US20030077623A1 - Polynucleotides and polypeptides involved in post-transcriptional gene silencing - Google Patents

Polynucleotides and polypeptides involved in post-transcriptional gene silencing Download PDF

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US20030077623A1
US20030077623A1 US10/174,363 US17436302A US2003077623A1 US 20030077623 A1 US20030077623 A1 US 20030077623A1 US 17436302 A US17436302 A US 17436302A US 2003077623 A1 US2003077623 A1 US 2003077623A1
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Karlene Butler
Rebecca Cahoon
Leslie Harvell
J. Rafalski
Hajime Sakai
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EIDP Inc
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Assigned to E. I. DU PONT DE NEMOURS AND COMPANY reassignment E. I. DU PONT DE NEMOURS AND COMPANY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SAKAI, HAJIME, RAFALSKI, J. ANTONI, BUTLER, KARLENE H., HARVELL, LESLIE T., CAHOON, REBECCA E.
Publication of US20030077623A1 publication Critical patent/US20030077623A1/en
Priority to US11/093,888 priority patent/US20050204427A1/en
Priority to US12/237,540 priority patent/US8022196B2/en
Priority to US13/208,396 priority patent/US8278431B2/en
Priority to US13/595,504 priority patent/US8748697B2/en
Priority to US14/284,623 priority patent/US9453236B2/en
Priority to US15/244,065 priority patent/US20160355834A1/en
Abandoned 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding polypeptides in plants and seeds involved in post-transcriptional gene silencing.
  • dsRNA double-stranded RNA
  • cRNA small complementary RNA
  • cRNA small complementary RNA
  • PTG-silenced plants Hamilton and Baulcombe (1999) Science 286:950-952
  • a protein similar to RNA-dependent RNA polymerase, the enzyme involved in cRNA synthesis is required for PTGS (Mourrain et al. (2000) Cell 101:533-542).
  • AGO1 protein Another protein identified to be required for PTGS is the ARGONAUTE (AGO1) protein (Bohmert et al. (1998) EMBO J 17:170-180; Fagard et al. (2000) Proc Natl Acad Sci USA 97:11650-11654).
  • AGO1 protein shares homology with the RDE1 and QDE-2 proteins which have been found to be required for RNAi in C. elegans and for quelling in Neurospora, respectively, thus reinforcing the notion that PTGS, RNAi, and quelling are similar processes at the mechanistic level.
  • AGO1/RDE1/QDE-2 proteins are similar to elF2C, a protein important for protein translation.
  • dsRNA mediates PTGS by disrupting proper positioning of elF2C in the translation machinery complex, thereby preventing translation of the target mRNA (Tabara et al. (1999) Cell 99:123-132; Fagard et al. (2000) Proc Natl Acad Sci USA 97:11650-11654).
  • PTGS is an important process, which if manipulated properly, may be used to control transgene expression.
  • AGO1 protein family includes the ZWILLE (ZLL) or PINHEAD (PNH) protein involved in plant development (Moussian et al. (1998) EMBO J 17:1799-1809; Lynn et al. (1999) Development 126:469-481), and the RDE-1 protein involved in transposon silencing (Tabara et al. (1999) Cell 99:123-132). These sequences may be used to manipulate PTGS.
  • the polynucleotides disclosed herein may also be used to manipulate transposon activity, meristem activity, plant architecture and development, and proliferation of undifferentiated plant cells in culture, which would be useful in callus propagation.
  • the present invention includes isolated polynucleotides comprising: (a) a first nucleotide sequence encoding a first polypeptide having post-transcriptional gene silencing activity wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80% sequence identity, or (b) a second nucleotide sequence encoding a second polypeptide having post-transcriptional gene silencing activity wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 85% sequence identity.
  • the identity be at least 85%, it is more preferred that the identity is at least 90%, and it is even more preferred that the identity be at least 95%.
  • the identity be at least 90%, and it is more preferred that the identity be at least 95%.
  • the present invention includes isolated polynucleotides encoding the amino acid sequence of SEQ ID NO:8, 12, 14, 22, 28, 38, 40, 42 or 54 or nucleotide sequences comprising the nucleotide sequence of SEQ ID NO:7, 11, 13, 21, 27, 37, 39, 41 or 53.
  • the present invention also includes isolated polynucleotides comprising the complement of nucleotide sequences of the present invention.
  • the present invention also includes:
  • an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80%, 85%, 90%, or 95% sequence identity, (b) a second nucleotide sequence encoding a second polypeptide, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID SEQ ID NO:8, 38 or 42 have at least 85%, 90%, or 95% sequence identity, or (c) the complement of the nucleotide sequence of (a) or (b); the first polypeptide preferably comprises the amino acid sequence of of SEQ ID NO:12, 14, 22, 28, 40 or 54; the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:8, 38 or 42; the first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:
  • a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence, and a cell, a plant, and a seed comprising the recombinant DNA construct;
  • a vector comprising any of the isolated polynucleotides of the present invention.
  • an isolated polynucleotide comprising a nucleotide sequence comprised by any of the polynucleotides of the first embodiment, wherein the nucleotide sequence contains at least 30, 40, or 60 nucleotides;
  • a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention, and the cell transformed by this method, advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium;
  • a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a plant from the transformed plant cell, a transgenic plant produced by this method, and seed obtained from this transgenic plant;
  • an isolated polypeptide comprising: (a) a first amino acid sequence, wherein the first amino acid sequence and and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80%, 85%, 90% or 95% sequence identity, or (b) a second amino acid sequence, wherein the second amino acid sequence and and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 85%, 90% or 95% sequence identity;
  • the first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54
  • the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:8, 38 or 42;
  • the polypeptide preferably has post-transcriptional gene silencing activity;
  • a method for isolating a polypeptide encoded by polynucleotides of the present invention comprising isolating the polypeptide from cultivated cells, from the culture medium, or from both the cultivated cells and the culture medium, wherein the cells contain a recombinant DNA construct comprising the polynucleotide operably linked to at least one regulatory sequence;
  • a virus preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the recombinant DNA constructs of the present invention;
  • a method of selecting an isolated polynucleotide that affects the level of expression in a host cell, preferably a plant cell, of a gene encoding a polypeptide having post-transcriptional gene silencing activity comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; (b) introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; (c) measuring the level of the polypeptide involved in post-transcriptional gene silencing or its activity in the host cell containing the isolated polynucleotide or the isolated recombinant DNA construct; and (d) comparing the level of the polypeptide involved in post-transcriptional gene silencing or its activity in the host cell containing the isolated polynucleotide or the isolated recombinant DNA construct with the level of the polypeptide involved in
  • a method of obtaining a nucleic acid fragment encoding a substantial portion of a polypeptide involved in post-transcriptional gene silencing comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, or 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 substantial portion of a polypeptide involved in post-transcriptional gene silencing;
  • a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a polypeptide involved in post-transcriptional gene silencing 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;
  • a method for positive selection of a transformed cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the polypeptide involved in post-transcriptional gene silencing polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means; and
  • a method of altering the level of expression of a polypeptide involved in post-transcriptional gene silencing in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the polypeptide involved in post-transcriptional gene silencing in the transformed host cell.
  • FIGS. 1A, 1B, 1 C and 1 D depict the amino acid sequence alignment of the polypeptides involved in post-transcriptional gene silencing encoded by the following: (a) nucleotide sequence derived from corn clone cle1f.pk002.k13 (SEQ ID NO:8), (b) nucleotide sequence derived from corn clone p0119.cmtmm21r (SEQ ID NO:22), (c) nucleotide sequence derived from soybean clone ssl1c.pk003.g3 (SEQ ID NO:40), (d) nucleotide sequence of a contig assembled from nucleotide sequences obtained from wheat clone wdk1c.pk012.i2 and PCR fragments (SEQ ID NO:42), and (e) nucleotide sequence from Oryza sativa (NCBI GenBank Identifier (GI) No. 6539559; SEQ ID NO:55
  • FIGS. 2A, 2B, 2 C, 2 D and 2 E depict the amino acid sequence alignment of the polypeptides involved in post-transcriptional gene silencing encoded by the following: (a) nucleotide sequence derived from corn clone csc1c.pk006.j19 (SEQ ID NO:12), (b) nucleotide sequence derived from corn clone ctn1c.pk003.i20 (SEQ ID NO:14), (c) nucleotide sequence of a contig assembled from nucleotide sequences obtained from rice clone rlm1n.pk001.m11 and PCR fragments (SEQ ID NO:28), (d) nucleotide sequence of a contig assembled from nucleotide sequences obtained from soybean clone sdc2c.pk001.p4 and PCR fragments (SEQ ID NO: 38), and (e) nucleotide sequence from Arabidopsis
  • 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 EST, FIS or PCR sequences (“Contig”), or sequences encoding the entire protein, or functionally active polypeptide, derived from an EST, an FIS, or a contig (“CGS”).
  • 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.
  • polynucleotides that encode polypeptides involved in post-transcriptional gene silencing may be used in plant cells to alter the post-transcriptional gene silencing pathway. More specifically, the polynucleotides of the instant invention may be used to create transgenic plants where the levels of polypeptides involved in post-transcriptional gene silencing are altered with respect to non-transgenic plants which would result in plants with an enhancement or a deficiency in post-transcriptional gene silencing.
  • the present invention has solved this problem by providing polynucleotide and deduced polypeptide sequences corresponding to novel polypeptides involved in post-transcriptional gene silencing from corn ( Zea mays ), rice ( Oryza sativa ), soybean ( Glycine max ) and wheat ( Triticum aestivum ).
  • polynucleotide polynucleotide sequence
  • nucleic acid sequence nucleic acid sequence
  • nucleic acid fragment nucleic acid fragment
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • An isolated polynucleotide of the present invention may include at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, or the complement of such sequences.
  • isolated refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment.
  • Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides.
  • the term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
  • nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.
  • a “recombinant DNA construct” comprises any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence.
  • 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 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.
  • the terms “substantially similar” and “corresponding substantially” are used interchangeably herein.
  • Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the 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 of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell.
  • a substantially similar nucleic acid fragment representing at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell.
  • the level of the 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 of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
  • antisense suppression and cosuppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using 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 of the 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 of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide.
  • an isolated polynucleotide comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, and the complement of such nucleotide sequences may be used to affect the expression and/or function of a polypeptide involved in post-transcriptional gene silencing in a host cell.
  • a method of using an isolated polynucleotide to affect the level of expression of a polypeptide in a host cell may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.
  • 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.
  • 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 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 identities 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.
  • 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 the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health).
  • BLAST Basic Local Alignment Search Tool
  • 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.
  • 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).
  • 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 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 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 a 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 the 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, recombinant DNA constructs, 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.
  • 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 may be composed of different elements derived from different promoters found in nature, or may 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 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.
  • 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).
  • 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.
  • the primary transcript 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 polypeptides by the cell.
  • cDNA refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I.
  • 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.
  • operably linked refers to the association of two or more nucleic acid fragments on a single polynucleotide 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 of the 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 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).
  • a “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.
  • altered levels or “altered expression” 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” or the term “mature” when used in describing a 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” or the term “precursor” when used in describing a 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).
  • a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
  • any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).
  • a “mitochondrial signal peptide” is an amino acid sequence which directs a precursor protein into the mitochondria (Zhang and Glaser (2002) Trends Plant Sci 7:14-21).
  • 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; Ishida Y. et al. (1996) Nature Biotech. 14:745-750) 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).
  • isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
  • a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990.
  • plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′regulatory sequences and a dominant selectable marker.
  • plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • “Stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance.
  • “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance.
  • Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms.
  • transformation refers to both stable transformation and transient transformation.
  • recombinant construct refers to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodology well known to one skilled in the art. Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used, the choice of vector is dependent upon the method that will be used to transform host plants as is well known to those skilled in the art.
  • “Motifs” or “subsequences” refer to short regions of conserved sequences of nucleic acids or amino acids that comprise part of a longer sequence. For example, it is expected that such conserved subsequences would be important for function, and could be used to identify new homologues in plants. It is expected that some or all of the elements may be found in a homologue. Also, it is expected that one or two of the conserved amino acids in any given motif may differ in a true homologue.
  • PCR or “polymerase chain reaction” is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).
  • the present invention includes an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 200 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:24 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (c) a third nucleotide sequence encoding a third polypeptide comprising
  • the first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52
  • the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2 4
  • the third polypeptide preferably comprises the amino acid sequence of SEQ ID NO:34
  • the fourth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:10
  • the fifth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:50
  • the sixth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 or SEQ ID NO:54
  • the seventh polypeptide preferably comprises the amino acid sequence of SEQ ID NO:26
  • the eighth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:14 or SEQ
  • the first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:47, or SEQ ID NO:51
  • the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:23
  • the third nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:33
  • the fourth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:9
  • the fifth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:21, or SEQ ID NO:49
  • the sixth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:27, SEQ ID NO:39, or SEQ ID NO:53
  • This invention also includes the isolated complement of such polynucleotides, wherein the complement and the polynucleotide preferably consist of the same number of nucleotides, and the nucleotide sequences of the complement and the polynucleotide preferably have 100% complementarity.
  • nucleic acid fragments encoding at least a portion of several polypeptides involved in post-transcriptional gene silencing 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.
  • 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 polypeptides involved in post-transcriptional gene silencing 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).
  • an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • 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.
  • 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.
  • 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.
  • a polynucleotide comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, 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.
  • 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).
  • this invention includes viruses and host cells comprising either the recombinant DNA constructs of the invention as described herein or isolated polynucleotides of the invention as described herein.
  • host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.
  • 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 PTGS in those plants. Since some of the AGO1 family members have also been shown to be involved in transposon silencing, meristem development, and differentiation of meristematic tissue, the polynucleotides disclosed herein may also be used to manipulate transposon activity, meristem activity, plant architecture and development, and proliferation of undifferentiated plant cells in culture, which would be useful in callus propagation.
  • Overexpression of the proteins of the instant invention may be accomplished by first constructing a recombinant DNA construct 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 recombinant DNA construct 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 recombinant DNA construct may also comprise one or more introns in order to facilitate gene expression.
  • Plasmid vectors comprising the instant isolated polynucleotide(s) (or recombinant DNA construct(s)) may 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 recombinant DNA construct or 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.
  • Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.
  • the recombinant DNA construct(s) described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), nuclear localization signals (Raikhel (1992) Plant Phys.
  • a recombinant DNA construct 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.
  • a recombinant DNA construct 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 recombinant DNA constructs could be introduced into plants via transformation wherein expression of the 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.
  • the present invention includes an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 100 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (b) a second amino acid sequence comprising at least 200 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:24 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (c) a third amino acid sequence comprising at least 100 amino acids, wherein the third amino acid sequence and the amino acid sequence of SEQ ID NO:34 have at least 80%, 85%, 90%, or 95% identity based on the C
  • the first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52
  • the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:24
  • the third amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:34
  • the fourth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:10
  • the fifth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:50
  • the sixth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 or SEQ ID NO:54
  • the seventh amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:26
  • the eighth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID
  • 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 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 recombinant DNA constructs for production of the instant polypeptides.
  • This recombinant DNA construct could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded polypeptides involved in post-transcriptional gene silencing.
  • An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 6).
  • All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used 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.
  • RFLP restriction fragment length polymorphism
  • Southern blots Mantonis
  • 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.
  • 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).
  • 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). Although current methods of FISH mapping favor use of large clones (several kb 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.
  • FISH fluorescence in situ hybridization
  • 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.
  • 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.
  • 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 one of 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.
  • cDNA libraries representing mRNAs from various corn ( Zea mays ), soybean ( Glycine max ), rice ( Oryza sativa ), and wheat ( Triticum aestivum ) tissues were prepared. The characteristics of the libraries are described below.
  • 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, Calif.). 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 DH10B 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.
  • FIS data is generated utilizing a modified transposition protocol.
  • Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed M13 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.
  • Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, Calif.) which is based upon the Saccharomyces cerevisiae Ty1 transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772).
  • the in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules.
  • the transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.) via electroporation.
  • the transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res.
  • Phred/Phrap is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files.
  • the Phrap sequence assembly program uses these quality values to increase the accuracy of the assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle).
  • the cDNA fragment corresponds to a portion of the 3′-terminus of the gene and does not cover the entire open reading frame.
  • the first of these methods results in the production of a fragment of DNA containing a portion of the desired gene sequence while the second method results in the production of a fragment containing the entire open reading frame.
  • Both of these methods use two rounds of PCR amplification to obtain fragments from one or more libraries. The libraries some times are chosen based on previous knowledge that the specific gene should be found in a certain tissue and some times are randomly-chosen. Reactions to obtain the same gene may be performed on several libraries in parallel or on a pool of libraries.
  • Library pools are normally prepared using from 3 to 5 different libraries and normalized to a uniform dilution.
  • both methods use a vector-specific (forward) primer corresponding to a portion of the vector located at the 5′-terminus of the clone coupled with a gene-specific (reverse) primer.
  • the first method uses a sequence that is complementary to a portion of the already known gene sequence while the second method uses a gene-specific primer complementary to a portion of the 3′-untranslated region (also referred to as UTR).
  • UTR 3′-untranslated region
  • a nested set of primers is used for both methods.
  • the resulting DNA fragment is ligated into a pBluescript vector using a commercial kit and following the manufacturer's protocol.
  • This kit is selected from many available from several vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.).
  • the plasmid DNA is isolated by alkaline lysis method and submitted for sequencing and assembly using Phred/Phrap, as above.
  • cDNA clones encoding polypeptides involved in post-transcriptional gene silencing were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health) 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 of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases).
  • BLAST Basic Local Alignment Search Tool
  • Altschul et al. (1993) J. Mol. Biol. 215:403-410 see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes
  • 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 NCBI.
  • BLASTX National Center for Biotechnology Information
  • the P-value (probability) of observing a match of a cDNA 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 of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.
  • ESTs submitted for analysis are compared to the genbank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402.) against the Du Pont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction. Once the most 5-prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1.
  • Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm.
  • the tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.
  • the following three Arabidopsis thaliana sequences each represent the same 1048 amino acid sequence: GI No.11386626; GI No. 2149640; and GI No.15221177.
  • the following two Arabidopsis thaliana sequences each represent the same 988 amino acid sequence: GI No. 5107374 and GI No.12643935.
  • FIGS. 1 A- 1 D present an alignment of the amino acid sequences set forth in SEQ ID NOs:8, 22, 40, and 42, and the Oryza sativa sequence (NCBI GI No. 6539559; SEQ ID NO:55).
  • FIGS. 2 A- 2 E present an alignment of the amino acid sequences set forth in SEQ ID NOs:12, 14, 28, and 38, and the Arabidopsis thaliana sequence (NCBI GI No. 2149640; SEQ ID NO:56).
  • the data in Table 5 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:8, 12, 14, 22, 28, 38, 40, and 42, the Oryza sativa sequence (NCBI GI No.
  • Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a polypeptide involved in post-transcriptional gene silencing. These sequences represent the first corn and wheat sequences indicated to encode polypeptides involved in post-transcriptional gene silencing (AGO1 protein family) known to Applicants.
  • a recombinant DNA construct comprising a cDNA encoding the instant polypeptide 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) of the cDNA clone, plant cDNA or plant cDNA libraries using appropriate oligonucleotide primers.
  • Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the 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 NcoI and SmaI and fractionated on an agarose gel.
  • the appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103.
  • Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Boulevard., Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
  • the DNA segment from pML103 contains a 1.05 kb SaII-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SaII fragment from the 3′ end of the 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 XL1-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 recombinant DNA construct encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, and the 10 kD zein 3′ region.
  • the recombinant DNA construct 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 of the 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.
  • the pat gene in p35S/Ac is under the control of the 35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the 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 rpm) 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) of the 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 Calif.), 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 bialaphos (5 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 bialaphos. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the bialaphos-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 8:833-839).
  • Plants in which PTGS has been elevated or diminished can be assayed by making the following two sexual crosses: (1) a first transgenic plant, transformed with a gene encoding a polypeptide involved in PTGS, is crossed with a second transgenic plant that contains an active reporter transgene, such as the GUS gene, and (2), the first transgenic plant is crossed with a third transgenic plant that contains a post-transcriptionally silenced reporter gene. If PTGS has been elevated, reporter gene expression in the progeny plants from the first cross should be reduced. If PTGS has been diminished, reporter gene expression in progeny plants from the second cross should be increased.
  • a seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the ⁇ subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the 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 Ncol (which includes the ATG translation initiation codon), SmaI, KpnI and XbaI. The entire cassette is flanked by HindIII sites.
  • the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone, plant cDNA or plant cDNA libraries, using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the 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, 3-5 mm in length dissected from surface sterilized, immature seeds of the 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 be 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) Nature (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 of the 35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli ; Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens .
  • the seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptide and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
  • Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60 ⁇ 15 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.
  • 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 cDNA fragment of the gene may be generated by polymerase chain reaction (PCR) of the cDNA clone, plant cDNA or plant cDNA libraries, using appropriate oligonucleotide primers.
  • PCR polymerase chain reaction
  • 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 EcoRI and HindIII sites in pET-3a at their original positions.
  • oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamHI site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the NdeI site at the position of translation initiation was converted to an NcoI 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% low melting agarose gel. Buffer and agarose contain 10 ⁇ g/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies, Madison, Wis.) 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 (NEB), Beverly, Mass.).
  • 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 polypeptide 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. 189:113-130). 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 0.2 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 of the supernatant determined.
  • One ⁇ g of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.
  • the polypeptides encoded by the polynucleotides of the instant invention may be expressed in a yeast ( Saccharomyces cerevisiae ) strain YPH. Plasmid DNA, plant cDNA or plant cDNA libraries, may be used as template to amplify the portion encoding the polypeptide involved in post-transcriptional gene silencing. Amplification may be performed using the GC melt kit (Clontech) with a 1 M final concentration of GC melt reagent and using a Perkin Elmer 9700 thermocycler. The amplified insert may then be incubated with a modified pRS315 plasmid (NCBI General Identifier No. 984798; Sikorski, R. S.
  • Plasmid pRS315 has been previously modified by the insertion of a bidirectional gal1/10 promoter between the Xho I and Hind III sites.
  • the plasmid may then be transformed into the YPH yeast strain using standard procedures where the insert recombines through gap repair to form the desired transformed yeast strain (Hua, S. B. et al. (1997) Plasmid 38:91-96).
  • Yeast cells may be prepared according to a modification of the methods of Pompon et al. (Pompon, D. et al. (1996) Meth. Enz. 272:51-64). Briefly, a yeast colony will be grown overnight (to saturation) in SG (-Leucine) medium at 30° C. with good aeration. A 1:50 dilution of this culture will be made into 500 mL of YPGE medium with adenine supplementation and allowed to grow at 30° C. with good aeration to an OD 600 of 1.6 (24-30 h). Fifty mL of 20% galactose will be added, and the culture allowed to grow overnight at 30° C.
  • the cells will be recovered by centrifugation at 5,500 rpm for five minutes in a Sorvall GS-3 rotor.
  • the cell pellet resuspended in 500 mL of 0.1 M potassium phosphate buffer (pH 7.0) and then allowed to grow at 30° C. for another 24 hours.
  • the cells may be recovered by centrifugation as described above and the presence of the polypeptide of the instant invention determined by HPLC/mass spectrometry or any other suitable method.
  • the cDNA fragment of the gene may be generated by polymerase chain reaction (PCR) of the CDNA clone, plant cDNA or plant cDNA libraries, using appropriate oligonucleotide primers.
  • the cDNAs encoding the instant polypeptides may be introduced into the baculovirus genome itself.
  • the cDNAs may be placed under the control of the polyhedron promoter, the IE1 promoter, or any other one of the baculovirus promoters.
  • the cDNA, together with appropriate leader sequences is then inserted into a baculovirus transfer vector using standard molecular cloning techniques. Following transformation of E. coli DH5 ⁇ , isolated colonies are chosen and plasmid DNA is prepared and is analyzed by restriction enzyme analysis. Colonies containing the appropriate fragment are isolated, propagated, and plasmid DNA is prepared for cotransfection.
  • Spodoptera frugiperda cells are propagated in ExCell® 401 media (JRH Biosciences, Lenexa, Kan.) supplemented with 3.0% fetal bovine serum.
  • Lipofectin® 50 ⁇ L at 0.1 mg/mL, Gibco/BRL
  • Sf-9 cells are co-transfected with the viral DNA/transfer vector solution.
  • the supernatant fluid from the co-transfection experiment is collected at 5 days post-transfection and recombinant viruses are isolated employing standard plaque purification protocols, wherein only polyhedrin-positive plaques are selected (O'Reilly et al. (1992), Baculovirus Expression Vectors: A Laboratory Manual , W. H. Freeman and Company, New York.).
  • Sf-9 cells in 35 mM petri dishes (50% monolayer) are inoculated with 100 ⁇ L of a serial dilution of the viral suspension, and supernatant fluids are collected at 5 days post infection. In order to prepare larger quantities of virus for characterization, these supernatant fluids are used to inoculate larger tissue cultures for large-scale propagation of recombinant viruses.
  • DNA Zea mays 1 ccacgcgtcc gatgaaatct gtcgtggagt acttcaagga aatgtatggt ttcaccattc 60 agcatcctca tcttccttgc cttcaggttg gaaaccaaaa gaaggcgaac tatttaccaa 120 tggaggcctg caagatcgtt gaaggccaga gatacacgaa gaggttgaat gaaaaacaga 180 tcacatcgtt gctaaaggtt acatgccaaa ggcctcgaga acaagagatg crizttttac 240 agacagttca tcaaaatgga tatgagcaag atccatatgc gaaggaattt gggatcaaca 300 ttagtgagaa

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Abstract

This invention relates to isolated nucleic acid fragments encoding polypeptides involved in post-transcriptional gene silencing. The invention also relates to construction of a recombinant DNA construct encoding all or a portion of the polypeptide involved in post-transcriptional gene silencing, in sense or antisense orientation, wherein expression of the recombinant DNA construct results in production of altered levels in a transformed host cell of the the polypeptide involved in post-transcriptional gene silencing.

