Methionine metabolic enzymes

This application claims priority benefit of U.S. Provisional Application No. 60/099,519 filed Sep. 8, 1998, now pending.

This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in methionine metabolism in plants and seeds.

Human food and animal feed derived from many grains are deficient in the sulfur amino acids, methionine and cysteine, which are required in an animal diet. In corn, the sulfur amino acids are the third most limiting amino acids, after lysine and tryptophan, for the dietary requirements of many animals. The use of soybean meal, which is rich in lysine and tryptophan, to supplement corn in animal feed is limited by the low sulfur amino acid content of the legume. Thus, an increase in the sulfur amino acid content of either corn or soybean would improve the nutritional quality of the mixtures and reduce the need for further supplementation through addition of more expensive methionine.

One genetic engineering approach to increase the sulfur amino acid content of seeds is to isolate genes coding for proteins that are rich in the sulfur-containing amino acids methionine and cysteine, to link the genes to strong seed-specific regulatory sequences, to transform the chimeric gene into crops plants and to identify transformants wherein the gene is sufficiently highly expressed to cause an increase in total sulfur amino acid content. However, increasing the sulfur amino acid content of seeds by expression of sulfur-rich proteins may be limited by the ability of the plant to synthesize methionine, by the synthesis and stability of the methionine-rich protein, and by effects of over-accumulation of the methionine-rich protein on the viability of the transgenic seeds.

An alternative approach would be to increase the production and accumulation of the free amino acid, methionine, via genetic engineering technology. However, little guidance is available on the control of the biosynthesis and accumulation of methionine in plants, particularly in the seeds of plants.

Methionine-tRNA ligase (EC 6.1.1.10), also called Methionyl-tRNA synthetase, or L-Methione:tRNA(Met) ligase (AMP-forming) belongs to the class-I aminoacyl-tRNA synthetases. This is a cytoplasmic enzyme which catalyzes the ligation of methionine to transfer RNA in protein translation. Methionine-tRNA ligase is the first enzyme sustaining the methionine pathway in translation initiation in Escherichia coli. This enzyme is probably essential for cell survival, being required not only for elongation of protein synthesis but also for the initiation of all mRNA translation through initiator tRNA-aminoacylation. The active site of methionyl-tRNA synthetase possesses two functions: synthetic, which provides Met-tRNA for protein synthesis, and editing, which rejects inadvertently misactivated homocysteine. During editing, the side chain —SH group of homocysteine reacts with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. There is a specific —SH binding subsite, distinct from the methionine binding subsite, in the synthetic/editing active site of methionyl-tRNA synthetase (Jakubowski (1996) Biochemistry 35:8252-8259).

Methionyl-tRNA formyltransferase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) produces formylmethionyl-tRNA. This residue, which already has an amide bond, is the first residue in a polypeptide which initiates with methionine. The structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation (Lee et al. (1991) J. Biol. Chem. 266:18012-18017). Corn, Rice and Soybean ESTs encoding polypeptides with similarities to methionyl-tRNA formyltransferase are found in the NCBI database having General Identifier Nos. 485595, 569773, 571553, 3107255, 4292436 4313709, 4573024, 4966952, 5018236, 5055738, 5555461, 5606916, 5649988 and 5666614.

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 500 amino acids having at least 90% identity, preferably at least 400 amino acids having at least 95% identity, based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn methionyl-tRNA fomyltransferase of SEQ ID NO:2, a rice methionyl-tRNA formyltransferase of SEQ ID NO:4, a soybean methionyl-tRNA formyltransferase of SEQ ID NO:6, a wheat methionyl-tRNA formyltransferase of SEQ ID NO:8, a corn methionyl-tRNA synthetase of SEQ ID NO:10, and a soybean methionyl-tRNA synthetase of SEQ ID NO:12. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above. It is preferred that the isolated polynucleotides of the claimed invention comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9 and 11 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10 and 12. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11 and the complement of such nucleotide sequences.

The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.

The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eucaryotic, such as a yeast or a plant cell, or procaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.

The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.

The present invention relates to a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide of at least 500 amino acids having at least 90% homology, preferably at least 400 amino acids having at least 95% homology, based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10 and 12.

The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in a plant cell, the method comprising the steps of:

constructing an isolated polynucleotide or chimeric gene of the present invention;

introducing the isolated polynucleotide into a plant cell;

measuring the level a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in the plant cell containing the polynucleotide; and

comparing the level of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in the plant cell containing the isolated polynucleotide with the level of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in a plant 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 a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase gene, preferably a plant methionyl-tRNA formyltransferase or methionyl-tRNA synthetase gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11 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 thioredoxin polypeptide amino acid sequence.

The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a subsantial portion of the amino acid sequence encoding a to a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide comprising the steps of:

probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and

sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

The invention can be more fully understood from the following detailed description and the accompanying Drawing and Sequence Listing which form a part of this application.

