeast, bacteria, plant, and virus.
19. A virus comprising the isolated polynucleotide of Claim 11.
20. A polypeptide of at least 205 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a glutamyl-tRNA synthetase polypeptide of SEQ ID NOs: 10, 12 and 14.
21. An isolated polynucleotide comprising a nucleotide sequence encoding a first polypeptide of at least 79 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a glutamyl-tRNA synthetase polypeptide of SEQ ID NO: 16, or an isolated polynucleotide comprising the complement of the nucleotide sequence.
22. The isolated polynucleotide of Claim 21 , wherein the isolated nucleotide sequence consists of a nucleic acid sequence of SEQ ID NO: 15 that codes for the polypeptide of SEQ ID NO: 16.
23. The isolated polynucleotide of Claim 21 wherein the isolated polynucleotide is
DNA.
24. The isolated polynucleotide of Claim 21 wherein the isolated polynucleotide is RNA.
25. A chimeric gene comprising the isolated polynucleotide of Claim 21 operably linked to suitable regulatory sequences.
26. An isolated host cell comprising the chimeric gene of Claim 25.
27. An isolated host cell comprising an isolated polynucleotide of Claim 21.
28. The isolated host cell of Claim 27 wherein the isolated host is selected from the group consisting of yeast, bacteria, plant, and virus.
29. A virus comprising the isolated polynucleotide of Claim 21.
30. A polypeptide of at least 79 amino acids that has at least 80%> identity based on the Clustal method of alignment when compared to a glutamyl-tRNA synthetase polypeptide of SEQ ID NO:16.
31. An isolated polynucleotide comprising a nucleotide sequence encoding a first polypeptide of at least 243 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of histidyl-tRNA synthetase polypeptides of SEQ ID NO:sl8, 20 and 22, or an isolated polynucleotide comprising the complement ofthe nucleotide sequence.
32. The isolated polynucleotide of Claim 31 , wherein the isolated nucleotide sequence consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17, 19 and 21 that codes for the polypeptide selected from the group consisting of SEQ ID NOs: 18, 20 and 22.
33. The isolated polynucleotide of Claim 31 wherein the isolated polynucleotide is DNA.
34. The isolated polynucleotide of Claim 31 wherein the isolated polynucleotide is RNA.
35. A chimeric gene comprising the isolated polynucleotide of Claim 31 operably linked to suitable regulatory sequences.
36. An isolated host cell comprising the chimeric gene of Claim 35.
37. An isolated host cell comprising an isolated polynucleotide of Claim 31.
38. The isolated host cell of Claim 37 wherein the isolated host is selected from the group consisting of yeast, bacteria, plant, and virus.
39. A virus comprising the isolated polynucleotide of Claim 31.
40. A polypeptide of at least 243 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of histidyl-tRNA synthetase polypeptides of SEQ ID NOs: 18, 20 and 22.
41. A method of selecting an isolated polynucleotide that affects the level of expression of a aminoacyl-tRNA synthetase polypeptide in a plant cell, the method comprising the steps of:
(a) constructing an isolated polynucleotide comprising a nucleotide sequence of at least one of 30 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences;
(b) introducing the isolated polynucleotide into a plant cell; and
(c) measuring the level of an aminoacyl t-RNA synthetase polypeptide in the plant cell containing the polynucleotide.
42. The method of Claim 41 wherein the isolated polynucleotide consists of a nucleotide sequence selected from the group consisting of SEQ ID NOsJ, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37 that codes for the polypeptide selected from the group consisting of SEQ ID NOsJ, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.
43. A method of selecting an isolated polynucleotide that affects the level of expression of a aminoacyl-tRNA synthetase polypeptide in a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of any of Claims 1, 11, 21 or 31;
(b) introducing the isolated polynucleotide into a plant cell;
(c) measuring the level of an aminoacyl-tRNA synthetase polypeptide in the plant cell containing the polynucleotide; and (d) comparing the level of aminoacyl-tRNA synthetase polypeptide in the plant cell containing the isolated polynucleotide with the level of aminoacyl-tRNA synthetase polypeptide in a plant cell that does not contain the polynucleotide.
44. A method of obtaining a nucleic acid fragment encoding a aminoacyl-tRNA synthetase polypeptide comprising the steps of: (a) synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 30 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOsJ, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences; and
(b) amplifying a nucleic acid sequence using the oligonucleotide primer.
45. A method of obtaining a nucleic acid fragment encoding the amino acid sequence encoding a aminoacyl-tRNA synthetase polypeptide comprising the steps of:
(a) probing a cDNA or genomic library with an isolated polynucleotide comprising a nucleotide sequence of at least one of 30 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOsJ, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such nucleotide sequences;
(b) identifying a DNA clone that hybridizes with the isolated polynucleotide;
(c) isolating the identified DNA clone; and
(d) sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
46. A method for evaluating at least one compound for its ability to inhibit the activity of a aminoacyl-tRNA synthetase, the method comprising the steps of:
(a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a aminoacyl-tRNA synthetase, operably linked to suitable regulatory sequences;
(b) growing the transformed host cell under conditions that are suitable for expression ofthe chimeric gene wherein expression ofthe chimeric gene results in production ofthe aminoacyl-tRNA synthetase encoded by the operably linked nucleic acid fragment in the transformed host cell; (c) optionally purifying the aminoacyl-tRNA synthetase expressed by the transformed host cell;
(d) treating the aminoacyl-tRNA synthetase with a compound to be tested; and (e) determining the activity ofthe aminoacyl-tRNA synthetase that has been treated with a test compound.
47. A composition comprising an isolated polynucleotide of Claim 1.
48. A composition comprising an isolated polynucleotide of Claim 11.
49. A composition comprising an isolated polynucleotide of Claim 21.
50. A composition comprising an isolated polynucleotide of Claim 31.
51. A composition comprising a polypeptide of Claim 10, Claim 20, Claim 30, or Claim 40.
52. An isolated polynucleotide comprising the nucleotide sequence comprising at least one of 30 contiguous nucleotides of nucleic acid sequences selected from the group consisting of SEQ ID NOsJ, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and the complement of such sequences.
53. An expression cassette comprising an isolated polynucleotide of Claim 1, Claim 11, Claim 21, or Claim 31 operably linked to a promoter.
54. A method for positive selection of a transformed cell comprising:
(a) transforming a plant cell with an expression cassette of Claim 53; and
(b) growing the transformed plant cell under conditions allowing expression of the polynucleotide in an amount sufficient to complement an amino-acyl t-RNA synthesis auxotroph in a plant cell to provide a positive selection means.
55. A method for positive selection of a transformed cell comprising:
(a) transforming a plant cell with a chimeric gene of Claim 5, Claim 15, Claim 25 or Claim 35; and
(b) growing the transformed plant cell under conditions allowing expression of the polynucleotide in an amount sufficient to complement an amino-acyl t-RNA synthesis auxotroph in a plant cell to provide a positive selection means.
56. The method of Claim 54 wherein the plant cell is a monocot.
57. The method of Claim 56 wherein the plant cell is a dicot.
EP99971858A 1998-11-10 1999-11-09 PLANT AMINOACYL-tRNA SYNTHETASES Withdrawn EP1131453A2 (en)

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