Description

  • This application claims the benefit of U.S. Provisional Application No. 60/298,973, filed Jun. 18, 2001, the entire content of which is herein incorporated by reference.[0001]
    • FIELD OF THE INVENTION
  • This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding polypeptides in plants and seeds involved in post-transcriptional gene silencing. [0002]
    • BACKGROUND OF THE INVENTION
  • Post-transcriptional gene silencing (PTGS), which operates at the level of sequence-specific RNA degradation, has emerged as a major phenomenon through which transgene expression in plants is down-regulated. It was first recognized in plants, and similar mechanisms since then have been observed in non-plant systems, where it is known by different names, to wit, quelling in the fungus [0003] Neurospora crassa (Romano and Macino (1992) Mol Microbiol 6:3343-3353), and RNA interference (RNAi) in worms, flies, and mammals (Bosher and Labouesse (2000) Nat Cell Biol 2:E31-36).
  • Although the mechanism remains to be fully elucidated, it appears that double-stranded RNA (dsRNA) serve as key intermediates in PTGS (Bass (2000) [0004] Cell 101:235-238). The involvement of dsRNA is supported by identification of small complementary RNA (cRNA), 21-25 nucleotides long, which can bind the target RNA to form dsRNA, in PTG-silenced plants (Hamilton and Baulcombe (1999) Science 286:950-952), and the finding that a protein similar to RNA-dependent RNA polymerase, the enzyme involved in cRNA synthesis, is required for PTGS (Mourrain et al. (2000) Cell 101:533-542).
  • Another protein identified to be required for PTGS is the ARGONAUTE (AGO1) protein (Bohmert et al. (1998) [0005] EMBO J 17:170-180; Fagard et al. (2000) Proc Natl Acad Sci USA 97:11650-11654). AGO1 protein shares homology with the RDE1 and QDE-2 proteins which have been found to be required for RNAi in C. elegans and for quelling in Neurospora, respectively, thus reinforcing the notion that PTGS, RNAi, and quelling are similar processes at the mechanistic level. AGO1/RDE1/QDE-2 proteins are similar to elF2C, a protein important for protein translation. It is therefore hypothesized that dsRNA mediates PTGS by disrupting proper positioning of elF2C in the translation machinery complex, thereby preventing translation of the target mRNA (Tabara et al. (1999) Cell 99:123-132; Fagard et al. (2000) Proc Natl Acad Sci USA 97:11650-11654).
  • It is apparent that PTGS is an important process, which if manipulated properly, may be used to control transgene expression. Disclosed herein are sequences very homologous to the AGO1 protein family, which includes the ZWILLE (ZLL) or PINHEAD (PNH) protein involved in plant development (Moussian et al. (1998) [0006] EMBO J 17:1799-1809; Lynn et al. (1999) Development 126:469-481), and the RDE-1 protein involved in transposon silencing (Tabara et al. (1999) Cell 99:123-132). These sequences may be used to manipulate PTGS. Since some of the AGO1 family members have also been shown to be involved in transposon silencing, meristem development, and differentiation of meristematic tissue, the polynucleotides disclosed herein may also be used to manipulate transposon activity, meristem activity, plant architecture and development, and proliferation of undifferentiated plant cells in culture, which would be useful in callus propagation.
    • SUMMARY OF INVENTION
  • The present invention includes isolated polynucleotides comprising: (a) a first nucleotide sequence encoding a first polypeptide having post-transcriptional gene silencing activity wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80% sequence identity, or (b) a second nucleotide sequence encoding a second polypeptide having post-transcriptional gene silencing activity wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 85% sequence identity. For the first polypeptide, it is preferred that the identity be at least 85%, it is more preferred that the identity is at least 90%, and it is even more preferred that the identity be at least 95%. For the second polypeptide, it is preferred that the identity be at least 90%, and it is more preferred that the identity be at least 95%. More preferably, the present invention includes isolated polynucleotides encoding the amino acid sequence of SEQ ID NO:8, 12, 14, 22, 28, 38, 40, 42 or 54 or nucleotide sequences comprising the nucleotide sequence of SEQ ID NO:7, 11, 13, 21, 27, 37, 39, 41 or 53. The present invention also includes isolated polynucleotides comprising the complement of nucleotide sequences of the present invention. [0007]
  • The present invention also includes: [0008]
  • in a preferred first embodiment, an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80%, 85%, 90%, or 95% sequence identity, (b) a second nucleotide sequence encoding a second polypeptide, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID SEQ ID NO:8, 38 or 42 have at least 85%, 90%, or 95% sequence identity, or (c) the complement of the nucleotide sequence of (a) or (b); the first polypeptide preferably comprises the amino acid sequence of of SEQ ID NO:12, 14, 22, 28, 40 or 54; the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:8, 38 or 42; the first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:11, 13, 21, 27, 39 or 53; the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:7, 37 or 41; the first and second polypeptides preferably have post-transcriptional gene silencing activity; [0009]
  • in a preferred second embodiment, a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence, and a cell, a plant, and a seed comprising the recombinant DNA construct; [0010]
  • in a preferred third embodiment, a vector comprising any of the isolated polynucleotides of the present invention; [0011]
  • in a preferred fourth embodiment, an isolated polynucleotide comprising a nucleotide sequence comprised by any of the polynucleotides of the first embodiment, wherein the nucleotide sequence contains at least 30, 40, or 60 nucleotides; [0012]
  • in a preferred fifth embodiment, a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention, and the cell transformed by this method, advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium; [0013]
  • in a preferred sixth embodiment, a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a plant from the transformed plant cell, a transgenic plant produced by this method, and seed obtained from this transgenic plant; [0014]
  • in a preferred seventh embodiment, an isolated polypeptide comprising: (a) a first amino acid sequence, wherein the first amino acid sequence and and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80%, 85%, 90% or 95% sequence identity, or (b) a second amino acid sequence, wherein the second amino acid sequence and and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 85%, 90% or 95% sequence identity; the first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54, and the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:8, 38 or 42; the polypeptide preferably has post-transcriptional gene silencing activity; [0015]
  • in a preferred eight embodiment, a method for isolating a polypeptide encoded by polynucleotides of the present invention comprising isolating the polypeptide from cultivated cells, from the culture medium, or from both the cultivated cells and the culture medium, wherein the cells contain a recombinant DNA construct comprising the polynucleotide operably linked to at least one regulatory sequence; [0016]
  • in a preferred ninth embodiment, a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the recombinant DNA constructs of the present invention; [0017]
  • in a preferred tenth embodiment, a method of selecting an isolated polynucleotide that affects the level of expression in a host cell, preferably a plant cell, of a gene encoding a polypeptide having post-transcriptional gene silencing activity, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; (b) introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; (c) measuring the level of the polypeptide involved in post-transcriptional gene silencing or its activity in the host cell containing the isolated polynucleotide or the isolated recombinant DNA construct; and (d) comparing the level of the polypeptide involved in post-transcriptional gene silencing or its activity in the host cell containing the isolated polynucleotide or the isolated recombinant DNA construct with the level of the polypeptide involved in post-transcriptional gene silencing or its activity in the host cell that does not contain the isolated polynucleotide or the isolated recombinant DNA construct; [0018]
  • in a preferred eleventh embodiment, a method of obtaining a nucleic acid fragment encoding a substantial portion of a polypeptide involved in post-transcriptional gene silencing comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, or 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 substantial portion of a polypeptide involved in post-transcriptional gene silencing; [0019]
  • in a preferred twelfth embodiment, a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a polypeptide involved in post-transcriptional gene silencing 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; [0020]
  • in a preferred thirteenth embodiment, a method for positive selection of a transformed cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the polypeptide involved in post-transcriptional gene silencing polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means; and [0021]
  • in a preferred fourteenth embodiment, a method of altering the level of expression of a polypeptide involved in post-transcriptional gene silencing in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the polypeptide involved in post-transcriptional gene silencing in the transformed host cell.[0022]
    • BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING
  • The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application. [0023]
  • FIGS. 1A, 1B, [0024] 1C and 1D depict the amino acid sequence alignment of the polypeptides involved in post-transcriptional gene silencing encoded by the following: (a) nucleotide sequence derived from corn clone cle1f.pk002.k13 (SEQ ID NO:8), (b) nucleotide sequence derived from corn clone p0119.cmtmm21r (SEQ ID NO:22), (c) nucleotide sequence derived from soybean clone ssl1c.pk003.g3 (SEQ ID NO:40), (d) nucleotide sequence of a contig assembled from nucleotide sequences obtained from wheat clone wdk1c.pk012.i2 and PCR fragments (SEQ ID NO:42), and (e) nucleotide sequence from Oryza sativa (NCBI GenBank Identifier (GI) No. 6539559; SEQ ID NO:55). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences.
  • FIGS. 2A, 2B, [0025] 2C, 2D and 2E depict the amino acid sequence alignment of the polypeptides involved in post-transcriptional gene silencing encoded by the following: (a) nucleotide sequence derived from corn clone csc1c.pk006.j19 (SEQ ID NO:12), (b) nucleotide sequence derived from corn clone ctn1c.pk003.i20 (SEQ ID NO:14), (c) nucleotide sequence of a contig assembled from nucleotide sequences obtained from rice clone rlm1n.pk001.m11 and PCR fragments (SEQ ID NO:28), (d) nucleotide sequence of a contig assembled from nucleotide sequences obtained from soybean clone sdc2c.pk001.p4 and PCR fragments (SEQ ID NO: 38), and (e) nucleotide sequence from Arabidopsis thaliana (NCBI GenBank Identifier (GI) No. 2149640; SEQ ID NO:56). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences.
  • 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 EST, FIS or PCR sequences (“Contig”), or sequences encoding the entire protein, or functionally active polypeptide, derived from an EST, an FIS, or a contig (“CGS”). 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. [0026]
    TABLE 1
    Polypeptides Involved in Post-Transcriptional Gene Silencing
    SEQ ID NO:
    Polypeptide (Nucleo- (Amino
    (Plant Source) Clone Designation Status tide) Acid)
    Zwille Homolog p0102.cerba57r FIS 1 2
    (Corn)
    Zwille Homolog ses2w.pk0009.g6 FIS 3 4
    (Soybean)
    Zwille Homolog ssm.pk0063.a4 FIS 5 6
    (Soybean)
    Argonaute Homolog cle1f.pk002.k13 CGS 7 8
    (Corn) (FIS)
    Argonaute Homolog cpf1c.pk008.j24 FIS 9 10
    (Corn)
    Argonaute Homolog csc1c.pk006.j19 CGS 11 12
    (Corn) (FIS)
    Argonaute Homolog ctn1c.pk003.i20 CGS 13 14
    (Corn) (FIS)
    Argonaute Homolog Contig of contig 15 16
    (Corn) p0002.cgevj06r
    p0125.czaab55r
    (FIS)
    p0125.czaat57r
    Argonaute Homolog p0102.cerae32ra EST 17 18
    (Corn)
    Argonaute Homolog p0107.cbcbd69r EST 19 20
    (Corn)
    Argonaute Homolog p0119.cmtmm21r CGS 21 22
    (Corn) (FIS)
    Argonaute Homolog rca1n.pk018.b3 FIS 23 24
    (Rice)
    Argonaute Homolog rl0n.pk124.g8 FIS 25 26
    (Rice)
    Argonaute Homolog Contig of CGS 27 28
    (Rice) rlm1n.pk004.m11
    (FIS)
    PCR fragment
    sequence
    Argonaute Homolog rls6.pk0082.d4 FIS 29 30
    (Rice)
    Argonaute Homolog rsl1n.pk004.d12 FIS 31 32
    (Rice)
    Argonaute Homolog rtc1c.pk008.k19.f EST 33 34
    (Rice)
    Argonaute Homolog sdc1c.pk0004.d11 FIS 35 36
    (Soybean)
    Argonaute Homolog Contig of CGS 37 38
    (Soybean) sdc2c.pk001.p4
    (FIS)
    PCR fragment
    sequence
    Argonaute Homolog ssl1c.pk003.g3 CGS 39 40
    (Soybean) (FIS)
    Argonaute Homolog Contig of CGS 41 42
    (Wheat) wdk1c.pk012.i2
    (FIS)
    PCR fragment
    sequence
    Argonaute Homolog wlm96.pk029.c23 FIS 43 44
    (Wheat)
    Argonaute Homolog wne1g.pk003.f8 EST 45 46
    (Wheat)
    Argonaute Homolog wr1.pk0073.c7 EST 47 48
    (Wheat)
    Argonaute Homolog wre1n.pk0001.h6 FIS 49 50
    (Wheat)
    Argonaute Homolog wre1n.pk162.h10 EST 51 52
    (Wheat)
    Argonaute Homolog rdi2c.pk002.d14:fis CGS 53 54
    (Rice)
  • 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 [0027] 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.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The problem to be solved, therefore, was to identify polynucleotides that encode polypeptides involved in post-transcriptional gene silencing. These polynucleotides may be used in plant cells to alter the post-transcriptional gene silencing pathway. More specifically, the polynucleotides of the instant invention may be used to create transgenic plants where the levels of polypeptides involved in post-transcriptional gene silencing are altered with respect to non-transgenic plants which would result in plants with an enhancement or a deficiency in post-transcriptional gene silencing. The present invention has solved this problem by providing polynucleotide and deduced polypeptide sequences corresponding to novel polypeptides involved in post-transcriptional gene silencing from corn ([0028] Zea mays), rice (Oryza sativa), soybean (Glycine max) and wheat (Triticum aestivum).
  • In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. 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, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, or the complement of such sequences. [0029]
  • The term “isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides. [0030]
  • The term “recombinant” means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques. A “recombinant DNA construct” comprises any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence. [0031]
  • 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. [0032]
  • 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. The terms “substantially similar” and “corresponding substantially” are used interchangeably herein. [0033]
  • Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the 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 of the 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 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the 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 of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment. [0034]
  • For example, it is well known in the art that antisense suppression and cosuppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using 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, an isolated polynucleotide comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, and the complement of such nucleotide sequences may be used to affect the expression and/or function of a polypeptide involved in post-transcriptional gene silencing in a host cell. A method of using an isolated polynucleotide to affect the level of expression of a polypeptide in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide. [0035]
  • 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. [0036]
  • 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 (isolated polynucleotides of the 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 identities 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. [0037]
  • It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying related polypeptide sequences. Useful examples of percent identities are 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%. 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 ClustalV method of alignment (Higgins and Sharp (1989) [0038] CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the ClustalV 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) [0039] J. Mol. Biol. 215:403-410; see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health). 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 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 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. [0040]
  • “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 a 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 the 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. [0041]
  • “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, recombinant DNA constructs, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. [0042]
  • “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. [0043]
  • “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 may be composed of different elements derived from different promoters found in nature, or may 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) [0044] 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.
  • “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) [0045] Mol. Biotechnol. 3:225-236).
  • “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) [0046] 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 polypeptides by the cell. “cDNA” refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I. “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. [0047]
  • The term “operably linked” refers to the association of two or more nucleic acid fragments on a single polynucleotide 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. [0048]
  • 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). [0049]
  • A “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function. [0050]
  • “Altered levels” or “altered expression” 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. [0051]
  • “Mature protein” or the term “mature” when used in describing a 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” or the term “precursor” when used in describing a 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. [0052]
  • 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) [0053] 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). A “mitochondrial signal peptide” is an amino acid sequence which directs a precursor protein into the mitochondria (Zhang and Glaser (2002) Trends Plant Sci 7:14-21).
  • “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) [0054] Meth. Enzymol. 143:277; Ishida Y. et al. (1996) Nature Biotech. 14:745-750) 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). Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • “Stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. The term “transformation” as used herein refers to both stable transformation and transient transformation. [0055]
  • The terms “recombinant construct”, “expression construct” and “recombinant expression construct” are used interchangeably herein. These terms refer to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodology well known to one skilled in the art. Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used, the choice of vector is dependent upon the method that will be used to transform host plants as is well known to those skilled in the art. [0056]
  • Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. [0057] Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).
  • “Motifs” or “subsequences” refer to short regions of conserved sequences of nucleic acids or amino acids that comprise part of a longer sequence. For example, it is expected that such conserved subsequences would be important for function, and could be used to identify new homologues in plants. It is expected that some or all of the elements may be found in a homologue. Also, it is expected that one or two of the conserved amino acids in any given motif may differ in a true homologue. [0058]
  • “PCR” or “polymerase chain reaction” is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159). [0059]
  • The present invention includes an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 200 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:24 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (c) a third nucleotide sequence encoding a third polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the third polypeptide and the amino acid sequence of SEQ ID NO:34 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (d) a fourth nucleotide sequence encoding a fourth polypeptide comprising at least 150 amino acids, wherein the amino acid sequence of the fourth polypeptide and the amino acid sequence of SEQ ID NO:10 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (e) a fifth nucleotide sequence encoding a fifth polypeptide comprising at least 200 amino acids, wherein the amino acid sequence of the fifth polypeptide and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:50 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (f) a sixth nucleotide sequence encoding a sixth polypeptide comprising at least 300 amino acids, wherein the amino acid sequence of the sixth polypeptide and the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 or SEQ ID NO:54 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (g) a seventh nucleotide sequence encoding a seventh polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the seventh polypeptide and the amino acid sequence of SEQ ID NO:26 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (h) an eighth nucleotide sequence encoding an eighth polypeptide comprising at least 200 amino acids, wherein the amino acid sequence of the eighth polypeptide and the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:32 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (i) a ninth nucleotide sequence encoding a ninth polypeptide comprising at least 250 amino acids, wherein the amino acid sequence of the ninth polypeptide and the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:12 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (j) a tenth nucleotide sequence encoding a tenth polypeptide comprising at least 300 amino acids, wherein the amino acid sequence of the tenth polypeptide and the amino acid sequence of SEQ ID NO:42 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (k) an eleventh nucleotide sequence encoding an eleventh polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the eleventh polypeptide and the amino acid sequence of SEQ ID NO:46 have at least 90% or 95% identity based on the ClustalV alignment method, (l) a twelfth nucleotide sequence encoding a twelfth polypeptide comprising at least 150 amino acids, wherein the amino acid sequence of the twelfth polypeptide and the amino acid sequence of SEQ ID NO:4 have at least 90% or 95% identity based on the ClustalV alignment method, (m) a thirteenth nucleotide sequence encoding a thirteenth polypeptide comprising at least 250 amino acids, wherein the amino acid sequence of the thirteenth polypeptide and the amino acid sequence of SEQ ID NO:38 have at least 90% or 95% identity based on the ClustalV alignment method, or (n) the complement of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth nucleotide sequence, wherein the complement and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth nucleotide sequence contain the same number of nucleotides and are 100% complementary. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52, the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2 4, the third polypeptide preferably comprises the amino acid sequence of SEQ ID NO:34, the fourth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:10, the fifth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:50, the sixth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 or SEQ ID NO:54, the seventh polypeptide preferably comprises the amino acid sequence of SEQ ID NO:26, the eighth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:32, the ninth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:12, the tenth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:42, the eleventh polypeptide preferably comprises the amino acid sequence of SEQ ID NO:46, the twelfth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:4, and the thirteenth polypeptide preferably comprises the amino acid sequence of SEQ ID NO:38. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:47, or SEQ ID NO:51, the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:23, the third nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:33, the fourth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:9, the fifth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:21, or SEQ ID NO:49, the sixth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:27, SEQ ID NO:39, or SEQ ID NO:53, the seventh nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:25, the eighth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:13 or SEQ ID NO:31, the ninth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:11, the tenth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:41, the eleventh nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:45, the twelfth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:3, and the thirteenth nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:37. The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth polypeptides preferably are polypeptides involved in post-transcriptional gene silencing. [0060]
  • This invention also includes the isolated complement of such polynucleotides, wherein the complement and the polynucleotide preferably consist of the same number of nucleotides, and the nucleotide sequences of the complement and the polynucleotide preferably have 100% complementarity. [0061]
  • Nucleic acid fragments encoding at least a portion of several polypeptides involved in post-transcriptional gene silencing 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). [0062]
  • For example, genes encoding other polypeptides involved in post-transcriptional gene silencing, 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, an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, 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. [0063]
  • 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) [0064] 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 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, 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.
  • 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) [0065] Adv. Immunol. 36:1-34; Maniatis).
  • In another preferred embodiment, this invention includes viruses and host cells comprising either the recombinant DNA constructs of the invention as described herein or isolated polynucleotides of the invention as described herein. Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants. [0066]
  • As was noted above, 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 PTGS in those plants. Since some of the AGO1 family members have also been shown to be involved in transposon silencing, meristem development, and differentiation of meristematic tissue, the polynucleotides disclosed herein may also be used to manipulate transposon activity, meristem activity, plant architecture and development, and proliferation of undifferentiated plant cells in culture, which would be useful in callus propagation. [0067]
  • Overexpression of the proteins of the instant invention may be accomplished by first constructing a recombinant DNA construct 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 recombinant DNA construct 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 recombinant DNA construct may also comprise one or more introns in order to facilitate gene expression. [0068]
  • Plasmid vectors comprising the instant isolated polynucleotide(s) (or recombinant DNA construct(s)) may 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 recombinant DNA construct or 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) [0069] 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.
  • 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 recombinant DNA construct(s) described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) [0070] Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), nuclear localization signals (Raikhel (1992) Plant Phys. 100:1627-1632) or mitochondrial signal sequences (Zhang and Glaser (2002) Trends Plant Sci 7:14-21) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use 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 recombinant DNA construct 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 recombinant DNA construct 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 recombinant DNA constructs could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated. [0071]
  • 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 a 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. [0072]
  • 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 recombinant DNA constructs 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. 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. [0073]
  • In another preferred embodiment, the present invention includes an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 100 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (b) a second amino acid sequence comprising at least 200 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:24 have at least 70%, 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (c) a third amino acid sequence comprising at least 100 amino acids, wherein the third amino acid sequence and the amino acid sequence of SEQ ID NO:34 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (d) a fourth amino acid sequence comprising at least 150 amino acids, wherein the fourth amino acid sequence and the amino acid sequence of SEQ ID NO:10 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (e) a fifth amino acid sequence comprising at least 200 amino acids, wherein the fifth amino acid sequence and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:50 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (f) a sixth amino acid sequence comprising at least 300 amino acids, wherein the sixth amino acid sequence and the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 or SEQ ID NO:54 have at least 80%, 85%, 90%, or 95% identity based on the ClustalV alignment method, (g) a seventh amino acid sequence comprising at least 100 amino acids, wherein the seventh amino acid sequence and the amino acid sequence of SEQ ID NO:26 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (h) an eighth amino acid sequence comprising at least 200 amino acids, wherein the eighth amino acid sequence and the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:32 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (i) a ninth amino acid sequence comprising at least 250 amino acids, wherein the ninth amino acid sequence and the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:12 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (j) a tenth amino acid sequence comprising at least 300 amino acids, wherein the tenth amino acid sequence and the amino acid sequence of SEQ ID NO:42 have at least 85%, 90%, or 95% identity based on the ClustalV alignment method, (k) an eleventh amino acid sequence comprising at least 100 amino acids, wherein the eleventh amino acid sequence and the amino acid sequence of SEQ ID NO:46 have at least 90% or 95% identity based on the ClustalV alignment method, (l) a twelfth amino acid sequence comprising at least 150 amino acids, wherein the twelfth amino acid sequence and the amino acid sequence of SEQ ID NO:4 have at least 90% or 95% identity based on the ClustalV alignment method, or (m) a thirteenth amino acid sequence comprising at least 250 amino acids, wherein the thirteenth amino acid sequence and the amino acid sequence of SEQ ID NO:38 have at least 90% or 95% identity based on the ClustalV alignment method. The first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52, the second amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:24, the third amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:34, the fourth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:10, the fifth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:50, the sixth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 or SEQ ID NO:54, the seventh amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:26, the eighth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:32, the ninth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:12, the tenth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:42, the eleventh amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:46, the twelfth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:4, and the thirteenth amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:38. The polypeptide preferably is a polypeptide involved in post-transcriptional gene silencing. [0074]
  • 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 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 recombinant DNA constructs for production of the instant polypeptides. This recombinant DNA construct could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded polypeptides involved in post-transcriptional gene silencing. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 6). [0075]
  • All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used 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) [0076] 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) [0077] 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: [0078] 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) [0079] Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several kb 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) [0080] 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) [0081] 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 one of 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 one of 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.
    • EXAMPLES
  • The present invention is further defined in the following Examples, in which 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. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. [0082]
  • The disclosure of each reference set forth herein is incorporated herein by reference in its entirety. [0083]
    • Examples 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones
  • cDNA libraries representing mRNAs from various corn ([0084] Zea mays), soybean (Glycine max), rice (Oryza sativa), and wheat (Triticum aestivum) tissues were prepared. The characteristics of the libraries are described below.
    TABLE 2
    cDNA Libraries from Corn, Rice, Soybean, and Wheat
    Library Tissue Clone
    cle1f Corn Leaf at VE-V5 Stage** cle1f.pk002.k13
    cpf1c Corn Treated with Chemicals Related to cpf1c.pk008.j24
    Protein Synthesis***
    csc1c Corn 20 Day Seedling (Germination Cold csc1c.pk006.j19
    Stress)
    ctn1c Corn Tassel, Night Harvested ctn1c.pk003.i20
    p0002 Corn Tassel, Premeiotic Cells to Early p0002.cgevj06r
    Uninucleate Stage
    p0102 Corn Early Meiosis Tassels* p0102.cerae32ra
    p0102.cerba57r
    p0107 Corn Whole Kernels 7 Days After p0107.cbcbd69r
    Pollination*
    p0119 Corn V12 Stage** Ear Shoot With Husk, p0119.cmtmm21r
    Night Harvested*
    p0125 Corn Anther Prophase I* p0125.czaab55r
    p0125.czaat57r
    rca1n Rice Callus* rca1n.pk018.b3
    rdi2c Rice (Oryza sativa, Nipponbare) developing rdi2c.pk002.d14
    inflorescence at rachis branch-floral organ
    primordia formation
    rl0n Rice 15 Day Old Leaf* rl0n.pk124.g8
    rlm1n Rice Leaf 15 Days After Germination, rlm1n.pk001.m11
    Harvested 2-72 Hours Following Infection
    With Magnaporta grisea (4360-R-62 and
    4360-R-67)*
    rls6 Susceptible Rice Leaf 15 Days After rls6.pk0082.d4
    Germination, 6 Hours After Infection of
    Strain Magnaporthe grisea 4360-R-67
    (AVR2-YAMO)
    rsl1n Rice 15-Day-Old Seedling* rsl1n.pk004.d12
    rtc1c Rice Leaf Inoculated with Magnaporthe rtc1c.pk008.k19.f
    grisea Strain 0184 at 4, 8, and 24 Hours
    sdc1c Soybean Developing Cotyledon (3-5 mm) sdc1c.pk0004.d11
    sdc2c Soybean Developing Cotyledon (6-7 mm) sdc2c.pk001.p4
    ses2w Soybean Embryogenic Suspension 2 Weeks ses2w.pk0009.g6
    After Subculture
    ssl1c Soybean Seed 25 Days After Fertilization ssl1c.pk003.g3
    ssm Soybean Shoot Meristem ssm.pk0063.a4
    wdk1c Wheat Developing Kernel, 3 Days After wdk1c.pk012.i2
    Anthesis
    wlm96 Wheat Seedlings 96 Hours After Inoculation wlm96.pk029.c23
    With Erysiphe graminis f. sp tritici
    wne1g Wheat Nebulized Genomic Library wne1g.pk003.f8
    wr1 Wheat Root From 7 Day Old Light Grown wr1.pk0073.c7
    Seedling
    wre1n Wheat Root From 7 Day Old Etiolated wre1n.pk0001.h6
    Seedling* wre1n.pk162.h10
  • 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, Calif.). 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 DH10B 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) [0085] Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
  • Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol. Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed M13 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made. [0086]
  • Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, Calif.) which is based upon the [0087] Saccharomyces cerevisiae Ty1 transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.) via electroporation. The transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agar plates of only those subclones containing the integrated transposon. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared via alkaline lysis, and templates are sequenced (ABI Prism dye-terminator ReadyReaction mix) outward from the transposition event site, utilizing unique primers specific to the binding sites within the transposon.
  • Sequence data is collected (ABI Prism Collections) and assembled using Phred/Phrap (P. Green, University of Washington, Seattle). Phred/Phrap is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files. The Phrap sequence assembly program uses these quality values to increase the accuracy of the assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle). [0088]
  • In some of the clones the cDNA fragment corresponds to a portion of the 3′-terminus of the gene and does not cover the entire open reading frame. In order to obtain the upstream information one of two different protocols are used. The first of these methods results in the production of a fragment of DNA containing a portion of the desired gene sequence while the second method results in the production of a fragment containing the entire open reading frame. Both of these methods use two rounds of PCR amplification to obtain fragments from one or more libraries. The libraries some times are chosen based on previous knowledge that the specific gene should be found in a certain tissue and some times are randomly-chosen. Reactions to obtain the same gene may be performed on several libraries in parallel or on a pool of libraries. Library pools are normally prepared using from 3 to 5 different libraries and normalized to a uniform dilution. In the first round of amplification both methods use a vector-specific (forward) primer corresponding to a portion of the vector located at the 5′-terminus of the clone coupled with a gene-specific (reverse) primer. The first method uses a sequence that is complementary to a portion of the already known gene sequence while the second method uses a gene-specific primer complementary to a portion of the 3′-untranslated region (also referred to as UTR). In the second round of amplification a nested set of primers is used for both methods. The resulting DNA fragment is ligated into a pBluescript vector using a commercial kit and following the manufacturer's protocol. This kit is selected from many available from several vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA is isolated by alkaline lysis method and submitted for sequencing and assembly using Phred/Phrap, as above. [0089]
    • Example 2 Identification of cDNA Clones
  • cDNA clones encoding polypeptides involved in post-transcriptional gene silencing were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) [0090] J. Mol. Biol. 215:403-410; see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health) 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 of the 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 NCBI. For convenience, the P-value (probability) of observing a match of a cDNA 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 of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.