FIG. 1 depicts the methionine metabolism biochemical pathway. In the presence of water and NAD + , adenosylhomocysteinase (EC 3.3.1.1) catalyzes the conversion of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. Using FADH, and S-adenosyl-L-methionine as cofactors, methionine synthase (EC 2.1.1.13) catalyzes the conversion of L-homocysteine and 5-methyltetrahydrofolate to L-methionine and tetrahydrofolate. In the presence of Mg +2 , and using energy from the conversion of ATP to AMP, methionyl tRNA ligase (EC 6.1.1.10) adds tRNA to L-methionine to produce L-methionyl-tRNA and pyrophosphate. Using water as a cofactor, methionyl-tRNA formyltransferase (EC 2.1.2.9) transfers the formyl group from 10-formyltetrahydrofolate to N-methionyl-tRNA and releases tetrahydrofolate.

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. 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.

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. 13:3021-3030 (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.

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 of the present invention may include at least 40 contiguous nucleotides, preferably at least 30 contiguous nucleotides, of the nucleic acid sequence of the SEQ ID NOs:1, 3, 5, 7, 9 and 11.

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 of the 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 of the 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 of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the 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.

For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucelic 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 of the 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 of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Consequently, a polynucleotide comprising a nucleotide sequence of at least 40 (preferably 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in a plant cell.

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 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×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 of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the 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 (polynucleotides) encode amino acid sequences that are 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragment encode amino acid sequences that are 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are 95% identical to the amino acid sequences reported herein. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the 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, WINDOW=5 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 of the 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. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten 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 of the sequences as reported herein, may now use all or a substantial portion of the 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 affecting 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 of the amino acid sequences set forth herein. The skilled artisan is well aware of the “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 of the 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 of the 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 of the 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 15:1-82. 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 of the translation start sequence. The translation leader sequence may affect processing of the 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 of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

“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 of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the 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. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the 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 of the 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 of the 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. Pat. 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.

“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. Pat. No. 4,945,050, incorporated 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 methionine metabolic enzymes 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 of the 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 methionyl-tRNA formyltransferases or methionyl-tRNA synthetases, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the 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 of the 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 of the 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 of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the 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 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the 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 86:5673-5677; 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 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least 40 (preferably 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11 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 methionyl-tRNA formyltransferase or methionyl-tRNA synthetase polypeptide.

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the 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) Adv. Immunol. 36:1-34; Maniatis).

The nucleic acid fragments of the 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 methionine-containing products in those cells. Because methionyl-tRNA synthetase is essential for cell survival, inhibitors of this enzyme may be herbicidal targets. Manipulation of the methionyl-tRNA synthetase and formylmethionyl-tRNA contents of corn and soybean, among others, may lead to increase methionine content in the seeds of these crops.

Overexpression of the proteins of the 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 of the 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) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), 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.

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 of the 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 of the 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 of the 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. Pat. 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 of the 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 of the 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 of the 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 of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the 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 of the encoded methionine metabolic enzyme. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 7).

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 methionine metabolism. Accordingly, inhibition of the activity of one or more of the 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 of the nucleic acid fragments of the 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 of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the 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 of the DNA polymorphisms is noted and used to calculate the position of the 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 4:37-41. 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. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), 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 of the 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 86:9402-9406; 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 of the 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 of the 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 of the instant polypeptides disclosed herein.

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 of the 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.

Zea mays

1

tcaaaaacga ctccaggcta gaggggctca tcaagatggc cgatctggtc agttagtccc 60

taatcttcca cctatcttcc ttttttcttt ctcgattgct tactactact tcgaatgcat 120

ttcgaattga ctcacggatt tcctcatcct ctcctgccat gctgctgctg cttggatctg 180

atctgcgcgc caagaccata aacctggccg cgatctgcac gccggccgac tacaacactc 240

gcccactcga caccatctac agcaacttca tcgacgcact cccagtggtc aagtactgct 300

ccgagaacag caagcgtctc atccacttct ccacctgcga ggtctacggc aagaccatcg 360

gcagcttcct ccccaaagac cacccgctcc gcaaggagcc tgaattttat gttcttaaag 420

aagatgagtc gccctgtatt tttggtccaa tcgtgaaaca aagatggtcc tatgcatgcg 480

caaaacaact tattgagagg cttgtatttg ctgaaggtgc agaaaatggc cttgatttca 540

caatcgtgag acctttcaat tggattggac caaggatgga cttcattcct ggtgttgatg 600

gtcctagtga aggtgtgcct cgggttttgg cttgcttcag taacaatctg ctccggagag 660

agcccttgaa gcttgttgat gggggtcagt ctcagagaac ctttgtctac atcaaggatg 720

ccatcgaagc tgtagtgctg atgattgaaa atcctgctcg agccaatggc cacatcttca 780

atgttgggaa tccgaacaat gaagttaccg ttagggagtt ggcccaaatg atgacagagg 840

tctacgcaaa tgtctcagga gaggcaccgc tggatgagcc catgattgac gtgagctcga 900

gtcagttcta cggcgaagga tacgatgata gcgacaagag gatccccgac atgactataa 960

tcaacaagca gctaggttgg aacccaaaga cgcctctcaa ggatctgctg gagacaacgc 1020

tgacgtatca gcacaagaca tacaaagaag ctgtgaaaag gcaaatgtcg caggcttcag 1080

cgtcgactta gatagcgtgc ccctggctct cgtctagagc ctagttgcag ctgttaagat 1140

gtttcggaag cgcaggtcac acatacgcta gtgatctttt ttttttgagg ccctagatgt 1200

ccgtgttgtt agataatcaa gggatgctgt atcagtatga tatatttgtg gcagagttga 1260

tgtattccat cgttcgtcgt actagttggg ttggttgccg agagctgtta tatattgctg 1320

tttgagtgta gaactagttg ctcagttata tgttattggt tcatgagatt agcgcccaca 1380

tcaatctgtt gatagcacat acatacatac tatagcaggg caatgctctg ctctgttggt 1440

ttggctccta tgatgaatct cattcgtcag ccatcaagtt tctg 1484

2

344

Zea mays

2

Ser Leu Ile Phe His Leu Ser Ser Phe Phe Leu Ser Arg Leu Leu Thr

1 5 10 15

Thr Thr Ser Asn Ala Phe Arg Ile Asp Ser Arg Ile Ser Ser Ser Ser

20 25 30

Pro Ala Met Leu Leu Leu Leu Gly Ser Asp Leu Arg Ala Lys Thr Ile

35 40 45

Asn Leu Ala Ala Ile Cys Thr Pro Ala Asp Tyr Asn Thr Arg Pro Leu

50 55 60

Asp Thr Ile Tyr Ser Asn Phe Ile Asp Ala Leu Pro Val Val Lys Tyr

65 70 75 80

Cys Ser Glu Asn Ser Lys Arg Leu Ile His Phe Ser Thr Cys Glu Val

85 90 95

Tyr Gly Lys Thr Ile Gly Ser Phe Leu Pro Lys Asp His Pro Leu Arg

100 105 110

Lys Glu Pro Glu Phe Tyr Val Leu Lys Glu Asp Glu Ser Pro Cys Ile

115 120 125

Phe Gly Pro Ile Val Lys Gln Arg Trp Ser Tyr Ala Cys Ala Lys Gln

130 135 140

Leu Ile Glu Arg Leu Val Phe Ala Glu Gly Ala Glu Asn Gly Leu Asp

145 150 155 160

Phe Thr Ile Val Arg Pro Phe Asn Trp Ile Gly Pro Arg Met Asp Phe

165 170 175

Ile Pro Gly Val Asp Gly Pro Ser Glu Gly Val Pro Arg Val Leu Ala

180 185 190

Cys Phe Ser Asn Asn Leu Leu Arg Arg Glu Pro Leu Lys Leu Val Asp

195 200 205

Gly Gly Gln Ser Gln Arg Thr Phe Val Tyr Ile Lys Asp Ala Ile Glu

210 215 220

Ala Val Val Leu Met Ile Glu Asn Pro Ala Arg Ala Asn Gly His Ile

225 230 235 240

Phe Asn Val Gly Asn Pro Asn Asn Glu Val Thr Val Arg Glu Leu Ala

245 250 255

Gln Met Met Thr Glu Val Tyr Ala Asn Val Ser Gly Glu Ala Pro Leu

260 265 270

Asp Glu Pro Met Ile Asp Val Ser Ser Ser Gln Phe Tyr Gly Glu Gly

275 280 285

Tyr Asp Asp Ser Asp Lys Arg Ile Pro Asp Met Thr Ile Ile Asn Lys