  • ESTs submitted for analysis are compared to the genbank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) [0091] Nucleic Acids Res. 25:3389-3402.) against the Du Pont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction. Once the most 5-prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.
    • Example 3 Characterization of cDNA Clones Encoding Polypeptides Involved in Post-Transcriptional Gene Silencing
  • The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity of the polypeptides encoded by the cDNAs to polypeptides involved in post-transcriptional gene silencing and AGO1 family members from [0092] Neurospora crassa (NCBI GenBank Identifier (GI) No. 7248733), Arabidopsis thaliana (NCBI GI Nos. 3885334, 6692120, 11386626, 2149640, 5107374, 12643935 and 15221177), and Oryza sativa (NCBI GI No. 6539559). The following three Arabidopsis thaliana sequences each represent the same 1048 amino acid sequence: GI No.11386626; GI No. 2149640; and GI No.15221177. The following two Arabidopsis thaliana sequences each represent the same 988 amino acid sequence: GI No. 5107374 and GI No.12643935. Shown in Table 3 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), the sequences of contigs assembled from two or more EST, FIS or PCR sequencess (“Contig”), or sequences encoding an entire protein, or functionally active polypeptide, derived from an FIS or a contig (“CGS”):
    TABLE 3
    BLAST Results for Sequences Encoding Polypeptides Homologous
    to Polypeptides Involved in Post-Transcriptional Gene Silencing
    (AGO1 Protein Family)
    BLAST Results
    Clone Status NCBI GI No. BLAST pLog Score
    p0102.cerba57r FIS 12643935  >180.00
    ses2w.pk0009.g6 FIS 5107374 >180.00
    ssm.pk0063.a4 FIS 5107374 >180.00
    cle1f.pk002.k13 (FIS) CGS 6539559 >180.00
    cpf1c.pk008.j24 FIS 2149640 >180.00
    csc1c.pk006.j19 (FIS) CGS 2149640 >180.00
    ctn1c.pk003.i20 (FIS) CGS 2149640 >180.00
    Contig of Contig 11386626  >180.00
    p0002.cgevj06r
    p0125.czaab55r (FIS)
    p0125.czaat57r
    p0102.cerae32ra EST 5107374 31.10
    p0107.cbcbd69r EST 2149640 57.15
    p0119.cmtmm21r (FIS) CGS 6539559 >180.00
    rca1n.pk018.b3 FIS 2149640 >180.00
    rl0n.pk124.g8 FIS 2149640 131.00
    Contig of CGS 11386626  >180.00
    rlm1n.pk001.m11 (FIS)
    PCR fragment sequence
    rls6.pk0082.d4 FIS 6539559 31.70
    rsl1n.pk004.d12 FIS 11386626  171.00
    rtc1c.pk008.k19.f EST 2149640 64.22
    sdc1c.pk0004.d11 FIS 6692120 76.05
    Contig of CGS 2149640 >180.00
    sdc2c.pk001.p4 (FIS)
    PCR fragment sequence
    ssl1c.pk003.g3 (FIS) CGS 3885334 >180.00
    Contig of CGS 6539559 >180.00
    wdk1c.pk012.i2 (FIS)
    PCR fragment sequence
    wlm96.pk029.c23 FIS 7248733 45.30
    wne1g.pk003.f8 EST 2149640 47.10
    wr1.pk0073.c7 EST 2149640 27.70
    wre1n.pk0001.h6 FIS 6539559 >180.00
    wre1n.pk162.h10 EST 2149640 30.70
    rdi2c.pk002.d14 (FIS) CGS 15221177  >180.00
  • FIGS. [0093] 1A-1D present an alignment of the amino acid sequences set forth in SEQ ID NOs:8, 22, 40, and 42, and the Oryza sativa sequence (NCBI GI No. 6539559; SEQ ID NO:55). FIGS. 2A-2E present an alignment of the amino acid sequences set forth in SEQ ID NOs:12, 14, 28, and 38, and the Arabidopsis thaliana sequence (NCBI GI No. 2149640; SEQ ID NO:56). The data in Table 5 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:8, 12, 14, 22, 28, 38, 40, and 42, the Oryza sativa sequence (NCBI GI No. 6539559; SEQ ID NO: 55), and the Arabidopsis thaliana sequence (NCBI GI No.2149640; SEQ ID NO: 56).
    TABLE 5
    Percent Identity of Amino Acid Sequences Deduced From the
    Nucleotide Sequences Encoding Polypeptides Homologous to
    Polypeptides Involved in Post-Transcriptional Gene Silencing
    (AGO1 Protein Family)
    SEQ ID NO. NCBI GI No. Percent Identity
    8 6539559 82.2
    12 2149640 72.1
    14 2149640 72.6
    22 6539559 73.2
    28 2149640 72.2
    38 2149640 78.2
    40 6539559 68.8
    42 6539559 83.7
    54 2149640 73.3
  • 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 ClustalV method of alignment (Higgins and Sharp (1989) [0094] CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the ClustalV 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 polypeptide involved in post-transcriptional gene silencing. These sequences represent the first corn and wheat sequences indicated to encode polypeptides involved in post-transcriptional gene silencing (AGO1 protein family) known to Applicants.
    • Example 4 Expression of Recombinant DNA Constructs in Monocot Cells
  • A recombinant DNA construct comprising a cDNA encoding the instant polypeptide 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) of the cDNA clone, plant cDNA or plant cDNA libraries using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the 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 NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SaII-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SaII fragment from the 3′ end of the 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 [0095] E. coli XL1-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 recombinant DNA construct encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, and the 10 kD zein 3′ region.
  • The recombinant DNA construct 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 of the 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) [0096] 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. The pat gene in p35S/Ac is under the control of the 35S promoter from cauliflower mosaic virus (Odell et al. (1985) [0097] Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
  • The particle bombardment method (Klein et al. (1987) [0098] 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 rpm) 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) of the 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 Calif.), 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. [0099]
  • Seven days after bombardment the tissue can be transferred to N6 medium that contains bialaphos (5 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 bialaphos. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the bialaphos-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium. [0100]
  • 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) [0101] Bio/Technology 8:833-839).
  • Plants in which PTGS has been elevated or diminished can be assayed by making the following two sexual crosses: (1) a first transgenic plant, transformed with a gene encoding a polypeptide involved in PTGS, is crossed with a second transgenic plant that contains an active reporter transgene, such as the GUS gene, and (2), the first transgenic plant is crossed with a third transgenic plant that contains a post-transcriptionally silenced reporter gene. If PTGS has been elevated, reporter gene expression in the progeny plants from the first cross should be reduced. If PTGS has been diminished, reporter gene expression in progeny plants from the second cross should be increased. Also, if PTGS has been diminished, a correlated decrease in the methylation state of the reporter transgene in the progeny of the second cross would be expected (Fagard et al. (2000) [0102] Proc Natl Acad Sci USA 97:11650-11654).
    • Example 5 Expression of Recombinant DNA Constructs in Dicot Cells
  • A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean [0103] Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the 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 Ncol (which includes the ATG translation initiation codon), SmaI, KpnI and XbaI. The entire cassette is flanked by HindIII sites.
  • The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone, plant cDNA or plant cDNA libraries, using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the 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. [0104]
  • 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 of the 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. [0105]
  • Soybean embryogenic suspension cultures can be 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. [0106]
  • Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) [0107] Nature (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 of the 35S promoter from cauliflower mosaic virus (Odell et al. (1985) [0108] Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptide and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
  • To 50 μL of a 60 mg/[0109] 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 of the 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 60×15 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. [0110]
  • 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. [0111]
    • Example 6 Expression of Recombinant DNA Constructs in Microbial Cells
  • The cDNA fragment of the gene may be generated by polymerase chain reaction (PCR) of the cDNA clone, plant cDNA or plant cDNA libraries, using appropriate oligonucleotide primers. The cDNAs encoding the instant polypeptides can be inserted into the T7 [0112] 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 EcoRI and HindIII sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamHI site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the NdeI site at the position of translation initiation was converted to an NcoI 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% low melting agarose gel. Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies, Madison, Wis.) 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 (NEB), Beverly, Mass.). 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 polypeptide are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis. [0113]
  • For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into [0114] E. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). 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 0.2 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 of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.
    • Example 7 Expression of Recombinant DNA Constructs in Yeast Cells
  • The polypeptides encoded by the polynucleotides of the instant invention may be expressed in a yeast ([0115] Saccharomyces cerevisiae) strain YPH. Plasmid DNA, plant cDNA or plant cDNA libraries, may be used as template to amplify the portion encoding the polypeptide involved in post-transcriptional gene silencing. Amplification may be performed using the GC melt kit (Clontech) with a 1 M final concentration of GC melt reagent and using a Perkin Elmer 9700 thermocycler. The amplified insert may then be incubated with a modified pRS315 plasmid (NCBI General Identifier No. 984798; Sikorski, R. S. and Hieter, P. (1989) Genetics 122:19-27) that has been digested with Not I and Spe I. Plasmid pRS315 has been previously modified by the insertion of a bidirectional gal1/10 promoter between the Xho I and Hind III sites. The plasmid may then be transformed into the YPH yeast strain using standard procedures where the insert recombines through gap repair to form the desired transformed yeast strain (Hua, S. B. et al. (1997) Plasmid 38:91-96).
  • Yeast cells may be prepared according to a modification of the methods of Pompon et al. (Pompon, D. et al. (1996) [0116] Meth. Enz. 272:51-64). Briefly, a yeast colony will be grown overnight (to saturation) in SG (-Leucine) medium at 30° C. with good aeration. A 1:50 dilution of this culture will be made into 500 mL of YPGE medium with adenine supplementation and allowed to grow at 30° C. with good aeration to an OD600 of 1.6 (24-30 h). Fifty mL of 20% galactose will be added, and the culture allowed to grow overnight at 30° C. The cells will be recovered by centrifugation at 5,500 rpm for five minutes in a Sorvall GS-3 rotor. The cell pellet resuspended in 500 mL of 0.1 M potassium phosphate buffer (pH 7.0) and then allowed to grow at 30° C. for another 24 hours.
  • The cells may be recovered by centrifugation as described above and the presence of the polypeptide of the instant invention determined by HPLC/mass spectrometry or any other suitable method. [0117]
    • Example 8 Expression of Recombinant DNA Constructs in Insect Cells
  • The cDNA fragment of the gene may be generated by polymerase chain reaction (PCR) of the CDNA clone, plant cDNA or plant cDNA libraries, using appropriate oligonucleotide primers. The cDNAs encoding the instant polypeptides may be introduced into the baculovirus genome itself. For this purpose the cDNAs may be placed under the control of the polyhedron promoter, the IE1 promoter, or any other one of the baculovirus promoters. The cDNA, together with appropriate leader sequences is then inserted into a baculovirus transfer vector using standard molecular cloning techniques. Following transformation of [0118] E. coli DH5α, isolated colonies are chosen and plasmid DNA is prepared and is analyzed by restriction enzyme analysis. Colonies containing the appropriate fragment are isolated, propagated, and plasmid DNA is prepared for cotransfection.
  • [0119] Spodoptera frugiperda cells (Sf-9) are propagated in ExCell® 401 media (JRH Biosciences, Lenexa, Kan.) supplemented with 3.0% fetal bovine serum. Lipofectin® (50 μL at 0.1 mg/mL, Gibco/BRL) is added to a 50 μL aliquot of the transfer vector containing the toxin gene (500 ng) and linearized polyhedrin-negative AcNPV (2.5 μg, Baculogold® viral DNA, Pharmigen, San Diego, Calif.). Sf-9 cells (approximate 50% monolayer) are co-transfected with the viral DNA/transfer vector solution. The supernatant fluid from the co-transfection experiment is collected at 5 days post-transfection and recombinant viruses are isolated employing standard plaque purification protocols, wherein only polyhedrin-positive plaques are selected (O'Reilly et al. (1992), Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman and Company, New York.). Sf-9 cells in 35 mM petri dishes (50% monolayer) are inoculated with 100 μL of a serial dilution of the viral suspension, and supernatant fluids are collected at 5 days post infection. In order to prepare larger quantities of virus for characterization, these supernatant fluids are used to inoculate larger tissue cultures for large-scale propagation of recombinant viruses.
  • 1 56 1 2042 DNA Zea mays 1 ccacgcgtcc gatgaaatct gtcgtggagt acttcaagga aatgtatggt ttcaccattc 60 agcatcctca tcttccttgc cttcaggttg gaaaccaaaa gaaggcgaac tatttaccaa 120 tggaggcctg caagatcgtt gaaggccaga gatacacgaa gaggttgaat gaaaaacaga 180 tcacatcgtt gctaaaggtt acatgccaaa ggcctcgaga acaagagatg gatattttac 240 agacagttca tcaaaatgga tatgagcaag atccatatgc gaaggaattt gggatcaaca 300 ttagtgagaa gctaacctat gttgaagccc gagtccttcc tgcaccttgg ctgaagtatc 360 atgacactgg aaaagagaaa gagtgcttac cacaggttgg tcagtggaac atggtaaaca 420 agaaagtgat aaacggatgc aaggtgagcc actgggcatg tataaacttc tcaaggagtg 480 ttccagaagc cacagctcgg ggattttgcc aggaattggc acaaatgtgt caaatttcgg 540 gcatggaatt taacagtgag cccgtgatgc caatatattc agctagacca gatcaagtag 600 tgaaggcact taaaagtgtg tataatattg cactgaacaa actcaagggt aaagaacttg 660 aacttcttct ggctatactc cccgacaaca atggtccgtt atatggtgac atcaaacgta 720 tttgtgaaac tgatttggga ttgatatcac aatgttgctt aaccaagcat gtttttaaga 780 tcagcaaaca gtacttggca aatgtctcac tgaaaattaa tgttaagatg ggaggaagaa 840 acactgtgct cctggacgca ataagttgga gcattccttt ggtcagtgac atcccaacta 900 ttatatttgg tgcagatgta acacaccctg aaaccgggga ggactcaagt ccatcaatcg 960 ctgccgttgt tgcttctcaa gattggccag aagttacaaa gtatgctgga ttggtttgtg 1020 ctcaggcaca ccggcaagag ctcattcagg acctttacaa aacatggcac gatcctcaga 1080 gaggcactgt aacaggcggc atgatcaggg agctgttaat atccttcagg aaggccactg 1140 ggcagaagcc attgagaata atattctaca gggacggtgt tagtgaaggc cagttctatc 1200 aagttctcct ttacgagtta gatgccatcc gtaaggcatg cgcatcccta gaaccaaatt 1260 accagcctcc tgtaacattt gtggtggttc aaaaacgtca tcatacgaga ctatttacaa 1320 acaatcacaa agacagaagt agcatggaca agagtggaaa tattttgcca ggaactgttg 1380 ttgattctaa gatatgccac ccaacagagt ttgatttcta cctctgtagt catgctggaa 1440 tccagggaac aagtaggccc gctcactacc atgtcctctg ggatgagaac aatttcacag 1500 cagacgaaat gcaaacactg acaaacaacc tttgctacac ttatgcccgg tgcacacgct 1560 cggtttctgt tgtccctcct gcatactacg cacacctggc agcattccgg gcgcggttct 1620 acatggaacc agagatgtcg gagaaccaga cgtcgaagag ctccaatggc acgaacggag 1680 gcttggtgaa gcccctgcct gctgtgaagg agaaggtgaa aagggtgatg ttctactgct 1740 gacgaggtga ccgctttaac aaccattcac atgctgtagc taacttggta gggttcagta 1800 ggggattaga ttagctttct ccaggaacga agaggaaacg ggatgcgtat ttggatcatg 1860 aacaatcaat ctgttagcga tcgctgtaaa atactcggaa atgcctgtat aatagttctt 1920 gttggttcag atgcatgcat ccaatgttcc agtgtactat gaaaaggggg tgtagaagaa 1980 accttctggt gttttctagg ttgaaaaaaa aaaaaaaaaa aaaaaacaaa aaaaaaaaaa 2040 aa 2042 2 577 PRT Zea mays 2 Pro Met Lys Ser Val Val Glu Tyr Phe Lys Glu Met Tyr Gly Phe Thr 1 5 10 15 Ile Gln His Pro His Leu Pro Cys Leu Gln Val Gly Asn Gln Lys Lys 20 25 30 Ala Asn Tyr Leu Pro Met Glu Ala Cys Lys Ile Val Glu Gly Gln Arg 35 40 45 Tyr Thr Lys Arg Leu Asn Glu Lys Gln Ile Thr Ser Leu Leu Lys Val 50 55 60 Thr Cys Gln Arg Pro Arg Glu Gln Glu Met Asp Ile Leu Gln Thr Val 65 70 75 80 His Gln Asn Gly Tyr Glu Gln Asp Pro Tyr Ala Lys Glu Phe Gly Ile 85 90 95 Asn Ile Ser Glu Lys Leu Thr Tyr Val Glu Ala Arg Val Leu Pro Ala 100 105 110 Pro Trp Leu Lys Tyr His Asp Thr Gly Lys Glu Lys Glu Cys Leu Pro 115 120 125 Gln Val Gly Gln Trp Asn Met Val Asn Lys Lys Val Ile Asn Gly Cys 130 135 140 Lys Val Ser His Trp Ala Cys Ile Asn Phe Ser Arg Ser Val Pro Glu 145 150 155 160 Ala Thr Ala Arg Gly Phe Cys Gln Glu Leu Ala Gln Met Cys Gln Ile 165 170 175 Ser Gly Met Glu Phe Asn Ser Glu Pro Val Met Pro Ile Tyr Ser Ala 180 185 190 Arg Pro Asp Gln Val Val Lys Ala Leu Lys Ser Val Tyr Asn Ile Ala 195 200 205 Leu Asn Lys Leu Lys Gly Lys Glu Leu Glu Leu Leu Leu Ala Ile Leu 210 215 220 Pro Asp Asn Asn Gly Pro Leu Tyr Gly Asp Ile Lys Arg Ile Cys Glu 225 230 235 240 Thr Asp Leu Gly Leu Ile Ser Gln Cys Cys Leu Thr Lys His Val Phe 245 250 255 Lys Ile Ser Lys Gln Tyr Leu Ala Asn Val Ser Leu Lys Ile Asn Val 260 265 270 Lys Met Gly Gly Arg Asn Thr Val Leu Leu Asp Ala Ile Ser Trp Ser 275 280 285 Ile Pro Leu Val Ser Asp Ile Pro Thr Ile Ile Phe Gly Ala Asp Val 290 295 300 Thr His Pro Glu Thr Gly Glu Asp Ser Ser Pro Ser Ile Ala Ala Val 305 310 315 320 Val Ala Ser Gln Asp Trp Pro Glu Val Thr Lys Tyr Ala Gly Leu Val 325 330 335 Cys Ala Gln Ala His Arg Gln Glu Leu Ile Gln Asp Leu Tyr Lys Thr 340 345 350 Trp His Asp Pro Gln Arg Gly Thr Val Thr Gly Gly Met Ile Arg Glu 355 360 365 Leu Leu Ile Ser Phe Arg Lys Ala Thr Gly Gln Lys Pro Leu Arg Ile 370 375 380 Ile Phe Tyr Arg Asp Gly Val Ser Glu Gly Gln Phe Tyr Gln Val Leu 385 390 395 400 Leu Tyr Glu Leu Asp Ala Ile Arg Lys Ala Cys Ala Ser Leu Glu Pro 405 410 415 Asn Tyr Gln Pro Pro Val Thr Phe Val Val Val Gln Lys Arg His His 420 425 430 Thr Arg Leu Phe Thr Asn Asn His Lys Asp Arg Ser Ser Met Asp Lys 435 440 445 Ser Gly Asn Ile Leu Pro Gly Thr Val Val Asp Ser Lys Ile Cys His 450 455 460 Pro Thr Glu Phe Asp Phe Tyr Leu Cys Ser His Ala Gly Ile Gln Gly 465 470 475 480 Thr Ser Arg Pro Ala His Tyr His Val Leu Trp Asp Glu Asn Asn Phe 485 490 495 Thr Ala Asp Glu Met Gln Thr Leu Thr Asn Asn Leu Cys Tyr Thr Tyr 500 505 510 Ala Arg Cys Thr Arg Ser Val Ser Val Val Pro Pro Ala Tyr Tyr Ala 515 520 525 His Leu Ala Ala Phe Arg Ala Arg Phe Tyr Met Glu Pro Glu Met Ser 530 535 540 Glu Asn Gln Thr Ser Lys Ser Ser Asn Gly Thr Asn Gly Gly Leu Val 545 550 555 560 Lys Pro Leu Pro Ala Val Lys Glu Lys Val Lys Arg Val Met Phe Tyr 565 570 575 Cys 3 2827 DNA Glycine max 3 ttctagaaca gtaaacaggt ctatcatagc agaactagtg aggctgtata aagagtctga 60 cttggggatg agacttccag catatgatgg cagaaaaagt ttgtacactg cagggcagct 120 tccctttgct tggagagagt ttaagattaa gcttatagat gaagaggatg gagttaatgg 180 ccctaaaagg gaaagagagt acagggtggt gatcaagttc gttgctcggg ctaacttgta 240 tcacttggga cagtttctag ctggtaggcg tgctgatgca ccgcaagagg cacttcaaat 300 tcttgacatt gtattaagag agctgtcaac taagaggtat tgccctattg ggaggtcctt 360 cttttcacct gatattagaa caccgcaacg gcttggagag ggattagaat catggtgtgg 420 attttaccag agtataaggc ctacacaaat gggcctttcc cttaatattg atatggcgtc 480 tgctgcgttt attgagcctc ttccagtagt ggaatttgtt ggccagctat tagcaaaaga 540 tgtgctgtca aggccattgt cagatgctga tcgcattaag attaagaaag cccttagagg 600 agttaaagtt gaagtaacac acagaggaag tgtgagaaga aaatatcgtg tttctggatt 660 gacttctcaa ccaaccagag aacttgtgtt tcctgttgat gagaactcaa ctatgaaatc 720 agtagttgaa tacttccaag agatgtatgg tttcactatt caatatactc accttccttg 780 ccttcaagta ggaaaccaaa agaaggctaa ctatttacct atggaggcct gcaaaattgt 840 tgaggggcaa cgttatacaa aaagattgaa tgagaagcaa attacagctc tgttgaaagt 900 tacttgccag agacctcgcg atcgggaaaa tgacatttta cggaccgttc aacataatgc 960 ttatgatcaa gatccttatg caaaggaatt tggaattaaa atcagtgaaa agctagcttc 1020 tgttgaagca cgaattcttc cggccccttg gcttaaatat cacgaaagtg ggaaagagaa 1080 gaactgttta ccccaagttg gtcagtggaa tatgatgaac aagaaaatga ttaatggaat 1140 gactgttagc cggtgggcat gcataaattt ttcaaggagc gtgcaagata gtgttgctcg 1200 cactttttgt aatgaacttg ctcaaatgtg tcaagtatct ggcatggaat ttaatccaga 1260 gtctgttatt cccatctaca atgccaaacc tgaacaggtg gaaaaagctt tgaaacatgt 1320 ttaccatgtg tcagggagca aaattaaagg aaaggaattg gagcttttgt tagcaatatt 1380 gccagacaat aacgggtctc tctatggtga tctcaagcga atttgtgaaa ctgaccttgg 1440 tttaatttca caatgctgtc tgacaaagca tgtcttcaaa atcactaaac agtacttggc 1500 taatgtgtct ctgaagatca atgtgaagat gggaggtaga aacactgtac ttcttgatgc 1560 tgtaagcagc agaataccat tggttagtga catgccaacc ataattttcg gagcagatgt 1620 aacccaccct gaaaatggag aagaattgag cccttcaata gcagctgtag tcgcatccca 1680 ggactggccc gaagtgacaa aatatgccgg tttagtatgt gctcaagctc ataggcagga 1740 acttatacaa gatttgtaca aaacttggca agaccctgtt cgtggcacag ttagtggtgg 1800 catgatccga gatttactgg tttccttcag aaaggcaaca ggacaaaagc cactacgaat 1860 tatattttac agggatggtg taagtgaagg acaattttac caagttttac tttatgagtt 1920 agatgcaatt cggaaggcat gtgcttcctt agaaccaaac taccagcctc cagtaacttt 1980 catagttgtg caaaaaagac atcatacccg gttatttgca aacaactaca gggacagaag 2040 cagtacagat cggagtggga atatattgcc tgggactgtt gttgatacca aaatctgcca 2100 tccaacagaa tttgattttt atctctgcag ccatgctggc atccagggta ctagtcggcc 2160 agctcattat catgtcctgt gggatgaaaa caacttcaca cctgatggaa ttcagtctct 2220 gacaaacaac ctttgttata catatgccag gtgtacacgc tcagtatcag ttgttcctcc 2280 agcatattat gcacatttag cagcgtttcg agcacgtttc tatatggaac cagatatgca 2340 agacaatggc tctgcaggtg acggtaatgg tcatggtgcc aaagcaacac gagcagctgg 2400 tgattatagt gtcaagccat tgccagactt gaaagaaaat gtgaagagag tcatgtttta 2460 ctgttagact gcttagtggc ttggccttgg tagaatgata gatatatggg gcaagcatca 2520 acatgataag caagttttca aatcatggag tgcaatgttc acctcacatt actttgtaca 2580 ttagtcgtgt aggttttgct gtggtagatc catgattaca gttcttgagc catagtttag 2640 aatgaatttc tacaagcatt attaggtttt atatagatgc caaatttagc attgtaaaaa 2700 atattctctg tcaatctttg tagaaaattt tgccataagg cctttacaga tgctggagta 2760 gaaatttcct tcatctttgc aaggagggga agttttttcc tagtaaaaaa aaaaaaaaaa 2820 aaaaaaa 2827 4 821 PRT Glycine max 4 Ser Arg Thr Val Asn Arg Ser Ile Ile Ala Glu Leu Val Arg Leu Tyr 1 5 10 15 Lys Glu Ser Asp Leu Gly Met Arg Leu Pro Ala Tyr Asp Gly Arg Lys 20 25 30 Ser Leu Tyr Thr Ala Gly Gln Leu Pro Phe Ala Trp Arg Glu Phe Lys 35 40 45 Ile Lys Leu Ile Asp Glu Glu Asp Gly Val Asn Gly Pro Lys Arg Glu 50 55 60 Arg Glu Tyr Arg Val Val Ile Lys Phe Val Ala Arg Ala Asn Leu Tyr 65 70 75 80 His Leu Gly Gln Phe Leu Ala Gly Arg Arg Ala Asp Ala Pro Gln Glu 85 90 95 Ala Leu Gln Ile Leu Asp Ile Val Leu Arg Glu Leu Ser Thr Lys Arg 100 105 110 Tyr Cys Pro Ile Gly Arg Ser Phe Phe Ser Pro Asp Ile Arg Thr Pro 115 120 125 Gln Arg Leu Gly Glu Gly Leu Glu Ser Trp Cys Gly Phe Tyr Gln Ser 130 135 140 Ile Arg Pro Thr Gln Met Gly Leu Ser Leu Asn Ile Asp Met Ala Ser 145 150 155 160 Ala Ala Phe Ile Glu Pro Leu Pro Val Val Glu Phe Val Gly Gln Leu 165 170 175 Leu Ala Lys Asp Val Leu Ser Arg Pro Leu Ser Asp Ala Asp Arg Ile 180 185 190 Lys Ile Lys Lys Ala Leu Arg Gly Val Lys Val Glu Val Thr His Arg 195 200 205 Gly Ser Val Arg Arg Lys Tyr Arg Val Ser Gly Leu Thr Ser Gln Pro 210 215 220 Thr Arg Glu Leu Val Phe Pro Val Asp Glu Asn Ser Thr Met Lys Ser 225 230 235 240 Val Val Glu Tyr Phe Gln Glu Met Tyr Gly Phe Thr Ile Gln Tyr Thr 245 250 255 His Leu Pro Cys Leu Gln Val Gly Asn Gln Lys Lys Ala Asn Tyr Leu 260 265 270 Pro Met Glu Ala Cys Lys Ile Val Glu Gly Gln Arg Tyr Thr Lys Arg 275 280 285 Leu Asn Glu Lys Gln Ile Thr Ala Leu Leu Lys Val Thr Cys Gln Arg 290 295 300 Pro Arg Asp Arg Glu Asn Asp Ile Leu Arg Thr Val Gln His Asn Ala 305 310 315 320 Tyr Asp Gln Asp Pro Tyr Ala Lys Glu Phe Gly Ile Lys Ile Ser Glu 325 330 335 Lys Leu Ala Ser Val Glu Ala Arg Ile Leu Pro Ala Pro Trp Leu Lys 340 345 350 Tyr His Glu Ser Gly Lys Glu Lys Asn Cys Leu Pro Gln Val Gly Gln 355 360 365 Trp Asn Met Met Asn Lys Lys Met Ile Asn Gly Met Thr Val Ser Arg 370 375 380 Trp Ala Cys Ile Asn Phe Ser Arg Ser Val Gln Asp Ser Val Ala Arg 385 390 395 400 Thr Phe Cys Asn Glu Leu Ala Gln Met Cys Gln Val Ser Gly Met Glu 405 410 415 Phe Asn Pro Glu Ser Val Ile Pro Ile Tyr Asn Ala Lys Pro Glu Gln 420 425 430 Val Glu Lys Ala Leu Lys His Val Tyr His Val Ser Gly Ser Lys Ile 435 440 445 Lys Gly Lys Glu Leu Glu Leu Leu Leu Ala Ile Leu Pro Asp Asn Asn 450 455 460 Gly Ser Leu Tyr Gly Asp Leu Lys Arg Ile Cys Glu Thr Asp Leu Gly 465 470 475 480 Leu Ile Ser Gln Cys Cys Leu Thr Lys His Val Phe Lys Ile Thr Lys 485 490 495 Gln Tyr Leu Ala Asn Val Ser Leu Lys Ile Asn Val Lys Met Gly Gly 500 505 510 Arg Asn Thr Val Leu Leu Asp Ala Val Ser Ser Arg Ile Pro Leu Val 515 520 525 Ser Asp Met Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro Glu 530 535 540 Asn Gly Glu Glu Leu Ser Pro Ser Ile Ala Ala Val Val Ala Ser Gln 545 550 555 560 Asp Trp Pro Glu Val Thr Lys Tyr Ala Gly Leu Val Cys Ala Gln Ala 565 570 575 His Arg Gln Glu Leu Ile Gln Asp Leu Tyr Lys Thr Trp Gln Asp Pro 580 585 590 Val Arg Gly Thr Val Ser Gly Gly Met Ile Arg Asp Leu Leu Val Ser 595 600 605 Phe Arg Lys Ala Thr Gly Gln Lys Pro Leu Arg Ile Ile Phe Tyr Arg 610 615 620 Asp Gly Val Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Tyr Glu Leu 625 630 635 640 Asp Ala Ile Arg Lys Ala Cys Ala Ser Leu Glu Pro Asn Tyr Gln Pro 645 650 655 Pro Val Thr Phe Ile Val Val Gln Lys Arg His His Thr Arg Leu Phe 660 665 670 Ala Asn Asn Tyr Arg Asp Arg Ser Ser Thr Asp Arg Ser Gly Asn Ile 675 680 685 Leu Pro Gly Thr Val Val Asp Thr Lys Ile Cys His Pro Thr Glu Phe 690 695 700 Asp Phe Tyr Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser Arg Pro 705 710 715 720 Ala His Tyr His Val Leu Trp Asp Glu Asn Asn Phe Thr Pro Asp Gly 725 730 735 Ile Gln Ser Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys Thr 740 745 750 Arg Ser Val Ser Val Val Pro Pro Ala Tyr Tyr Ala His Leu Ala Ala 755 760 765 Phe Arg Ala Arg Phe Tyr Met Glu Pro Asp Met Gln Asp Asn Gly Ser 770 775 780 Ala Gly Asp Gly Asn Gly His Gly Ala Lys Ala Thr Arg Ala Ala Gly 785 790 795 800 Asp Tyr Ser Val Lys Pro Leu Pro Asp Leu Lys Glu Asn Val Lys Arg 805 810 815 Val Met Phe Tyr Cys 820 5 1501 DNA Glycine max 5 gttttgccaa cagttagttc aaatatgcca aatctcaggc atggaattta gtcaagaccc 60 tgtgattcca atatattcag caaaacctga tctggtaaag aaagccttga agtatgtaca 120 ttctgctgta cttgataaac ttggtgggaa agaactagag ttgttgattg ccattcttcc 180 agacaacaat ggctctctgt atggcgatct caaaagaatc tgtgaaaccg atctggggtt 240 gatttctcag tgctgtctta caaaacacgt attcaagatc aataggcagt atttggcaaa 300 tgtggcacta aagatcaatg tcaagatggg aggaaggaac acagtacttt tggatgccct 360 aagttggagg atcccattgg ttagtgacat tccaacaata atttttggag cagatgtaac 420 acatccagaa tctggagagg acccttgtcc atccattgct gctgttgtag cctcccagga 480 ctggccggaa gtaacaaagt acgcaggatt ggtatgcgct cagcctcatc gtgaggaact 540 cattcaagat ctttttaaat gttggaagga tcctcatcat ggtatagttt atggtggcat 600 gatcagagag ctgttactct cttttaagaa ggcaaccgga caaaaaccat tgaggataat 660 attttacagg gatggggtaa gtgaaggaca gttctaccag gttttgttgt atgagcttga 720 tgccatccgt aaggcttgtg catctttgga acctagttac caacctccgg taacatttgt 780 tgtggttcaa aagcgacatc acactagact cttctcaaac aatcatgacg acagaaatag 840 cactgataag agtgggaata tcttacctgg tactgtggtg gattctaaga tctgtcatcc 900 tacggaattc gacttctatt tatgcagtca tgcgggaatt cagggtacaa gtagaccagc 960 tcattatcat gttctgtggg acgagaacaa tttcactgct gatgagatcc aatctctgac 1020 caacaacttg tgctacacct atgcaagatg tacacgatca gtttctgtag tgcctcctgc 1080 gtactatgct catttggcag cttacagagc tcgattctac atggaaccta atgtccatga 1140 aattgctaaa tctcgaggtg caaggtcaaa agatgagtca gttcggccac tacctgctct 1200 gaaagagaag gtgaagaatg taatgtttta ttgttgaatg agacaaaata gagagacatc 1260 taagtagaga aacagcagca tatgtaggaa aaggaaatta aattagcaga gctcagaaag 1320 ctcaatatgt acaacctaac gtgttcataa ttcataattc tccgcatgga aaattttgac 1380 aaagtctagg ttgtttttca gtatttctag tgcttaggga aggtaataac ttatgtagaa 1440 attatttgtg tatcggtttt cgagcttcaa gacaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500 a 1501 6 411 PRT Glycine max 6 Phe Cys Gln Gln Leu Val Gln Ile Cys Gln Ile Ser Gly Met Glu Phe 1 5 10 15 Ser Gln Asp Pro Val Ile Pro Ile Tyr Ser Ala Lys Pro Asp Leu Val 20 25 30 Lys Lys Ala Leu Lys Tyr Val His Ser Ala Val Leu Asp Lys Leu Gly 35 40 45 Gly Lys Glu Leu Glu Leu Leu Ile Ala Ile Leu Pro Asp Asn Asn Gly 50 55 60 Ser Leu Tyr Gly Asp Leu Lys Arg Ile Cys Glu Thr Asp Leu Gly Leu 65 70 75 80 Ile Ser Gln Cys Cys Leu Thr Lys His Val Phe Lys Ile Asn Arg Gln 85 90 95 Tyr Leu Ala Asn Val Ala Leu Lys Ile Asn Val Lys Met Gly Gly Arg 100 105 110 Asn Thr Val Leu Leu Asp Ala Leu Ser Trp Arg Ile Pro Leu Val Ser 115 120 125 Asp Ile Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro Glu Ser 130 135 140 Gly Glu Asp Pro Cys Pro Ser Ile Ala Ala Val Val Ala Ser Gln Asp 145 150 155 160 Trp Pro Glu Val Thr Lys Tyr Ala Gly Leu Val Cys Ala Gln Pro His 165 170 175 Arg Glu Glu Leu Ile Gln Asp Leu Phe Lys Cys Trp Lys Asp Pro His 180 185 190 His Gly Ile Val Tyr Gly Gly Met Ile Arg Glu Leu Leu Leu Ser Phe 195 200 205 Lys Lys Ala Thr Gly Gln Lys Pro Leu Arg Ile Ile Phe Tyr Arg Asp 210 215 220 Gly Val Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Tyr Glu Leu Asp 225 230 235 240 Ala Ile Arg Lys Ala Cys Ala Ser Leu Glu Pro Ser Tyr Gln Pro Pro 245 250 255 Val Thr Phe Val Val Val Gln Lys Arg His His Thr Arg Leu Phe Ser 260 265 270 Asn Asn His Asp Asp Arg Asn Ser Thr Asp Lys Ser Gly Asn Ile Leu 275 280 285 Pro Gly Thr Val Val Asp Ser Lys Ile Cys His Pro Thr Glu Phe Asp 290 295 300 Phe Tyr Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser Arg Pro Ala 305 310 315 320 His Tyr His Val Leu Trp Asp Glu Asn Asn Phe Thr Ala Asp Glu Ile 325 330 335 Gln Ser Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys Thr Arg 340 345 350 Ser Val Ser Val Val Pro Pro Ala Tyr Tyr Ala His Leu Ala Ala Tyr 355 360 365 Arg Ala Arg Phe Tyr Met Glu Pro Asn Val His Glu Ile Ala Lys Ser 370 375 380 Arg Gly Ala Arg Ser Lys Asp Glu Ser Val Arg Pro Leu Pro Ala Leu 385 390 395 400 Lys Glu Lys Val Lys Asn Val Met Phe Tyr Cys 405 410 7 3096 DNA Zea mays 7 gtttcggtgg ggttcttgcc gctgcggttg ttcgtgcggc gcggatttag ggagggttct 60 gaggcgaggg cttttgcccc cctcgagcga tttgcagctt tgggtccgat acagtgctca 120 tcaaggctca ctaaatggag tctcacaatg gcgaggccaa tgacttgcct ccaccacctc 180 ctctgattgc tggtgttgaa ccacttaaag ctgatgaaac aaagatgcca ttgaaaccta 240 ggagtctggt ccagagaaat ggatttggca gaaaggggca gccaataaag ctgataacaa 300 atcacttcaa agtttctctt gtgaatgctg aagaattttt ctaccattac tatgtcaatt 360 tgaagtatga agatgataca ccggttgatc gcaaagggtc aggaaggaaa gtgattgaaa 420 aactgcagca aacttatgct gctgaacttg caaataaaga ttttgcctat gatggtgaga 480 agagcctgtt cacaattggt gctcttcctc aagttaaaaa tgagtttact gtcgtggttg 540 aagatttttc aactggaaag actcctgcaa acggcagtcc aggaaatgac agtcctcccg 600 gaagtgacag gaaaagggtc agaaggcctt acaatacaaa gacctataag gtcgagctct 660 cttttgcagc aaaaattcct atgagtgcaa tctcacaggc cttaagaggt caggaatcag 720 agcacactca ggaagcaatt cgagtgattg acattattct gaggcagcac tcagctaagc 780 agggttgcct attagtaagg caatcattct tccacaacaa tccttccaat tttgttgacc 840 tgggtggtgg tgtagtgggc tgtagaggtt ttcattctag ttttcgagca acccagagtg 900 gactttcact caatatcgat gtgtcgacta caatgatagt gaaacctggt cctgtcattg 960 attttctgct tgacaatcag aaagttggtg attcaagcat gattgattgg gctaagggca 1020 agcgtgcact gaagaacttg aggataaaaa taagtccagc gaaccaagaa cagaagattg 1080 ttggtctcag cgaaagaact tgtcgtgagc aattattcac actgaaacat aaaaatggta 1140 acaatggtga ctctgaagag atcactgttt atgattactt cgtaaagcag cgtggcatag 1200 tgctgcaata ctctggtgat cttccttgca tcaatgtggg aaaactaaag cggccaacat 1260 attttccaat tgagttatgc agtcttgtgc ctttacaaag atacactaaa gctttgaaca 1320 cacttcagag gtcatcactc gtggagaaat ctaggcagaa accgcaggaa aggatgtctg 1380 ttttatctga tgtgctgcaa agaagcaact atgatgcaga gcccatgttg aaggcatgcg 1440 ggattacaat tgctagaaat ttcacagaag ttgatggtag ggtattgcag ccacctaagc 1500 ttaaagctgg gaatggtgaa gacattttta cacgcaatgg tagatggaac ttcaacaata 1560 agaggctcat tagagcttgt agtgtcgaga aatgggcggt ggtaaacttt tctgcacgat 1620 gcaatgtcag ggatcttgtc cgggatctca tcaagtgtgg aggcatgaag ggcattatgg 1680 ttgatgctcc ttttgctgta tttgatgaga atccttcaat gagacggtca cctgctataa 1740 gaagggttga agacatgttt gaacaagtga aaactaagct tcctggagca ccaaagtttc 1800 ttttgtgtgt tctagctgaa aggaagaatt ctgatattta tgggccttgg aagaagaaat 1860 gccttgctga atttgggatc gttacacaat gtgtggcacc aactagagtg aacgaccagt 1920 atcttacaaa tgtcctactt aagataaatg caaagctggg tggcatgaat tcgttgctcc 1980 aaattgaaac atccccagca attcctcttg tatccaaggt cccaactata atcttgggaa 2040 tggatgtgtc acacggttct cctggacatt ctgatgtacc atctattgct gctgttgtta 2100 gttctcgtga atggcctctt atctcgaaat acagagcttc tgtccgcacc caatcaccta 2160 aaatggaaat gattgactca ttgtttaagc cacgggaagc tgaagatgat ggtctgatcc 2220 gggagtgtct gattgacttc tacaccagtt ctgggaagag aaagcctgac caagttatca 2280 tattcaggga cggtgttagc gaaagtcagt ttaatcaggt gctgaacatt gagttgcaac 2340 aaatcatcga ggcttgcaaa tttcttgatg agaaatggaa tcccaagttc acgttgatta 2400 ttgcccagaa gaatcatcac actaaatttt tcattcctgg aaagccagat aatgtcccac 2460 caggaactgt ggtggacaac aaagtctgcc atccaaagaa cttcgatttc tacatgtgtg 2520 cgcatgctgg aatgatcggg actacgaggc caactcacta ccacatcctg catgatgaga 2580 taggcttcag tcctgatgat ctgcaggagc tggtgcattc gctctcttat gtgtaccaaa 2640 ggagcacaac agccatatca gtcgttgctc ccatctgcta cgcacatctg gcagctgctc 2700 aggttggcca gttcataaag ttcgatgaga tgtcggagac gtcctccagt catggcgggc 2760 atacttcggc gggcagcgtt ccggtccagg agctgccgcg cctgcatgag aaagtgagga 2820 gctcgatgtt cttttgctga gccgtggttt tacttttttg gtggatggtg aacccctcta 2880 gttatgtcgg tagacgctct tggatgacgc tctagttgtg gtccaggaag gctcgagctg 2940 gtacgatgtt aaatgttagt tttttaagcg tcgctgcggc tatgttggtg cctcaggaag 3000 acttggaacc tggttaggat gtcgttaaat ctacccctta tcgttcctgg ttaaaaaaaa 3060 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 3096 8 901 PRT Zea mays 8 Met Glu Ser His Asn Gly Glu Ala Asn Asp Leu Pro Pro Pro Pro Pro 1 5 10 15 Leu Ile Ala Gly Val Glu Pro Leu Lys Ala Asp Glu Thr Lys Met Pro 20 25 30 Leu Lys Pro Arg Ser Leu Val Gln Arg Asn Gly Phe Gly Arg Lys Gly 35 40 45 Gln Pro Ile Lys Leu Ile Thr Asn His Phe Lys Val Ser Leu Val Asn 50 55 60 Ala Glu Glu Phe Phe Tyr His Tyr Tyr Val Asn Leu Lys Tyr Glu Asp 65 70 75 80 Asp Thr Pro Val Asp Arg Lys Gly Ser Gly Arg Lys Val Ile Glu Lys 85 90 95 Leu Gln Gln Thr Tyr Ala Ala Glu Leu Ala Asn Lys Asp Phe Ala Tyr 100 105 110 Asp Gly Glu Lys Ser Leu Phe Thr Ile Gly Ala Leu Pro Gln Val Lys 115 120 125 Asn Glu Phe Thr Val Val Val Glu Asp Phe Ser Thr Gly Lys Thr Pro 130 135 140 Ala Asn Gly Ser Pro Gly Asn Asp Ser Pro Pro Gly Ser Asp Arg Lys 145 150 155 160 Arg Val Arg Arg Pro Tyr Asn Thr Lys Thr Tyr Lys Val Glu Leu Ser 165 170 175 Phe Ala Ala Lys Ile Pro Met Ser Ala Ile Ser Gln Ala Leu Arg Gly 180 185 190 Gln Glu Ser Glu His Thr Gln Glu Ala Ile Arg Val Ile Asp Ile Ile 195 200 205 Leu Arg Gln His Ser Ala Lys Gln Gly Cys Leu Leu Val Arg Gln Ser 210 215 220 Phe Phe His Asn Asn Pro Ser Asn Phe Val Asp Leu Gly Gly Gly Val 225 230 235 240 Val Gly Cys Arg Gly Phe His Ser Ser Phe Arg Ala Thr Gln Ser Gly 245 250 255 Leu Ser Leu Asn Ile Asp Val Ser Thr Thr Met Ile Val Lys Pro Gly 260 265 270 Pro Val Ile Asp Phe Leu Leu Asp Asn Gln Lys Val Gly Asp Ser Ser 275 280 285 Met Ile Asp Trp Ala Lys Gly Lys Arg Ala Leu Lys Asn Leu Arg Ile 290 295 300 Lys Ile Ser Pro Ala Asn Gln Glu Gln Lys Ile Val Gly Leu Ser Glu 305 310 315 320 Arg Thr Cys Arg Glu Gln Leu Phe Thr Leu Lys His Lys Asn Gly Asn 325 330 335 Asn Gly Asp Ser Glu Glu Ile Thr Val Tyr Asp Tyr Phe Val Lys Gln 340 345 350 Arg Gly Ile Val Leu Gln Tyr Ser Gly Asp Leu Pro Cys Ile Asn Val 355 360 365 Gly Lys Leu Lys Arg Pro Thr Tyr Phe Pro Ile Glu Leu Cys Ser Leu 370 375 380 Val Pro Leu Gln Arg Tyr Thr Lys Ala Leu Asn Thr Leu Gln Arg Ser 385 390 395 400 Ser Leu Val Glu Lys Ser Arg Gln Lys Pro Gln Glu Arg Met Ser Val 405 410 415 Leu Ser Asp Val Leu Gln Arg Ser Asn Tyr Asp Ala Glu Pro Met Leu 420 425 430 Lys Ala Cys Gly Ile Thr Ile Ala Arg Asn Phe Thr Glu Val Asp Gly 435 440 445 Arg Val Leu Gln Pro Pro Lys Leu Lys Ala Gly Asn Gly Glu Asp Ile 450 455 460 Phe Thr Arg Asn Gly Arg Trp Asn Phe Asn Asn Lys Arg Leu Ile Arg 465 470 475 480 Ala Cys Ser Val Glu Lys Trp Ala Val Val Asn Phe Ser Ala Arg Cys 485 490 495 Asn Val Arg Asp Leu Val Arg Asp Leu Ile Lys Cys Gly Gly Met Lys 500 505 510 Gly Ile Met Val Asp Ala Pro Phe Ala Val Phe Asp Glu Asn Pro Ser 515 520 525 Met Arg Arg Ser Pro Ala Ile Arg Arg Val Glu Asp Met Phe Glu Gln 530 535 540 Val Lys Thr Lys Leu Pro Gly Ala Pro Lys Phe Leu Leu Cys Val Leu 545 550 555 560 Ala Glu Arg Lys Asn Ser Asp Ile Tyr Gly Pro Trp Lys Lys Lys Cys 565 570 575 Leu Ala Glu Phe Gly Ile Val Thr Gln Cys Val Ala Pro Thr Arg Val 580 585 590 Asn Asp Gln Tyr Leu Thr Asn Val Leu Leu Lys Ile Asn Ala Lys Leu 595 600 605 Gly Gly Met Asn Ser Leu Leu Gln Ile Glu Thr Ser Pro Ala Ile Pro 610 615 620 Leu Val Ser Lys Val Pro Thr Ile Ile Leu Gly Met Asp Val Ser His 625 630 635 640 Gly Ser Pro Gly His Ser Asp Val Pro Ser Ile Ala Ala Val Val Ser 645 650 655 Ser Arg Glu Trp Pro Leu Ile Ser Lys Tyr Arg Ala Ser Val Arg Thr 660 665 670 Gln Ser Pro Lys Met Glu Met Ile Asp Ser Leu Phe Lys Pro Arg Glu 675 680 685 Ala Glu Asp Asp Gly Leu Ile Arg Glu Cys Leu Ile Asp Phe Tyr Thr 690 695 700 Ser Ser Gly Lys Arg Lys Pro Asp Gln Val Ile Ile Phe Arg Asp Gly 705 710 715 720 Val Ser Glu Ser Gln Phe Asn Gln Val Leu Asn Ile Glu Leu Gln Gln 725 730 735 Ile Ile Glu Ala Cys Lys Phe Leu Asp Glu Lys Trp Asn Pro Lys Phe 740 745 750 Thr Leu Ile Ile Ala Gln Lys Asn His His Thr Lys Phe Phe Ile Pro 755 760 765 Gly Lys Pro Asp Asn Val Pro Pro Gly Thr Val Val Asp Asn Lys Val 770 775 780 Cys His Pro Lys Asn Phe Asp Phe Tyr Met Cys Ala His Ala Gly Met 785 790 795 800 Ile Gly Thr Thr Arg Pro Thr His Tyr His Ile Leu His Asp Glu Ile 805 810 815 Gly Phe Ser Pro Asp Asp Leu Gln Glu Leu Val His Ser Leu Ser Tyr 820 825 830 Val Tyr Gln Arg Ser Thr Thr Ala Ile Ser Val Val Ala Pro Ile Cys 835 840 845 Tyr Ala His Leu Ala Ala Ala Gln Val Gly Gln Phe Ile Lys Phe Asp 850 855 860 Glu Met Ser Glu Thr Ser Ser Ser His Gly Gly His Thr Ser Ala Gly 865 870 875 880 Ser Val Pro Val Gln Glu Leu Pro Arg Leu His Glu Lys Val Arg Ser 885 890 895 Ser Met Phe Phe Cys 900 9 2446 DNA Zea mays 9 gcacgagatc aaatttgctg ctcgcgctga tctccaccat ttggctatgt ttcttgctgg 60 gaggcagcca gatgcccctc aagaggctct tcaagtactt gacatcgtgc tacgtgaaat 120 gcctactgcc aagtattgtc ctgttggtag atcattttat tctcccaagt tagggagacc 180 tcagcaactt ggtgaaggtt tggaaacttg gcgtggtttc taccaaagca taaggcccac 240 acagatgggt ctttctctga atattgatat gtcctctact gcattttttg aggccctccc 300 tgtaattgat tttgtttctc agcttcttaa tagagatatc tcagttagac cattgtctga 360 ttctgatcgc gtgaagatta aaaaagccct acgaggtgtg aaagtggagg tcacacaccg 420 tggaaacatg cgtaggaaat atcggatatc tggccttact ccacaagcaa caagggagtt 480 atcattccct attgatgatc gtggtactgt taagactgtg gtgcaatact tcctggagac 540 ttatggtttc agtattcagc acaccacttt accttgtttg caagtgggca atcagcaaag 600 accaaattat ctgcctatgg aggtctgtaa gatagttgag gggcagcgct actcaaaacg 660 acttaatgat aaacagatca ctgctctact gaaggtgact tgccaacgtc cccaagcgcg 720 tgagaaggac atcttggaga ctgtgtatca caatgcctac tccaaggatc cttatgccca 780 ggaatttggt ataacgattg atgagcgtct tgcatcggtt gaagctcgtg ttctgcctcc 840 cccaaggctg aaataccatg atagtggcag agaaagggat gtattgccaa aagttggcca 900 gtggaacatg atgaataaga aaatggtcaa tggtggtaga gttagcagct gggcatgcat 960 taacttctca cggaatgtgc aagatggtgc tgccgggggt ttctgtcatg aattggcttt 1020 gatgtgccaa gtatcaggaa tggattttgt acttgaacct gtgctgtcac cttgctatgc 1080 aaggcctgaa cttgttgaaa gagcactaaa gggacgctat caagatgcga tgaacatact 1140 cgggcctcag ggccgagaac tcgacttgct gattgttata ctgcctgaca ataatggttc 1200 tctttacggg gatgtcaaaa ggatctgtga gactaatctt ggattggtct cccaatgctg 1260 tctgactaaa catgttttca aggtgaacaa gcagcagtat cttgcaaatg ttgccctgaa 1320 aataaatgtg aaggttgggg gaaggaatac tgtgcttgtt gatgctttgg caaggagaat 1380 cccccttgtc agtgacatag cgactattat ctttggtgct gatgtgaccc atccccatcc 1440 tggggaagat tctagtcctt ccattgcagc tgtggttgct tctcaagact ggcctgaggt 1500 tacaaagtat gcaggattgg tgagtgctca agcccatcgt caagaattga tacaggatct 1560 tttcaaggta tggcaagatc ccgaaagggg gactgtctct ggtggcatga tcagggagct 1620 tctcatatct ttctggaggg caactggaca gaaaccaaag aggatcatat tctacaggga 1680 tggcgtcagt gagggacaat tctaccaagt tctgttgtat gaacttgatg ccattagaaa 1740 ggcctgtgcg tcattggagt ctgactacca gcctccagtt acttttgtcg tggtccagaa 1800 gcgtcatcac accaggttgt ttgctaataa tcacaatgat aatcgtgctg tcgataaaag 1860 cgggaacata ctgcctggca ccgtggtgga ctcgaagatc tgccatccaa ctgagtttga 1920 tttctacctg tgcagccatg ctggcattca gggaacaagc cgccctgccc attaccatgt 1980 tctgtgggat gagaacaact ttacggctga tgggttgcaa actctcacca acaacttgtg 2040 ttacacgtat gctaggtgca cacgctcagt atcgattgtt cctcctgcat actatgctca 2100 cctggcagcc ttccgagctc ggttctacat ggagccagat acgagtgaca gtggatctat 2160 ggcaagccgt ggccctccac caggggggcg caacaccaag gctgccggtg ttgggaatgt 2220 tgctgtgagg ccattacctg ccctcaagga aaacgtgaag cgggtcatgt tctactgcta 2280 agactgatgc tgttaaggca gagctacctt ttattattac agtatatcgt gaagactaga 2340 gtattttttt ccacgtactt gatgatgctg agctaccttt taaaaaaaaa aaaaaaaaaa 2400 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2446 10 757 PRT Zea mays 