290 295 300

Gln Leu Gly Trp Asn Pro Lys Thr Pro Leu Lys Asp Leu Leu Glu Thr

305 310 315 320

Thr Leu Thr Tyr Gln His Lys Thr Tyr Lys Glu Ala Val Lys Arg Gln

325 330 335

Met Ser Gln Ala Ser Ala Ser Thr

340

3

1586

Oryza sativa

3

gcacgagaga gagtgtgggc gctcttaaat aggagagaaa aagaaaacca ccgccaccga 60

catcgcctcg cctcgtaacc cccaaatcca gcgcctccct tcctccccaa taatcgaatc 120

caatccatct tccacgcgca ttccggatcc gcggaccaag ggaggatgtc gtcgtcgtcg 180

tcgccgcccg ccgcctccgc cgccgcgagg ctggatctgg acggcaaccc catcgcgccc 240

ctcaccatct gcatgatcgg cgccggcggc ttcatcggct cccacctctg cgagaagctc 300

atggccgaga ccgcccacgt cgtctacgcc gtcgacgtct actgcgacaa gatccgccac 360

ctcgtcgacc ccgcccctcc ccacctccat ggccgcatct ccttccaccg cctcaacatc 420

aagaacgact ccaggctcga gggcctcatc aagatggccg atctgacgat caacctggcg 480

gcgatctgca cgccggccga ctacaacacg cgccccctcg acaccatcta cagcaacttc 540

atcgacgcgc tccctgtggt caagtactgc tctgagaaca acaagcgtct catccacttc 600

tccacgtgcg aggtctacgg caagaccatc ggcagcttcc tccccaccga ccaccctctc 660

cgcaaggaac ctgaatttta tgtactgaaa gaagatgaat ccccctgcat ttttggtcca 720

attgtgaaac agcgatggtc ttatgcatgt gcgaagcagc ttattgagag gcttattttt 780

gctgaaggag cagaaaatgg ccttgagttc acaattgtga gacctttcaa ttggattgga 840

ccaagaatgg actttattcc tggtgttgat ggtcctagtg agggtgttcc tcgggttttg 900

gcatgtttca gtaacaatct cctgcgtcgg gagcccctga aacttgttga tggtggccag 960

tcccagagaa cttttgttta cattaaggat gccattgaag ctgttcactt gatgattgaa 1020

aatcctgctc gtgccaatgg tcaaatcttc aatgtcggga atcctaacaa tgaagtcact 1080

gttaggcaat tggctgaaat gatgacagag gtctatgcaa atgtctcagg agagccacca 1140

ctggatgaac ctatgattga cgtgagttcg aagcagttct atggtgaagg atatgatgat 1200

agtgataaga ggattcctga catgaccata atcaacaagc agcttggttg gaatccgaag 1260

acccctctca aggatttgct ggagacaaca ttgacctacc agcacaagac ttacaaagaa 1320

gctatcaaaa ggcaaatgtc acaggcttca gcgtcgagtt agacaacacg ctccaatcca 1380

tagatggtga tgttggctct tgtagagcac tgtcagtggc tataatcgtt tggaagtgga 1440

atacgtatac tcaagtaatc ctttttgtgc atctagtcac tcctagatcc ctaccaactt 1500

ttaggtttcc tcctgtgtaa gccggcaagg ggttatccac catatctgtg gcagagtcga 1560

tgtattcatt tgtacctttg gttgct 1586

4

427

Oryza sativa

4

Pro Pro Asn Pro Ala Pro Pro Phe Leu Pro Asn Asn Arg Ile Gln Ser

1 5 10 15

Ile Phe His Ala His Ser Gly Ser Ala Asp Gln Gly Arg Met Ser Ser

20 25 30

Ser Ser Ser Pro Pro Ala Ala Ser Ala Ala Ala Arg Leu Asp Leu Asp

35 40 45

Gly Asn Pro Ile Ala Pro Leu Thr Ile Cys Met Ile Gly Ala Gly Gly

50 55 60

Phe Ile Gly Ser His Leu Cys Glu Lys Leu Met Ala Glu Thr Ala His

65 70 75 80

Val Val Tyr Ala Val Asp Val Tyr Cys Asp Lys Ile Arg His Leu Val

85 90 95

Asp Pro Ala Pro Pro His Leu His Gly Arg Ile Ser Phe His Arg Leu

100 105 110

Asn Ile Lys Asn Asp Ser Arg Leu Glu Gly Leu Ile Lys Met Ala Asp

115 120 125

Leu Thr Ile Asn Leu Ala Ala Ile Cys Thr Pro Ala Asp Tyr Asn Thr

130 135 140

Arg Pro Leu Asp Thr Ile Tyr Ser Asn Phe Ile Asp Ala Leu Pro Val

145 150 155 160

Val Lys Tyr Cys Ser Glu Asn Asn Lys Arg Leu Ile His Phe Ser Thr

165 170 175

Cys Glu Val Tyr Gly Lys Thr Ile Gly Ser Phe Leu Pro Thr Asp His

180 185 190

Pro Leu Arg Lys Glu Pro Glu Phe Tyr Val Leu Lys Glu Asp Glu Ser

195 200 205

Pro Cys Ile Phe Gly Pro Ile Val Lys Gln Arg Trp Ser Tyr Ala Cys

210 215 220

Ala Lys Gln Leu Ile Glu Arg Leu Ile Phe Ala Glu Gly Ala Glu Asn

225 230 235 240

Gly Leu Glu Phe Thr Ile Val Arg Pro Phe Asn Trp Ile Gly Pro Arg

245 250 255

Met Asp Phe Ile Pro Gly Val Asp Gly Pro Ser Glu Gly Val Pro Arg

260 265 270

Val Leu Ala Cys Phe Ser Asn Asn Leu Leu Arg Arg Glu Pro Leu Lys

275 280 285

Leu Val Asp Gly Gly Gln Ser Gln Arg Thr Phe Val Tyr Ile Lys Asp

290 295 300

Ala Ile Glu Ala Val His Leu Met Ile Glu Asn Pro Ala Arg Ala Asn

305 310 315 320

Gly Gln Ile Phe Asn Val Gly Asn Pro Asn Asn Glu Val Thr Val Arg

325 330 335

Gln Leu Ala Glu Met Met Thr Glu Val Tyr Ala Asn Val Ser Gly Glu

340 345 350

Pro Pro Leu Asp Glu Pro Met Ile Asp Val Ser Ser Lys Gln Phe Tyr

355 360 365

Gly Glu Gly Tyr Asp Asp Ser Asp Lys Arg Ile Pro Asp Met Thr Ile

370 375 380

Ile Asn Lys Gln Leu Gly Trp Asn Pro Lys Thr Pro Leu Lys Asp Leu

385 390 395 400

Leu Glu Thr Thr Leu Thr Tyr Gln His Lys Thr Tyr Lys Glu Ala Ile

405 410 415

Lys Arg Gln Met Ser Gln Ala Ser Ala Ser Ser

420 425

5

1338

Glycine max

5

cgaaggcctc atcaagatgg cagatctcac tattaatctg gctgccattt gcactcccgc 60

ggattacaac acccgccctc tcgacaccat ttacagcaat ttcatcgacg cgctccccgt 120

ggtgaaatac tgttccgaga acaacaagcg ccttatccat ttctctactt gcgaagtgta 180

cggaaagacg attggagcct ttctccctaa agatagtcct cttcgtaagg atccggcata 240

ctatgttctt aaagaagacg agtctccttg catttttggt tctattgaaa aacagaggtg 300

gtcttatgcc tgtgcgaaac agttgattga gaggctgatt tatgctgagg gtgctgaaaa 360

tggcttggag ttcacaattg tgaggccttt taactggatt ggacctcgaa tggatttcat 420

tcctggcatt gatggtccaa gtgagggtgt tcctcgggtt cttgcatgct ttagcaataa 480

tcttctcaga ggagagcccc tcaagcttgt ggacggtggc cagtcccaga gaacctttgt 540

ttacattaaa gatgctattg aagctgtctt gctgatgatt gaaaaccctg ccagggccaa 600

tggccacata tttaatgtgg gtaacccaaa caatgaggtt actgttaggc agcttgctga 660

aataatgatt aaggtttatt caaaggtaag tggcgaacaa actcctgaaa cacctaccgt 720

tgatgtgagc tcaaaagaat tttatggtga gggatatgat gatagtgaca agagaatccc 780

tgacatgacc ataataaaca ggcagcttgg atggaatccc aagacttcac tttgggacct 840

gcttgagtcc actctgacat atcagcacag gacatatgct gaagccgtta agaaagtcat 900

tgcaaaaccc gttgcaagtt aagcttttta cagtgatggt gatgtgctac ctttgagaag 960

ggtgttcttg tcttgcccaa gtgttctgcc atattatact ggttgatttt atataaaatc 1020

gtaaattttc ctcctattgg tttcgttagc ctgtaggcaa tatagccccc acatggagaa 1080

gtaggaattt atacaagggc aatctgtgct ctatgtaatt ttaatgtaga gaccatgcgc 1140

tgctaaacat gttgagacac tttatatatt acctagggaa gacttttttc tgtgttttta 1200

aggatctctc gattcattgc tttgttgtct ttccaggtca gctgtaagtt gtatcattct 1260

atcattctgt ttgaccttaa gccatgtgtt ctaaattatt gtcattaatt tattttatga 1320

atattttttt ttgttact 1338

6

306

Glycine max

6

Glu Gly Leu Ile Lys Met Ala Asp Leu Thr Ile Asn Leu Ala Ala Ile

1 5 10 15

Cys Thr Pro Ala Asp Tyr Asn Thr Arg Pro Leu Asp Thr Ile Tyr Ser

20 25 30

Asn Phe Ile Asp Ala Leu Pro Val Val Lys Tyr Cys Ser Glu Asn Asn

35 40 45

Lys Arg Leu Ile His Phe Ser Thr Cys Glu Val Tyr Gly Lys Thr Ile

50 55 60

Gly Ala Phe Leu Pro Lys Asp Ser Pro Leu Arg Lys Asp Pro Ala Tyr

65 70 75 80

Tyr Val Leu Lys Glu Asp Glu Ser Pro Cys Ile Phe Gly Ser Ile Glu

85 90 95

Lys Gln Arg Trp Ser Tyr Ala Cys Ala Lys Gln Leu Ile Glu Arg Leu

100 105 110

Ile Tyr Ala Glu Gly Ala Glu Asn Gly Leu Glu Phe Thr Ile Val Arg

115 120 125