10 Ile Lys Phe Ala Ala Arg Ala Asp Leu His His Leu Ala Met Phe Leu 1 5 10 15 Ala Gly Arg Gln Pro Asp Ala Pro Gln Glu Ala Leu Gln Val Leu Asp 20 25 30 Ile Val Leu Arg Glu Met Pro Thr Ala Lys Tyr Cys Pro Val Gly Arg 35 40 45 Ser Phe Tyr Ser Pro Lys Leu Gly Arg Pro Gln Gln Leu Gly Glu Gly 50 55 60 Leu Glu Thr Trp Arg Gly Phe Tyr Gln Ser Ile Arg Pro Thr Gln Met 65 70 75 80 Gly Leu Ser Leu Asn Ile Asp Met Ser Ser Thr Ala Phe Phe Glu Ala 85 90 95 Leu Pro Val Ile Asp Phe Val Ser Gln Leu Leu Asn Arg Asp Ile Ser 100 105 110 Val Arg Pro Leu Ser Asp Ser Asp Arg Val Lys Ile Lys Lys Ala Leu 115 120 125 Arg Gly Val Lys Val Glu Val Thr His Arg Gly Asn Met Arg Arg Lys 130 135 140 Tyr Arg Ile Ser Gly Leu Thr Pro Gln Ala Thr Arg Glu Leu Ser Phe 145 150 155 160 Pro Ile Asp Asp Arg Gly Thr Val Lys Thr Val Val Gln Tyr Phe Leu 165 170 175 Glu Thr Tyr Gly Phe Ser Ile Gln His Thr Thr Leu Pro Cys Leu Gln 180 185 190 Val Gly Asn Gln Gln Arg Pro Asn Tyr Leu Pro Met Glu Val Cys Lys 195 200 205 Ile Val Glu Gly Gln Arg Tyr Ser Lys Arg Leu Asn Asp Lys Gln Ile 210 215 220 Thr Ala Leu Leu Lys Val Thr Cys Gln Arg Pro Gln Ala Arg Glu Lys 225 230 235 240 Asp Ile Leu Glu Thr Val Tyr His Asn Ala Tyr Ser Lys Asp Pro Tyr 245 250 255 Ala Gln Glu Phe Gly Ile Thr Ile Asp Glu Arg Leu Ala Ser Val Glu 260 265 270 Ala Arg Val Leu Pro Pro Pro Arg Leu Lys Tyr His Asp Ser Gly Arg 275 280 285 Glu Arg Asp Val Leu Pro Lys Val Gly Gln Trp Asn Met Met Asn Lys 290 295 300 Lys Met Val Asn Gly Gly Arg Val Ser Ser Trp Ala Cys Ile Asn Phe 305 310 315 320 Ser Arg Asn Val Gln Asp Gly Ala Ala Gly Gly Phe Cys His Glu Leu 325 330 335 Ala Leu Met Cys Gln Val Ser Gly Met Asp Phe Val Leu Glu Pro Val 340 345 350 Leu Ser Pro Cys Tyr Ala Arg Pro Glu Leu Val Glu Arg Ala Leu Lys 355 360 365 Gly Arg Tyr Gln Asp Ala Met Asn Ile Leu Gly Pro Gln Gly Arg Glu 370 375 380 Leu Asp Leu Leu Ile Val Ile Leu Pro Asp Asn Asn Gly Ser Leu Tyr 385 390 395 400 Gly Asp Val Lys Arg Ile Cys Glu Thr Asn Leu Gly Leu Val Ser Gln 405 410 415 Cys Cys Leu Thr Lys His Val Phe Lys Val Asn Lys Gln Gln Tyr Leu 420 425 430 Ala Asn Val Ala Leu Lys Ile Asn Val Lys Val Gly Gly Arg Asn Thr 435 440 445 Val Leu Val Asp Ala Leu Ala Arg Arg Ile Pro Leu Val Ser Asp Ile 450 455 460 Ala Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro His Pro Gly Glu 465 470 475 480 Asp Ser Ser Pro Ser Ile Ala Ala Val Val Ala Ser Gln Asp Trp Pro 485 490 495 Glu Val Thr Lys Tyr Ala Gly Leu Val Ser Ala Gln Ala His Arg Gln 500 505 510 Glu Leu Ile Gln Asp Leu Phe Lys Val Trp Gln Asp Pro Glu Arg Gly 515 520 525 Thr Val Ser Gly Gly Met Ile Arg Glu Leu Leu Ile Ser Phe Trp Arg 530 535 540 Ala Thr Gly Gln Lys Pro Lys Arg Ile Ile Phe Tyr Arg Asp Gly Val 545 550 555 560 Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Tyr Glu Leu Asp Ala Ile 565 570 575 Arg Lys Ala Cys Ala Ser Leu Glu Ser Asp Tyr Gln Pro Pro Val Thr 580 585 590 Phe Val Val Val Gln Lys Arg His His Thr Arg Leu Phe Ala Asn Asn 595 600 605 His Asn Asp Asn Arg Ala Val Asp Lys Ser Gly Asn Ile Leu Pro Gly 610 615 620 Thr Val Val Asp Ser Lys Ile Cys His Pro Thr Glu Phe Asp Phe Tyr 625 630 635 640 Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser Arg Pro Ala His Tyr 645 650 655 His Val Leu Trp Asp Glu Asn Asn Phe Thr Ala Asp Gly Leu Gln Thr 660 665 670 Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys Thr Arg Ser Val 675 680 685 Ser Ile Val Pro Pro Ala Tyr Tyr Ala His Leu Ala Ala Phe Arg Ala 690 695 700 Arg Phe Tyr Met Glu Pro Asp Thr Ser Asp Ser Gly Ser Met Ala Ser 705 710 715 720 Arg Gly Pro Pro Pro Gly Gly Arg Asn Thr Lys Ala Ala Gly Val Gly 725 730 735 Asn Val Ala Val Arg Pro Leu Pro Ala Leu Lys Glu Asn Val Lys Arg 740 745 750 Val Met Phe Tyr Cys 755 11 3808 DNA Zea mays 11 ctgctttctc cagtgagccg cacccgcact accgctgacg ctaattaacc acaagcgacc 60 gtcgccttcc cccacctcct cccttcctca aaaaaaggcg gaggcgggag tggtggtggt 120 gctcgtgggc acgcagcgga gcaccctgta cagcagcagc gctgcggcag tagagagtgc 180 cattggtgga gctggtaact agccctcccc cctccgttcc cgtcccgcgc gcagccgtct 240 gccgagcctg ctcagtgccc atcatggtga ggaagaagag aactggccct ggtggctctg 300 gagaaacttc tggagagtct tcaggagcct ctggacaagg ttcctcacag cagcctgagc 360 gaactcaaca acctggggga ggacgtggct gggtgcctca acagggtggc catggtggtg 420 ggcaacacca gggtcgtgat cgacattatc agggacgtgg aggaccaggg ccacatcacc 480 ttggtagtgg ggcacctgag tatcacccgc gtgaatacca gggacgtggt ggtgaatatc 540 agggacatgg tggtgagtac cagggacggg gtggtgacta ccagggacgt ggtggtggcc 600 gctccagagg tggaatgcca cagccatact atggtgggca taggggaggt aatgttggac 660 gcaatgttcc tccaggtccg tccaggacag ttcccgagct gcaccaagcc ccatatgtcc 720 agtatccagc cccggtggtt tcgccctccc catcgggacc tggctcatcc tcacagccta 780 tggcagaggt gagctctgga caagtccagc aacagtttca gcaacttgcc gatcgtggtc 840 agagttccac gagccaagaa attcaagtgg caccagcatc aagcaaatcg gttcgattcc 900 cgttacggcc cggcaagggc acttatgggg acaggtgcat tgtgaaggca aatcattttt 960 ttgctgagct tcctgacaaa gaccttcacc aatatgatgt atctataaca cctgaggtta 1020 cttcacgtgg cgtcaatcgt gctgtcatgg gtgagcttgt aacaatatat agacaatccc 1080 atttgggtgg gcgtctacct gcatacgatg gaagaaagag cctgtatact gctggaccat 1140 tgccatttac ttctatggca tttgaaatta ccttgcaaga tgaggaagat agtcttggcg 1200 gtcgccaagg tggacatagg cgtgagagag tatttagggt ggtgatcaaa tttgcagccc 1260 gtgctgatct ccaccatctg gctatgtttc tagctggaag gcaagcagat gcccctcagg 1320 aagctcttca agtgcttgac attgtactac gtgaattgcc taccgcgagg tattctcctg 1380 tcggtaggtc attttactct cccaacttag ggagacgtca aaaacttggt gagggattgg 1440 aaagttggcg tggtttttac caaagcataa ggccgacaca gatgggcctt tcactgaata 1500 ttgatatgtc ctctactgca tttatcgagc ctctccctgt gatcgatttt gttgctcagc 1560 ttcttaacag agatatctca gttaggccat tgtctgattc tgatcgcgtg aagattaaaa 1620 aagccctaag aggtgtgaag gttgaggtga ctcacagggg aaacatgcgc agaaaatatc 1680 gcatttctgg cctcacctca caagcaacaa gagagctatc attccctgtt gatgatcgtg 1740 gtactgtgaa gactgtggtg caatacttca tggagactta tggttttagt atccagcaca 1800 ccactttacc atgcttgcaa gtgggtaatc aacaaagacc aaattatctg cctatggagg 1860 tttgcaagat agttgaagga cagcgttact caaagcgact caatgagaaa caaatcactg 1920 ctctactgaa agtgacctgc cagcgccctc aagagcgcga gctggacatc ttacagactg 1980 tgcatcacaa tgcatactat gaagacccct atgcactgga atttggtata agaattgatg 2040 aacgtcttgc tgcagttgaa gctcgtgttc tgccaccacc aagacttaaa taccatgata 2100 gtggccgaga gaaggatgtt ttgcccagag ttggccaatg gaacatgatg aataagaaaa 2160 tggttaatgg tggcagagtg agcaactggg catgtattaa cttctctcgg aatgtgcaag 2220 atagtgccgc taggggtttc tctcatgagt tggcagtcat gtgccaaata tcaggaatgg 2280 attttgctct tgagcctgtg ctgcctccag tgactgcaag gccagaacat gttgagagag 2340 cgttaaaggc acgttatcaa gatgcaatga acatactgag gccacaggga agggaacttg 2400 atctgctgat cgtaatactg cctgacaaca atggttctct ttatggggat ctcaaaagga 2460 tctgtgagac tgaactcgga ttggtctccc agtgttgtct gactaaacat gtttttaaga 2520 tgagcaagca gtaccttgca aatgttgcac tcaaaataaa tgttaaggtt gggggaagga 2580 atactgtact tttagatgct ttgtcaagga gaatccccct tgtcagtgac agaccgacca 2640 taatatttgg tgctgatgtt acccatccac atcctggaga agattccagt ccttccattg 2700 cagccgttgt tgcttcgcaa gactggcccg aggtcacgaa atacgctgga ctagtgagtg 2760 cgcaagccca tcgccaggag ctgatacagg atcttttcaa agtatggcag gacccgcaga 2820 gaaggacggt aactggcggc atgataaagg aacttctcat ttctttcaag agggcaactg 2880 gacagaagcc ccagaggatc atattctaca gggatggtgt cagtgaggga cagttctatc 2940 aagtattgct gtacgaactt gatgccatta gaaaggcctg tgcgtccctg gagcccaact 3000 accagcctcc agttactttt gtcgtggtac agaagcgcca tcacactagg ctgtttgcga 3060 acaaccacag tgatcagcgc acagtcgata gaagcggaaa catactgcct ggcaccgtgg 3120 tcgattcgaa gatttgccat cctactgagt ttgacttcta cctgtgtagc catgctggca 3180 ttcagggaac gagccgccct gctcactacc atgtcctgtg ggacgagaac aagttcacag 3240 ctgacgagct gcagaccctg acgaacaacc tgtgctacac gtacgctagg tgcacccgct 3300 ccgtgtccat cgtgcccccg gcgtactacg ctcatctggc agccttccga gctcgcttct 3360 acatggagcc agacacctct gacagcgggt cactggccag cggtgcccgt ggccccccac 3420 ccggtgcggc acgcagcagc acgagagggg ccgggagtgt cgaggtcagg cccctacctg 3480 ctctcaagga gaacgtgaag cgtgtcatgt tttactgctg agacgctggt gggctgcctt 3540 cgccaaggaa aatgccctgg agcattccca tgtacccgca ctgtttcggt gatacagtac 3600 tatctaacgc cgattttgcg cgttaagact tccagtgatc tgggaaattt cttgtacgac 3660 tgttgtagtg ttgtgtattc gtaatgtgat gacgcggcag ttcttctagg agcttagtgc 3720 cgtgtaaaat atctgttgta agttgtaacc tgtcaccctc tagtgttatg tcatgatgaa 3780 ccaaattaaa aaaaaaaaaa aaaaaaaa 3808 12 1085 PRT Zea mays 12 Met Val Arg Lys Lys Arg Thr Gly Pro Gly Gly Ser Gly Glu Thr Ser 1 5 10 15 Gly Glu Ser Ser Gly Ala Ser Gly Gln Gly Ser Ser Gln Gln Pro Glu 20 25 30 Arg Thr Gln Gln Pro Gly Gly Gly Arg Gly Trp Val Pro Gln Gln Gly 35 40 45 Gly His Gly Gly Gly Gln His Gln Gly Arg Asp Arg His Tyr Gln Gly 50 55 60 Arg Gly Gly Pro Gly Pro His His Leu Gly Ser Gly Ala Pro Glu Tyr 65 70 75 80 His Pro Arg Glu Tyr Gln Gly Arg Gly Gly Glu Tyr Gln Gly His Gly 85 90 95 Gly Glu Tyr Gln Gly Arg Gly Gly Asp Tyr Gln Gly Arg Gly Gly Gly 100 105 110 Arg Ser Arg Gly Gly Met Pro Gln Pro Tyr Tyr Gly Gly His Arg Gly 115 120 125 Gly Asn Val Gly Arg Asn Val Pro Pro Gly Pro Ser Arg Thr Val Pro 130 135 140 Glu Leu His Gln Ala Pro Tyr Val Gln Tyr Pro Ala Pro Val Val Ser 145 150 155 160 Pro Ser Pro Ser Gly Pro Gly Ser Ser Ser Gln Pro Met Ala Glu Val 165 170 175 Ser Ser Gly Gln Val Gln Gln Gln Phe Gln Gln Leu Ala Asp Arg Gly 180 185 190 Gln Ser Ser Thr Ser Gln Glu Ile Gln Val Ala Pro Ala Ser Ser Lys 195 200 205 Ser Val Arg Phe Pro Leu Arg Pro Gly Lys Gly Thr Tyr Gly Asp Arg 210 215 220 Cys Ile Val Lys Ala Asn His Phe Phe Ala Glu Leu Pro Asp Lys Asp 225 230 235 240 Leu His Gln Tyr Asp Val Ser Ile Thr Pro Glu Val Thr Ser Arg Gly 245 250 255 Val Asn Arg Ala Val Met Gly Glu Leu Val Thr Ile Tyr Arg Gln Ser 260 265 270 His Leu Gly Gly Arg Leu Pro Ala Tyr Asp Gly Arg Lys Ser Leu Tyr 275 280 285 Thr Ala Gly Pro Leu Pro Phe Thr Ser Met Ala Phe Glu Ile Thr Leu 290 295 300 Gln Asp Glu Glu Asp Ser Leu Gly Gly Arg Gln Gly Gly His Arg Arg 305 310 315 320 Glu Arg Val Phe Arg Val Val Ile Lys Phe Ala Ala Arg Ala Asp Leu 325 330 335 His His Leu Ala Met Phe Leu Ala Gly Arg Gln Ala Asp Ala Pro Gln 340 345 350 Glu Ala Leu Gln Val Leu Asp Ile Val Leu Arg Glu Leu Pro Thr Ala 355 360 365 Arg Tyr Ser Pro Val Gly Arg Ser Phe Tyr Ser Pro Asn Leu Gly Arg 370 375 380 Arg Gln Lys Leu Gly Glu Gly Leu Glu Ser Trp Arg Gly Phe Tyr Gln 385 390 395 400 Ser Ile Arg Pro Thr Gln Met Gly Leu Ser Leu Asn Ile Asp Met Ser 405 410 415 Ser Thr Ala Phe Ile Glu Pro Leu Pro Val Ile Asp Phe Val Ala Gln 420 425 430 Leu Leu Asn Arg Asp Ile Ser Val Arg Pro Leu Ser Asp Ser Asp Arg 435 440 445 Val Lys Ile Lys Lys Ala Leu Arg Gly Val Lys Val Glu Val Thr His 450 455 460 Arg Gly Asn Met Arg Arg Lys Tyr Arg Ile Ser Gly Leu Thr Ser Gln 465 470 475 480 Ala Thr Arg Glu Leu Ser Phe Pro Val Asp Asp Arg Gly Thr Val Lys 485 490 495 Thr Val Val Gln Tyr Phe Met Glu Thr Tyr Gly Phe Ser Ile Gln His 500 505 510 Thr Thr Leu Pro Cys Leu Gln Val Gly Asn Gln Gln Arg Pro Asn Tyr 515 520 525 Leu Pro Met Glu Val Cys Lys Ile Val Glu Gly Gln Arg Tyr Ser Lys 530 535 540 Arg Leu Asn Glu Lys Gln Ile Thr Ala Leu Leu Lys Val Thr Cys Gln 545 550 555 560 Arg Pro Gln Glu Arg Glu Leu Asp Ile Leu Gln Thr Val His His Asn 565 570 575 Ala Tyr Tyr Glu Asp Pro Tyr Ala Leu Glu Phe Gly Ile Arg Ile Asp 580 585 590 Glu Arg Leu Ala Ala Val Glu Ala Arg Val Leu Pro Pro Pro Arg Leu 595 600 605 Lys Tyr His Asp Ser Gly Arg Glu Lys Asp Val Leu Pro Arg Val Gly 610 615 620 Gln Trp Asn Met Met Asn Lys Lys Met Val Asn Gly Gly Arg Val Ser 625 630 635 640 Asn Trp Ala Cys Ile Asn Phe Ser Arg Asn Val Gln Asp Ser Ala Ala 645 650 655 Arg Gly Phe Ser His Glu Leu Ala Val Met Cys Gln Ile Ser Gly Met 660 665 670 Asp Phe Ala Leu Glu Pro Val Leu Pro Pro Val Thr Ala Arg Pro Glu 675 680 685 His Val Glu Arg Ala Leu Lys Ala Arg Tyr Gln Asp Ala Met Asn Ile 690 695 700 Leu Arg Pro Gln Gly Arg Glu Leu Asp Leu Leu Ile Val Ile Leu Pro 705 710 715 720 Asp Asn Asn Gly Ser Leu Tyr Gly Asp Leu Lys Arg Ile Cys Glu Thr 725 730 735 Glu Leu Gly Leu Val Ser Gln Cys Cys Leu Thr Lys His Val Phe Lys 740 745 750 Met Ser Lys Gln Tyr Leu Ala Asn Val Ala Leu Lys Ile Asn Val Lys 755 760 765 Val Gly Gly Arg Asn Thr Val Leu Leu Asp Ala Leu Ser Arg Arg Ile 770 775 780 Pro Leu Val Ser Asp Arg Pro Thr Ile Ile Phe Gly Ala Asp Val Thr 785 790 795 800 His Pro His Pro Gly Glu Asp Ser Ser Pro Ser Ile Ala Ala Val Val 805 810 815 Ala Ser Gln Asp Trp Pro Glu Val Thr Lys Tyr Ala Gly Leu Val Ser 820 825 830 Ala Gln Ala His Arg Gln Glu Leu Ile Gln Asp Leu Phe Lys Val Trp 835 840 845 Gln Asp Pro Gln Arg Arg Thr Val Thr Gly Gly Met Ile Lys Glu Leu 850 855 860 Leu Ile Ser Phe Lys Arg Ala Thr Gly Gln Lys Pro Gln Arg Ile Ile 865 870 875 880 Phe Tyr Arg Asp Gly Val Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu 885 890 895 Tyr Glu Leu Asp Ala Ile Arg Lys Ala Cys Ala Ser Leu Glu Pro Asn 900 905 910 Tyr Gln Pro Pro Val Thr Phe Val Val Val Gln Lys Arg His His Thr 915 920 925 Arg Leu Phe Ala Asn Asn His Ser Asp Gln Arg Thr Val Asp Arg Ser 930 935 940 Gly Asn Ile Leu Pro Gly Thr Val Val Asp Ser Lys Ile Cys His Pro 945 950 955 960 Thr Glu Phe Asp Phe Tyr Leu Cys Ser His Ala Gly Ile Gln Gly Thr 965 970 975 Ser Arg Pro Ala His Tyr His Val Leu Trp Asp Glu Asn Lys Phe Thr 980 985 990 Ala Asp Glu Leu Gln Thr Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala 995 1000 1005 Arg Cys Thr Arg Ser Val Ser Ile Val Pro Pro Ala Tyr Tyr Ala His 1010 1015 1020 Leu Ala Ala Phe Arg Ala Arg Phe Tyr Met Glu Pro Asp Thr Ser Asp 1025 1030 1035 1040 Ser Gly Ser Leu Ala Ser Gly Ala Arg Gly Pro Pro Pro Gly Ala Ala 1045 1050 1055 Arg Ser Ser Thr Arg Gly Ala Gly Ser Val Glu Val Arg Pro Leu Pro 1060 1065 1070 Ala Leu Lys Glu Asn Val Lys Arg Val Met Phe Tyr Cys 1075 1080 1085 13 3714 DNA Zea mays unsure (1789) n = A, C, G or T 13 gccttccccc cctcccctcc tcaaaaaagg cggagaggtg gtggtgctcg tgggcacgca 60 gtggagcacc cagtacagca gcagcgctgc ggcagtggag ttaggagctt agcactccgc 120 ctccgttccc atcccgcgcg cagccgtcgg ccgagcctgc tcagtgccca tcatggtgag 180 gaagaagaga actggccctg gcggctctgg agaaacttct ggagagtctt caggagcttc 240 tggacaaggt tcctcacagc ggcctgaacg gactcaacaa cctggggcag gacgtggctg 300 ggtgcctcag cagggtggcc gtggtggcgg gcaacaccag ggtcgtggtg gacattatca 360 aggccgtgga gggccaggtc cacatcaccc tggtggactg cctgagtatc accagcgtga 420 ataccaggga cgaggtggtg agtaccaggg acagtaccag gggcgtggtg gtgcccgctc 480 cagaggtgga atttcacagc catactatgg tgggcatagg ggaggtagtg ttggacgaaa 540 tgttcctcca ggtccatcca gaacagttcc cgagctgcac caagccccat acgtccagta 600 tcaagccccg gtgatttcac catccccatc gggacctggc tcatcctcac agcctatggc 660 agaggtgagc tctggacaag tccagcaaca gtttgagcaa cttgccattc atggtcagag 720 ttccatgagt caagaagttc aagtggcacc agcatcaagc aaatcggttc gattcccatt 780 acgccccggc aagggcactt atggggacag gtgcattgtg aaggcgaatc atttttttgc 840 tgagcttcct gacaaagacc ttcaccaata tgatgtaact ataacacctg aagttacttc 900 acgtggcgtt aatcgtgctg tcatgggaga gcttgtaaca ctatatagac aatcccattt 960 gggcgggcgt ctacctgcgt acgatggaag aaagagcctt tataccgctg gaccattgcc 1020 ttttacttct atgacatttg aaattacctt gcaagatgag gaagatagtg ttggcggtgg 1080 ccagggcgga caaaggcgcg agagagtatt tagggtggtg atcaaatttg cggcccgtgc 1140 tgatctccat catctggcta tgtttctagc tggaaggcaa gcagacgctc ctcaagaagc 1200 tcttcaagtg cttgacattg tactacgtga attgcctact gcgaggtatt ctcctgttgg 1260 taggtcattt tattctccca acttagggag acgtcagcaa cttggtgagg gtttggaaag 1320 ttggcgcggt ttttaccaaa gcataaggcc gacacagatg ggcctttcac tgaatattga 1380 tatgtcctct actgcattta tcgagcctct ccctgtgatt gattttgttg ctcagcttct 1440 taatagagat atttcagtta ggccattgtc tgattctgat cgcgtgaaga tcaaaaaagc 1500 cttaagaggt gtgaaggttg aggtcactca caggggaaac atgcgcagaa agtatcgcat 1560 ttctggcctc acctcacaag caacaagaga gctatcattc cctgttgatg atcgtggtac 1620 tgtgaagact gtggtccaat acttcatgga gacttatggt tttagcatcc agcacaccac 1680 tttaccgtgc ttgcaagtgg gcaatcaaca aagaccaaat tatctgccta tggaggtttg 1740 caagatagtt gaaggacagc gttactcaaa gcgactcaat gagaaacana tcactgcttt 1800 actgaaagtg acctgccagc gccctcaaga gcgtgagctg gacattttac agactgtgca 1860 tcacaatgcg tactatgaag acccgtatgc acaggaattt ggtataagaa ttgatgaacg 1920 ccttgctgca gttgaagctc gtgttctgcc accaccaagg cttaaatacc atgatagtgg 1980 ccgagagaag gatgttttgc ccagagttgg ccaatggaac atgatgaata agaaaatggt 2040 aaatggtggc agagtcagca actgggcatg tattaacttc tctcggaatg tgcaagatag 2100 tgccgctagg ggtttctgtc atgaactggc aatcatgtgc caaatatcag gaatggattt 2160 ttcccttgag cctgtgctgc ctccagtgac tgcaaggcca gaacatgttg aaagagcgtt 2220 gaaggcacgt tatcaagatg caatgaacat actgaggcca caggggaggg aacttgatct 2280 gctgattgta atactgcctg acattaatgg ttccttatat ggggatctca aaaggatctg 2340 tgagactgat ctcggattgg tctcccagtg ttgtctgact aaacatgttt ttaagatgag 2400 caagcagtat cttgcaaatg ttgcactcaa aataaatgtt aaggttggtg gaaggaatac 2460 tgtacttgta gatgctttga caaggagaat cccccttgtc agtgacagac cgaccataat 2520 atttggtgct gatgttaccc atccacatcc tggagaagat tccagtcctt ccattgcagc 2580 tgtggttgct tcgcaagact ggcctgaggt caccaaatat gctggactag tgagtgccca 2640 agcccatcgc caggagctga tacaggatct tttcaaagta tggcaagatc cacagagaag 2700 gacagtaact ggtggcatga taaaggaact tctcatttct ttcaagagag caactggaca 2760 gaagccccag aggatcatat tctacaggga tggtgtcagt gagggacagt tctatcaagt 2820 attgttgtat gaacttgatg ccatcagaaa ggcatgtgca tccttggagc ccaactacca 2880 gcctccagtt acttttgtcg tggtgcagaa acgacatcac actaggctgt ttgctaataa 2940 ccacaacgat cagcgtacag ttgatagaag cggaaacata ctgcctggca ccgtggttga 3000 ttcgaagatt tgccatccta ctgaatttga tttctacctg tgtagccatg ctggcattca 3060 gggaacaagc cgccctgctc attaccatgt cctgtgggac gagaacaagt tcacagctga 3120 tgagctgcag actctgacaa acaacctatg ctacacgtac gctaggtgca cccgctccgt 3180 gtcaattgtg cccccggcat actatgctca tctggcagcc ttccgagctc gcttctacat 3240 ggagccagat acctctgaca gtggctcaat ggccagtggt gcccgtggcc ctccaccagg 3300 tgcggcacgc agcatgagag gagcggggag tgttgcggtc aggcccctac ctgctctcaa 3360 ggaaaacgtg aagcgtgtca tgttttactg ctgagatgct gagctacctt caccaagaaa 3420 atatcctgac ttgttccatg tacccgcact gtttcggtga tactatctga caccgaattt 3480 atgcattaag tcttccagtg gtctggagat tttaagtaac gcctgttttt attcgtgagt 3540 tgtaacgctg cagttcgagg agcttcagtg ctgtatgatg tgtaaactat ttgttgtaag 3600 ttgtaaccaa ttgttgtaag ttgtaaccag ccactatgtt ataatcctgt ttgtttcagc 3660 taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 3714 14 1073 PRT Zea mays UNSURE (539) Xaa = ANY AMINO ACID 14 Met Val Arg Lys Lys Arg Thr Gly Pro Gly Gly Ser Gly Glu Thr Ser 1 5 10 15 Gly Glu Ser Ser Gly Ala Ser Gly Gln Gly Ser Ser Gln Arg Pro Glu 20 25 30 Arg Thr Gln Gln Pro Gly Ala Gly Arg Gly Trp Val Pro Gln Gln Gly 35 40 45 Gly Arg Gly Gly Gly Gln His Gln Gly Arg Gly Gly His Tyr Gln Gly 50 55 60 Arg Gly Gly Pro Gly Pro His His Pro Gly Gly Leu Pro Glu Tyr His 65 70 75 80 Gln Arg Glu Tyr Gln Gly Arg Gly Gly Glu Tyr Gln Gly Gln Tyr Gln 85 90 95 Gly Arg Gly Gly Ala Arg Ser Arg Gly Gly Ile Ser Gln Pro Tyr Tyr 100 105 110 Gly Gly His Arg Gly Gly Ser Val Gly Arg Asn Val Pro Pro Gly Pro 115 120 125 Ser Arg Thr Val Pro Glu Leu His Gln Ala Pro Tyr Val Gln Tyr Gln 130 135 140 Ala Pro Val Ile Ser Pro Ser Pro Ser Gly Pro Gly Ser Ser Ser Gln 145 150 155 160 Pro Met Ala Glu Val Ser Ser Gly Gln Val Gln Gln Gln Phe Glu Gln 165 170 175 Leu Ala Ile His Gly Gln Ser Ser Met Ser Gln Glu Val Gln Val Ala 180 185 190 Pro Ala Ser Ser Lys Ser Val Arg Phe Pro Leu Arg Pro Gly Lys Gly 195 200 205 Thr Tyr Gly Asp Arg Cys Ile Val Lys Ala Asn His Phe Phe Ala Glu 210 215 220 Leu Pro Asp Lys Asp Leu His Gln Tyr Asp Val Thr Ile Thr Pro Glu 225 230 235 240 Val Thr Ser Arg Gly Val Asn Arg Ala Val Met Gly Glu Leu Val Thr 245 250 255 Leu Tyr Arg Gln Ser His Leu Gly Gly Arg Leu Pro Ala Tyr Asp Gly 260 265 270 Arg Lys Ser Leu Tyr Thr Ala Gly Pro Leu Pro Phe Thr Ser Met Thr 275 280 285 Phe Glu Ile Thr Leu Gln Asp Glu Glu Asp Ser Val Gly Gly Gly Gln 290 295 300 Gly Gly Gln Arg Arg Glu Arg Val Phe Arg Val Val Ile Lys Phe Ala 305 310 315 320 Ala Arg Ala Asp Leu His His Leu Ala Met Phe Leu Ala Gly Arg Gln 325 330 335 Ala Asp Ala Pro Gln Glu Ala Leu Gln Val Leu Asp Ile Val Leu Arg 340 345 350 Glu Leu Pro Thr Ala Arg Tyr Ser Pro Val Gly Arg Ser Phe Tyr Ser 355 360 365 Pro Asn Leu Gly Arg Arg Gln Gln Leu Gly Glu Gly Leu Glu Ser Trp 370 375 380 Arg Gly Phe Tyr Gln Ser Ile Arg Pro Thr Gln Met Gly Leu Ser Leu 385 390 395 400 Asn Ile Asp Met Ser Ser Thr Ala Phe Ile Glu Pro Leu Pro Val Ile 405 410 415 Asp Phe Val Ala Gln Leu Leu Asn Arg Asp Ile Ser Val Arg Pro Leu 420 425 430 Ser Asp Ser Asp Arg Val Lys Ile Lys Lys Ala Leu Arg Gly Val Lys 435 440 445 Val Glu Val Thr His Arg Gly Asn Met Arg Arg Lys Tyr Arg Ile Ser 450 455 460 Gly Leu Thr Ser Gln Ala Thr Arg Glu Leu Ser Phe Pro Val Asp Asp 465 470 475 480 Arg Gly Thr Val Lys Thr Val Val Gln Tyr Phe Met Glu Thr Tyr Gly 485 490 495 Phe Ser Ile Gln His Thr Thr Leu Pro Cys Leu Gln Val Gly Asn Gln 500 505 510 Gln Arg Pro Asn Tyr Leu Pro Met Glu Val Cys Lys Ile Val Glu Gly 515 520 525 Gln Arg Tyr Ser Lys Arg Leu Asn Glu Lys Xaa Ile Thr Ala Leu Leu 530 535 540 Lys Val Thr Cys Gln Arg Pro Gln Glu Arg Glu Leu Asp Ile Leu Gln 545 550 555 560 Thr Val His His Asn Ala Tyr Tyr Glu Asp Pro Tyr Ala Gln Glu Phe 565 570 575 Gly Ile Arg Ile Asp Glu Arg Leu Ala Ala Val Glu Ala Arg Val Leu 580 585 590 Pro Pro Pro Arg Leu Lys Tyr His Asp Ser Gly Arg Glu Lys Asp Val 595 600 605 Leu Pro Arg Val Gly Gln Trp Asn Met Met Asn Lys Lys Met Val Asn 610 615 620 Gly Gly Arg Val Ser Asn Trp Ala Cys Ile Asn Phe Ser Arg Asn Val 625 630 635 640 Gln Asp Ser Ala Ala Arg Gly Phe Cys His Glu Leu Ala Ile Met Cys 645 650 655 Gln Ile Ser Gly Met Asp Phe Ser Leu Glu Pro Val Leu Pro Pro Val 660 665 670 Thr Ala Arg Pro Glu His Val Glu Arg Ala Leu Lys Ala Arg Tyr Gln 675 680 685 Asp Ala Met Asn Ile Leu Arg Pro Gln Gly Arg Glu Leu Asp Leu Leu 690 695 700 Ile Val Ile Leu Pro Asp Ile Asn Gly Ser Leu Tyr Gly Asp Leu Lys 705 710 715 720 Arg Ile Cys Glu Thr Asp Leu Gly Leu Val Ser Gln Cys Cys Leu Thr 725 730 735 Lys His Val Phe Lys Met Ser Lys Gln Tyr Leu Ala Asn Val Ala Leu 740 745 750 Lys Ile Asn Val Lys Val Gly Gly Arg Asn Thr Val Leu Val Asp Ala 755 760 765 Leu Thr Arg Arg Ile Pro Leu Val Ser Asp Arg Pro Thr Ile Ile Phe 770 775 780 Gly Ala Asp Val Thr His Pro His Pro Gly Glu Asp Ser Ser Pro Ser 785 790 795 800 Ile Ala Ala Val Val Ala Ser Gln Asp Trp Pro Glu Val Thr Lys Tyr 805 810 815 Ala Gly Leu Val Ser Ala Gln Ala His Arg Gln Glu Leu Ile Gln Asp 820 825 830 Leu Phe Lys Val Trp Gln Asp Pro Gln Arg Arg Thr Val Thr Gly Gly 835 840 845 Met Ile Lys Glu Leu Leu Ile Ser Phe Lys Arg Ala Thr Gly Gln Lys 850 855 860 Pro Gln Arg Ile Ile Phe Tyr Arg Asp Gly Val Ser Glu Gly Gln Phe 865 870 875 880 Tyr Gln Val Leu Leu Tyr Glu Leu Asp Ala Ile Arg Lys Ala Cys Ala 885 890 895 Ser Leu Glu Pro Asn Tyr Gln Pro Pro Val Thr Phe Val Val Val Gln 900 905 910 Lys Arg His His Thr Arg Leu Phe Ala Asn Asn His Asn Asp Gln Arg 915 920 925 Thr Val Asp Arg Ser Gly Asn Ile Leu Pro Gly Thr Val Val Asp Ser 930 935 940 Lys Ile Cys His Pro Thr Glu Phe Asp Phe Tyr Leu Cys Ser His Ala 945 950 955 960 Gly Ile Gln Gly Thr Ser Arg Pro Ala His Tyr His Val Leu Trp Asp 965 970 975 Glu Asn Lys Phe Thr Ala Asp Glu Leu Gln Thr Leu Thr Asn Asn Leu 980 985 990 Cys Tyr Thr Tyr Ala Arg Cys Thr Arg Ser Val Ser Ile Val Pro Pro 995 1000 1005 Ala Tyr Tyr Ala His Leu Ala Ala Phe Arg Ala Arg Phe Tyr Met Glu 1010 1015 1020 Pro Asp Thr Ser Asp Ser Gly Ser Met Ala Ser Gly Ala Arg Gly Pro 1025 1030 1035 1040 Pro Pro Gly Ala Ala Arg Ser Met Arg Gly Ala Gly Ser Val Ala Val 1045 1050 1055 Arg Pro Leu Pro Ala Leu Lys Glu Asn Val Lys Arg Val Met Phe Tyr 1060 1065 1070 Cys 15 3072 DNA Zea mays unsure (411) n = A, C, G or T 15 ctccaaatac gtaacaaact tctacgtgag cgccagggca gttgcgccca tgggaaggca 60 tccgcctgtc gatgaggcga tggacttcaa cggcaacgga cgggacgagg caaacccgag 120 cggctctgag gcggggaacc acaacgagca ccgcggcgac gacccctcgc gcgttggcca 180 gagcctgccc gccgatatcc gccaaaatgg gcagccaacc ctcggggagg agatcaccgc 240 gccgctgtgg gaggagttcg aggcgctcgg catccacgtc cgccgctccg agcccgtgtt 300 cccgccgcgc ccagggtacg gcgccgcggg gacgccgtac gtcgtcaggg ccaacctctt 360 cctcggtcgc ctcgtcgacg aggccctgca tcagtacaac gtaaccattt ngcccgagcc 420 gacgcccaag gccgcgtaca gagagatcat gacgaagctg ttgtccgaga accagcacac 480 ggatttcgac ggccgcttct ccgtgtacga tgatggtgac tcgctcttca cagccggtgc 540 gctgccgttc gacaccaagg agttcgaggt ccccctctct gcaggcggcg acgaaaagat 600 ggacaggaag tacaaggtga tgatcaacca tgccgcaacg attagtctgc tacagctgag 660 gatgctgtta gcgggctatc ccacggacat ccccgcgcag gcgctcgtgg tcctcgacac 720 cgtgctgcgt gacgtcttca acgaacgcaa tgacatggaa tgcgtcgtga ttgacaaaaa 780 ggatcgcaca ctgggtgttg acgcatggaa ggggctctat ctgagcatca ggccaacaca 840 aaactgcttg tctctgattg cagacgtgtc ctcatctgta ttcgttcaac ccctgctatt 900 gattgaattc gttcagaaga tcctaaagat agatgccgtg gataggaact tgactaaacc 960 tgagtatgac aagctcttga aggccctcag gggtgtgagg attcaagtca cacacagaga 1020 taatagacgc cgagtatggt caaagaaaaa agataataga cgccaactct ctacgtacag 1080 agttgctggc ttgtcagtga atcctactaa tgatttgagt tttgaatcaa aggttggagt 1140 cacaacgact gtgattgatt acttcagaga aatatacggc ctggaactga aatacaaata 1200 tctcccatgc gtcaatgctg gcagcgagca ggatccaatc tattttccta tagaggtttg 1260 caagatagct cccaagcagt gttaccagaa gaagctggaa ggtagtcagt tttctactcc 1320 aaggaagtca gcctggatcc atcctgaagc cgagcaatcc tgtcctcaga ttgttgagca 1380 gaggcagtac aaacaaacca aacgtgcaaa tgaatttgac ttagaatttg atggcaatct 1440 tacaacagtt gctgctagag ttctgctgcc tccaaatctt aagtatgatg attctgtatc 1500 acagaaaaca tggtttccac tggatgggta ctggaatatg aaagacaaga aagtaataaa 1560 tggtgccaag atcagaaact gggcatgtct taatttttgt gaagatttat ccaaggaaga 1620 tattaagaag ttttgcttta agctggctga aatgtctcgt attactggac tggactttgc 1680 cgatttgaag ctcccaatat tcactgcacg tccagatcga gttgaagatg gtattcgtag 1740 gtgctatcag gaagcgaaga acaagctaag ggatcagaag attgatttac tgcttgctat 1800 actaccagat aaaaaagaca gtttatatgg aaatattaaa aggatctgtg agacagatat 1860 tggtcttgtg tcacagtgtt gtcgaaggtc aagagtctta gtgaataata atcagatatt 1920 ggcaaatatt gctattaaga tcaatgccaa ggttggagga agaatctcag tattcgatga 1980 cgtacagaag agtttaccgg ttgtttcaaa taagccaaca attatatttg gtgctcatgt 2040 ttctcaccct tctgttgtag atggttctac tggcccttct attgcttctg tcgttgcatc 2100 ccaagactgg catgaggtgt ctaagtataa tggtgttgtt cgtgcacaag gtcacactga 2160 agagatcggt ggccttgaag acattgtcaa ggagctcctt catgcatttg caaacgagtc 2220 caaggagaag ctccagcagc tgatattcta cagggatggc ataagtgagg gtcaattcaa 2280 tcgaattttg gagaaagaaa tcccagcgat agaaaaggct tggaacgcac tgtatgacaa 2340 tgagaagcca caaatcacct tcgttgttgt gcagaagagg cataaactga ggctgttccc 2400 cgtggacgac aactataaga tccgttctgc taagaagaaa attgttgagc ctggcacagt 2460 ggttgatagt gagatctgtc acccagcaga atttgatttc ttcctttgca gccaatctgg 2520 tggtatcaaa ggcccaaggc gtcctgtgag gtaccttgta ctgcgagatg ataacaactt 2580 cacggcagat gaactgcagg ctctcacaaa taacctgtgc tacacttatt caggcggcaa 2640 tcgttcgttg tcggtcgctc ctcccgcata ctacgcccaa aagctcgcac atcgggcccg 2700 cgtctacctc gccaaaggct cggacaataa tgcagcagct gctaatggtg gtcggaagca 2760 aattccagag ataaagaatg agctgaaggg gtccatgttc tactgctagt cctttgcctg 2820 ctgaacggac gatgcattgt tctatagtga aagacttgag tgtgctctga gtctctgact 2880 gacatctgga gaaggatggc atctgcaata gtcgccgtgt tctttttagt acactagaat 2940 aaatggatgt tttttgtgga cgcccatgtt gaactagttt tcttttccag taagtacttc 3000 agaatgagtg agataaatat atcattcagc gtctggtggt ctggcattgg aaaaaaaaaa 3060 aaaaaaaaaa ag 3072 16 919 PRT Zea mays UNSURE (121) Xaa = ANY AMINO ACID 16 Met Gly Arg His Pro Pro Val Asp Glu Ala Met Asp Phe Asn Gly Asn 1 5 10 15 Gly Arg Asp Glu Ala Asn Pro Ser Gly Ser Glu Ala Gly Asn His Asn 20 25 30 Glu His Arg Gly Asp Asp Pro Ser Arg Val Gly Gln Ser Leu Pro Ala 35 40 45 Asp Ile Arg Gln Asn Gly Gln Pro Thr Leu Gly Glu Glu Ile Thr Ala 50 55 60 Pro Leu Trp Glu Glu Phe Glu Ala Leu Gly Ile His Val Arg Arg Ser 65 70 75 80 Glu Pro Val Phe Pro Pro Arg Pro Gly Tyr Gly Ala Ala Gly Thr Pro 85 90 95 Tyr Val Val Arg Ala Asn Leu Phe Leu Gly Arg Leu Val Asp Glu Ala 100 105 110 Leu His Gln Tyr Asn Val Thr Ile Xaa Pro Glu Pro Thr Pro Lys Ala 115 120 125 Ala Tyr Arg Glu Ile Met Thr Lys Leu Leu Ser Glu Asn Gln His Thr 130 135 140 Asp Phe Asp Gly Arg Phe Ser Val Tyr Asp Asp Gly Asp Ser Leu Phe 145 150 155 160 Thr Ala Gly Ala Leu Pro Phe Asp Thr Lys Glu Phe Glu Val Pro Leu 165 170 175 Ser Ala Gly Gly Asp Glu Lys Met Asp Arg Lys Tyr Lys Val Met Ile 180 185 190 Asn His Ala Ala Thr Ile Ser Leu Leu Gln Leu Arg Met Leu Leu Ala 195 200 205 Gly Tyr Pro Thr Asp Ile Pro Ala Gln Ala Leu Val Val Leu Asp Thr 210 215 220 Val Leu Arg Asp Val Phe Asn Glu Arg Asn Asp Met Glu Cys Val Val 225 230 235 240 Ile Asp Lys Lys Asp Arg Thr Leu Gly Val Asp Ala Trp Lys Gly Leu 245 250 255 Tyr Leu Ser Ile Arg Pro Thr Gln Asn Cys Leu Ser Leu Ile Ala Asp 260 265 270 Val Ser Ser Ser Val Phe Val Gln Pro Leu Leu Leu Ile Glu Phe Val 275 280 285 Gln Lys Ile Leu Lys Ile Asp Ala Val Asp Arg Asn Leu Thr Lys Pro 290 295 300 Glu Tyr Asp Lys Leu Leu Lys Ala Leu Arg Gly Val Arg Ile Gln Val 305 310 315 320 Thr His Arg Asp Asn Arg Arg Arg Val Trp Ser Lys Lys Lys Asp Asn 325 330 335 Arg Arg Gln Leu Ser Thr Tyr Arg Val Ala Gly Leu Ser Val Asn Pro 340 345 350 Thr Asn Asp Leu Ser Phe Glu Ser Lys Val Gly Val Thr Thr Thr Val 355 360 365 Ile Asp Tyr Phe Arg Glu Ile Tyr Gly Leu Glu Leu Lys Tyr Lys Tyr 370 375 380 Leu Pro Cys Val Asn Ala Gly Ser Glu Gln Asp Pro Ile Tyr Phe Pro 385 390 395 400 Ile Glu Val Cys Lys Ile Ala Pro Lys Gln Cys Tyr Gln Lys Lys Leu 405 410 415 Glu Gly Ser Gln Phe Ser Thr Pro Arg Lys Ser Ala Trp Ile His Pro 420 425 430 Glu Ala Glu Gln Ser Cys Pro Gln Ile Val Glu Gln Arg Gln Tyr Lys 435 440 445 Gln Thr Lys Arg Ala Asn Glu Phe Asp Leu Glu Phe Asp Gly Asn Leu 450 455 460 Thr Thr Val Ala Ala Arg Val Leu Leu Pro Pro Asn Leu Lys Tyr Asp 465 470 475 480 Asp Ser Val Ser Gln Lys Thr Trp Phe Pro Leu Asp Gly Tyr Trp Asn 485 490 495 Met Lys Asp Lys Lys Val Ile Asn Gly Ala Lys Ile Arg Asn Trp Ala 500 505 510 Cys Leu Asn Phe Cys Glu Asp Leu Ser Lys Glu Asp Ile Lys Lys Phe 515 520 525 Cys Phe Lys Leu Ala Glu Met Ser Arg Ile Thr Gly Leu Asp Phe Ala 530 535 540 Asp Leu Lys Leu Pro Ile Phe Thr Ala Arg Pro Asp Arg Val Glu Asp 545 550 555 560 Gly Ile Arg Arg Cys Tyr Gln Glu Ala Lys Asn Lys Leu Arg Asp Gln 565 570 575 Lys Ile Asp Leu Leu Leu Ala Ile Leu Pro Asp Lys Lys Asp Ser Leu 580 585 590 Tyr Gly Asn Ile Lys Arg Ile Cys Glu Thr Asp Ile Gly Leu Val Ser 595 600 605 Gln Cys Cys Arg Arg Ser Arg Val Leu Val Asn Asn Asn Gln Ile Leu 610 615 620 Ala Asn Ile Ala Ile Lys Ile Asn Ala Lys Val Gly Gly Arg Ile Ser 625 630 635 640 Val Phe Asp Asp Val Gln Lys Ser Leu Pro Val Val Ser Asn Lys Pro 645 650 655 Thr Ile Ile Phe Gly Ala His Val Ser His Pro Ser Val Val Asp Gly 660 665 670 Ser Thr Gly Pro Ser Ile Ala Ser Val Val Ala Ser Gln Asp Trp His 675 680 685 Glu Val Ser Lys Tyr Asn Gly Val Val Arg Ala Gln Gly His Thr Glu 690 695 700 Glu Ile Gly Gly Leu Glu Asp Ile Val Lys Glu Leu Leu His Ala Phe 705 710 715 720 Ala Asn Glu Ser Lys Glu Lys Leu Gln Gln Leu Ile Phe Tyr Arg Asp 725 730 735 Gly Ile Ser Glu Gly Gln Phe Asn Arg Ile Leu Glu Lys Glu Ile Pro 740 745 750 Ala Ile Glu Lys Ala Trp Asn Ala Leu Tyr Asp Asn Glu Lys Pro Gln 755 760 765 Ile Thr Phe Val Val Val Gln Lys Arg His Lys Leu Arg Leu Phe Pro 770 775 780 Val Asp Asp Asn Tyr Lys Ile Arg Ser Ala Lys Lys Lys Ile Val Glu 785 790 795 800 Pro Gly Thr Val Val Asp Ser Glu Ile Cys His Pro Ala Glu Phe Asp 805 810 815 Phe Phe Leu Cys Ser Gln Ser Gly Gly Ile Lys Gly Pro Arg Arg Pro 820 825 830 Val Arg Tyr Leu Val Leu Arg Asp Asp Asn Asn Phe Thr Ala Asp Glu 835 840 845 Leu Gln Ala Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ser Gly Gly Asn 850 855 860 Arg Ser Leu Ser Val Ala Pro Pro Ala Tyr Tyr Ala Gln Lys Leu Ala 865 870 875 880 His Arg Ala Arg Val Tyr Leu Ala Lys Gly Ser Asp Asn Asn Ala Ala 885 890 895 Ala Ala Asn Gly Gly Arg Lys Gln Ile Pro Glu Ile Lys Asn Glu Leu 900 905 910 Lys Gly Ser Met Phe Tyr Cys 915 17 400 DNA Zea mays unsure (286) n = A, C, G or T 17 caagaaggca caagggtgtc agttgtgcat tactttaaac aacgatataa ctactactta 60 caatacactc actggccatg ccttcaagct ggccgtgttg acaagcagat ctatttacct 120 atagaggttt gcagcatagt tcagggacaa cgctactcca gtaagctgaa tgagaatcaa 180 gtcaggaaca tcctgcagtt tacctgcgag cgaccagcag ataggcaaac tagaactttt 240 gaggtattca agaattacaa atctgatgga tcaacttatg caaaanaatt tggccttacg 300 tttgatggat caacttacgn ntnggatgct cgagttgctc ccagtccaag gcttaaatac 360 catgatccga naaaaaaagt ttnggcaacc tccatcggaa 400 18 126 PRT Zea mays UNSURE (96) Xaa = ANY AMINO ACID 18 Gln Glu Gly Thr Arg Val Ser Val Val His Tyr Phe Lys Gln Arg Tyr 1 5 10 15 Asn Tyr Tyr Leu Gln Tyr Thr His Trp Pro Cys Leu Gln Ala Gly Arg 20 25 30 Val Asp Lys Gln Ile Tyr Leu Pro Ile Glu Val Cys Ser Ile Val Gln 35 40 45 Gly Gln Arg Tyr Ser Ser Lys Leu Asn Glu Asn Gln Val Arg Asn Ile 50 55 60 Leu Gln Phe Thr Cys Glu Arg Pro Ala Asp Arg Gln Thr Arg Thr Phe 65 70 75 80 Glu Val Phe Lys Asn Tyr Lys Ser Asp Gly Ser Thr Tyr Ala Lys Xaa 85 90 95 Phe Gly Leu Thr Phe Asp Gly Ser Thr Tyr Xaa Xaa Asp Ala Arg Val 100 105 110 Ala Pro Ser Pro Arg Leu Lys Tyr His Asp Pro Xaa Lys Lys 115 120 125 19 550 DNA Zea mays unsure (479) n = A, C, G or T 19 cggacgcgtg ggcaagattg tagaagggca gagatactct aagaagctta atgacagaca 60 agtgacgaac atacttagag caacttgtaa acgtccccag gagagagaga agagcatacg 120 tgatatggtt ctgcataaca agtatgcaga tgataagttt gctcaggagt ttggcatcga 180 agttagcagt gatctagtga ctgttccagc ccgtgtgctg cctccacccc tgttgaaata 240 tcatgactct ggtagggaga aaacttgtgc accaagtgtt ggacaatgga acatgatcaa 300 taagaaaatg atcaatggtg gaactattga taactggact tgtttgaact tttcacgcat 360 gcgccctgat gaagtacaaa ggttctgtat ggatctgact catatgtgca atgccactgg 420 aatggttgtc aatccacgcc catttattga aatccggtct gctgctccta accatatana 480 naatgctttg ananatgttc acaagaaaac cncccaaata cttgcccaca aacatgggaa 540 atcnactcca 550 20 163 PRT Zea mays UNSURE (160) Xaa = ANY AMINO ACID 20 Gly Arg Val Gly Lys Ile Val Glu Gly Gln Arg Tyr Ser Lys Lys Leu 1 5 10 15 Asn Asp Arg Gln Val Thr Asn Ile Leu Arg Ala Thr Cys Lys Arg Pro 20 25 30 Gln Glu Arg Glu Lys Ser Ile Arg Asp Met Val Leu His Asn Lys Tyr 35 40 45 Ala Asp Asp Lys Phe Ala Gln Glu Phe Gly Ile Glu Val Ser Ser Asp 50 55 60 Leu Val Thr Val Pro Ala Arg Val Leu Pro Pro Pro Leu Leu Lys Tyr 65 70 75 80 His Asp Ser Gly Arg Glu Lys Thr Cys Ala Pro Ser Val Gly Gln Trp 85 90 95 Asn Met Ile Asn Lys Lys Met Ile Asn Gly Gly Thr Ile Asp Asn Trp 100 105 110 Thr Cys Leu Asn Phe Ser Arg Met Arg Pro Asp Glu Val Gln Arg Phe 115 120 125 Cys Met Asp Leu Thr His Met Cys Asn Ala Thr Gly Met Val Val Asn 130 135 140 Pro Arg Pro Phe Ile Glu Ile Arg Ser Ala Ala Pro Asn His Ile Xaa 145 150 155 160 Asn Ala Leu 21 3009 DNA Zea mays 21 ctcgcctcgt ccgtcctcct gcctacttcc ttgcttttgg taggtgctgc ttgttttatc 60 ttgaaatggg ctctcatgat ggcgaggatg aagagttgcc acccccccct ccggtgccac 120 cagatgtgat tcccattaaa gctgaagatg ctgtgggtga atcaccagca aaccatatat 180 taaagccaaa gagattactg atggacaggc ctggtatagg aagaaaaggg cagccgaccc 240 agctctattc aaatcacttt aaagtcgctg tgaagagtac agaagacgtc ttctttcact 300 actatgtaaa cctgaagtat gaggatgatc gacccgttga tggtaaaggg atcggcagaa 360 aggtgattga taaactgcag cagacatatc gtgcagagct ttctaacaag gactttgcat 420 atgatggaga aaagagcctg tttacagttg gtggtcttcc acaaaaaaag aatgagttca 480 ccgttgtctt ggaggacgta tctactggaa agactgctgc caatgggagc cctggaggta 540 atgacagtcc tggaggtggt gataggaaga gagtgaggag gccataccag acgaaaactt 600 tcaaagtgga gataaatttt gcagcagagg ttcctatgag tgctattggt caagtcatta 660 gaggcgaaga atctgagaac tccctggagg cgcttcgtgt tcttgatatc atactgaggc 720 agcattccgc agaacaaggc tgccttttgg ttaagcaatc atttttctac aacaaccctt 780 catgctttgt tgacttgggt ggtggtgtga tgggttgtcg tggatttcat tcaagcttcc 840 gtggcacaca gagtggactt tccctcaatg ttgatgtctc aacaacaatg atcgtgaaac 900 ctggccctgt tattgatttt cttctttcta accagaatgt taatgatcct agcagaattg 960 attggcaaaa ggccaagcgt gctctcaagg gcttgaggat tagaaccact cctgcaaatt 1020 cagaattcaa gatttttggt ctcagcgaga ggatctgcaa agaacaaacg tttccgctga 1080 ggcagagaaa tggtagcaac ggagattgtg ataccattga aataactgtc tatgactact 1140 atgcaaagaa aggaatcgat ctaaagtatt ctggtgattt cccctgtata aatacaggga 1200 aggcaaagcg cccaacatat tttccaatcg agctatgctc gcttgttccg cttcaaagat 1260 acaccaaagc tttgtctacg ctacaaaggt catcccttgt ggagaagtct agacagaagc 1320 ctgaagaaag gatgaccgtt ctaaatgatg cactgcaacg cagtaactac gattctgacc 1380 ccatgttgag ggcatgtggt gtttcagttg ctccaaaatt tacccaagtt gaaggaagga 1440 tccttcaagc cccaaagctg aaagccggca atggtgatga tatcttttca cgaaatggac 1500 ggtggaattt cactaatagg aagttttatg aaacctgctc tgtgaataag tgggcggtcg 1560 ttaatttctc tgcacgttgt gatgttcgga atcttatccg tgacctgatg aggaatgcat 1620 ctgcaaaggg aattcaaatg gaggaacctt ttgatgtgtt tgaagagagt ccctctatga 1680 ggcgtgcacc tgtgtcaaga agggtggatg atatgtttgg gcagataaaa tcaaaacttc 1740 ctggagctcc taggttcctc ttgtgccttc tccctgagag gaaaaattgt gaaatctatg 1800 gtccttggaa gagaaagtgc ctggccgagt ttggtattgt cacacagtgt ctagctccat 1860 taagagtcaa tgatccgtac ctgcttaatt tgctgatgaa gatcaatgca aagcttggtg 1920 gtctgaactc gttgctgcaa gttgaagcat cttcgtcaat accacatgtg tcgcaagtac 1980 ccaccatcat cttaggtatg gatgtttcac atggtcatcc aggacaagat agaccttcgg 2040 ttgcagcggt ggttagttct cgtcaatggc ctcttatctc tagatataga gcatcagtgc 2100 acacccaatc tgccagacta gaaatgatgt cctcgttgtt taagccgcgg ggtactgatg 2160 atgatggcct catccgggaa tcactgatcg acttccacac tagctctgga aagcgaaaac 2220 cagaacacat aattattttc agggatggag tcagtgaaag tcagtttacc caggtcatca 2280 acattgagct ggatcagatc atcgaggcat gtaagtttct ggatgagaag tggtcaccca 2340 agttcactgt gattgttgct caaaagaacc accacaccaa gttctttcag acggcatcac 2400 cagacaatgt tcttcctgga actgtggtgg atagtaaagt ttgccatcct aagaacttcg 2460 acttctacat gtgtgcacat gctgggatga ttggaacaac aaggccgacc cactatcatg 2520 ttctgcacga cgagataggt ttcagtgccg acgagatgca ggagtttgtt cattcgctct 2580 cttacgtgta ccagaggagc acgacagcca tctcagttgt tgctccagtg tgctacgccc 2640 acctcgctgc agcccaggtg agcacgttcc tgagattgga ggagatgtca gacgcgtcct 2700 ccagccaggg aggagggcat acctcggctg gcagtgctcc tgtgccggag ctgcctcgcc 2760 tgcatgacaa agtcaggagc tccatgttct tctgctagct gatgtgcgtg cgcatcagga 2820 tcgagctcca tgttttgtgt tagtaaggcc tagttagtaa ggctgtagaa agaatgttta 2880 atgtttgcat gctaaagtcc aaacaatcaa aaccactact atatctacca gagcactgat 2940 cgatcaaaca acaagagtca gcatcaatca atcaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000 aaaaaaaaa 3009 22 910 PRT Zea mays 22 Met Gly Ser His Asp Gly Glu Asp Glu Glu Leu Pro Pro Pro Pro Pro 1 5 10 15 Val Pro Pro Asp Val Ile Pro Ile Lys Ala Glu Asp Ala Val Gly Glu 20 25 30 Ser Pro Ala Asn His Ile Leu Lys Pro Lys Arg Leu Leu Met Asp Arg 35 40 45 Pro Gly Ile Gly Arg Lys Gly Gln Pro Thr Gln Leu Tyr Ser Asn His 50 55 60 Phe Lys Val Ala Val Lys Ser Thr Glu Asp Val Phe Phe His Tyr Tyr 65 70 75 80 Val Asn Leu Lys Tyr Glu Asp Asp Arg Pro Val Asp Gly Lys Gly Ile 85 90 95 Gly Arg Lys Val Ile Asp Lys Leu Gln Gln Thr Tyr Arg Ala Glu Leu 100 105 110 Ser Asn Lys Asp Phe Ala Tyr Asp Gly Glu Lys Ser Leu Phe Thr Val 115 120 125 Gly Gly Leu Pro Gln Lys Lys Asn Glu Phe Thr Val Val Leu Glu Asp 130 135 140 Val Ser Thr Gly Lys Thr Ala Ala Asn Gly Ser Pro Gly Gly Asn Asp 145 150 155 160 Ser Pro Gly Gly Gly Asp Arg Lys Arg Val Arg Arg Pro Tyr Gln Thr 165 170 175 Lys Thr Phe Lys Val Glu Ile Asn Phe Ala Ala Glu Val Pro Met Ser 180 185 190 Ala Ile Gly Gln Val Ile Arg Gly Glu Glu Ser Glu Asn Ser Leu Glu 195 200 205 Ala Leu Arg Val Leu Asp Ile Ile Leu Arg Gln His Ser Ala Glu Gln 210 215 220 Gly Cys Leu Leu Val Lys Gln Ser Phe Phe Tyr Asn Asn Pro Ser Cys 225 230 235 240 Phe Val Asp Leu Gly Gly Gly Val Met Gly Cys Arg Gly Phe His Ser 245 250 255 Ser Phe Arg Gly Thr Gln Ser Gly Leu Ser Leu Asn Val Asp Val Ser 260 265 270 Thr Thr Met Ile Val Lys Pro Gly Pro Val Ile Asp Phe Leu Leu Ser 275 280 285 Asn Gln Asn Val Asn Asp Pro Ser Arg Ile Asp Trp Gln Lys Ala Lys 290 295 300 Arg Ala Leu Lys Gly Leu Arg Ile Arg Thr Thr Pro Ala Asn Ser Glu 305 310 315 320 Phe Lys Ile Phe Gly Leu Ser Glu Arg Ile Cys Lys Glu Gln Thr Phe 325 330 335 Pro Leu Arg Gln Arg Asn Gly Ser Asn Gly Asp Cys Asp Thr Ile Glu 340 345 350 Ile Thr Val Tyr Asp Tyr Tyr Ala Lys Lys Gly Ile Asp Leu Lys Tyr 355 360 365 Ser Gly Asp Phe Pro Cys Ile Asn Thr Gly Lys Ala Lys Arg Pro Thr 370 375 380 Tyr Phe Pro Ile Glu Leu Cys Ser Leu Val Pro Leu Gln Arg Tyr Thr 385 390 395 400 Lys Ala Leu Ser Thr Leu Gln Arg Ser Ser Leu Val Glu Lys Ser Arg 405 410 415 Gln Lys Pro Glu Glu Arg Met Thr Val Leu Asn Asp Ala Leu Gln Arg 420 425 430 Ser Asn Tyr Asp Ser Asp Pro Met Leu Arg Ala Cys Gly Val Ser Val 435 440 445 Ala Pro Lys Phe Thr Gln Val Glu Gly Arg Ile Leu Gln Ala Pro Lys 450 455 460 Leu Lys Ala Gly Asn Gly Asp Asp Ile Phe Ser Arg Asn Gly Arg Trp 465 470 475 480 Asn Phe Thr Asn Arg Lys Phe Tyr Glu Thr Cys Ser Val Asn Lys Trp 485 490 495 Ala Val Val Asn Phe Ser Ala Arg Cys Asp Val Arg Asn Leu Ile Arg 500 505 510 Asp Leu Met Arg Asn Ala Ser Ala Lys Gly Ile Gln Met Glu Glu Pro 515 520 525 Phe Asp Val Phe Glu Glu Ser Pro Ser Met Arg Arg Ala Pro Val Ser 530 535 540 Arg Arg Val Asp Asp Met Phe Gly Gln Ile Lys Ser Lys Leu Pro Gly 545 550 555 560 Ala Pro Arg Phe Leu Leu Cys Leu Leu Pro Glu Arg Lys Asn Cys Glu 565 570 575 Ile Tyr Gly Pro Trp Lys Arg Lys Cys Leu Ala Glu Phe Gly Ile Val 580 585 590 Thr Gln Cys Leu Ala Pro Leu Arg Val Asn Asp Pro Tyr Leu Leu Asn 595 600 605 Leu Leu Met Lys Ile Asn Ala Lys Leu Gly Gly Leu Asn Ser Leu Leu 610 615 620 Gln Val Glu Ala Ser Ser Ser Ile Pro His Val Ser Gln Val Pro Thr 625 630 635 640 Ile Ile Leu Gly Met Asp Val Ser His Gly His Pro Gly Gln Asp Arg 645 650 655 Pro Ser Val Ala Ala Val Val Ser Ser Arg Gln Trp Pro Leu Ile Ser 660 665 670 Arg Tyr Arg Ala Ser Val His Thr Gln Ser Ala Arg Leu Glu Met Met 675 680 685 Ser Ser Leu Phe Lys Pro Arg Gly Thr Asp Asp Asp Gly Leu Ile Arg 690 695 700 Glu Ser Leu Ile Asp Phe His Thr Ser Ser Gly Lys Arg Lys Pro Glu 705 710 715 720 His Ile Ile Ile Phe Arg Asp Gly Val Ser Glu Ser Gln Phe Thr Gln 725 730 735 Val Ile Asn Ile Glu Leu Asp Gln Ile Ile Glu Ala Cys Lys Phe Leu 740 745 750 Asp Glu Lys Trp Ser Pro Lys Phe Thr Val Ile Val Ala Gln Lys Asn 755 760 765 His His Thr Lys Phe Phe Gln Thr Ala Ser Pro Asp Asn Val Leu Pro 770 775 780 Gly Thr Val Val Asp Ser Lys Val Cys His Pro Lys Asn Phe Asp Phe 785 790 795 800 Tyr Met Cys Ala His Ala Gly Met Ile Gly Thr Thr Arg Pro Thr His 805 810 815 Tyr His Val Leu His Asp Glu Ile Gly Phe Ser Ala Asp Glu Met Gln 820 825 830 Glu Phe Val His Ser Leu Ser Tyr Val Tyr Gln Arg Ser Thr Thr Ala 835 840 845 Ile Ser Val Val Ala Pro Val Cys Tyr Ala His Leu Ala Ala Ala Gln 850 855 860 Val Ser Thr Phe Leu Arg Leu Glu Glu Met Ser Asp Ala Ser Ser Ser 865 870 875 880 Gln Gly Gly