Pro Phe Asn Trp Ile Gly Pro Arg Met Asp Phe Ile Pro Gly Ile Asp

130 135 140

Gly Pro Ser Glu Gly Val Pro Arg Val Leu Ala Cys Phe Ser Asn Asn

145 150 155 160

Leu Leu Arg Gly Glu Pro Leu Lys Leu Val Asp Gly Gly Gln Ser Gln

165 170 175

Arg Thr Phe Val Tyr Ile Lys Asp Ala Ile Glu Ala Val Leu Leu Met

180 185 190

Ile Glu Asn Pro Ala Arg Ala Asn Gly His Ile Phe Asn Val Gly Asn

195 200 205

Pro Asn Asn Glu Val Thr Val Arg Gln Leu Ala Glu Ile Met Ile Lys

210 215 220

Val Tyr Ser Lys Val Ser Gly Glu Gln Thr Pro Glu Thr Pro Thr Val

225 230 235 240

Asp Val Ser Ser Lys Glu Phe Tyr Gly Glu Gly Tyr Asp Asp Ser Asp

245 250 255

Lys Arg Ile Pro Asp Met Thr Ile Ile Asn Arg Gln Leu Gly Trp Asn

260 265 270

Pro Lys Thr Ser Leu Trp Asp Leu Leu Glu Ser Thr Leu Thr Tyr Gln

275 280 285

His Arg Thr Tyr Ala Glu Ala Val Lys Lys Val Ile Ala Lys Pro Val

290 295 300

Ala Ser

305

7

664

Triticum aestivum

7

gcacgagcaa caatctcctc cgtagagagc ccctgaagct tgtcgatggc ggcgagtccc 60

agagaacttt tgtttacatc aaggatgcca ttgaagctgt tcttctgatg attgaaaacc 120

ctgctcgagc caatggtcat atcttcaacg ttgggaaccc cgacaatgaa gtcactgtta 180

gggagttggc cgaaatgatg acagaggtct acgctaaggt ctcaggagag cccccgctgg 240

aggagcctgt ggtcgacgtg agcgcgaaag aattctacgg cgaagggtac gacgacagcg 300

acaagaggat ccctgacatg accctgatca acaagcagct agggtggaac cccaagaccc 360

ctctcaagga cctgctggag acgacgctga cctaccagca caagacctac aaggaggctg 420

tgaagacgca gatgtgtctg gccacggcga cgccttcaag ctagacgatg acggcgcggc 480

catgctgctc ttgatctttt ggaagccaga tacttatatc gtagtgaaac cgtcgtcgtc 540

gtttttgtcg cgcctctaga tcacacccac ccatcttctt acctacctag attcgtcatt 600

gtgtaaccct atattgggca aagggaactg agtatctatt atacctgtgg tggcagtgtt 660

tgtt 664

8

153

Triticum aestivum

8

Thr Ser Asn Asn Leu Leu Arg Arg Glu Pro Leu Lys Leu Val Asp Gly

1 5 10 15

Gly Glu Ser Gln Arg Thr Phe Val Tyr Ile Lys Asp Ala Ile Glu Ala

20 25 30

Val Leu Leu Met Ile Glu Asn Pro Ala Arg Ala Asn Gly His Ile Phe

35 40 45

Asn Val Gly Asn Pro Asp Asn Glu Val Thr Val Arg Glu Leu Ala Glu

50 55 60

Met Met Thr Glu Val Tyr Ala Lys Val Ser Gly Glu Pro Pro Leu Glu

65 70 75 80

Glu Pro Val Val Asp Val Ser Ala Lys Glu Phe Tyr Gly Glu Gly Tyr

85 90 95

Asp Asp Ser Asp Lys Arg Ile Pro Asp Met Thr Leu Ile Asn Lys Gln

100 105 110

Leu Gly Trp Asn Pro Lys Thr Pro Leu Lys Asp Leu Leu Glu Thr Thr

115 120 125

Leu Thr Tyr Gln His Lys Thr Tyr Lys Glu Ala Val Lys Thr Gln Met

130 135 140

Cys Leu Ala Thr Ala Thr Pro Ser Ser

145 150

9

2281

Zea mays

9

ccacgcgtcc gaaaaatttt tagctgatag gcttgttgaa ggaacatgcc cgaacaaagt 60

ctgcaacgca tcagcaagag gagatcagtg tgaaacatgc agcaccttgt tgaatccaac 120

tgaacttatt gacccaaagt gcaaggtttg taagaacact ccacgtattc gtgacacgga 180

tcacttattt ttggagcttc ctctattgag agacaagctg gtaaactaca tcaatgaaac 240

ttcagttgct ggtttgtgga gtcaaaatgc tattcaagca acaaatgcat ggctgaagga 300

agggctaaag ccacgctgca tcaccagaga tcttaaatgg ggtgttcctg tcccccatga 360

gaagtataaa gacaaggtgt tctatgtttg gtttgatgca cctatcggct acgtgtctat 420

tacggcatcc tatacacctg agtgggagaa gtggtggaag aatcctgata atgtagaatt 480

gtttcagttt atgggcaaag ataatgtgcc gtttcacacg atcatgttcc cttcaacgct 540

actcggaact ggggaaaagt ggacaatgat gaaaacgata agtgttactg aatatttgaa 600

ttatgaagca ggcaaatttt ccaagagtaa aggtattgga gtctttggta atgatgcaaa 660

ggatacaaat attcctcctg aagtatggcg atactatttg cttatgaatc gccctgaggc 720

atcagataca ctctttactt gggctgattt gcaagccaaa ttgaacagcg agttgctgaa 780

caacttggga aacttcatta atcgtgtgct aagttttgtt gcaaaaccag ctggagctgg 840

atatggttct gttgtacccg atgcacctaa tgcgggcgcg cacacattga caaaaacact 900

tgcagaaaaa accagtaaat gggttgaaca atatctcgac gcaatggaaa aggttaaact 960

gaaacaagga ctcaagagtg caatgggcat ttctagtgat ggaaatgcat atttgcaaga 1020

gagccagttt tggaaacttt acaaggaaga tcctgcagca tgtgcaatcg tgatgaaaac 1080

ttcagttggc cttgtttacc tgcttgcctg tctgctggag cctttcatgc cctctttttc 1140

tgatgaagtg ttacgtcaac taaatttatc tccagaagaa aatctgtcat taagtgaaga 1200

aaagggagaa attgcgaagg caaaaactcc ttggaatttt gtaccagcag gtcacagaat 1260

agggaaaccc gcacctctgt tcaaggaatt gaaagatgaa gacgtagctc tccatagaga 1320

aaaatatgca gggagtcaag ctgagagaag ctcaaaagca gcagctgatg ctgaagccaa 1380

caaagttgct aaccagctca agggcacaaa attatctgat ggaggaacaa aaaaggaacc 1440

gaagaaacaa tctggtggct caaaatcgaa gacagctgag gctgacatta cagttgcaaa 1500

gttggatatt agagtggggc ttatcagaaa agcagagaag catccagatg ctgattccct 1560

ttacgtggag gagattgatg ttggagagga cgcgccgaga acagtagtca gtggccttgt 1620

taaattcata cctcttgaag aaatgcagag ccggaaagtt tgcgtgctct gcaatctgaa 1680

accggtggca atgcgtggca taaaatcaca tgcaatggtt ctggctgcat cgaatgagga 1740

ccacacgaag gttgagttgg tggaacctcc agagtctgct gctgtggggg agcgtgttac 1800

ctttgctggg tacgcgggtg agcccgaggc ttccctcagt ggcaagagca agacgtggga 1860

gaagcttgcc gccgagttgc acagcaacgg tgagctggtg gcgtgctaca gagatgtgcc 1920

cttcactacc tcggctggag tctgcagggt gaagacaata gcgaacggag agattcgcta 1980

gtaccacatc accgaactgc agccagctga gccggtctgt gtctggtttc gctgaaagcg 2040

attgttcctg actttcatct tcgaaaattt tggtatttcc atttgttcaa ataccgcaaa 2100

gcaaccatga tttagaaaaa tacagagagt gccctttctg catcctcttt ttatcagtta 2160

gtacttggct cctgtcgtct cactcggagg ctccatgctt cttgctgtat ctgtatttcc 2220

ttagaagtga ttctttacct gctcttattt atagttccat atgaaaggaa caaagtgtat 2280

c 2281

10

659

Zea mays

10

His Ala Ser Glu Lys Phe Leu Ala Asp Arg Leu Val Glu Gly Thr Cys

1 5 10 15

Pro Asn Lys Val Cys Asn Ala Ser Ala Arg Gly Asp Gln Cys Glu Thr

20 25 30

Cys Ser Thr Leu Leu Asn Pro Thr Glu Leu Ile Asp Pro Lys Cys Lys

35 40 45

Val Cys Lys Asn Thr Pro Arg Ile Arg Asp Thr Asp His Leu Phe Leu

50 55 60

Glu Leu Pro Leu Leu Arg Asp Lys Leu Val Asn Tyr Ile Asn Glu Thr

65 70 75 80

Ser Val Ala Gly Leu Trp Ser Gln Asn Ala Ile Gln Ala Thr Asn Ala

85 90 95

Trp Leu Lys Glu Gly Leu Lys Pro Arg Cys Ile Thr Arg Asp Leu Lys

100 105 110

Trp Gly Val Pro Val Pro His Glu Lys Tyr Lys Asp Lys Val Phe Tyr

115 120 125

Val Trp Phe Asp Ala Pro Ile Gly Tyr Val Ser Ile Thr Ala Ser Tyr

130 135 140

Thr Pro Glu Trp Glu Lys Trp Trp Lys Asn Pro Asp Asn Val Glu Leu

145 150 155 160

Phe Gln Phe Met Gly Lys Asp Asn Val Pro Phe His Thr Ile Met Phe

165 170 175

Pro Ser Thr Leu Leu Gly Thr Gly Glu Lys Trp Thr Met Met Lys Thr

180 185 190

Ile Ser Val Thr Glu Tyr Leu Asn Tyr Glu Ala Gly Lys Phe Ser Lys

195 200 205

Ser Lys Gly Ile Gly Val Phe Gly Asn Asp Ala Lys Asp Thr Asn Ile

210 215 220

Pro Pro Glu Val Trp Arg Tyr Tyr Leu Leu Met Asn Arg Pro Glu Ala

225 230 235 240

Ser Asp Thr Leu Phe Thr Trp Ala Asp Leu Gln Ala Lys Leu Asn Ser

245 250 255