Gly His Thr Ser Ala Gly Ser Ala Pro Val Pro Glu Leu 885 890 895 Pro Arg Leu His Asp Lys Val Arg Ser Ser Met Phe Phe Cys 900 905 910 23 2429 DNA Oryza sativa 23 aaaccattca agttcttgat gttgtcctta gggagtcacc atcttggaat tatgtcacag 60 tgtccagatc cttcttctct acccagtttg gtcaccgggg tgacattggt gagggacttg 120 agtgttggag aggttactat cagagcctgc gcccaacaca gatgggcctt tcgctgaata 180 tagatatatc tgcaacgtcc ttttttaagc ctgtgacagt gatccaattt gtggaggagt 240 tcctgaacat acgtgacacc tcaagacctt tgtcagaccg ggatcgtgtg aagataaaga 300 aagcattacg tggggttcgc attgaaacaa accaccaaga ggaccaaatc agaagataca 360 agataacagg gattaccccc attcctatga gccagctgat atttcctgtt gatgataatg 420 ggacaaggaa gactgttgtt cagtacttct gggataggta caattacaga ctgaagtacg 480 cttcttggcc ctgcctacag tctggcagtg attctcgccc tgtatactta cctatggagg 540 tgtgcaagat tgtagaaggg cagaggtact ccaagaagct taatgacaaa caagtgacca 600 acatccttag agcaacctgt caacgccccc agcagaggga acagagcatt catgagatgg 660 ttctccacaa caagtataca gaggataggt ttgctcagga gttcggtatc aaggtctgca 720 atgacctagt ctctgttcca gcccgtgtgc tgcctccacc catgttgaag tatcatgatt 780 ctggaaggga gaaaacttgt gcacccagtg ttggacagtg gaacatgatt aacaagaaaa 840 tgatcaatgg aggaactgtg gataactgga catgtctgag tttttcacga atgcgtcctg 900 aggaggtaca aaggttctgt ggtgacctga ttcagatgtg caatgccact ggaatgtctt 960 tcaatccaag accagtcgtg gatgtccggt caacaaatcc taacaatata gagaatgctc 1020 tgagggatgt tcacaggaga acatcagaac tgctagccag agagggaaag ggaggcctgc 1080 agcttttaat tgtaattctg cctgaagtta gtggttctta tgggaaaatt aaaagggtct 1140 gtgagactga ccttggcatt gtatctcaat gttgtttgcc aaggcatgcc agcaggccga 1200 acaagcaata tttggaaaat gttgcactca aaatcaatgt caaggtcgga gggcgcaaca 1260 ctgttcttga gcgagccttt atccgcaatg gcataccatt tgtgtcagaa gtcccaacaa 1320 tcatctttgg cgctgatgtc acacaccctc cacctggaga ggactctgca tcatctattg 1380 ctgcggttgt ggcatctatg gattggcctg aaatcaccaa ataccgaggt ctggtctctg 1440 ctcaaccaca tagacaggag ataatagaag atctctttag tgttggtaaa gatccagtga 1500 aggttgtaaa tggtgggatg atcagggagt tgcttatcgc attccgcaag aagactggca 1560 gaaggcctga gaggataatc ttctatagag atggtgtaag tgaaggtcag ttcagccatg 1620 tgcttcttca tgaaatggat gccatcagaa aggcttgtgc atctttggag gagggatatc 1680 taccacctgt cacatttgta gtagttcaga aaaggcatca cacaaggctt ttcccagagg 1740 ttcatgggag gcgagacatg actgacaaga gcggaaacat ccttcctgga actgtcgtgg 1800 accgtcagat ttgccatcct acagagttcg atttctacct gtgtagccat gctggcatac 1860 agggtactag caggccaact cattaccatg tcctttacga tgagaaccat tttacagccg 1920 atgcacttca gtccctgacc aacaatcttt gctataccta tgcgcgatgc acccgggcag 1980 tgtctgtggt cccaccggcc tactatgctc atcttgctgc attccgcgct cgctactacg 2040 tggaaggaga gagttcggat ggtggctcga cccctggcag cagcgggcag gctgtggcgc 2100 gagagggccc tgtggaggtg cgccagcttc ccaagatcaa ggagaacgtc aaggacgtca 2160 tgttctactg ctgaggagat tgttggcaag gagagcccaa tattctggta gttttttggt 2220 tggtagactt gtttgtgtcc ttggtttgga gctggttgct tgtagttcca tttgctgttt 2280 ccgagtagcc ggattgtgac tgagcttttg tggtctttaa ggccttaact ctgcttgaga 2340 caatgcaagt cttttaaatt tccctgtggc taaaaaagaa gaaaaacaag aaaaaaaaaa 2400 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2429 24 723 PRT Oryza sativa 24 Thr Ile Gln Val Leu Asp Val Val Leu Arg Glu Ser Pro Ser Trp Asn 1 5 10 15 Tyr Val Thr Val Ser Arg Ser Phe Phe Ser Thr Gln Phe Gly His Arg 20 25 30 Gly Asp Ile Gly Glu Gly Leu Glu Cys Trp Arg Gly Tyr Tyr Gln Ser 35 40 45 Leu Arg Pro Thr Gln Met Gly Leu Ser Leu Asn Ile Asp Ile Ser Ala 50 55 60 Thr Ser Phe Phe Lys Pro Val Thr Val Ile Gln Phe Val Glu Glu Phe 65 70 75 80 Leu Asn Ile Arg Asp Thr Ser Arg Pro Leu Ser Asp Arg Asp Arg Val 85 90 95 Lys Ile Lys Lys Ala Leu Arg Gly Val Arg Ile Glu Thr Asn His Gln 100 105 110 Glu Asp Gln Ile Arg Arg Tyr Lys Ile Thr Gly Ile Thr Pro Ile Pro 115 120 125 Met Ser Gln Leu Ile Phe Pro Val Asp Asp Asn Gly Thr Arg Lys Thr 130 135 140 Val Val Gln Tyr Phe Trp Asp Arg Tyr Asn Tyr Arg Leu Lys Tyr Ala 145 150 155 160 Ser Trp Pro Cys Leu Gln Ser Gly Ser Asp Ser Arg Pro Val Tyr Leu 165 170 175 Pro Met Glu Val Cys Lys Ile Val Glu Gly Gln Arg Tyr Ser Lys Lys 180 185 190 Leu Asn Asp Lys Gln Val Thr Asn Ile Leu Arg Ala Thr Cys Gln Arg 195 200 205 Pro Gln Gln Arg Glu Gln Ser Ile His Glu Met Val Leu His Asn Lys 210 215 220 Tyr Thr Glu Asp Arg Phe Ala Gln Glu Phe Gly Ile Lys Val Cys Asn 225 230 235 240 Asp Leu Val Ser Val Pro Ala Arg Val Leu Pro Pro Pro Met Leu Lys 245 250 255 Tyr His Asp Ser Gly Arg Glu Lys Thr Cys Ala Pro Ser Val Gly Gln 260 265 270 Trp Asn Met Ile Asn Lys Lys Met Ile Asn Gly Gly Thr Val Asp Asn 275 280 285 Trp Thr Cys Leu Ser Phe Ser Arg Met Arg Pro Glu Glu Val Gln Arg 290 295 300 Phe Cys Gly Asp Leu Ile Gln Met Cys Asn Ala Thr Gly Met Ser Phe 305 310 315 320 Asn Pro Arg Pro Val Val Asp Val Arg Ser Thr Asn Pro Asn Asn Ile 325 330 335 Glu Asn Ala Leu Arg Asp Val His Arg Arg Thr Ser Glu Leu Leu Ala 340 345 350 Arg Glu Gly Lys Gly Gly Leu Gln Leu Leu Ile Val Ile Leu Pro Glu 355 360 365 Val Ser Gly Ser Tyr Gly Lys Ile Lys Arg Val Cys Glu Thr Asp Leu 370 375 380 Gly Ile Val Ser Gln Cys Cys Leu Pro Arg His Ala Ser Arg Pro Asn 385 390 395 400 Lys Gln Tyr Leu Glu Asn Val Ala Leu Lys Ile Asn Val Lys Val Gly 405 410 415 Gly Arg Asn Thr Val Leu Glu Arg Ala Phe Ile Arg Asn Gly Ile Pro 420 425 430 Phe Val Ser Glu Val Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His 435 440 445 Pro Pro Pro Gly Glu Asp Ser Ala Ser Ser Ile Ala Ala Val Val Ala 450 455 460 Ser Met Asp Trp Pro Glu Ile Thr Lys Tyr Arg Gly Leu Val Ser Ala 465 470 475 480 Gln Pro His Arg Gln Glu Ile Ile Glu Asp Leu Phe Ser Val Gly Lys 485 490 495 Asp Pro Val Lys Val Val Asn Gly Gly Met Ile Arg Glu Leu Leu Ile 500 505 510 Ala Phe Arg Lys Lys Thr Gly Arg Arg Pro Glu Arg Ile Ile Phe Tyr 515 520 525 Arg Asp Gly Val Ser Glu Gly Gln Phe Ser His Val Leu Leu His Glu 530 535 540 Met Asp Ala Ile Arg Lys Ala Cys Ala Ser Leu Glu Glu Gly Tyr Leu 545 550 555 560 Pro Pro Val Thr Phe Val Val Val Gln Lys Arg His His Thr Arg Leu 565 570 575 Phe Pro Glu Val His Gly Arg Arg Asp Met Thr Asp Lys Ser Gly Asn 580 585 590 Ile Leu Pro Gly Thr Val Val Asp Arg Gln Ile Cys His Pro Thr Glu 595 600 605 Phe Asp Phe Tyr Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser Arg 610 615 620 Pro Thr His Tyr His Val Leu Tyr Asp Glu Asn His Phe Thr Ala Asp 625 630 635 640 Ala Leu Gln Ser Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys 645 650 655 Thr Arg Ala Val Ser Val Val Pro Pro Ala Tyr Tyr Ala His Leu Ala 660 665 670 Ala Phe Arg Ala Arg Tyr Tyr Val Glu Gly Glu Ser Ser Asp Gly Gly 675 680 685 Ser Thr Pro Gly Ser Ser Gly Gln Ala Val Ala Arg Glu Gly Pro Val 690 695 700 Glu Val Arg Gln Leu Pro Lys Ile Lys Glu Asn Val Lys Asp Val Met 705 710 715 720 Phe Tyr Cys 25 1619 DNA Oryza sativa 25 cttacattct ggaagggtga gagtatgcac gccagaggat ggggcgtgga acatgaaaga 60 caagaaagta gttaacggtg ctacaattaa aagctgggca tgtgtcaact tgtgcgaggg 120 tttggataat cgtgttgttg aagcattctg ccttcaattg gtcagaacgt ccaaaataac 180 tggactggac tttgcgaatg tgagccttcc aatattgaaa gctgatcctc ataatgttaa 240 aactgatctt cctatgcgct atcaggaagc atgcagctgg tcgagggata acaagattga 300 cctcctactt gttgtaatga cagatgataa aaataatgcc agcttatatg gtgacgttaa 360 aagaatctgt gaaacagaaa tcggtgtatt gtcacagtgt tgtcgagcga agcaagtcta 420 caaggagagg aatgttcagt actgcgcaaa tgttgctctt aagatcaatg ccaaggctgg 480 aggaaggaac tcggtatttc ttaatgtaga agcaagttta ccggttgttt caaagagccc 540 aactattata tttggtgctg atgttaccca tcctgggtcc tttgatgaaa gtaccccttc 600 cattgcttcg gttgttgctt ccgcagactg gcctgaggtg accaagtata attctgttgt 660 tcgtatgcaa gcttctcgta aggagattat acaagatctt gatagcattg ttagggaact 720 tctcaatgca ttcaaaaggg actccaagat ggagccgaag cagctcattt tctacaggga 780 cggcgtaagc gagggtcagt tccagcaagt tgtagagagc gaaataccgg agatagaaaa 840 ggcttggaag tctctgtatg ctggcaagcc acgaattacc ttcatagtgg tgcagaagag 900 gcatcataca aggctgttcc ccaacaatta caatgatcca cgcggcatgg atgggactgg 960 aaatgttcgt ccaggcacag tagttgatac agtgatctgt caccctcgag agtttgattt 1020 cttcctgtgc agccaagccg ggatcaaagg gacaagccgt cctagccatt accatgtgct 1080 gcgcgacgac aacaacttca ccgcagatca gcttcagtct gtcacaaaca acctgtgcta 1140 cttatataca agctgcactc gctcggtgtc tattccacct cctgtttact acgctcataa 1200 gctcgcattc cgcgctcgtt tctacctcac ccaagttccc gtcgccggtg gagatccagg 1260 tgctgctaag ttccagtggg tacttccaga gattaaggaa gaggtgaaaa agtccatgtt 1320 cttttgctag tcgtccttgt gcccccctga aactgaagcc tggagccagc cggcaagctc 1380 tggaaatgct ctgaataatc aaacttggaa gaataagcac ctgcccaggt tgccattcgt 1440 ttccatgtgg catggaggat ggcatcctga aaaggatatt gtcatgtttg tgtggttttt 1500 aaacgacatt gaagtttatc tccggtgtta ctatctcagc actttggatg ttttattttg 1560 ttatgtctga agatatagac acaaaacttc atttttgttt caaaaaaaaa aaaaaaaaa 1619 26 442 PRT Oryza sativa 26 Leu His Ser Gly Arg Val Arg Val Cys Thr Pro Glu Asp Gly Ala Trp 1 5 10 15 Asn Met Lys Asp Lys Lys Val Val Asn Gly Ala Thr Ile Lys Ser Trp 20 25 30 Ala Cys Val Asn Leu Cys Glu Gly Leu Asp Asn Arg Val Val Glu Ala 35 40 45 Phe Cys Leu Gln Leu Val Arg Thr Ser Lys Ile Thr Gly Leu Asp Phe 50 55 60 Ala Asn Val Ser Leu Pro Ile Leu Lys Ala Asp Pro His Asn Val Lys 65 70 75 80 Thr Asp Leu Pro Met Arg Tyr Gln Glu Ala Cys Ser Trp Ser Arg Asp 85 90 95 Asn Lys Ile Asp Leu Leu Leu Val Val Met Thr Asp Asp Lys Asn Asn 100 105 110 Ala Ser Leu Tyr Gly Asp Val Lys Arg Ile Cys Glu Thr Glu Ile Gly 115 120 125 Val Leu Ser Gln Cys Cys Arg Ala Lys Gln Val Tyr Lys Glu Arg Asn 130 135 140 Val Gln Tyr Cys Ala Asn Val Ala Leu Lys Ile Asn Ala Lys Ala Gly 145 150 155 160 Gly Arg Asn Ser Val Phe Leu Asn Val Glu Ala Ser Leu Pro Val Val 165 170 175 Ser Lys Ser Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro Gly 180 185 190 Ser Phe Asp Glu Ser Thr Pro Ser Ile Ala Ser Val Val Ala Ser Ala 195 200 205 Asp Trp Pro Glu Val Thr Lys Tyr Asn Ser Val Val Arg Met Gln Ala 210 215 220 Ser Arg Lys Glu Ile Ile Gln Asp Leu Asp Ser Ile Val Arg Glu Leu 225 230 235 240 Leu Asn Ala Phe Lys Arg Asp Ser Lys Met Glu Pro Lys Gln Leu Ile 245 250 255 Phe Tyr Arg Asp Gly Val Ser Glu Gly Gln Phe Gln Gln Val Val Glu 260 265 270 Ser Glu Ile Pro Glu Ile Glu Lys Ala Trp Lys Ser Leu Tyr Ala Gly 275 280 285 Lys Pro Arg Ile Thr Phe Ile Val Val Gln Lys Arg His His Thr Arg 290 295 300 Leu Phe Pro Asn Asn Tyr Asn Asp Pro Arg Gly Met Asp Gly Thr Gly 305 310 315 320 Asn Val Arg Pro Gly Thr Val Val Asp Thr Val Ile Cys His Pro Arg 325 330 335 Glu Phe Asp Phe Phe Leu Cys Ser Gln Ala Gly Ile Lys Gly Thr Ser 340 345 350 Arg Pro Ser His Tyr His Val Leu Arg Asp Asp Asn Asn Phe Thr Ala 355 360 365 Asp Gln Leu Gln Ser Val Thr Asn Asn Leu Cys Tyr Leu Tyr Thr Ser 370 375 380 Cys Thr Arg Ser Val Ser Ile Pro Pro Pro Val Tyr Tyr Ala His Lys 385 390 395 400 Leu Ala Phe Arg Ala Arg Phe Tyr Leu Thr Gln Val Pro Val Ala Gly 405 410 415 Gly Asp Pro Gly Ala Ala Lys Phe Gln Trp Val Leu Pro Glu Ile Lys 420 425 430 Glu Glu Val Lys Lys Ser Met Phe Phe Cys 435 440 27 3549 DNA Oryza sativa 27 gttggacaac gggtactact cccatcaagc tttagccatg atgagaaaga aaaaaactga 60 accccgtaat gctggggaaa gttctggaac tcaacaagcc actggagctc ctggacgggg 120 tccttcacag cgacctgaga gagctcaaca gcatggaggt ggtggttggc agcctgccaa 180 tcctcaatat gctcaacaag ctggtcgtgg tggtggacaa caccagggac gtggtggacg 240 ttaccagggt cgtggagggc caacatcaca tcaaccaggt ggtggtccgg ttgaatatca 300 agcacatgag tactatggcc gtggtgtcca acggcaagga ggaatgccac aacacaggag 360 tggcagtggt ggacatggag ttcctgccag tccatcaaga acagttcccg agctgcacca 420 agcctcacaa gaccagtacc aagctacggt ggttgcacca tcaccatcaa gaactggccc 480 atcttcgctg cctgttgagg ccagcagcga agaagtccaa catcagtttc aggaacttgc 540 catccagggt caaagcccca ctagccaggc cattcaacca gcaccaccat cgagcaaatc 600 agtgagattt ccaatgcgcc ctggcaaggg tacatttggt gataggtgca tcgtgaaagc 660 caaccatttc tttgctgaat tgcctgacaa agaccttcac cagtatgatg tgtctataac 720 tcctgaggtt ccttcacgtg gtgtcaatcg tgctgtcatt ggagaaattg taacacaata 780 taggcagtct catttgggtg gccgtcttcc agtctatgat ggaaggaaga gcttatacac 840 agctggtcca ttaccattta cttctaggac ctttgacgtt attctgcagg atgaggaaga 900 gagccttgct gttgggcaag gtgcacagag gcgtgagaga ccatttaagg tcgtgatcaa 960 atttgctgca cgcgctgatc tccaccattt agccatgttt ttagctggaa ggcaagcgga 1020 tgctcctcaa gaagctcttc aagttcttga cattgttcta cgtgaattgc ctactgcaag 1080 gtactctcca gttgcaaggt cattttattc gcctaactta ggaaggcgcc aacaacttgg 1140 cgagggcctg gaaagttggc gtggttttta ccaaagcata cgacccacgc agatgggact 1200 ttctctgaat attgatatgt catcgacagc attcattgag cctctacctg tgattgactt 1260 tgttgcacag cttttgaaca gagacatctc agttagacca ttatctgatg ctgatcgtgt 1320 gaagatcaag aaggccctaa ggggtgtaaa ggttgaggtc acacatagag gcaatatgcg 1380 caggaagtat cgcatttctg gccttacctc gcaagcaaca cgagagttgt cttttcccat 1440 tgataatcat ggtactgtga agacggtggt gcaatacttc caggagacat atggatttaa 1500 cattaagcac acaactttgc cttgcttgca agtgggcaat caacaaaggc caaattatct 1560 accaatggag gtctgtaaga ttgtggaggg acagcgttac tcaaaaagac taaatgagaa 1620 gcagataact gctcttctta aagtgacctg ccagcgccct caagagcgtg agctggacat 1680 tttgcagact gtgcatcaca atgcatacca tcaggatcca tatgcacagg agtttggcat 1740 aaggatcgat gagcgacttg catctgttga agctcgtgtt ctaccacccc cctggcttaa 1800 gtaccacgat agtggcagag agaaggatgt cttgccaaga attggccaat ggaatatgat 1860 gaataagaaa atggtcaatg gtggtagagt taacaactgg acatgcatca atttttctcg 1920 tcatgtccaa gataatgctg ctaggagttt ctgtcgcgag cttgctatta tgtgccaaat 1980 atctgggatg gacttctcaa ttgatcctgt ggttcctcta gtgactgcaa gacctgaaca 2040 tgtggaaaga gcgctcaagg cacgctatca agaggccatg aatatactga aaccacaggg 2100 cggggagctt gacctgctga ttgcaatatt gcctgacaat aatggttctc tttatggcga 2160 tctcaaaagg atatgtgaga ctgatcttgg attggtctcg caatgctgtc ttacgaagca 2220 tgtttttaag atgagcaaac agtatttagc aaacgttgcc cttaaaatca atgttaaggt 2280 gggaggaaga aatacagtac ttgttgatgc tttgacaagg aggattcccc ttgtcagtga 2340 cagaccaact atcatatttg gtgcggatgt tactcatcct catcctggag aagattccag 2400 tccttccatt gcagctgtgg ttgcttctca agactggcct gaagtcacta agtatgctgg 2460 attggtgagt gcccaagccc atcgtcaaga attgatacaa gatcttttca aagtatggca 2520 agacccgcat agaggaactg ttactggtgg catgatcaag gagcttctca tttctttcaa 2580 gagggctact ggacagaaac ctcagaggat aatattttac agggatggtg tcagcgaggg 2640 gcagttttat caagttttgt tgtatgagct tgatgccatt agaaaggctt gtgcatccct 2700 ggaacccaac tatcagcctc cagttacctt tgtggtggtc cagaagcggc atcacacaag 2760 gttgtttgct aataatcaca acgaccagcg tactgttgat agaagtggaa acattctgcc 2820 tggaactgtt gttgactcaa agatttgcca tccaaccgag tttgatttct acctgtgtag 2880 ccatgctggc atacagggaa caagccgtcc tgctcattat catgttctgt gggatgagaa 2940 caaatttact gcagacgagt tgcaaaccct cacgaacaac ttgtgctaca cgtatgcaag 3000 gtgcactcgc tctgtatcaa ttgtgcctcc tgcgtactat gctcatctgg cagccttccg 3060 agctcgcttt tacatggagc cagagacatc tgacagtgga tcaatggcga gtggagctgc 3120 aacgagccgt ggccttccac caggtgtgcg cagcgccagg gttgctggaa atgtagccgt 3180 caggcctcta cctgctctca aggaaaacgt gaagcgtgtc atgttttact gctaagagct 3240 tgggctgtac cccgtatgcg ccaaggaatg tagtactatg ttatgttatt ttagcacttg 3300 cactctgtcg ttgatcccgt taaaacgggt atgctaccat aagctgttgg actattctgg 3360 gtattgtagt actacttgtt ttgtatttgt gtttgtgacg ctgcagagcg tgaacaacgc 3420 aagtctggta cttgtatcgt tgtgtttgtg ggaacctaaa tcttgttgga cctttgttgt 3480 gcttgaagaa ccaagttaaa taatcctgtc agtataggga tttaattgca aaaaaaaaaa 3540 aaaaaaaaa 3549 28 1065 PRT Oryza sativa 28 Met Met Arg Lys Lys Lys Thr Glu Pro Arg Asn Ala Gly Glu Ser Ser 1 5 10 15 Gly Thr Gln Gln Ala Thr Gly Ala Pro Gly Arg Gly Pro Ser Gln Arg 20 25 30 Pro Glu Arg Ala Gln Gln His Gly Gly Gly Gly Trp Gln Pro Ala Asn 35 40 45 Pro Gln Tyr Ala Gln Gln Ala Gly Arg Gly Gly Gly Gln His Gln Gly 50 55 60 Arg Gly Gly Arg Tyr Gln Gly Arg Gly Gly Pro Thr Ser His Gln Pro 65 70 75 80 Gly Gly Gly Pro Val Glu Tyr Gln Ala His Glu Tyr Tyr Gly Arg Gly 85 90 95 Val Gln Arg Gln Gly Gly Met Pro Gln His Arg Ser Gly Ser Gly Gly 100 105 110 His Gly Val Pro Ala Ser Pro Ser Arg Thr Val Pro Glu Leu His Gln 115 120 125 Ala Ser Gln Asp Gln Tyr Gln Ala Thr Val Val Ala Pro Ser Pro Ser 130 135 140 Arg Thr Gly Pro Ser Ser Leu Pro Val Glu Ala Ser Ser Glu Glu Val 145 150 155 160 Gln His Gln Phe Gln Glu Leu Ala Ile Gln Gly Gln Ser Pro Thr Ser 165 170 175 Gln Ala Ile Gln Pro Ala Pro Pro Ser Ser Lys Ser Val Arg Phe Pro 180 185 190 Met Arg Pro Gly Lys Gly Thr Phe Gly Asp Arg Cys Ile Val Lys Ala 195 200 205 Asn His Phe Phe Ala Glu Leu Pro Asp Lys Asp Leu His Gln Tyr Asp 210 215 220 Val Ser Ile Thr Pro Glu Val Pro Ser Arg Gly Val Asn Arg Ala Val 225 230 235 240 Ile Gly Glu Ile Val Thr Gln Tyr Arg Gln Ser His Leu Gly Gly Arg 245 250 255 Leu Pro Val Tyr Asp Gly Arg Lys Ser Leu Tyr Thr Ala Gly Pro Leu 260 265 270 Pro Phe Thr Ser Arg Thr Phe Asp Val Ile Leu Gln Asp Glu Glu Glu 275 280 285 Ser Leu Ala Val Gly Gln Gly Ala Gln Arg Arg Glu Arg Pro Phe Lys 290 295 300 Val Val Ile Lys Phe Ala Ala Arg Ala Asp Leu His His Leu Ala Met 305 310 315 320 Phe Leu Ala Gly Arg Gln Ala Asp Ala Pro Gln Glu Ala Leu Gln Val 325 330 335 Leu Asp Ile Val Leu Arg Glu Leu Pro Thr Ala Arg Tyr Ser Pro Val 340 345 350 Ala Arg Ser Phe Tyr Ser Pro Asn Leu Gly Arg Arg Gln Gln Leu Gly 355 360 365 Glu Gly Leu Glu Ser Trp Arg Gly Phe Tyr Gln Ser Ile Arg Pro Thr 370 375 380 Gln Met Gly Leu Ser Leu Asn Ile Asp Met Ser Ser Thr Ala Phe Ile 385 390 395 400 Glu Pro Leu Pro Val Ile Asp Phe Val Ala Gln Leu Leu Asn Arg Asp 405 410 415 Ile Ser Val Arg Pro Leu Ser Asp Ala Asp Arg Val Lys Ile Lys Lys 420 425 430 Ala Leu Arg Gly Val Lys Val Glu Val Thr His Arg Gly Asn Met Arg 435 440 445 Arg Lys Tyr Arg Ile Ser Gly Leu Thr Ser Gln Ala Thr Arg Glu Leu 450 455 460 Ser Phe Pro Ile Asp Asn His Gly Thr Val Lys Thr Val Val Gln Tyr 465 470 475 480 Phe Gln Glu Thr Tyr Gly Phe Asn Ile Lys His Thr Thr Leu Pro Cys 485 490 495 Leu Gln Val Gly Asn Gln Gln Arg Pro Asn Tyr Leu Pro Met Glu Val 500 505 510 Cys Lys Ile Val Glu Gly Gln Arg Tyr Ser Lys Arg Leu Asn Glu Lys 515 520 525 Gln Ile Thr Ala Leu Leu Lys Val Thr Cys Gln Arg Pro Gln Glu Arg 530 535 540 Glu Leu Asp Ile Leu Gln Thr Val His His Asn Ala Tyr His Gln Asp 545 550 555 560 Pro Tyr Ala Gln Glu Phe Gly Ile Arg Ile Asp Glu Arg Leu Ala Ser 565 570 575 Val Glu Ala Arg Val Leu Pro Pro Pro Trp Leu Lys Tyr His Asp Ser 580 585 590 Gly Arg Glu Lys Asp Val Leu Pro Arg Ile Gly Gln Trp Asn Met Met 595 600 605 Asn Lys Lys Met Val Asn Gly Gly Arg Val Asn Asn Trp Thr Cys Ile 610 615 620 Asn Phe Ser Arg His Val Gln Asp Asn Ala Ala Arg Ser Phe Cys Arg 625 630 635 640 Glu Leu Ala Ile Met Cys Gln Ile Ser Gly Met Asp Phe Ser Ile Asp 645 650 655 Pro Val Val Pro Leu Val Thr Ala Arg Pro Glu His Val Glu Arg Ala 660 665 670 Leu Lys Ala Arg Tyr Gln Glu Ala Met Asn Ile Leu Lys Pro Gln Gly 675 680 685 Gly Glu Leu Asp Leu Leu Ile Ala Ile Leu Pro Asp Asn Asn Gly Ser 690 695 700 Leu Tyr Gly Asp Leu Lys Arg Ile Cys Glu Thr Asp Leu Gly Leu Val 705 710 715 720 Ser Gln Cys Cys Leu Thr Lys His Val Phe Lys Met Ser Lys Gln Tyr 725 730 735 Leu Ala Asn Val Ala Leu Lys Ile Asn Val Lys Val Gly Gly Arg Asn 740 745 750 Thr Val Leu Val Asp Ala Leu Thr Arg Arg Ile Pro Leu Val Ser Asp 755 760 765 Arg Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro His Pro Gly 770 775 780 Glu Asp Ser Ser Pro Ser Ile Ala Ala Val Val Ala Ser Gln Asp Trp 785 790 795 800 Pro Glu Val Thr Lys Tyr Ala Gly Leu Val Ser Ala Gln Ala His Arg 805 810 815 Gln Glu Leu Ile Gln Asp Leu Phe Lys Val Trp Gln Asp Pro His Arg 820 825 830 Gly Thr Val Thr Gly Gly Met Ile Lys Glu Leu Leu Ile Ser Phe Lys 835 840 845 Arg Ala Thr Gly Gln Lys Pro Gln Arg Ile Ile Phe Tyr Arg Asp Gly 850 855 860 Val Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Tyr Glu Leu Asp Ala 865 870 875 880 Ile Arg Lys Ala Cys Ala Ser Leu Glu Pro Asn Tyr Gln Pro Pro Val 885 890 895 Thr Phe Val Val Val Gln Lys Arg His His Thr Arg Leu Phe Ala Asn 900 905 910 Asn His Asn Asp Gln Arg Thr Val Asp Arg Ser Gly Asn Ile Leu Pro 915 920 925 Gly Thr Val Val Asp Ser Lys Ile Cys His Pro Thr Glu Phe Asp Phe 930 935 940 Tyr Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser Arg Pro Ala His 945 950 955 960 Tyr His Val Leu Trp Asp Glu Asn Lys Phe Thr Ala Asp Glu Leu Gln 965 970 975 Thr Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys Thr Arg Ser 980 985 990 Val Ser Ile Val Pro Pro Ala Tyr Tyr Ala His Leu Ala Ala Phe Arg 995 1000 1005 Ala Arg Phe Tyr Met Glu Pro Glu Thr Ser Asp Ser Gly Ser Met Ala 1010 1015 1020 Ser Gly Ala Ala Thr Ser Arg Gly Leu Pro Pro Gly Val Arg Ser Ala 1025 1030 1035 1040 Arg Val Ala Gly Asn Val Ala Val Arg Pro Leu Pro Ala Leu Lys Glu 1045 1050 1055 Asn Val Lys Arg Val Met Phe Tyr Cys 1060 1065 29 772 DNA Oryza sativa 29 gttctaaccg ccggccgccg ccctccccgc acgacgccga cgccgccctc ctcgcccaac 60 gccggctcag ccccttctcc tccccgcccg acgccgcccc ttctcctccc cgtcccacgc 120 cgaccccgcc cgacgccggc gccactctgc ttgtccccgg ccggcgccga gcctgctcct 180 ccccgcccga cgccggcgcc gccgacgctg ctctgctcct ccccgaccgg cgccgacctg 240 ctcctcccag cccggagccc gacgccggca catctcatcc agatgtccga taacatggct 300 gccaaaattg gtgaaattgt ccaagtacat aatgataatc ctgtaaagag agtacctatt 360 gcacgaccta gctttggccg tgaaggaaag caaatcaagc tgctctcaaa ccacttcact 420 gtgaagctta gtggaattga tgcggttttc taccaataca gtgtttccat caaatctgag 480 gatgataagg tgattgatgg aaagggtatt ggccgaaagg tcatggataa agtgctgcaa 540 acatacagct ctgagcttgc tgggaaggaa tttgcgtatg atggtgaaaa atgtctattt 600 actgtggggc ctcttccaca gaacaacttt gagttcactg ttatcttgga ggaaacatct 660 tcaagagctg ctggtgggag tctagggcat ggaagcccta atcaaggtga catcaaaaaa 720 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gaaaaaaaaa aaaaaaaaaa aa 772 30 238 PRT Oryza sativa 30 Val Leu Thr Ala Gly Arg Arg Pro Pro Arg Thr Thr Pro Thr Pro Pro 1 5 10 15 Ser Ser Pro Asn Ala Gly Ser Ala Pro Ser Pro Pro Arg Pro Thr Pro 20 25 30 Pro Leu Leu Leu Pro Val Pro Arg Arg Pro Arg Pro Thr Pro Ala Pro 35 40 45 Leu Cys Leu Ser Pro Ala Gly Ala Glu Pro Ala Pro Pro Arg Pro Thr 50 55 60 Pro Ala Pro Pro Thr Leu Leu Cys Ser Ser Pro Thr Gly Ala Asp Leu 65 70 75 80 Leu Leu Pro Ala Arg Ser Pro Thr Pro Ala His Leu Ile Gln Met Ser 85 90 95 Asp Asn Met Ala Ala Lys Ile Gly Glu Ile Val Gln Val His Asn Asp 100 105 110 Asn Pro Val Lys Arg Val Pro Ile Ala Arg Pro Ser Phe Gly Arg Glu 115 120 125 Gly Lys Gln Ile Lys Leu Leu Ser Asn His Phe Thr Val Lys Leu Ser 130 135 140 Gly Ile Asp Ala Val Phe Tyr Gln Tyr Ser Val Ser Ile Lys Ser Glu 145 150 155 160 Asp Asp Lys Val Ile Asp Gly Lys Gly Ile Gly Arg Lys Val Met Asp 165 170 175 Lys Val Leu Gln Thr Tyr Ser Ser Glu Leu Ala Gly Lys Glu Phe Ala 180 185 190 Tyr Asp Gly Glu Lys Cys Leu Phe Thr Val Gly Pro Leu Pro Gln Asn 195 200 205 Asn Phe Glu Phe Thr Val Ile Leu Glu Glu Thr Ser Ser Arg Ala Ala 210 215 220 Gly Gly Ser Leu Gly His Gly Ser Pro Asn Gln Gly Asp Ile 225 230 235 31 1238 DNA Oryza sativa 31 tcacaatgct gttgcacgaa gcaggtgttc aaaatgaaca aacaaattct tgcaaatctt 60 gctctgaaga taaatgtcaa ggttgggggc aggaacactg tgctggtgga tgctgtgtca 120 aggcgtattc ctctggtaac cgacagacct acaattatat ttggtgctga tgttacccat 180 cctcatcctg gagaggacag cagtccctca attgctgctg ttgtagcctc ccaagattgg 240 cctgaggtga caaagtatgc tgggttggtt tctgctcaag cccaccgaca agagctgata 300 gaagatctat ataaaatctg gcaggatcca cagagaggaa cagttagtgg tggcatgatc 360 cgtgagctgc ttatatcctt caaaagatca actggtgaga agccccagcg aataatattt 420 tacagggatg gcgttagtga aggccaattt taccaagttc tactttatga attgaatgca 480 atccgaaaag catgtgcctc cctggagaca aattaccaac caaaggtgac tttcattgtg 540 gttcagaaac gtcaccacac aagattattt gcacataatc acaacgatca gaactcagtt 600 gacaggagcg ggaacatact ccctggtacg gttgtagatt caaagatctg tcatccaact 660 gagtttgact tctacctgtg tagccatgct ggcattaagg gtactagtcg tccagctcat 720 tatcatgtct tgtgggatga aaacaacttc acagctgatg cattgcagat tcttaccaac 780 aacctttgct acacctatgc aaggtgcact cgctctgtat caattgttcc acctgcttat 840 tatgctcatc tggctgcctt ccgtgctcgt ttctatatgg aaccagatac atctgacagc 900 agctctgtcg ttagtgggcc tggtgtacgt gggccacttt ctggctcatc aacatcacgt 960 actcgggccc ctggtggtgc agctgttaag ccacttcctg ctctgaagga tagtgtgaag 1020 agggtcatgt tctactgctg aagctagggc ctacatagct aaagctcttc gtttcttggc 1080 aacctgccta tgatggttgt aattatgtgt caaaaaatcc cataataatc tgccagctgc 1140 tatcttctcc attgtactat gctggtcatg tttgccaaag ttaccctata tgtatgtata 1200 ttatgctatt gtttttttaa aaaaaaaaaa aaaaaaaa 1238 32 346 PRT Oryza sativa 32 Ser Gln Cys Cys Cys Thr Lys Gln Val Phe Lys Met Asn Lys Gln Ile 1 5 10 15 Leu Ala Asn Leu Ala Leu Lys Ile Asn Val Lys Val Gly Gly Arg Asn 20 25 30 Thr Val Leu Val Asp Ala Val Ser Arg Arg Ile Pro Leu Val Thr Asp 35 40 45 Arg Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro His Pro Gly 50 55 60 Glu Asp Ser Ser Pro Ser Ile Ala Ala Val Val Ala Ser Gln Asp Trp 65 70 75 80 Pro Glu Val Thr Lys Tyr Ala Gly Leu Val Ser Ala Gln Ala His Arg 85 90 95 Gln Glu Leu Ile Glu Asp Leu Tyr Lys Ile Trp Gln Asp Pro Gln Arg 100 105 110 Gly Thr Val Ser Gly Gly Met Ile Arg Glu Leu Leu Ile Ser Phe Lys 115 120 125 Arg Ser Thr Gly Glu Lys Pro Gln Arg Ile Ile Phe Tyr Arg Asp Gly 130 135 140 Val Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Tyr Glu Leu Asn Ala 145 150 155 160 Ile Arg Lys Ala Cys Ala Ser Leu Glu Thr Asn Tyr Gln Pro Lys Val 165 170 175 Thr Phe Ile Val Val Gln Lys Arg His His Thr Arg Leu Phe Ala His 180 185 190 Asn His Asn Asp Gln Asn Ser Val Asp Arg Ser Gly Asn Ile Leu Pro 195 200 205 Gly Thr Val Val Asp Ser Lys Ile Cys His Pro Thr Glu Phe Asp Phe 210 215 220 Tyr Leu Cys Ser His Ala Gly Ile Lys Gly Thr Ser Arg Pro Ala His 225 230 235 240 Tyr His Val Leu Trp Asp Glu Asn Asn Phe Thr Ala Asp Ala Leu Gln 245 250 255 Ile Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys Thr Arg Ser 260 265 270 Val Ser Ile Val Pro Pro Ala Tyr Tyr Ala His Leu Ala Ala Phe Arg 275 280 285 Ala Arg Phe Tyr Met Glu Pro Asp Thr Ser Asp Ser Ser Ser Val Val 290 295 300 Ser Gly Pro Gly Val Arg Gly Pro Leu Ser Gly Ser Ser Thr Ser Arg 305 310 315 320 Thr Arg Ala Pro Gly Gly Ala Ala Val Lys Pro Leu Pro Ala Leu Lys 325 330 335 Asp Ser Val Lys Arg Val Met Phe Tyr Cys 340 345 33 551 DNA Oryza sativa unsure (23) n = A, C, G or T 33 ttgccatggc ctaccatacg acnaaccaga ttacgctcat atggccatgg aggccagtgc 60 aagaattggc caatggaata tgatgaataa gaaaatggtc aatggtggta gagttaacaa 120 ctggacatgc atcaattttt ctcgtcatgt ccaagataat gctgctagga gtttctgtcg 180 cgagcttgct attatgtgcc aaatatctgg gatggacttc tcaattgatc ctgtggttcc 240 tctagtgact gcaagacctg aacatgtgga aagagcgctc aaggcacgct atcaagaggc 300 catgaatata ctgaaaccac agggcgggga gcttgacctg ctgattgcaa tattgcctga 360 caataatggt tctctttatg gcgatctcaa aaggatatgt gagactgatc ttggattggt 420 ctcgcaatgc tgtcttacga agcatgtttt taagatgagc aaacagtatt taacaaacgt 480 tgcccttaaa atcaatgtta aggngggaag gaaaaaatac aagtactttg ttggatgcct 540 ttgacnaagg g 551 34 169 PRT Oryza sativa UNSURE (8) Xaa = ANY AMINO ACID 34 Cys His Gly Leu Pro Tyr Asp Xaa Pro Asp Tyr Ala His Met Ala Met 1 5 10 15 Glu Ala Ser Ala Arg Ile Gly Gln Trp Asn Met Met Asn Lys Lys Met 20 25 30 Val Asn Gly Gly Arg Val Asn Asn Trp Thr Cys Ile Asn Phe Ser Arg 35 40 45 His Val Gln Asp Asn Ala Ala Arg Ser Phe Cys Arg Glu Leu Ala Ile 50 55 60 Met Cys Gln Ile Ser Gly Met Asp Phe Ser Ile Asp Pro Val Val Pro 65 70 75 80 Leu Val Thr Ala Arg Pro Glu His Val Glu Arg Ala Leu Lys Ala Arg 85 90 95 Tyr Gln Glu Ala Met Asn Ile Leu Lys Pro Gln Gly Gly Glu Leu Asp 100 105 110 Leu Leu Ile Ala Ile Leu Pro Asp Asn Asn Gly Ser Leu Tyr Gly Asp 115 120 125 Leu Lys Arg Ile Cys Glu Thr Asp Leu Gly Leu Val Ser Gln Cys Cys 130 135 140 Leu Thr Lys His Val Phe Lys Met Ser Lys Gln Tyr Leu Thr Asn Val 145 150 155 160 Ala Leu Lys Ile Asn Val Lys Xaa Gly 165 35 966 DNA Glycine max 35 cttcggaagt tgagggatta cctcagtgga agcgtgcttt cgatccctag ggatgttttg 60 cacggcttgg atttggtggt gaaggaaaat ccttcgaagc agtgtgtttc cttggggcgg 120 tgcttcttcc ccatgaaccc tcctttgagg aagaaagatc ttaaccatgg cataattgcg 180 attggagggt ttcagcagag tcttaagtct acttctcagg gattgtcctt gtgcctggac 240 tattcggttt tgtcctttcg gaagaagctg ttggtgttgg attttctgca cgagcatatt 300 agggacttca atttaaggga gtttgggcgg ttcaggagac aagttgagca tgtacttatt 360 gggttgaagg ttaatgttaa acaccggaag acaaagcaga agtacactat tactaggttg 420 acacccaagg ttacgagaca tatcacattc cctattttgg atcccgaggg ccggaatccc 480 ccaaaggaag ctactctggt tggttacttt ctagagaagt atggtgtgaa cattgaatac 540 aaggacattc ctgccttgga ttttggaggc aacaagacga attttgtgcc tatggagttt 600 tgtgagttgg ttgaggggca gagatatccc aaagagaatt tggacaaata tgctgccaag 660 gacttaaaag acatgtcagt ggctcctcca agggtgaggc aaagtacaat acaagcaatg 720 gtaaactcag aggacggacc gtgcggaggt ggtgttatta aaaattttgg aatgagtgtc 780 aacacttcca tgacaaatgt gacaggacgt gtaattcagc ctccacaatt gaagctaggt 840 aatccaaatg gccagactgt tagtatgaca cttgaagtag agaaatgtca gtggaatcta 900 gtgggacgat caatggtgga aggcaagcca gttgagtgtt ggggcattct tgattttacc 960 tcgtgc 966 36 322 PRT Glycine max 36 Leu Arg Lys Leu Arg Asp Tyr Leu Ser Gly Ser Val Leu Ser Ile Pro 1 5 10 15 Arg Asp Val Leu His Gly Leu Asp Leu Val Val Lys Glu Asn Pro Ser 20 25 30 Lys Gln Cys Val Ser Leu Gly Arg Cys Phe Phe Pro Met Asn Pro Pro 35 40 45 Leu Arg Lys Lys Asp Leu Asn His Gly Ile Ile Ala Ile Gly Gly Phe 50 55 60 Gln Gln Ser Leu Lys Ser Thr Ser Gln Gly Leu Ser Leu Cys Leu Asp 65 70 75 80 Tyr Ser Val Leu Ser Phe Arg Lys Lys Leu Leu Val Leu Asp Phe Leu 85 90 95 His Glu His Ile Arg Asp Phe Asn Leu Arg Glu Phe Gly Arg Phe Arg 100 105 110 Arg Gln Val Glu His Val Leu Ile Gly Leu Lys Val Asn Val Lys His 115 120 125 Arg Lys Thr Lys Gln Lys Tyr Thr Ile Thr Arg Leu Thr Pro Lys Val 130 135 140 Thr Arg His Ile Thr Phe Pro Ile Leu Asp Pro Glu Gly Arg Asn Pro 145 150 155 160 Pro Lys Glu Ala Thr Leu Val Gly Tyr Phe Leu Glu Lys Tyr Gly Val 165 170 175 Asn Ile Glu Tyr Lys Asp Ile Pro Ala Leu Asp Phe Gly Gly Asn Lys 180 185 190 Thr Asn Phe Val Pro Met Glu Phe Cys Glu Leu Val Glu Gly Gln Arg 195 200 205 Tyr Pro Lys Glu Asn Leu Asp Lys Tyr Ala Ala Lys Asp Leu Lys Asp 210 215 220 Met Ser Val Ala Pro Pro Arg Val Arg Gln Ser Thr Ile Gln Ala Met 225 230 235 240 Val Asn Ser Glu Asp Gly Pro Cys Gly Gly Gly Val Ile Lys Asn Phe 245 250 255 Gly Met Ser Val Asn Thr Ser Met Thr Asn Val Thr Gly Arg Val Ile 260 265 270 Gln Pro Pro Gln Leu Lys Leu Gly Asn Pro Asn Gly Gln Thr Val Ser 275 280 285 Met Thr Leu Glu Val Glu Lys Cys Gln Trp Asn Leu Val Gly Arg Ser 290 295 300 Met Val Glu Gly Lys Pro Val Glu Cys Trp Gly Ile Leu Asp Phe Thr 305 310 315 320 Ser Cys 37 3613 DNA Glycine max 37 ttcttgcaag catctcattt ctctctttct ctctttctct ctctttggga gaaaacccac 60 tcttcttttc tctctcttgc acacatatac acactcctct tttttattcc cttcttcact 120 ccactgccca gcttcgccct gtccatcgct caccgtttgc agtagcttct ctacttttca 180 ctttctccct gagatcatgg tcagaaagag aagaactgaa ctacccagtg ggggtgaaag 240 ctctgaggct caacgccctg ctgaaaggag tgcaccaccc caacaacagg ctgctgctgc 300 tgccccagga ggggctggac cccaaggagg cagaggttgg ggtccccaag gaggacgagg 360 aggctatggt gggggccgca gtcgtgggat gccccaacag caatatggtg cccctcctga 420 atatcaaggt aggggaaggg gagggccttc tcagcaagga ggccgtggag ggtatggcgg 480 tggccgaagt ggtggtggta tgggcagtgg ccgtggcgta ggtccttcat atggtggccc 540 atccaggcca ccggcacccg agctgcacca agcaacctca gttcaattct atcaaactgg 600 ggtgagttct cagcctgcat tatctgaggc cagttcatca ctgccgccgc cggaacctgt 660 tgatttggaa cagtcaatgg cgcagatggt gcttcattct gaagctgctc cttctccgcc 720 tcctgcaagt aaatcatcaa tgaggttccc tcttcgacca ggaaagggta gctatggcac 780 caaatgtgtt gtcaaggcta atcatttctt tgccgagttg cccaacaaag atctgcatca 840 atatgatgta acaattactc ctgaagtgac atcaagagga gtgaaccgtg ctgttatgga 900 gcagttggtg aggctgtatc gggaatctca cttgggtaag agacttcctg cttacgatgg 960 gcgcaagagc ctctatactg ctggaccact tccttttatg tcaaaggagt tcagaattgt 1020 tcttgctgat gatgatgaag gagctggagg ccagaggagg gacagggaat tcaaggttgt 1080 gataaaattg gctgcacggg cagatcttca ccatttagga ctctttttac agggaaggca 1140 aactgatgct cctcaagagg ctttgcaggt ccttgacatt gttctgcgtg aactccctac 1200 tacaaggtat tgtcctgtag gaagatcatt ttattcacct gatttgggta gaagacagcc 1260 tttaggtgag ggattggaaa gctggcgtgg tttctaccag agtattcggc ctacacagat 1320 ggggctatcc ctgaacattg atatgtcttc cactgcattt attgagccat tgccggtaat 1380 tgacttcgta aatcaactgc tgaacagaga tgtatctgcc cggccattat ctgatgctga 1440 tcgtgttaag atcaagaaag ctcttcgagg tatcaaagtt gaagtaacac atcgtggaaa 1500 catgagaagg aaatatcgta tctctggtct gacttcacag gcaaccagag aattgacatt 1560 cccagtagat gaaaggggaa ccatgaaatc tgttgtggag tacttctatg agacatatgg 1620 gtttgtcatt caacatactc agtggccttg tctgcaagtt ggcaatacac agagacctaa 1680 ctatttgcca atggaggttt gcaagatagt ggaaggtcaa aggtactcaa aaaggcttaa 1740 tgagaggcaa atcaccgctt tgctgaaagt tacatgccag cgtcctgttg agagggagcg 1800 tgatatcatg cagacagtac accacaatgc ataccatgaa gatccttatg ccaaagaatt 1860 tgggatcaag atcagtgaga agcttgctca agttgaagct cgcatccttc ctgctccatg 1920 gctcaaatat cacgatacgg gcagagaaaa ggattgtctt cctcaagttg ggcaatggaa 1980 tatgatgaat aagaaaatgg ttaatggggg aacagttaac aactggttct gcataaactt 2040 ttcgaggaat gttcaagata gtgttgcccg cggtttttgc tatgaacttg ctcagatgtg 2100 ttatatatct ggaatggcat ttacacctga gccagtagtt cccccagtca gtgctcgccc 2160 tgatcaagtg gaaaaggttc ttaaaactcg gtatcacgat gccaagaata aactgcaagg 2220 aaaagagctt gatttactca ttgttatctt gccggataat aatggatcac tatatggtga 2280 cctcaaacgt atttgtgaga cagatctagg acttgtttca caatgttgct taactaagca 2340 tgtcttcaaa atgagcaagc agtaccttgc aaatgttgct ttgaaaatta atgtcaaagt 2400 tggagggaga aacactgtac tggttgatgc gctctcacga cgcattccct tggtcagtga 2460 cagacctaca attatttttg gagctgatgt gactcatcca catcctggag aggattcaag 2520 tccatcaatt gcagcagttg tggcttcgca agactatcct gaaattacaa agtatgctgg 2580 tttagtttgt gcccaagctc ataggcagga actcatccag gatcttttca aacaatggca 2640 agatccagtc agaggaacag tgactggtgg aatgatcaag gaacttctta tatcttttag 2700 gagagctaca ggacaaaagc cacaacgcat catattttat agggatggtg ttagtgaggg 2760 tcaattttat caggttctac tgtttgagct tgatgctatt cgaaaggcat gtgcatccct 2820 ggaacccaac tatcagcctc ctgtgacttt tgtggtggtt caaaagcgtc accacacaag 2880 gctctttgcc agcaaccatc acgataagag ttcttttgac aggagtggca acatattgcc 2940 tggtactgtt gttgactcca aaatctgcca tcccaccgaa tttgactttt atctctgcag 3000 ccatgctgga atacagggta caagccgtcc tgctcactac catgtgttgt gggatgaaaa 3060 caattttact gctgatgcct tgcaaacact caccaataat ctttgctaca catatgctcg 3120 gtgcacccga tctgtttcaa ttgtgcctcc tgcatactat gctcaccttg ctgcattccg 3180 tgcaaggttt tacatggaac ccgagacttc ggatagtggc tctatgacaa gtggtgctgt 3240 tgcaggccgt gggatgggtg gcggcggtgg tggtggtgta gggcgtagca cccgggcacc 3300 tggtgctaat gctgctgtga gaccattgcc tgcactcaaa gagaacgtta agagagttat 3360 gttttattgt taagaagata tgatatgcat gccaaagatt acttttagca accttgtttt 3420 gtggaggagt gctttttccc ttgctgcttt caaactatct ccagtggtgt ggtctgtgtc 3480 attagtattg agttttttga aactatttaa ggtgtgtggt gtgttgaata aggttgtcca 3540 gtgtggagtg gagtgtttta tctttgctat gagggtctga tatttgatgc aaaaaaaaaa 3600 aaaaaaaaaa aaa 3613 38 1058 PRT Glycine max 38 Met Val Arg Lys Arg Arg Thr Glu Leu Pro Ser Gly Gly Glu Ser Ser 1 5 10 15 Glu Ala Gln Arg Pro Ala Glu Arg Ser Ala Pro Pro Gln Gln Gln Ala 20 25 30 Ala Ala Ala Ala Pro Gly Gly Ala Gly Pro Gln Gly Gly Arg Gly Trp 35 40 45 Gly Pro Gln Gly Gly Arg Gly Gly Tyr Gly Gly Gly Arg Ser Arg Gly 50 55 60 Met Pro Gln Gln Gln Tyr Gly Ala Pro Pro Glu Tyr Gln Gly Arg Gly 65 70 75 80 Arg Gly Gly Pro Ser Gln Gln Gly Gly Arg Gly Gly Tyr Gly Gly Gly 85 90 95 Arg Ser Gly Gly Gly Met Gly Ser Gly Arg Gly Val Gly Pro Ser Tyr 100 105 110 Gly Gly Pro Ser Arg Pro Pro Ala Pro Glu Leu His Gln Ala Thr Ser 115 120 125 Val Gln Phe Tyr Gln Thr Gly Val Ser Ser Gln Pro Ala Leu Ser Glu 130 135 140 Ala Ser Ser Ser Leu Pro Pro Pro Glu Pro Val Asp Leu Glu Gln Ser 145 150 155 160 Met Ala Gln Met Val Leu His Ser Glu Ala Ala Pro Ser Pro Pro Pro 165 170 175 Ala Ser Lys Ser Ser Met Arg Phe Pro Leu Arg Pro Gly Lys Gly Ser 180 185 190 Tyr Gly Thr Lys Cys Val Val Lys Ala Asn His Phe Phe Ala Glu Leu 195 200 205 Pro Asn Lys Asp Leu His Gln Tyr Asp Val Thr Ile Thr Pro Glu Val 210 215 220 Thr Ser Arg Gly Val Asn Arg Ala Val Met Glu Gln Leu Val Arg Leu 225 230 235 240 Tyr Arg Glu Ser His Leu Gly Lys Arg Leu Pro Ala Tyr Asp Gly Arg 245 250 255 Lys Ser Leu Tyr Thr Ala Gly Pro Leu Pro Phe Met Ser Lys Glu Phe 260 265 270 Arg Ile Val Leu Ala Asp Asp Asp Glu Gly Ala Gly Gly Gln Arg Arg 275 280 285 Asp Arg Glu Phe Lys Val Val Ile Lys Leu Ala Ala Arg Ala Asp Leu 290 295 300 His His Leu Gly Leu Phe Leu Gln Gly Arg Gln Thr Asp Ala Pro Gln 305 310 315 320 Glu Ala Leu Gln Val Leu Asp Ile Val Leu Arg Glu Leu Pro Thr Thr 325 330 335 Arg Tyr Cys Pro Val Gly Arg Ser Phe Tyr Ser Pro Asp Leu Gly Arg 340 345 350 Arg Gln Pro Leu Gly Glu Gly Leu Glu Ser Trp Arg Gly Phe Tyr Gln 355 360 365 Ser Ile Arg Pro Thr Gln Met Gly Leu Ser Leu Asn Ile Asp Met Ser 370 375 380 Ser Thr Ala Phe Ile Glu Pro Leu Pro Val Ile Asp Phe Val Asn Gln 385 390 395 400 Leu Leu Asn Arg Asp Val Ser Ala Arg Pro Leu Ser Asp Ala Asp Arg 405 410 415 Val Lys Ile Lys Lys Ala Leu Arg Gly Ile Lys Val Glu Val Thr His 420 425 430 Arg Gly Asn Met Arg Arg Lys Tyr Arg Ile Ser Gly Leu Thr Ser Gln 435 440 445 Ala Thr Arg Glu Leu Thr Phe Pro Val Asp Glu Arg Gly Thr Met Lys 450 455 460 Ser Val Val Glu Tyr Phe Tyr Glu Thr Tyr Gly Phe Val Ile Gln His 465 470 475 480 Thr Gln Trp Pro Cys Leu Gln Val Gly Asn Thr Gln Arg Pro Asn Tyr 485 490 495 Leu Pro Met Glu Val Cys Lys Ile Val Glu Gly Gln Arg Tyr Ser Lys 500 505 510 Arg Leu Asn Glu Arg Gln Ile Thr Ala Leu Leu Lys Val Thr Cys Gln 515 520 525 Arg Pro Val Glu Arg Glu Arg Asp Ile Met Gln Thr Val His His Asn 530 535 540 Ala Tyr His Glu Asp Pro Tyr Ala Lys Glu Phe Gly Ile Lys Ile Ser 545 550 555 560 Glu Lys Leu Ala Gln Val Glu Ala Arg Ile Leu Pro Ala Pro Trp Leu 565 570 575 Lys Tyr His Asp Thr Gly Arg Glu Lys Asp Cys Leu Pro Gln Val Gly 580 585 590 Gln Trp Asn Met Met Asn Lys Lys Met Val Asn Gly Gly Thr Val Asn 595 600 605 Asn Trp Phe Cys Ile Asn Phe Ser Arg Asn Val Gln Asp Ser Val Ala 610 615 620 Arg Gly Phe Cys Tyr Glu Leu Ala Gln Met Cys Tyr Ile Ser Gly Met 625 630 635 640 Ala Phe Thr Pro Glu Pro Val Val Pro Pro Val Ser Ala Arg Pro Asp 645 650 655 Gln Val Glu Lys Val Leu Lys Thr Arg Tyr His Asp Ala Lys Asn Lys 660 665 670 Leu Gln Gly Lys Glu Leu Asp Leu Leu Ile Val Ile Leu Pro Asp Asn 675 680 685 Asn Gly Ser Leu Tyr Gly Asp Leu Lys Arg Ile Cys Glu Thr Asp Leu 690 695 700 Gly Leu Val Ser Gln Cys Cys Leu Thr Lys His Val Phe Lys Met Ser 705 710 715 720 Lys Gln Tyr Leu Ala Asn Val Ala Leu Lys Ile Asn Val Lys Val Gly 725 730 735 Gly Arg Asn Thr Val Leu Val Asp Ala Leu Ser Arg Arg Ile Pro Leu 740 745 750 Val Ser Asp Arg Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro 755 760 765 His Pro Gly Glu Asp Ser Ser Pro Ser Ile Ala Ala Val Val Ala Ser 770 775 780 Gln Asp Tyr Pro Glu Ile Thr Lys Tyr Ala Gly Leu Val Cys Ala Gln 785 790 795 800 Ala His Arg Gln Glu Leu Ile Gln Asp Leu Phe Lys Gln Trp Gln Asp 805 810 815 Pro Val Arg Gly Thr Val Thr Gly Gly Met Ile Lys Glu Leu Leu Ile 820 825 830 Ser Phe Arg Arg Ala Thr Gly Gln Lys Pro Gln Arg Ile Ile Phe Tyr 835 840 845 Arg Asp Gly Val Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Phe Glu 850 855 860 Leu Asp Ala Ile Arg Lys Ala Cys Ala Ser Leu Glu Pro Asn Tyr Gln 865 870 875 880 Pro Pro Val Thr Phe Val Val Val Gln Lys Arg His His Thr Arg Leu 885 890 895 Phe Ala Ser Asn His His Asp Lys Ser Ser Phe Asp Arg Ser Gly Asn 900 905 910 Ile Leu Pro Gly Thr Val Val Asp Ser Lys Ile Cys His Pro Thr Glu 915 920 925 Phe Asp Phe Tyr Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser Arg 930 935 940 Pro Ala His Tyr His Val Leu Trp Asp Glu Asn Asn Phe Thr Ala Asp 945 950 955 960 Ala Leu Gln Thr Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys 965 970 975 Thr Arg Ser Val Ser Ile Val Pro Pro Ala Tyr Tyr Ala His Leu Ala 980 985 990 Ala Phe Arg Ala Arg Phe Tyr Met Glu Pro Glu Thr Ser Asp Ser Gly 995 1000 1005 Ser Met Thr Ser Gly Ala Val Ala Gly Arg Gly Met Gly Gly Gly Gly 1010 1015 1020 Gly Gly Gly Val Gly Arg Ser Thr Arg Ala Pro Gly Ala Asn Ala Ala 1025 1030 1035 1040 Val Arg Pro Leu Pro Ala Leu Lys Glu Asn Val Lys Arg Val Met Phe 1045 1050 1055 Tyr Cys 39 3239 DNA Glycine max 39 ttctaaactc actctctcac tttctcactc cctcactccc tccgttgacg tttttgtttt 60 ctttttctct gtgttctgaa gaagttttag ggtttcgttt tgtttctctc ttcggccact 120 tcaggctatg gattcatttg agccagatgg aaatgggaag gagtcactgc caccaccacc 180 tcctgttgtt ccctctgata ttgtacctct caaagcagag gaggtgctct gtacccctac 240 cgagcataat aagaaaaagg cttcccgact tccaatagcc agatctggtc tgggatcaaa 300 aggaaataaa atacaattac taaccaatca cttcaaagtt aatgttgcta aaaatgatgg 360 gcatttcttc cattatagtg tggcttttac ttatgaagat ggacgccctg tagaaggtaa 420 gggtgtaggg agaaagataa tagatagggt gcaggagaca tatcattctg acttaaatgg 480 taaggacttt gcatatgatg gggagaaaag tctgtttact gttggctctc ttcctcaaaa 540 caagcttgag tttgaagttg ttcttgagga tgtcacctct aacaggaata atggcaattg 600 cagccctgat ggtctagggg acaatgagag tgacagaaag aggatgcgac gtccttatcg 660 ttcgaagtca ttcaaagtag agataagctt tgctgcaaaa attccaatgc aggccattgc 720 cagtgcctta cgcgggcaag agactgagaa ttttcaagaa gccatcagag ttcttgatat 780 cattttgagg cagcatgctg ctaagcaagg ctgcttactt gtacgccaat cctttttcca 840 caataatcca aataattttg ctgatgtagg aggtggtgtc ctaggctgta gaggattcca 900 ctcaagcttt agaactacac agagtggcct gtctcttaac atagatgtgt caactacaat 960 gataatttct cctgggcctg tggtggattt cttaatttcc aatcaaaatg tgagagatcc 1020 ttttcaactt gactgggcta aggccaaaag gaccctaaaa aatctgagga ttaaaactag 1080 cccatccaat caagaattca aaatttctgg gctcagtgaa ctcccatgca gagagcagac 1140 ttttactttg aaaggtaaag gtggggggga tggtgaagat ggtaatgagg aaatcactgt 1200 atatgattat tttgttaagg ttcgtaagat agatctccga tactctgctg accttccatg 1260 tatcaatgtt ggcaagccta aacgaccaac atttttcccc attgaggttt gtgaattggt 1320 atcattgcaa cgatatacaa aagctctgtc cacgcttcaa agggcttcat tagtggagaa 1380 gtcgaggcag aagccacaag agaggatgaa aattttgtct gatgcactga gaacaagcaa 1440 ctatggtgct gaacctatgc tccggaattg tggaatttct ataagcactg gcttcactga 1500 agtggagggc cgggtgttgc ctgcaccaag gttgaagttt ggcaatggtg aggatctcaa 1560 tcctaggaat gggagatgga atgtcagcag agtgaaattt gtggaaccat caaagataga 1620 aagatgggct gttgctaact tttctgcacg ctgtgatgta cgaggacttg tacgggacct 1680 cattagaatt ggagatatga aaggaattac tatagaacaa ccatttgacg tgtttgatga 1740 gaatccacag tttaggcgtg ccccccctat ggttagagtg gagaaaatgt tcgagcatat 1800 ccaatctaaa cttcctgggg ctcctcagtt ccttctctgt ttgcttcctg atcggaaaaa 1860 ttgtgatatt tatggtccat ggaaaaagaa gaatcttgct gattttggaa tcataaatca 1920 gtgtatgtgt cctttaaggg tcaatgacca gtacctgact aatgttatgt tgaagatcaa 1980 tgccaagctt ggtgggttga attcattgtt aggcgttgaa cattctcctt ctcttcctgt 2040 tgtttccaaa gctcccaccc tcattctggg aatggacgtg tcacatggct cacctgggca 2100 gactgacatt ccttcaattg ctgcggtggt cagctctaga cactggcctc tgatatcaaa 2160 gtatagggca tgtgttcgta cgcaatctgc aaagatggaa atgattgata atttgttcaa 2220 gctagtatct gaaaaggaag atgaaggcat cataagggaa cttttgcttg atttctatac 2280 aacttctggg aggagaaaac cggaaaatat aatcatattc agggatgggg ttagtgagtc 