Glu Leu Leu Asn Asn Leu Gly Asn Phe Ile Asn Arg Val Leu Ser Phe

260 265 270

Val Ala Lys Pro Ala Gly Ala Gly Tyr Gly Ser Val Val Pro Asp Ala

275 280 285

Pro Asn Ala Gly Ala His Thr Leu Thr Lys Thr Leu Ala Glu Lys Thr

290 295 300

Ser Lys Trp Val Glu Gln Tyr Leu Asp Ala Met Glu Lys Val Lys Leu

305 310 315 320

Lys Gln Gly Leu Lys Ser Ala Met Gly Ile Ser Ser Asp Gly Asn Ala

325 330 335

Tyr Leu Gln Glu Ser Gln Phe Trp Lys Leu Tyr Lys Glu Asp Pro Ala

340 345 350

Ala Cys Ala Ile Val Met Lys Thr Ser Val Gly Leu Val Tyr Leu Leu

355 360 365

Ala Cys Leu Leu Glu Pro Phe Met Pro Ser Phe Ser Asp Glu Val Leu

370 375 380

Arg Gln Leu Asn Leu Ser Pro Glu Glu Asn Leu Ser Leu Ser Glu Glu

385 390 395 400

Lys Gly Glu Ile Ala Lys Ala Lys Thr Pro Trp Asn Phe Val Pro Ala

405 410 415

Gly His Arg Ile Gly Lys Pro Ala Pro Leu Phe Lys Glu Leu Lys Asp

420 425 430

Glu Asp Val Ala Leu His Arg Glu Lys Tyr Ala Gly Ser Gln Ala Glu

435 440 445

Arg Ser Ser Lys Ala Ala Ala Asp Ala Glu Ala Asn Lys Val Ala Asn

450 455 460

Gln Leu Lys Gly Thr Lys Leu Ser Asp Gly Gly Thr Lys Lys Glu Pro

465 470 475 480

Lys Lys Gln Ser Gly Gly Ser Lys Ser Lys Thr Ala Glu Ala Asp Ile

485 490 495

Thr Val Ala Lys Leu Asp Ile Arg Val Gly Leu Ile Arg Lys Ala Glu

500 505 510

Lys His Pro Asp Ala Asp Ser Leu Tyr Val Glu Glu Ile Asp Val Gly

515 520 525

Glu Asp Ala Pro Arg Thr Val Val Ser Gly Leu Val Lys Phe Ile Pro

530 535 540

Leu Glu Glu Met Gln Ser Arg Lys Val Cys Val Leu Cys Asn Leu Lys

545 550 555 560

Pro Val Ala Met Arg Gly Ile Lys Ser His Ala Met Val Leu Ala Ala

565 570 575

Ser Asn Glu Asp His Thr Lys Val Glu Leu Val Glu Pro Pro Glu Ser

580 585 590

Ala Ala Val Gly Glu Arg Val Thr Phe Ala Gly Tyr Ala Gly Glu Pro

595 600 605

Glu Ala Ser Leu Ser Gly Lys Ser Lys Thr Trp Glu Lys Leu Ala Ala

610 615 620

Glu Leu His Ser Asn Gly Glu Leu Val Ala Cys Tyr Arg Asp Val Pro

625 630 635 640

Phe Thr Thr Ser Ala Gly Val Cys Arg Val Lys Thr Ile Ala Asn Gly

645 650 655

Glu Ile Arg

11

gtttgatgct ttactaggct atatatcagc actatctgat gatcaggaac aacctgattt 60

acttaaaact gtttcttcag gttggccagc ttcactgcac ttgattggta aggatattct 120

acgcttccat gctgtttact ggccagctat gctaatgtct gctggactaa gccttcctaa 180

aatggtattt ggccatggat tcttgacaaa ggatggaatg aagatgggca agtcactagg 240

gaatatactt gaaccaaacg atttggttaa taaatttggg acagatgcag ttagatactt 300

cttcctcaga gaggtggaat ttggcaatga tggagactat tcagaggaac gcttcatcaa 360

tattgtgaat gcacatcttg ccaatacaat tggaaatctt cttaatcgga cactangact 420

tctgaaaaag aactgccaat caattctggt ggtggatcca ctacagcng 469

12

Phe Asp Ala Leu Leu Gly Tyr Ile Ser Ala Leu Ser Asp Asp Gln Glu

1 5 10 15

Gln Pro Asp Leu Leu Lys Thr Val Ser Ser Gly Trp Pro Ala Ser Leu

20 25 30

His Leu Ile Gly Lys Asp Ile Leu Arg Phe His Ala Val Tyr Trp Pro

35 40 45

Ala Met Leu Met Ser Ala Gly Leu Ser Leu Pro Lys Met Val Phe Gly

50 55 60

His Gly Phe Leu Thr Lys Asp Gly Met Lys Met Gly Lys Ser Leu Gly

65 70 75 80

Asn Ile Leu Glu Pro Asn Asp Leu Val Asn Lys Phe Gly Thr Asp Ala

85 90 95

Val Arg Tyr Phe Phe Leu Arg Glu Val Glu Phe Gly Asn Asp Gly Asp

100 105 110

Tyr Ser Glu Glu Arg Phe Ile Asn Ile Val Asn Ala His Leu Ala Asn

115 120 125

Thr Ile Gly Asn Leu Leu Asn Arg Thr Leu Xaa Leu Leu Lys Lys Asn

130 135 140

Cys Gln Ser Ile Leu Val Val Asp

145 150