2340 acaattcaat caagttttga atattgaact cgatcgaatc attgaggctt gcaaatttct 2400 cgatgaaaat tgggagccaa aatttgtggt aattgttgct cagaagaacc accacactag 2460 atttttccag cctggctctc ccgacaatgt cccacctgga actgttatcg acaataaaat 2520 ttgtcatccc agaaattatg atttctacct atgtgcacat gctggaatga taggaactag 2580 taggcctacc cattatcatg tgctgcttga tcaggttggt ttctctccgg atcagctgca 2640 ggagcttgtc cattcattat catatgtgta tcagaggagc actactgcca tttctgttgt 2700 tgctccaata tgctatgcgc acttggctgc tactcagttg gggcagttca tgaaatttga 2760 ggacaaatct gaaacatctt caagccatgg tggattgagc ggtgcaagtg ctgttcccgt 2820 ccctcagttg cctcccttgc aagagaatgt ccgcaacaca atgttctttt gttgaagcct 2880 taatgctctg ccctgtctcc tcaagtggtg aaaatgctgt acataaaact atgtttctaa 2940 tcttgcaagt tatgcggacg aagtttatat tgtggtagac ttggtcttct tagccatatt 3000 tagtctttct agcacaagcc ttttcaaatg ttcggggacc ttaccttact ttttgtagca 3060 agactctctt tagcgcaacg tctttttgta gcaaggcttg atcttcagca cgtcttttgt 3120 taggcccccc cttttttaag gtttaattgc actttttacc tcgaatgctg taatttatag 3180 tgaattttac ttatctccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 3239 40 915 PRT Glycine max 40 Met Asp Ser Phe Glu Pro Asp Gly Asn Gly Lys Glu Ser Leu Pro Pro 1 5 10 15 Pro Pro Pro Val Val Pro Ser Asp Ile Val Pro Leu Lys Ala Glu Glu 20 25 30 Val Leu Cys Thr Pro Thr Glu His Asn Lys Lys Lys Ala Ser Arg Leu 35 40 45 Pro Ile Ala Arg Ser Gly Leu Gly Ser Lys Gly Asn Lys Ile Gln Leu 50 55 60 Leu Thr Asn His Phe Lys Val Asn Val Ala Lys Asn Asp Gly His Phe 65 70 75 80 Phe His Tyr Ser Val Ala Phe Thr Tyr Glu Asp Gly Arg Pro Val Glu 85 90 95 Gly Lys Gly Val Gly Arg Lys Ile Ile Asp Arg Val Gln Glu Thr Tyr 100 105 110 His Ser Asp Leu Asn Gly Lys Asp Phe Ala Tyr Asp Gly Glu Lys Ser 115 120 125 Leu Phe Thr Val Gly Ser Leu Pro Gln Asn Lys Leu Glu Phe Glu Val 130 135 140 Val Leu Glu Asp Val Thr Ser Asn Arg Asn Asn Gly Asn Cys Ser Pro 145 150 155 160 Asp Gly Leu Gly Asp Asn Glu Ser Asp Arg Lys Arg Met Arg Arg Pro 165 170 175 Tyr Arg Ser Lys Ser Phe Lys Val Glu Ile Ser Phe Ala Ala Lys Ile 180 185 190 Pro Met Gln Ala Ile Ala Ser Ala Leu Arg Gly Gln Glu Thr Glu Asn 195 200 205 Phe Gln Glu Ala Ile Arg Val Leu Asp Ile Ile Leu Arg Gln His Ala 210 215 220 Ala Lys Gln Gly Cys Leu Leu Val Arg Gln Ser Phe Phe His Asn Asn 225 230 235 240 Pro Asn Asn Phe Ala Asp Val Gly Gly Gly Val Leu Gly Cys Arg Gly 245 250 255 Phe His Ser Ser Phe Arg Thr Thr Gln Ser Gly Leu Ser Leu Asn Ile 260 265 270 Asp Val Ser Thr Thr Met Ile Ile Ser Pro Gly Pro Val Val Asp Phe 275 280 285 Leu Ile Ser Asn Gln Asn Val Arg Asp Pro Phe Gln Leu Asp Trp Ala 290 295 300 Lys Ala Lys Arg Thr Leu Lys Asn Leu Arg Ile Lys Thr Ser Pro Ser 305 310 315 320 Asn Gln Glu Phe Lys Ile Ser Gly Leu Ser Glu Leu Pro Cys Arg Glu 325 330 335 Gln Thr Phe Thr Leu Lys Gly Lys Gly Gly Gly Asp Gly Glu Asp Gly 340 345 350 Asn Glu Glu Ile Thr Val Tyr Asp Tyr Phe Val Lys Val Arg Lys Ile 355 360 365 Asp Leu Arg Tyr Ser Ala Asp Leu Pro Cys Ile Asn Val Gly Lys Pro 370 375 380 Lys Arg Pro Thr Phe Phe Pro Ile Glu Val Cys Glu Leu Val Ser Leu 385 390 395 400 Gln Arg Tyr Thr Lys Ala Leu Ser Thr Leu Gln Arg Ala Ser Leu Val 405 410 415 Glu Lys Ser Arg Gln Lys Pro Gln Glu Arg Met Lys Ile Leu Ser Asp 420 425 430 Ala Leu Arg Thr Ser Asn Tyr Gly Ala Glu Pro Met Leu Arg Asn Cys 435 440 445 Gly Ile Ser Ile Ser Thr Gly Phe Thr Glu Val Glu Gly Arg Val Leu 450 455 460 Pro Ala Pro Arg Leu Lys Phe Gly Asn Gly Glu Asp Leu Asn Pro Arg 465 470 475 480 Asn Gly Arg Trp Asn Val Ser Arg Val Lys Phe Val Glu Pro Ser Lys 485 490 495 Ile Glu Arg Trp Ala Val Ala Asn Phe Ser Ala Arg Cys Asp Val Arg 500 505 510 Gly Leu Val Arg Asp Leu Ile Arg Ile Gly Asp Met Lys Gly Ile Thr 515 520 525 Ile Glu Gln Pro Phe Asp Val Phe Asp Glu Asn Pro Gln Phe Arg Arg 530 535 540 Ala Pro Pro Met Val Arg Val Glu Lys Met Phe Glu His Ile Gln Ser 545 550 555 560 Lys Leu Pro Gly Ala Pro Gln Phe Leu Leu Cys Leu Leu Pro Asp Arg 565 570 575 Lys Asn Cys Asp Ile Tyr Gly Pro Trp Lys Lys Lys Asn Leu Ala Asp 580 585 590 Phe Gly Ile Ile Asn Gln Cys Met Cys Pro Leu Arg Val Asn Asp Gln 595 600 605 Tyr Leu Thr Asn Val Met Leu Lys Ile Asn Ala Lys Leu Gly Gly Leu 610 615 620 Asn Ser Leu Leu Gly Val Glu His Ser Pro Ser Leu Pro Val Val Ser 625 630 635 640 Lys Ala Pro Thr Leu Ile Leu Gly Met Asp Val Ser His Gly Ser Pro 645 650 655 Gly Gln Thr Asp Ile Pro Ser Ile Ala Ala Val Val Ser Ser Arg His 660 665 670 Trp Pro Leu Ile Ser Lys Tyr Arg Ala Cys Val Arg Thr Gln Ser Ala 675 680 685 Lys Met Glu Met Ile Asp Asn Leu Phe Lys Leu Val Ser Glu Lys Glu 690 695 700 Asp Glu Gly Ile Ile Arg Glu Leu Leu Leu Asp Phe Tyr Thr Thr Ser 705 710 715 720 Gly Arg Arg Lys Pro Glu Asn Ile Ile Ile Phe Arg Asp Gly Val Ser 725 730 735 Glu Ser Gln Phe Asn Gln Val Leu Asn Ile Glu Leu Asp Arg Ile Ile 740 745 750 Glu Ala Cys Lys Phe Leu Asp Glu Asn Trp Glu Pro Lys Phe Val Val 755 760 765 Ile Val Ala Gln Lys Asn His His Thr Arg Phe Phe Gln Pro Gly Ser 770 775 780 Pro Asp Asn Val Pro Pro Gly Thr Val Ile Asp Asn Lys Ile Cys His 785 790 795 800 Pro Arg Asn Tyr Asp Phe Tyr Leu Cys Ala His Ala Gly Met Ile Gly 805 810 815 Thr Ser Arg Pro Thr His Tyr His Val Leu Leu Asp Gln Val Gly Phe 820 825 830 Ser Pro Asp Gln Leu Gln Glu Leu Val His Ser Leu Ser Tyr Val Tyr 835 840 845 Gln Arg Ser Thr Thr Ala Ile Ser Val Val Ala Pro Ile Cys Tyr Ala 850 855 860 His Leu Ala Ala Thr Gln Leu Gly Gln Phe Met Lys Phe Glu Asp Lys 865 870 875 880 Ser Glu Thr Ser Ser Ser His Gly Gly Leu Ser Gly Ala Ser Ala Val 885 890 895 Pro Val Pro Gln Leu Pro Pro Leu Gln Glu Asn Val Arg Asn Thr Met 900 905 910 Phe Phe Cys 915 41 3151 DNA Triticum aestivum 41 gttgttcgag gagaggggag ggggagagac gagaagggga acggaaaaga aagccaagcc 60 ctctctcgcg gaggccaacg gcgaggcttc ctcccttgcg ccctcgcaga tcagttcagc 120 ggttcggctc ctcgggacca ttgttggttc gctgaaatgg agtcacacgg agaggacctg 180 ccaccaccac caccactccc gccaaatgca gagccgataa aagctgagtc ggctgatgac 240 ttgccaccac caccacccct gctgcctatc aaacctgaag aagcaaagaa gatctcaaag 300 cctaagaggg ccctgatcgc tcgtcctggt tttggcaaga ggggaaatcc tatacagctt 360 gtgacaaatc atttcaaagt ctcgttgaag acgacagacg agttcttcca tcattactat 420 gtaaatctga agtatgaaga tgacaggcct gttgatggaa aaggtgttgg tagaaaagtc 480 attgataagc ttgctcagac ttatccatcg gaactagccc ataaagactt tgcctatgat 540 ggtgaaaaga gtctttttac cattggtgcc ctcccacaaa ttaacaatga gtttgttgtg 600 gttcttgaag atgtttccag tggaaagact cctgcaaatg gcagccctgg aaacgacagt 660 ccagacaaga agagagtgaa aaggccatat caaactaaaa ccttcaaggt ggagttgagc 720 tttgctgcta gaatccccat gagtgctatt gcaatggcac tcaaaggcca ggaatcagag 780 cacacgcaag aagccattcg ggttattgat atcatattaa gacagcactc tgccaaacag 840 ggctgcctgt tagtccgcca gtcatttttt cacaacaatc cttcaaactt tgtggacttg 900 ggtgggggtg tgatgggctg ccgaggtttc cactcaagct ttcgagccac acagagcggg 960 ctttctctta atattgatgt ttctacaaca atgattgtga aacctggccc tgttgtcgat 1020 tttctgctgg ccaaccagaa ggttgaccac cctaataaaa ttgattgggc taaggccaag 1080 cgtgcactta agaatttaag gataaaaaca agcccagcaa atacagaata caagattgtt 1140 ggtttgagtg agaggaattg ttatgaacaa atgttttccc tcaagcaaag gaatggtggg 1200 aatggtgacc ctgaagcaat agaaatatct gtttatgatt actttgtgaa gaaccgtggc 1260 attgagctga ggtactctgg tgatttccct tgtataaatg ttgggaaacc taggcggcca 1320 acatattttc ccattgagct ctgccagctg gtccctttac aaaggtatac caaatctttg 1380 agtaccctac aaagatcatc tcttgttgag aagtccaggc agaagcctca agagaggatg 1440 tcagttttgt ctgatgtact gaaacgcagc agctatgata cagaacccat gttgaaggca 1500 tgtggaattt cgatagctca gggctttaca caggtggctg gtagggtact gcaggccccc 1560 aagctcaaag ctggaaatgg tgaagatatt ttcacaagga atggacgttg gaatttcaac 1620 aacaagaggc ttgctagagc ttgtgtggtg gacagatggg cagttgtaaa cttttcggct 1680 aggtgtaaca ccatgaacct tgtcaatgac ctcatcaagt gtgggggcat gaagggcatt 1740 acagtagaaa aacctcatat tgtaattgaa gagaatggtt caatgagacg tgcacctgct 1800 ccaaaaaggg ttgaggatat gtttgagcaa gtgaagtcta agcttcctgg ggctccgaag 1860 tttctcttgt gtattcttgc tgagaggaag aactcagatg tttatggtcc atggaagcga 1920 aaatgccttg ctgactttgg gattgtcact caatgtgtgg ccccaacaag ggtcaatgac 1980 caatatctga caaatgttct gctgaagatc aatgcaaaac ttggtggaat gaactcacta 2040 ctacaaattg aaatgtcccc aagtatacct cttgtatcaa aggtcccaac tctcatcttg 2100 ggaatggatg tgtcccatgg atcccctgga cagtctgata taccgtccat tgcagcagtt 2160 gttggttctc gggaatggcc tcttgtctcg aaatataggg cttcagtgcg ctcgcagtca 2220 ccaaagctcg aaatgataga ttcattgttc aagccacaag gaactgatga tgatggcctt 2280 gttcgggagt gtctcattga cttctacacc agttctggaa aaaggaaacc agatcagatc 2340 atcatcttca gggatggtgt tagtgagagc cagtttaatc aggtgctgaa cattgaattg 2400 gatcaaataa ttgaggcctg caagttcttg gatgaaaatt ggaaccccaa gttcacgctg 2460 attgttgccc agaaaaatca ccacaccaaa ttcttcatac ctggatctcc tgacaatgtc 2520 cctccaggca ctgttgtaga taatgcagtc tgccatccaa ggaattatga cttctacatg 2580 tgcgctcatg ctggaatgat tgggactaca aggccaacac actaccatat cctgcatgat 2640 gagatacact ttgctgcgga tgacctgcag gatcttgtgc actcgctctc atatgtgtac 2700 caaaggagca cgacagccat atcagttgtt tctccaatct gctatgcaca tcttgcggct 2760 gctcaggtgg cgcagttcat aaagtttgat gagatgtctg agacgtcgtc gagccagggc 2820 ggtggccaca cctctgccgg cagcgctcca gtgcaggagc tgcctcgcct ccatgagaaa 2880 gtccgcagca gcatgttctt ctgctgagcc agccagccag ccgcacttgc gcgttccaac 2940 ttttggtgat gcgcttggtt atctagtact agtagtatgt agtagtggcc tgtgatggcc 3000 tgttggactc ctgggatgtt gtgttcctaa gctggttgct gcacttggtg cctcagaacc 3060 tttgaatcct gtcagggtgc tgcagttgaa cctttactat cgaaccatct aatttgttgc 3120 tttcaaaaaa aaaaaaaaaa aaaaaaaaaa a 3151 42 916 PRT Triticum aestivum 42 Met Glu Ser His Gly Glu Asp Leu Pro Pro Pro Pro Pro Leu Pro Pro 1 5 10 15 Asn Ala Glu Pro Ile Lys Ala Glu Ser Ala Asp Asp Leu Pro Pro Pro 20 25 30 Pro Pro Leu Leu Pro Ile Lys Pro Glu Glu Ala Lys Lys Ile Ser Lys 35 40 45 Pro Lys Arg Ala Leu Ile Ala Arg Pro Gly Phe Gly Lys Arg Gly Asn 50 55 60 Pro Ile Gln Leu Val Thr Asn His Phe Lys Val Ser Leu Lys Thr Thr 65 70 75 80 Asp Glu Phe Phe His His Tyr Tyr Val Asn Leu Lys Tyr Glu Asp Asp 85 90 95 Arg Pro Val Asp Gly Lys Gly Val Gly Arg Lys Val Ile Asp Lys Leu 100 105 110 Ala Gln Thr Tyr Pro Ser Glu Leu Ala His Lys Asp Phe Ala Tyr Asp 115 120 125 Gly Glu Lys Ser Leu Phe Thr Ile Gly Ala Leu Pro Gln Ile Asn Asn 130 135 140 Glu Phe Val Val Val Leu Glu Asp Val Ser Ser Gly Lys Thr Pro Ala 145 150 155 160 Asn Gly Ser Pro Gly Asn Asp Ser Pro Asp Lys Lys Arg Val Lys Arg 165 170 175 Pro Tyr Gln Thr Lys Thr Phe Lys Val Glu Leu Ser Phe Ala Ala Arg 180 185 190 Ile Pro Met Ser Ala Ile Ala Met Ala Leu Lys Gly Gln Glu Ser Glu 195 200 205 His Thr Gln Glu Ala Ile Arg Val Ile Asp Ile Ile Leu Arg Gln His 210 215 220 Ser Ala Lys Gln Gly Cys Leu Leu Val Arg Gln Ser Phe Phe His Asn 225 230 235 240 Asn Pro Ser Asn Phe Val Asp Leu Gly Gly Gly Val Met Gly Cys Arg 245 250 255 Gly Phe His Ser Ser Phe Arg Ala Thr Gln Ser Gly Leu Ser Leu Asn 260 265 270 Ile Asp Val Ser Thr Thr Met Ile Val Lys Pro Gly Pro Val Val Asp 275 280 285 Phe Leu Leu Ala Asn Gln Lys Val Asp His Pro Asn Lys Ile Asp Trp 290 295 300 Ala Lys Ala Lys Arg Ala Leu Lys Asn Leu Arg Ile Lys Thr Ser Pro 305 310 315 320 Ala Asn Thr Glu Tyr Lys Ile Val Gly Leu Ser Glu Arg Asn Cys Tyr 325 330 335 Glu Gln Met Phe Ser Leu Lys Gln Arg Asn Gly Gly Asn Gly Asp Pro 340 345 350 Glu Ala Ile Glu Ile Ser Val Tyr Asp Tyr Phe Val Lys Asn Arg Gly 355 360 365 Ile Glu Leu Arg Tyr Ser Gly Asp Phe Pro Cys Ile Asn Val Gly Lys 370 375 380 Pro Arg Arg Pro Thr Tyr Phe Pro Ile Glu Leu Cys Gln Leu Val Pro 385 390 395 400 Leu Gln Arg Tyr Thr Lys Ser Leu Ser Thr Leu Gln Arg Ser Ser Leu 405 410 415 Val Glu Lys Ser Arg Gln Lys Pro Gln Glu Arg Met Ser Val Leu Ser 420 425 430 Asp Val Leu Lys Arg Ser Ser Tyr Asp Thr Glu Pro Met Leu Lys Ala 435 440 445 Cys Gly Ile Ser Ile Ala Gln Gly Phe Thr Gln Val Ala Gly Arg Val 450 455 460 Leu Gln Ala Pro Lys Leu Lys Ala Gly Asn Gly Glu Asp Ile Phe Thr 465 470 475 480 Arg Asn Gly Arg Trp Asn Phe Asn Asn Lys Arg Leu Ala Arg Ala Cys 485 490 495 Val Val Asp Arg Trp Ala Val Val Asn Phe Ser Ala Arg Cys Asn Thr 500 505 510 Met Asn Leu Val Asn Asp Leu Ile Lys Cys Gly Gly Met Lys Gly Ile 515 520 525 Thr Val Glu Lys Pro His Ile Val Ile Glu Glu Asn Gly Ser Met Arg 530 535 540 Arg Ala Pro Ala Pro Lys Arg Val Glu Asp Met Phe Glu Gln Val Lys 545 550 555 560 Ser Lys Leu Pro Gly Ala Pro Lys Phe Leu Leu Cys Ile Leu Ala Glu 565 570 575 Arg Lys Asn Ser Asp Val Tyr Gly Pro Trp Lys Arg Lys Cys Leu Ala 580 585 590 Asp Phe Gly Ile Val Thr Gln Cys Val Ala Pro Thr Arg Val Asn Asp 595 600 605 Gln Tyr Leu Thr Asn Val Leu Leu Lys Ile Asn Ala Lys Leu Gly Gly 610 615 620 Met Asn Ser Leu Leu Gln Ile Glu Met Ser Pro Ser Ile Pro Leu Val 625 630 635 640 Ser Lys Val Pro Thr Leu Ile Leu Gly Met Asp Val Ser His Gly Ser 645 650 655 Pro Gly Gln Ser Asp Ile Pro Ser Ile Ala Ala Val Val Gly Ser Arg 660 665 670 Glu Trp Pro Leu Val Ser Lys Tyr Arg Ala Ser Val Arg Ser Gln Ser 675 680 685 Pro Lys Leu Glu Met Ile Asp Ser Leu Phe Lys Pro Gln Gly Thr Asp 690 695 700 Asp Asp Gly Leu Val Arg Glu Cys Leu Ile Asp Phe Tyr Thr Ser Ser 705 710 715 720 Gly Lys Arg Lys Pro Asp Gln Ile Ile Ile Phe Arg Asp Gly Val Ser 725 730 735 Glu Ser Gln Phe Asn Gln Val Leu Asn Ile Glu Leu Asp Gln Ile Ile 740 745 750 Glu Ala Cys Lys Phe Leu Asp Glu Asn Trp Asn Pro Lys Phe Thr Leu 755 760 765 Ile Val Ala Gln Lys Asn His His Thr Lys Phe Phe Ile Pro Gly Ser 770 775 780 Pro Asp Asn Val Pro Pro Gly Thr Val Val Asp Asn Ala Val Cys His 785 790 795 800 Pro Arg Asn Tyr Asp Phe Tyr Met Cys Ala His Ala Gly Met Ile Gly 805 810 815 Thr Thr Arg Pro Thr His Tyr His Ile Leu His Asp Glu Ile His Phe 820 825 830 Ala Ala Asp Asp Leu Gln Asp Leu Val His Ser Leu Ser Tyr Val Tyr 835 840 845 Gln Arg Ser Thr Thr Ala Ile Ser Val Val Ser Pro Ile Cys Tyr Ala 850 855 860 His Leu Ala Ala Ala Gln Val Ala Gln Phe Ile Lys Phe Asp Glu Met 865 870 875 880 Ser Glu Thr Ser Ser Ser Gln Gly Gly Gly His Thr Ser Ala Gly Ser 885 890 895 Ala Pro Val Gln Glu Leu Pro Arg Leu His Glu Lys Val Arg Ser Ser 900 905 910 Met Phe Phe Cys 915 43 791 DNA Triticum aestivum 43 ctcgtgccga attcggcacg agaacttcct caaatacgca acgcatgccg cctgaaatat 60 cctgcaaccg acaccaaacg agggctcccc aggatcacaa ttgttgtctg tggtaaacgc 120 caccacactc gattctaccc taaaaacagc ggtgacgctg ataaatcatc gaatttgatg 180 gctggaactg ttgtcgatcg tggcgttaca gagactcgaa actgggactt ttacctacaa 240 gcccatgcat gtcttcaggg aacagcccgt gcctgtcatt actatgtgat aatagacgaa 300 attttccggt ccaataaggt taagggtggt cacaaaaatc acgctgatgc ccttgaggaa 360 ttgacaaaca atatgagtca tctgtttgga cgagcaacaa aagccgtcag tctttgtcct 420 cctgcttact atgctgattt actctgcaca agggtacgct gctacttatc tgaagttttc 480 gacccaagtg aggcccagag tgtgatgagt ggcggcacca accaaacgat cgaggacatt 540 gttattccgc cgagtatgag ggattccatg tactacatct aagctcattg catgagaatg 600 agaatcatta aaccataacc ttcggtgtta gttacagaat tagctgtgtc aagtcattat 660 agacgaaata ccatttctgt attgtagact ttgcgttccg aaatatttta tgcacacgca 720 aatgtatagc caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780 aaaaaaaaaa a 791 44 193 PRT Triticum aestivum 44 Leu Val Pro Asn Ser Ala Arg Glu Leu Pro Gln Ile Arg Asn Ala Cys 1 5 10 15 Arg Leu Lys Tyr Pro Ala Thr Asp Thr Lys Arg Gly Leu Pro Arg Ile 20 25 30 Thr Ile Val Val Cys Gly Lys Arg His His Thr Arg Phe Tyr Pro Lys 35 40 45 Asn Ser Gly Asp Ala Asp Lys Ser Ser Asn Leu Met Ala Gly Thr Val 50 55 60 Val Asp Arg Gly Val Thr Glu Thr Arg Asn Trp Asp Phe Tyr Leu Gln 65 70 75 80 Ala His Ala Cys Leu Gln Gly Thr Ala Arg Ala Cys His Tyr Tyr Val 85 90 95 Ile Ile Asp Glu Ile Phe Arg Ser Asn Lys Val Lys Gly Gly His Lys 100 105 110 Asn His Ala Asp Ala Leu Glu Glu Leu Thr Asn Asn Met Ser His Leu 115 120 125 Phe Gly Arg Ala Thr Lys Ala Val Ser Leu Cys Pro Pro Ala Tyr Tyr 130 135 140 Ala Asp Leu Leu Cys Thr Arg Val Arg Cys Tyr Leu Ser Glu Val Phe 145 150 155 160 Asp Pro Ser Glu Ala Gln Ser Val Met Ser Gly Gly Thr Asn Gln Thr 165 170 175 Ile Glu Asp Ile Val Ile Pro Pro Ser Met Arg Asp Ser Met Tyr Tyr 180 185 190 Ile 45 327 DNA Triticum aestivum 45 tgcgttctga catccattcg aggcccctct cagacgccga acgtgttaag atcaagaagg 60 cactgagagg agtaaaggtg gaagttactc atcgtggcaa catgcgaagg aagtaccgaa 120 tatctggtct gacaacccag gcaactcgag agctaacttt tcctgttgat gaagggggta 180 cagtaaagtc agtcgtacaa tactttcagg agacatatgg ctttgccatc cagcacacgt 240 acctgccttg cctccaagtt ggcaatcagc agcgtccaaa ttacttgggg gatcctctag 300 aggcgaccgg caggcataca agcttgg 327 46 95 PRT Triticum aestivum 46 Arg Ser Asp Ile His Ser Arg Pro Leu Ser Asp Ala Glu Arg Val Lys 1 5 10 15 Ile Lys Lys Ala Leu Arg Gly Val Lys Val Glu Val Thr His Arg Gly 20 25 30 Asn Met Arg Arg Lys Tyr Arg Ile Ser Gly Leu Thr Thr Gln Ala Thr 35 40 45 Arg Glu Leu Thr Phe Pro Val Asp Glu Gly Gly Thr Val Lys Ser Val 50 55 60 Val Gln Tyr Phe Gln Glu Thr Tyr Gly Phe Ala Ile Gln His Thr Tyr 65 70 75 80 Leu Pro Cys Leu Gln Val Gly Asn Gln Gln Arg Pro Asn Tyr Leu 85 90 95 47 571 DNA Triticum aestivum unsure (234) n = A, C, G or T 47 attagtgtta ccaaaagtcg gcaagtggga catgtggtgc aagaaaatgg tcaatggagg 60 agtagttaac acctgggcat gcattaactt tgcttgggaa gtcacagatg ctcatgctct 120 gaatttttgt gatgagttgg tgctgatgtg caatgtatcc gggatggact tcaggcctga 180 acctgtgctc cctgtaacag cttatgaccc taaatccgta gcacggtcac tcanaganac 240 accataaang tntcatgaac atacctggnc cacngcgcca aanactcgac ctgctgattc 300 naatattgct gacaagtant ggcacccttt atggtgacat caggagaata ttngggacag 360 atattgggag tggtctctca nngttgtctt gcaaaacatg tttttaancc caaaaaacat 420 atttnncaat gttgccctta aaataatgnt aangcnggag ganaaancgg tcntttangc 480 ttgaaaggaa cccccctatg ggaaaaaacg cnncnatttg ggcgnantag cntcaaaccn 540 gcaagggttc caccctccat gnnngtgtgg t 571 48 77 PRT Triticum aestivum 48 Leu Val Leu Pro Lys Val Gly Lys Trp Asp Met Trp Cys Lys Lys Met 1 5 10 15 Val Asn Gly Gly Val Val Asn Thr Trp Ala Cys Ile Asn Phe Ala Trp 20 25 30 Glu Val Thr Asp Ala His Ala Leu Asn Phe Cys Asp Glu Leu Val Leu 35 40 45 Met Cys Asn Val Ser Gly Met Asp Phe Arg Pro Glu Pro Val Leu Pro 50 55 60 Val Thr Ala Tyr Asp Pro Lys Ser Val Ala Arg Ser Leu 65 70 75 49 1565 DNA Triticum aestivum 49 actcgaatat gaggaccctc acactgtaat tgaagagagc ccgtcactga gacgagctcc 60 ggtggcacga agagtggagg agatgtttgc ccagataaag gccaagctac ctggagcacc 120 cttgtttctt ttgtgcctcc tccctgagag gaagaactgc gaagtttacg gtccttggaa 180 gaagaagtgt cttgctgatt tcggcatagt cacccaatgt ctagctccgc aaagagtcaa 240 tgaccagtac ttgagtaatc tgctactcaa gataaatgct aagctcggtg gactcaacac 300 actgcttcaa attgaagcag cccgtgcaat acccattgtg gggaaggtgc ctactatcat 360 cctgggcatg gatgtctcgc atggtcaacc tggccaatcc gacaggcctt ccattgctgc 420 ggtggtgagt tctcgtgagt ggcctctcat ctctaaatac agagcaacag tgcacactca 480 gtcacccaaa caggaggtga tggcttccct gtttaagcca cggggagctg aagatgatgg 540 ccttattcgg gaatctctta ttgacttgta cactagctct gggaagcgaa agccagacca 600 agttattatt ttcagggatg gagttagcga aagccagttt actcaggtga taaacattga 660 gcttgagcag atcattgagg catgcaagtg ccttgacgac aagtgggagc ccaagttcac 720 ggtcattgtt gctcagaaaa accatcatac caggtttttc cagacaaact cgccagaaaa 780 tgttcctcct ggcactgtgg tggataaaca agtgtgccat cccaagaact ttgacttcta 840 catgtgcgcg catgctggga tgattggcac gtcgaggcca acgcattacc atgttctgca 900 tgatgagatc ggcttcagtg gggatgagct ccaggagttt gtgcactcgc tctcctatgt 960 gtaccagagg agcacgacgg cgatatcagt agctgctccg atagcgtacg cgcatctggc 1020 ggcggcgcag gtgggcacct tcatgaagtt tgaggacatg tcggacacgt cgtcgagcca 1080 gggagggggc cacacgtctg cgggcagcgc cccggtgccg gagctgcctc ggctgcacga 1140 gaaagtgagg agctccatgt tcttctgctg atctgatgct gctcttgaac ttgatcgatg 1200 ccgctttctg tcagtggagg ttgaaccgtg cgtctgtata aataaaacct actagtacct 1260 atctatctat gtactatcta gatggcacct ggaactttag ctgttatcca gggtgcccgt 1320 aagtcggtcc gttgtgtcgg gtgccgctgg gaacgttccc atggatgtta ccgtttgtgg 1380 tgttggcgtt gttgaaccaa ccaacctgac cctagcttaa ccttgcttgg attggatgat 1440 gtgctagcta gctagagcta gagctagagt tagaccatgc atggctgatg gtatgtattg 1500 tgggatcata tctatctatc tccatcctga cttggtgata aaaaaaaaaa aaaaaaaaaa 1560 aaaaa 1565 50 389 PRT Triticum aestivum 50 Leu Glu Tyr Glu Asp Pro His Thr Val Ile Glu Glu Ser Pro Ser Leu 1 5 10 15 Arg Arg Ala Pro Val Ala Arg Arg Val Glu Glu Met Phe Ala Gln Ile 20 25 30 Lys Ala Lys Leu Pro Gly Ala Pro Leu Phe Leu Leu Cys Leu Leu Pro 35 40 45 Glu Arg Lys Asn Cys Glu Val Tyr Gly Pro Trp Lys Lys Lys Cys Leu 50 55 60 Ala Asp Phe Gly Ile Val Thr Gln Cys Leu Ala Pro Gln Arg Val Asn 65 70 75 80 Asp Gln Tyr Leu Ser Asn Leu Leu Leu Lys Ile Asn Ala Lys Leu Gly 85 90 95 Gly Leu Asn Thr Leu Leu Gln Ile Glu Ala Ala Arg Ala Ile Pro Ile 100 105 110 Val Gly Lys Val Pro Thr Ile Ile Leu Gly Met Asp Val Ser His Gly 115 120 125 Gln Pro Gly Gln Ser Asp Arg Pro Ser Ile Ala Ala Val Val Ser Ser 130 135 140 Arg Glu Trp Pro Leu Ile Ser Lys Tyr Arg Ala Thr Val His Thr Gln 145 150 155 160 Ser Pro Lys Gln Glu Val Met Ala Ser Leu Phe Lys Pro Arg Gly Ala 165 170 175 Glu Asp Asp Gly Leu Ile Arg Glu Ser Leu Ile Asp Leu Tyr Thr Ser 180 185 190 Ser Gly Lys Arg Lys Pro Asp Gln Val Ile Ile Phe Arg Asp Gly Val 195 200 205 Ser Glu Ser Gln Phe Thr Gln Val Ile Asn Ile Glu Leu Glu Gln Ile 210 215 220 Ile Glu Ala Cys Lys Cys Leu Asp Asp Lys Trp Glu Pro Lys Phe Thr 225 230 235 240 Val Ile Val Ala Gln Lys Asn His His Thr Arg Phe Phe Gln Thr Asn 245 250 255 Ser Pro Glu Asn Val Pro Pro Gly Thr Val Val Asp Lys Gln Val Cys 260 265 270 His Pro Lys Asn Phe Asp Phe Tyr Met Cys Ala His Ala Gly Met Ile 275 280 285 Gly Thr Ser Arg Pro Thr His Tyr His Val Leu His Asp Glu Ile Gly 290 295 300 Phe Ser Gly Asp Glu Leu Gln Glu Phe Val His Ser Leu Ser Tyr Val 305 310 315 320 Tyr Gln Arg Ser Thr Thr Ala Ile Ser Val Ala Ala Pro Ile Ala Tyr 325 330 335 Ala His Leu Ala Ala Ala Gln Val Gly Thr Phe Met Lys Phe Glu Asp 340 345 350 Met Ser Asp Thr Ser Ser Ser Gln Gly Gly Gly His Thr Ser Ala Gly 355 360 365 Ser Ala Pro Val Pro Glu Leu Pro Arg Leu His Glu Lys Val Arg Ser 370 375 380 Ser Met Phe Phe Cys 385 51 541 DNA Triticum aestivum unsure (33) n = A, C, G or T 51 gattccatgt cctttgggat ggaacaattt acngcggatg tttacgatct cagaacaatt 60 tgtgtacact acgcaaggtg cacccgttct gtatngattg tgcctccggc atactatgct 120 cacctcgcgg cttttcgagc tcggttctac atggaaccgg atacctccga tggtggctcg 180 gtcgcgagcg gtgccacgac aagccgtgcc cctcctggtg cacgcggcgg cagtagagct 240 gcagggaatg ttgctgttaa gcctctgcct gagctcaagg aaaacgtgaa gcgtgtcatg 300 ttttactgct gataagttgg ggcaacgcct ccggggtccg ggctatctat tccccgtgat 360 cccaactgaa gtgcctgctg atttaccaat cctttctttg cggcagaaaa tcaatcatca 420 gtcatcacat gagtgtatct atatatgtat cagtgctgcc atgtttcctg tgcaacctga 480 acatctcaat tcctcttttc atctacagat tttcaaatgg cattttccct gttaaaaaaa 540 a 541 52 103 PRT Triticum aestivum UNSURE (32) Xaa = ANY AMINO ACID 52 Asp Ser Met Ser Phe Gly Met Glu Gln Phe Thr Ala Asp Val Tyr Asp 1 5 10 15 Leu Arg Thr Ile Cys Val His Tyr Ala Arg Cys Thr Arg Ser Val Xaa 20 25 30 Ile Val Pro Pro Ala Tyr Tyr Ala His Leu Ala Ala Phe Arg Ala Arg 35 40 45 Phe Tyr Met Glu Pro Asp Thr Ser Asp Gly Gly Ser Val Ala Ser Gly 50 55 60 Ala Thr Thr Ser Arg Ala Pro Pro Gly Ala Arg Gly Gly Ser Arg Ala 65 70 75 80 Ala Gly Asn Val Ala Val Lys Pro Leu Pro Glu Leu Lys Glu Asn Val 85 90 95 Lys Arg Val Met Phe Tyr Cys 100 53 3705 DNA Oryza sativa unsure (3616) n = A, C, G or T 53 gagcagcagt gcggtagtgc aagcgctagt ggaggagttg ggaggaggcc ccctagggtt 60 tcccgagacc gcctcccccc gcgcctgcgc cgccgctcgc cgagcgcgcg ctccgtgccc 120 atcatggtga agaagaaaag aactgggtct ggcagcaccg gtgagagttc tggagaggct 180 ccaggagctc ctggccatgg ttcttcacag cgagctgaga gaggtcctca acagcatggg 240 ggaggacgtg gttgggtgcc tcaacatggt ggccgtggtg gtgggcaata ccagggccgt 300 ggtggacatt atcagggccg tggagggcaa ggttcacacc atccaggtgg agggcctcct 360 gagtatcagg gtcgtggagg gccaggttca catcatccag gtggtgggcc tcctgactat 420 cagggccgtg gaggatcagg ttcacatcac ccaggtggtg ggcctcccga gtatcaaccg 480 cgtgactatc aaggacgtgg tggtccacgc cccagaggtg gaatgccaca gccatactat 540 ggcggaccta gggggagtgg cggacgtagt gttccttcag gttcatcaag aacagttccc 600 gagctgcacc aagccccaca tgtccaatac caagccccga tggtttcacc aaccccatcg 660 ggagctggct catcctctca gcctgcggcg gaggtgagca gtggacaagt ccaacaacag 720 tttcagcaac ttgccacccg tgatcaaagt tcgaccagcc aagccattca aatagcacca 780 ccgtcaagca aatcagttag attcccgttg cgccctggca agggtacata tggggacagg 840 tgcattgtga aggcgaacca tttctttgct gaacttcctg ataaagacct tcaccaatac 900 gacgtatcta ttactcctga ggttacttca cgtggcgtga atcgtgctgt tatgtttgag 960 ttagtaacgc tgtatagata ttcccatttg ggcgggcgtc tacctgccta tgatggaagg 1020 aagagtcttt acacagctgg accattgcca tttgcttcta ggacatttga aattactctt 1080 caagatgagg aagatagtct tggtggtggc caaggcaccc aaaggcgtga gagactattt 1140 agggtggtga tcaagtttgc tgcccgtgct gatcttcacc atttggctat gtttctagct 1200 ggaaggcaag cagatgctcc tcaagaagcc cttcaagtcc ttgacattgt gttacgtgaa 1260 ttgcctacca caaggtactc accagttggt cggtcatttt attctcccaa tttagggaga 1320 cgccagcaac ttggtgaggg tttggaaagt tggcgtggtt tttaccaaag cataaggcct 1380 acccagatgg gtctctcact gaatattgat atgtcatcaa ctgcatttat tgagcctcta 1440 cctgtgattg actttgttgc tcagcttctg aacagagaca tctcagttag accattatct 1500 gattctgatc gtgtgaagat aaagaaagct ctaagaggtg tgaaggttga ggtgacgcat 1560 agaggaaaca tgcgtagaaa atatcgtata tctggactca cttcacaggc aacaagggag 1620 ttatcattcc ctgtcgatga tcgtggtact gtgaagactg tggtgcaata ttttctggag 1680 acatatggtt ttagtattca gcacaccact ttgccttgcc ttcaagtggg caatcagcaa 1740 aggcccaatt atctgcctat ggaggtttgt aagatcgttg agggacagcg ttactcgaag 1800 cggcttaacg agaaacagat tactgcgcta ttgaaagtga cttgccagcg acctcaagag 1860 cgtgaactgg atattttgcg gactgtatct cacaatgcat accatgaaga tcagtatgcg 1920 caggaatttg gcataaaaat tgatgagcgt cttgcatctg ttgaagctcg tgttctgcct 1980 cccccaaggc ttaaatacca tgatagtggg agagaaaagg atgtattgcc gagagttggc 2040 cagtggaaca tgatgaataa gaaaatggtc aatggtggga gagtcaacaa ctgggcatgt 2100 attaacttct ctagaaatgt gcaagatagt gctgccaggg gcttctgtca tgagctggct 2160 atcatgtgcc aaatatctgg aatggatttt gcactggaac ctgtgctgcc cccacttact 2220 gctagacctg aacatgtgga aagagcactg aaggcacgct atcaagatgc aatgaacatg 2280 ctcagaccgc agggcaggga acttgattta ctgattgtaa tactgcctga caataatggt 2340 tctctttatg gggatctcaa aagaatctgt gagactgatc ttggattggt ctcccaatgt 2400 tgtttgacaa aacatgtttt taaaatgagc aagcagtatc ttgcaaatgt tgcccttaaa 2460 ataaacgtta aggtgggggg aaggaatact gtacttgtgg atgctttgac aaggaggatt 2520 ccccttgtca gtgacagacc aactatcata tttggtgcgg atgttactca tcctcatcct 2580 ggagaagatt ccagtccttc cattgcagct gtggttgctt ctcaagactg gcctgaagtc 2640 actaagtatg ctggattggt gagtgcccaa gcccatcgtc aagaattgat acaagatctt 2700 ttcaaagtat ggcaagaccc gcatagagga actgttactg gtggcatgat caaggagctt 2760 ctcatttctt tcaagagggc tactggacag aaacctcaga ggataatatt ttacagggat 2820 ggtgtcagcg aggggcagtt ttatcaagtt ttgttgtatg agcttgatgc cattagaaag 2880 gcttgtgcat ccctggaacc caactatcag cctccagtta cctttgtggt ggtccagaag 2940 cggcatcaca caaggttgtt tgctaataat cacaacgacc agcgtactgt tgatagaagt 3000 ggaaacattc tgcctggaac tgttgttgac tcaaagattt gccatccaac cgagtttgat 3060 ttctacctgt gtagccatgc tggcatacag ggaacaagcc gtcctgctca ttatcatgtt 3120 ctgtgggatg agaacaaatt tactgcagac gagttgcaaa ccctcacgaa caacttgtgc 3180 tacacgtatg caaggtgcac tcgctctgta tcaattgtgc ctcctgcgta ctatgctcat 3240 ctggcagcct tccgagctcg cttttacatg gagccagaga catctgacag tggatcaatg 3300 gcgagtggag ctgcaacgag ccgtggcctt ccaccaggtg tgcgcagcgc cagggttgct 3360 ggaaatgtag ccgtcaggcc tctacctgct ctcaaggaaa acgtgaagcg tgtcatgttt 3420 tactgctaag agcttgggct gtaccccgta tgcgccaagg aatgtagtac tatgttatgt 3480 tattttagca cttgcactct gtcgttgatc ccgttaaaac gggtatgcta ccataagctg 3540 ttggactatt ctgggtattg tagtactact tgttttgtat ttgtgtttgt gacgctgcag 3600 agcgtgaaca acgcanaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660 aaaaaaaaaa aaaccaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 3705 54 1101 PRT Oryza sativa 54 Met Val Lys Lys Lys Arg Thr Gly Ser Gly Ser Thr Gly Glu Ser Ser 1 5 10 15 Gly Glu Ala Pro Gly Ala Pro Gly His Gly Ser Ser Gln Arg Ala Glu 20 25 30 Arg Gly Pro Gln Gln His Gly Gly Gly Arg Gly Trp Val Pro Gln His 35 40 45 Gly Gly Arg Gly Gly Gly Gln Tyr Gln Gly Arg Gly Gly His Tyr Gln 50 55 60 Gly Arg Gly Gly Gln Gly Ser His His Pro Gly Gly Gly Pro Pro Glu 65 70 75 80 Tyr Gln Gly Arg Gly Gly Pro Gly Ser His His Pro Gly Gly Gly Pro 85 90 95 Pro Asp Tyr Gln Gly Arg Gly Gly Ser Gly Ser His His Pro Gly Gly 100 105 110 Gly Pro Pro Glu Tyr Gln Pro Arg Asp Tyr Gln Gly Arg Gly Gly Pro 115 120 125 Arg Pro Arg Gly Gly Met Pro Gln Pro Tyr Tyr Gly Gly Pro Arg Gly 130 135 140 Ser Gly Gly Arg Ser Val Pro Ser Gly Ser Ser Arg Thr Val Pro Glu 145 150 155 160 Leu His Gln Ala Pro His Val Gln Tyr Gln Ala Pro Met Val Ser Pro 165 170 175 Thr Pro Ser Gly Ala Gly Ser Ser Ser Gln Pro Ala Ala Glu Val Ser 180 185 190 Ser Gly Gln Val Gln Gln Gln Phe Gln Gln Leu Ala Thr Arg Asp Gln 195 200 205 Ser Ser Thr Ser Gln Ala Ile Gln Ile Ala Pro Pro Ser Ser Lys Ser 210 215 220 Val Arg Phe Pro Leu Arg Pro Gly Lys Gly Thr Tyr Gly Asp Arg Cys 225 230 235 240 Ile Val Lys Ala Asn His Phe Phe Ala Glu Leu Pro Asp Lys Asp Leu 245 250 255 His Gln Tyr Asp Val Ser Ile Thr Pro Glu Val Thr Ser Arg Gly Val 260 265 270 Asn Arg Ala Val Met Phe Glu Leu Val Thr Leu Tyr Arg Tyr Ser His 275 280 285 Leu Gly Gly Arg Leu Pro Ala Tyr Asp Gly Arg Lys Ser Leu Tyr Thr 290 295 300 Ala Gly Pro Leu Pro Phe Ala Ser Arg Thr Phe Glu Ile Thr Leu Gln 305 310 315 320 Asp Glu Glu Asp Ser Leu Gly Gly Gly Gln Gly Thr Gln Arg Arg Glu 325 330 335 Arg Leu Phe Arg Val Val Ile Lys Phe Ala Ala Arg Ala Asp Leu His 340 345 350 His Leu Ala Met Phe Leu Ala Gly Arg Gln Ala Asp Ala Pro Gln Glu 355 360 365 Ala Leu Gln Val Leu Asp Ile Val Leu Arg Glu Leu Pro Thr Thr Arg 370 375 380 Tyr Ser Pro Val Gly Arg Ser Phe Tyr Ser Pro Asn Leu Gly Arg Arg 385 390 395 400 Gln Gln Leu Gly Glu Gly Leu Glu Ser Trp Arg Gly Phe Tyr Gln Ser 405 410 415 Ile Arg Pro Thr Gln Met Gly Leu Ser Leu Asn Ile Asp Met Ser Ser 420 425 430 Thr Ala Phe Ile Glu Pro Leu Pro Val Ile Asp Phe Val Ala Gln Leu 435 440 445 Leu Asn Arg Asp Ile Ser Val Arg Pro Leu Ser Asp Ser Asp Arg Val 450 455 460 Lys Ile Lys Lys Ala Leu Arg Gly Val Lys Val Glu Val Thr His Arg 465 470 475 480 Gly Asn Met Arg Arg Lys Tyr Arg Ile Ser Gly Leu Thr Ser Gln Ala 485 490 495 Thr Arg Glu Leu Ser Phe Pro Val Asp Asp Arg Gly Thr Val Lys Thr 500 505 510 Val Val Gln Tyr Phe Leu Glu Thr Tyr Gly Phe Ser Ile Gln His Thr 515 520 525 Thr Leu Pro Cys Leu Gln Val Gly Asn Gln Gln Arg Pro Asn Tyr Leu 530 535 540 Pro Met Glu Val Cys Lys Ile Val Glu Gly Gln Arg Tyr Ser Lys Arg 545 550 555 560 Leu Asn Glu Lys Gln Ile Thr Ala Leu Leu Lys Val Thr Cys Gln Arg 565 570 575 Pro Gln Glu Arg Glu Leu Asp Ile Leu Arg Thr Val Ser His Asn Ala 580 585 590 Tyr His Glu Asp Gln Tyr Ala Gln Glu Phe Gly Ile Lys Ile Asp Glu 595 600 605 Arg Leu Ala Ser Val Glu Ala Arg Val Leu Pro Pro Pro Arg Leu Lys 610 615 620 Tyr His Asp Ser Gly Arg Glu Lys Asp Val Leu Pro Arg Val Gly Gln 625 630 635 640 Trp Asn Met Met Asn Lys Lys Met Val Asn Gly Gly Arg Val Asn Asn 645 650 655 Trp Ala Cys Ile Asn Phe Ser Arg Asn Val Gln Asp Ser Ala Ala Arg 660 665 670 Gly Phe Cys His Glu Leu Ala Ile Met Cys Gln Ile Ser Gly Met Asp 675 680 685 Phe Ala Leu Glu Pro Val Leu Pro Pro Leu Thr Ala Arg Pro Glu His 690 695 700 Val Glu Arg Ala Leu Lys Ala Arg Tyr Gln Asp Ala Met Asn Met Leu 705 710 715 720 Arg Pro Gln Gly Arg Glu Leu Asp Leu Leu Ile Val Ile Leu Pro Asp 725 730 735 Asn Asn Gly Ser Leu Tyr Gly Asp Leu Lys Arg Ile Cys Glu Thr Asp 740 745 750 Leu Gly Leu Val Ser Gln Cys Cys Leu Thr Lys His Val Phe Lys Met 755 760 765 Ser Lys Gln Tyr Leu Ala Asn Val Ala Leu Lys Ile Asn Val Lys Val 770 775 780 Gly Gly Arg Asn Thr Val Leu Val Asp Ala Leu Thr Arg Arg Ile Pro 785 790 795 800 Leu Val Ser Asp Arg Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His 805 810 815 Pro His Pro Gly Glu Asp Ser Ser Pro Ser Ile Ala Ala Val Val Ala 820 825 830 Ser Gln Asp Trp Pro Glu Val Thr Lys Tyr Ala Gly Leu Val Ser Ala 835 840 845 Gln Ala His Arg Gln Glu Leu Ile Gln Asp Leu Phe Lys Val Trp Gln 850 855 860 Asp Pro His Arg Gly Thr Val Thr Gly Gly Met Ile Lys Glu Leu Leu 865 870 875 880 Ile Ser Phe Lys Arg Ala Thr Gly Gln Lys Pro Gln Arg Ile Ile Phe 885 890 895 Tyr Arg Asp Gly Val Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Tyr 900 905 910 Glu Leu Asp Ala Ile Arg Lys Ala Cys Ala Ser Leu Glu Pro Asn Tyr 915 920 925 Gln Pro Pro Val Thr Phe Val Val Val Gln Lys Arg His His Thr Arg 930 935 940 Leu Phe Ala Asn Asn His Asn Asp Gln Arg Thr Val Asp Arg Ser Gly 945 950 955 960 Asn Ile Leu Pro Gly Thr Val Val Asp Ser Lys Ile Cys His Pro Thr 965 970 975 Glu Phe Asp Phe Tyr Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser 980 985 990 Arg Pro Ala His Tyr His Val Leu Trp Asp Glu Asn Lys Phe Thr Ala 995 1000 1005 Asp Glu Leu Gln Thr Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg 1010 1015 1020 Cys Thr Arg Ser Val Ser Ile Val Pro Pro Ala Tyr Tyr Ala His Leu 1025 1030 1035 1040 Ala Ala Phe Arg Ala Arg Phe Tyr Met Glu Pro Glu Thr Ser Asp Ser 1045 1050 1055 Gly Ser Met Ala Ser Gly Ala Ala Thr Ser Arg Gly Leu Pro Pro Gly 1060 1065 1070 Val Arg Ser Ala Arg Val Ala Gly Asn Val Ala Val Arg Pro Leu Pro 1075 1080 1085 Ala Leu Lys Glu Asn Val Lys Arg Val Met Phe Tyr Cys 1090 1095 1100 55 904 PRT Oryza sativa 55 Met Glu Ser Asn Ser Gly Glu Ile Glu Glu Leu Pro Pro Pro Pro Pro 1 5 10 15 Leu Pro Pro Asn Ala Glu Pro Ile Lys Thr Asp Asp Thr Lys Lys Leu 20 25 30 Ser Lys Pro Lys Arg Ala Leu Met Ala Arg Ser Gly Cys Gly Lys Lys 35 40 45 Gly Gln Pro Ile Gln Leu Leu Thr Asn His Phe Lys Val Ser Leu Lys 50 55 60 Ala Ala Asp Glu Phe Phe His His Tyr Tyr Val Asn Leu Lys Tyr Glu 65 70 75 80 Asp Asp Arg Pro Val Asp Gly Lys Gly Ile Gly Arg Lys Val Leu Asp 85 90 95 Lys Leu Gln Gln Thr Tyr Ala Ser Glu Leu Ala Asn Lys Asp Phe Ala 100 105 110 Tyr Asp Gly Glu Lys Ser Leu Phe Thr Ile Gly Ala Leu Pro Gln Val 115 120 125 Asn Asn Glu Phe Thr Val Val Leu Glu Asp Phe Asn Thr Gly Lys Ser 130 135 140 Ser Ala Asn Gly Gly Ser Pro Gly Asn Asp Ser Pro Gly Asn Asp Arg 145 150 155 160 Lys Arg Val Arg Arg Pro Tyr Gln Thr Lys Thr Phe Lys Val Glu Leu 165 170 175 Asn Phe Ala Ala Lys Ile Pro Met Ser Ala Ile Ala Gln Ala Leu Arg 180 185 190 Gly Gln Glu Ser Glu Asn Thr Gln Glu Ala Ile Arg Val Ile Asp Ile 195 200 205 Ile Leu Arg Gln His Ser Ala Lys Gln Gly Cys Leu Leu Val Arg Gln 210 215 220 Ser Phe Phe His Asn Asn Pro Ser Asn Phe Val Asp Leu Gly Gly Gly 225 230 235 240 Val Met Gly Cys Arg Gly Phe His Ser Ser Phe Arg Ala Thr Gln Ser 245 250 255 Gly Leu Ser Leu Asn Ile Asp Val Ser Thr Thr Met Ile Val Lys Pro 260 265 270 Gly Pro Val Val Asp Phe Leu Leu Ala Asn Gln Lys Val Asp His Pro 275 280 285 Asn Lys Ile Asp Trp Ala Lys Ala Lys Arg Ala Leu Lys Asn Leu Arg 290 295 300 Ile Lys Thr Ser Pro Ala Asn Thr Glu Tyr Lys Ile Val Gly Leu Ser 305 310 315 320 Glu Arg Asn Cys Tyr Glu Gln Met Phe Thr Leu Lys Gln Arg Asn Gly 325 330 335 Asp Gly Glu Pro Glu Gly Val Glu Val Ser Val Tyr Glu Tyr Phe Val 340 345 350 Lys Asn Arg Gly Ile Glu Leu Arg Tyr Ser Gly Asp Phe Pro Cys Ile 355 360 365 Asn Val Gly Lys Pro Lys Arg Pro Thr Tyr Phe Pro Ile Glu Leu Cys 370 375 380 Ser Leu Val Pro Leu Gln Arg Tyr Thr Lys Ala Leu Ser Thr Leu Gln 385 390 395 400 Arg Ser Ser Leu Val Glu Lys Ser Arg Gln Lys Pro Glu Glu Arg Met 405 410 415 Ser Val Leu Ser Asp Val Leu Lys Arg Ser Asn Tyr Asp Ser Glu Pro 420 425 430 Met Leu Asn Ser Cys Gly Ile Ser Ile Ala Arg Gly Phe Thr Gln Val 435 440 445 Ala Gly Arg Val Leu Gln Ala Pro Lys Leu Lys Ala Gly Asn Gly Glu 450 455 460 Asp Leu Phe Ala Arg Asn Gly Arg Trp Asn Phe Asn Asn Lys Arg Leu 465 470 475 480 Ile Lys Ala Ser Ser Ile Glu Lys Trp Ala Val Val Asn Phe Ser Ala 485 490 495 Arg Cys Asn Ile Arg Asp Leu Val Arg Asp Ile Ile Lys Cys Gly Gly 500 505 510 Met Lys Gly Ile Lys Val Glu Asp Pro Phe Asp Val Ile Glu Glu Asp 515 520 525 Pro Ser Met Arg Arg Ala Pro Ala Ala Arg Arg Val Asp Gly Met Ile 530 535 540 Asp Lys Met Gln Lys Lys Leu Pro Gly Gln Pro Lys Phe Leu Leu Cys 545 550 555 560 Val Leu Ala Glu Arg Lys Asn Ser Asp Ile Tyr Gly Pro Trp Lys Arg 565 570 575 Lys Cys Leu Ala Glu Phe Gly Ile Ile Thr Gln Cys Val Ala Pro Thr 580 585 590 Arg Val Asn Asp Gln Tyr Ile Thr Asn Val Leu Leu Lys Ile Asn Ala 595 600 605 Lys Leu Gly Gly Leu Asn Ser Leu Leu Gln Ile Glu Thr Ser Pro Ser 610 615 620 Ile Pro Leu Val Ser Lys Val Pro Thr Ile Ile Leu Gly Met Asp Val 625 630 635 640 Ser His Gly Ser Pro Gly Gln Ser Asp Ile Pro Ser Ile Ala Ala Val 645 650 655 Val Ser Ser Arg Glu Trp Pro Leu Val Ser Lys Tyr Arg Ala Ser Val 660 665 670 Arg Ser Gln Ser Pro Lys Leu Glu Met Ile Asp Gly Leu Phe Lys Pro 675 680 685 Gln Gly Ala Gln Glu Asp Asp Gly Leu Ile Arg Glu Leu Leu Val Asp 690 695 700 Phe Tyr Thr Ser Thr Gly Lys Arg Lys Pro Asp Gln Val Ile Ile Phe 705 710 715 720 Arg Asp Gly Val Ser Glu Ser Gln Phe Thr Gln Val Leu Asn Ile Glu 725 730 735 Leu Asp Gln Ile Ile Glu Ala Cys Lys Phe Leu Asp Glu Asn Trp Ser 740 745 750 Pro Lys Phe Thr Leu Ile Val Ala Gln Lys Asn His His Thr Lys Phe 755 760 765 Phe Val Pro Gly Ser Gln Asn Asn Val Pro Pro Gly Thr Val Val Asp 770 775 780 Asn Ala Val Cys His Pro Arg Asn Asn Asp Phe Tyr Met Cys Ala His 785 790 795 800 Ala Gly Met Ile Gly Thr Thr Arg Pro Thr His Tyr His Ile Leu His 805 810 815 Asp Glu Ile Gly Phe Ser Ala Asp Asp Leu Gln Glu Leu Val His Ser 820 825 830 Leu Ser Tyr Val Tyr Gln Arg Ser Thr Thr Ala Ile Ser Val Val Ala 835 840 845 Pro Ile Cys Tyr Ala His Leu Ala Ala Ala Gln Val Ser Gln Phe Ile 850 855 860 Lys Phe Asp Glu Met Ser Glu Thr Ser Ser Ser His Gly Gly His Thr 865 870 875 880 Ser Ala Gly Ser Ala Pro Val Pro Glu Leu Pro Arg Leu His Asn Lys 885 890 895 Val Arg Ser Ser Met Phe Phe Cys 900 56 1048 PRT Arabidopsis thaliana 56 Met Val Arg Lys Arg Arg Thr Asp Ala Pro Ser Glu Gly Gly Glu Gly 1 5 10 15 Ser Gly Ser Arg Glu Ala Gly Pro Val Ser Gly Gly Gly Arg Gly Ser 20 25 30 Gln Arg Gly Gly Phe Gln Gln Gly Gly Gly Gln His Gln Gly Gly Arg 35 40 45 Gly Tyr Thr Pro Gln Pro Gln Gln Gly Gly Arg Gly Gly Arg Gly Tyr 50 55 60 Gly Gln Pro Pro Gln Gln Gln Gln Gln Tyr Gly Gly Pro Gln Glu Tyr 65 70 75 80 Gln Gly Arg Gly Arg Gly Gly Pro Pro His Gln Gly Gly Arg Gly Gly 85 90 95 Tyr Gly Gly Gly Arg Gly Gly Gly Pro Ser Ser Gly Pro Pro Gln Arg 100 105 110 Gln Ser Val Pro Glu Leu His Gln Ala Thr Ser Pro Thr Tyr Gln Ala 115 120 125 Val Ser Ser Gln Pro Thr Leu Ser Glu Val Ser Pro Thr Gln Val Pro 130 135 140 Glu Pro Thr Val Leu Ala Gln Gln Phe Glu Gln Leu Ser Val Glu Gln 145 150 155 160 Gly Ala Pro Ser Gln Ala Ile Gln Pro Ile Pro Ser Ser Ser Lys Ala 165 170 175 Phe Lys Phe Pro Met Arg Pro Gly Lys Gly Gln Ser Gly Lys Arg Cys 180 185 190 Ile Val Lys Ala Asn His Phe Phe Ala Glu Leu Pro Asp Lys Asp Leu 195 200 205 His His Tyr Asp Val Thr Ile Thr Pro Glu Val Thr Ser Arg Gly Val 210 215 220 Asn Arg Ala Val Met Lys Gln Leu Val Asp Asn Tyr Arg Asp Ser His 225 230 235 240 Leu Gly Ser Arg Leu Pro Ala Tyr Asp Gly Arg Lys Ser Leu Tyr Thr 245 250 255 Ala Gly Pro Leu Pro Phe Asn Ser Lys Glu Phe Arg Ile Asn Leu Leu 260 265 270 Asp Glu Glu Val Gly Ala Gly Gly Gln Arg Arg Glu Arg Glu Phe Lys 275 280 285 Val Val Ile Lys Leu Val Ala Arg Ala Asp Leu His His Leu Gly Met 290 295 300 Phe Leu Glu Gly Lys Gln Ser Asp Ala Pro Gln Glu Ala Leu Gln Val 305 310 315 320 Leu Asp Ile Val Leu Arg Glu Leu Pro Thr Ser Arg Tyr Ile Pro Val 325 330 335 Gly Arg Ser Phe Tyr Ser Pro Asp Ile Gly Lys Lys Gln Ser Leu Gly 340 345 350 Asp Gly Leu Glu Ser Trp Arg Gly Phe Tyr Gln Ser Ile Arg Pro Thr 355 360 365 Gln Met Gly Leu Ser Leu Asn Ile Asp Met Ser Ser Thr Ala Phe Ile 370 375 380 Glu Ala Asn Pro Val Ile Gln Phe Val Cys Asp Leu Leu Asn Arg Asp 385 390 395 400 Ile Ser Ser Arg Pro Leu Ser Asp Ala Asp Arg Val Lys Ile Lys Lys 405 410 415 Ala Leu Arg Gly Val Lys Val Glu Val Thr His Arg Gly Asn Met Arg 420 425 430 Arg Lys Tyr Arg Ile Ser Gly Leu Thr Ala Val Ala Thr Arg Glu Leu 435 440 445 Thr Phe Pro Val Asp Glu Arg Asn Thr Gln Lys Ser Val Val Glu Tyr 450 455 460 Phe His Glu Thr Tyr Gly Phe Arg Ile Gln His Thr Gln Leu Pro Cys 465 470 475 480 Leu Gln Val Gly Asn Ser Asn Arg Pro Asn Tyr Leu Pro Met Glu Val 485 490 495 Cys Lys Ile Val Glu Gly Gln Arg Tyr Ser Lys Arg Leu Asn Glu Arg 500 505 510 Gln Ile Thr Ala Leu Leu Lys Val Thr Cys Gln Arg Pro Ile Asp Arg 515 520 525 Glu Lys Asp Ile Leu Gln Thr Val Gln Leu Asn Asp Tyr Ala Lys Asp 530 535 540 Asn Tyr Ala Gln Glu Phe Gly Ile Lys Ile Ser Thr Ser Leu Ala Ser 545 550 555 560 Val Glu Ala Arg Ile Leu Pro Pro Pro Trp Leu Lys Tyr His Glu Ser 565 570 575 Gly Arg Glu Gly Thr Cys Leu Pro Gln Val Gly Gln Trp Asn Met Met 580 585 590 Asn Lys Lys Met Ile Asn Gly Gly Thr Val Asn Asn Trp Ile Cys Ile 595 600 605 Asn Phe Ser Arg Gln Val Gln Asp Asn Leu Ala Arg Thr Phe Cys Gln 610 615 620 Glu Leu Ala Gln Met Cys Tyr Val Ser Gly Met Ala Phe Asn Pro Glu 625 630 635 640 Pro Val Leu Pro Pro Val Ser Ala Arg Pro Glu Gln Val Glu Lys Val 645 650 655 Leu Lys Thr Arg Tyr His Asp Ala Thr Ser Lys Leu Ser Gln Gly Lys 660 665 670 Glu Ile Asp Leu Leu Ile Val Ile Leu Pro Asp Asn Asn Gly Ser Leu 675 680 685 Tyr Gly Asp Leu Lys Arg Ile Cys Glu Thr Glu Leu Gly Ile Val Ser 690 695 700 Gln Cys Cys Leu Thr Lys His Val Phe Lys Met Ser Lys Gln Tyr Met 705 710 715 720 Ala Asn Val Ala Leu Lys Ile Asn Val Lys Val Gly Gly Arg Asn Thr 725 730 735 Val Leu Val Asp Ala Leu Ser Arg Arg Ile Pro Leu Val Ser Asp Arg 740 745 750 Pro Thr Ile Ile Phe Gly Ala Asp Val Thr His Pro His Pro Gly Glu 755 760 765 Asp Ser Ser Pro Ser Ile Ala Ala Val Val Ala Ser Gln Asp Trp Pro 770 775 780 Glu Ile Thr Lys Tyr Ala Gly Leu Val Cys Ala Gln Ala His Arg Gln 785 790 795 800 Glu Leu Ile Gln Asp Leu Phe Lys Glu Trp Lys Asp Pro Gln Lys Gly 805 810 815 Val Val Thr Gly Gly Met Ile Lys Glu Leu Leu Ile Ala Phe Arg Arg 820 825 830 Ser Thr Gly His Lys Pro Leu Arg Ile Ile Phe Tyr Arg Asp Gly Val 835 840 845 Ser Glu Gly Gln Phe Tyr Gln Val Leu Leu Tyr Glu Leu Asp Ala Ile 850 855 860 Arg Lys Ala Cys Ala Ser Leu Glu Ala Gly Tyr Gln Pro Pro Val Thr 865 870 875 880 Phe Val Val Val Gln Lys Arg His His Thr Arg Leu Phe Ala Gln Asn 885 890 895 His Asn Asp Arg His Ser Val Asp Arg Ser Gly Asn Ile Leu Pro Gly 900 905 910 Thr Val Val Asp Ser Lys Ile Cys His Pro Thr Glu Phe Asp Phe Tyr 915 920 925 Leu Cys Ser His Ala Gly Ile Gln Gly Thr Ser Arg Pro Ala His Tyr 930 935 940 His Val Leu Trp Asp Glu Asn Asn Phe Thr Ala Asp Gly Leu Gln Ser 945 950 955 960 Leu Thr Asn Asn Leu Cys Tyr Thr Tyr Ala Arg Cys Thr Arg Ser Val 965 970 975 Ser Ile Val Pro Pro Ala Tyr Tyr Ala His Leu Ala Ala Phe Arg Ala 980 985 990 Arg Phe Tyr Met Glu Pro Glu Thr Ser Asp Ser Gly Ser Met Ala Ser 995 1000 1005 Gly Ser Met Ala Arg Gly Gly Gly Met Ala Gly Arg Ser Thr Arg Gly 1010 1015 1020 Pro Asn Val Asn Ala Ala Val Arg Pro Leu Pro Ala Leu Lys Glu Asn 1025 1030 1035 1040 Val Lys Arg Val Met Phe Tyr Cys 1045

Claims (19)

What is claimed is:
1. An isolated polynucleotide comprising:
(a) a first nucleotide sequence encoding a first polypeptide having post-transcriptional gene silencing activity, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80% sequence identity based on the ClustalV alignment method,
(b) a second nucleotide sequence encoding a second polypeptide having post-transcriptional gene silencing activity, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 85% sequence identity based on the ClustalV alignment method, or
(c) the complement of the nucleotide sequence of (a) or (b).
2. The polynucleotide of claim 1, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 85% sequence identity based on the ClustalV alignment method.
3. The polynucleotide of claim 1, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO: 12, 14, 22, 28, 40 or 54 have at least 90% sequence identity based on the ClustalV alignment method, and wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 90% sequence identity based on the ClustalV alignment method.
4. The polynucleotide of claim 1, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO: 12, 14, 22, 28, 40 or 54 have at least 95% sequence identity based on the ClustalV alignment method, and wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 95% sequence identity based on the ClustalV alignment method.
5. The polynucleotide of claim 1, wherein the amino acid sequence of the first polypeptide comprises the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54, and wherein the amino acid sequence of the second polypeptide comprises the amino acid sequence of SEQ ID NO:8, 38 or 42.
6. The polynucleotide of claim 1, wherein the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:11, 13, 21, 27, 39 or 53, and wherein the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:7, 37 or 41.
7. A vector comprising the polynucleotide of claim 1.
8. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.
9. A method for transforming a cell, comprising transforming a cell with the polynucleotide of claim 1.
10. A cell comprising the recombinant DNA construct of claim 8.
11. A method for production of a polypeptide having post-transcriptional gene silencing activity comprising the steps of cultivating the cell of claim 10 under conditions that allow for the synthesis of the polypeptide and isolating the polypeptide from the cultivated cells, from the culture medium, or from both the cultivated cells and the culture medium.
12. A method for producing a plant comprising transforming a plant cell with the polynucleotide of claim 1 and regenerating a plant from the transformed plant cell.
13. A plant comprising the recombinant DNA construct of claim 8.
14. A seed comprising the recombinant DNA construct of claim 8.
15. An isolated polypeptide having post-transcriptional gene silencing activity, wherein the polypeptide comprises:
(a) a first amino acid sequence, wherein the first amino acid sequence and and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80% sequence identity based on the ClustalV alignment method, or
(b) a second amino acid sequence, wherein the second amino acid sequence and and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 85% sequence identity based on the ClustalV alignment method.
16. The polypeptide of claim 15, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 85% sequence identity based on the ClustalV alignment method.
17. The polypeptide of claim 15, wherein the first amino acid sequence and the amino acid sequence SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 90% sequence identity based on the ClustalV alignment method, and wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 90% sequence identity based on the ClustalV alignment method.
18. The polypeptide of claim 15, wherein the first amino acid sequence and the amino acid sequence SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 95% sequence identity based on the ClustalV alignment method, and wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 95% sequence identity based on the ClustalV alignment method.
19. The polypeptide of claim 15, wherein the first amino acid sequence comprises the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54, and wherein the second amino acid sequence comprises the amino acid sequence of SEQ ID NO:8, 38 or 42.
US10/174,363 2001-06-18 2002-06-17 Polynucleotides and polypeptides involved in post-transcriptional gene silencing Abandoned US20030077623A1 (en)

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US10/174,363 US20030077623A1 (en) 2001-06-18 2002-06-17 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US11/093,888 US20050204427A1 (en) 2001-06-18 2005-03-30 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US12/237,540 US8022196B2 (en) 2001-06-18 2008-09-25 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US13/208,396 US8278431B2 (en) 2001-06-18 2011-08-12 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US13/595,504 US8748697B2 (en) 2001-06-18 2012-08-27 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US14/284,623 US9453236B2 (en) 2001-06-18 2014-05-22 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US15/244,065 US20160355834A1 (en) 2001-06-18 2016-08-23 Polynucleotides and polypeptides involved in post-transcriptional gene silencing

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US12/237,540 Expired - Fee Related US8022196B2 (en) 2001-06-18 2008-09-25 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US13/208,396 Expired - Fee Related US8278431B2 (en) 2001-06-18 2011-08-12 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
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US13/208,396 Expired - Fee Related US8278431B2 (en) 2001-06-18 2011-08-12 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US13/595,504 Expired - Fee Related US8748697B2 (en) 2001-06-18 2012-08-27 Polynucleotides and polypeptides involved in post-transcriptional gene silencing
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009000848A1 (en) * 2007-06-25 2008-12-31 Cropdesign N.V. Yield enhancement in plants by modulation of zmago1
US20120291155A1 (en) * 2009-10-21 2012-11-15 Daniel Grimanelli Means for inducing apoximis in cultivated plants having sexual reproduction and use thereof for the production of completely or partially apomictic plants

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US20030077623A1 (en) * 2001-06-18 2003-04-24 Butler Karlene H. Polynucleotides and polypeptides involved in post-transcriptional gene silencing
US20100238126A1 (en) * 2009-03-23 2010-09-23 Microsoft Corporation Pressure-sensitive context menus

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US20030077623A1 (en) * 2001-06-18 2003-04-24 Butler Karlene H. Polynucleotides and polypeptides involved in post-transcriptional gene silencing

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009000848A1 (en) * 2007-06-25 2008-12-31 Cropdesign N.V. Yield enhancement in plants by modulation of zmago1
US20120291155A1 (en) * 2009-10-21 2012-11-15 Daniel Grimanelli Means for inducing apoximis in cultivated plants having sexual reproduction and use thereof for the production of completely or partially apomictic plants
US9234209B2 (en) * 2009-10-21 2016-01-12 Institut De Recherche Pour Le Development (Ird) Means for inducing apoximis in cultivated plants having sexual reproduction and use thereof for the production of completely or partially apomictic plants

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US9453236B2 (en) 2016-09-27
US20120331581A1 (en) 2012-12-27
US8022196B2 (en) 2011-09-20
US8278431B2 (en) 2012-10-02
US20090055967A1 (en) 2009-02-26
US20140259217A1 (en) 2014-09-11
US20110321189A1 (en) 2011-12-29
US20050204427A1 (en) 2005-09-15
US8748697B2 (en) 2014-06-10

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