Patent application title: POLYNUCLEOTIDES AND POLYPEPTIDES INVOLVED IN POST-TRANSCRIPTIONAL GENE SILENCING
Inventors:
IPC8 Class: AC12N1582FI
USPC Class:
1 1
Class name:
Publication date: 2016-12-08
Patent application number: 20160355834
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.Claims:
1. A recombinant DNA construct comprising a polynucleotide operably
linked to at least one heterologous regulatory sequence, said
polynucleotide comprising: (a) a nucleotide sequence encoding a
polypeptide, wherein the amino acid sequence of the polypeptide has at
least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2,
or (b) the complement of the nucleotide sequence of (a), wherein a
transgenic plant expressing the polynucleotide has an altered level of
transposon silencing compared to a non-transgenic plant.
2. The recombinant DNA construct of claim 1, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
3. The recombinant DNA construct of claim 1, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1.
4. A vector comprising the recombinant DNA construct of claim 1.
5. The recombinant DNA construct of claim 1, wherein the at least one heterologous regulatory sequence is a promoter.
6. A method for transforming a cell, comprising transforming a cell with the recombinant DNA construct of claim 1.
7. A cell comprising the recombinant DNA construct of claim 1.
8. A method for production of a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2, the method comprising the steps of cultivating a cell comprising the recombinant DNA construct of claim 1 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.
9. A method for producing a transgenic plant comprising transforming a plant cell with the recombinant DNA construct of claim 1 and regenerating a transgenic plant from the transformed plant cell.
10. A plant comprising the recombinant DNA construct of claim 1.
11. A seed comprising the recombinant DNA construct of claim 1.
12. A cDNA comprising a nucleotide sequence encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.
13. The cDNA of claim 12, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
14. The cDNA of claim 12, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1.
Description:
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/284,623, filed on May 22, 2014, now pending,
which is divisional application of U.S. patent application Ser. No.
13/595,504, filed on Aug. 27, 2012, now U.S. Pat. No. 8,748,697, which is
a divisional application of U.S. patent application Ser. No. 13/208,396,
filed on Aug. 12, 2011, now U.S. Pat. No. 8,278,431, which is a
divisional application of U.S. patent application Ser. No. 12/237,540,
filed on Sep. 25, 2008, now U.S. Pat. No. 8,022,196, which is a
continuation application of U.S. patent application Ser. No. 11/093,888,
filed on Mar. 30, 2005, now abandoned, which is a continuation
application of U.S. patent application Ser. No. 10/174,363, filed on Jun.
17, 2002, now abandoned, which claims the benefit of U.S. Provisional
Application No. 60/298,973, filed Jun. 18, 2001, now expired, the entire
contents of each of which are herein incorporated by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 20140522_BB1454USDIV3_SequenceListing created on May 22, 2014 and having a size of 220 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] 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.
BACKGROUND OF THE INVENTION
[0004] 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 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).
[0005] Although the mechanism remains to be fully elucidated, it appears that double-stranded RNA (dsRNA) serve as key intermediates in PTGS (Bass (2000) 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).
[0006] 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 eIF2C, a protein important for protein translation. It is therefore hypothesized that dsRNA mediates PTGS by disrupting proper positioning of eIF2C 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).
[0007] 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) 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
[0008] 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.
[0009] The present invention also includes:
[0010] 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;
[0011] 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;
[0012] in a preferred third embodiment, a vector comprising any of the isolated polynucleotides of the present invention;
[0013] 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;
[0014] 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;
[0015] 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;
[0016] 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;
[0017] 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;
[0018] 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;
[0019] 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;
[0020] 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;
[0021] 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;
[0022] 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
[0023] 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.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING
[0024] 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.
[0025] FIGS. 1A, 1B, 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.
[0026] FIGS. 2A, 2B, 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.
[0027] 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. .sctn.1.821-1.825.
TABLE-US-00001 TABLE 1 Polypeptides Involved in Post-Transcriptional Gene Silencing SEQ ID NO: Polypeptide (Nucle- (Amino (Plant Source) Clone Designation Status otide) 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 (FIS) CGS 7 8 (Corn) Argonaute Homolog cpf1c.pk008.j24 FIS 9 10 (Corn) Argonaute Homolog csc1c.pk006.j19 (FIS) CGS 11 12 (Corn) Argonaute Homolog ctn1c.pk003.i20 (FIS) CGS 13 14 (Corn) 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.pk001.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 (FIS) CGS 39 40 (Soybean) 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)
[0028] The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. .sctn.1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0029] 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 (Zea mays), rice (Oryza sativa), soybean (Glycine max) and wheat (Triticum aestivum).
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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-a-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.
[0035] 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.
[0036] For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by 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.
[0037] 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.times.SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30 min, and then repeated twice with 0.2.times.SSC, 0.5% SDS at 50.degree. 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.times.SSC, 0.5% SDS was increased to 60.degree. C. Another preferred set of highly stringent conditions uses two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0038] 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.
[0039] 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) 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.
[0040] 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). 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.
[0041] "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.
[0042] "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.
[0043] "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.
[0044] "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.
[0045] "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) 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.
[0046] "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).
[0047] "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.
[0048] "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.
[0049] 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.
[0050] 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.
[0051] "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).
[0052] 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.
[0053] "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.
[0054] "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.
[0055] A "chloroplast transit peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. "Chloroplast transit sequence" refers to a nucleotide sequence that encodes a chloroplast transit peptide. A "signal peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632). 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).
[0056] "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). 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.
[0057] "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.
[0058] 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.
[0059] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis").
[0060] "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.
[0061] "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).
[0062] 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, (I) 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:24, 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.
[0063] 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.
[0064] 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).
[0065] 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.
[0066] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least 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.
[0067] 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).
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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. 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.
[0072] 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) 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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, (I) 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.
[0077] 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).
[0078] 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) 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).
[0079] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
[0080] 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).
[0081] 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.
[0082] A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
[0083] Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding 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
[0084] 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.
[0085] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.
Example 1
[0086] Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones 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.
TABLE-US-00002 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 cpf1c.pk008.j24 to Protein Synthesis*** csc1c Corn 20 Day Seedling (Germination csc1c.pk006.j19 Cold 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 p0107.cbcbd69r After Pollination* p0119 Corn V12 Stage** Ear Shoot With p0119.cmtmm21r Husk, Night Harvested* p0125 Corn Anther Prophase I* p0125.czaab55r p0125.czaat57r rca1n Rice Callus* rca1n.pk018.b3 rdi2c Rice (Oryza sativa, Nipponbare) rdi2c.pk002.d14 developing 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 rtc1c.pk008.k19.f Magnaporthe grisea Strain 0184 at 4, 8, and 24 Hours sdc1c Soybean Developing Cotyledon sdc1c.pk0004.d11 (3-5 mm) sdc2c Soybean Developing Cotyledon sdc2c.pk001.p4 (6-7 mm) ses2w Soybean Embryogenic Suspension ses2w.pk0009.g6 2 Weeks After Subculture ssl1c Soybean Seed 25 Days After ssl1c.pk003.g3 Fertilization ssm Soybean Shoot Meristem ssm.pk0063.a4 wdk1c Wheat Developing Kernel, 3 Days wdk1c.pk012.i2 After Anthesis wlm96 Wheat Seedlings 96 Hours After wlm96.pk029.c23 Inoculation With Erysiphe graminis f. sp tritici wne1g Wheat Nebulized Genomic Library wne1g.pk003.f8 wr1 Wheat Root From 7 Day Old Light wr1.pk0073.c7 Grown Seedling wre1n Wheat Root From 7 Day Old wre1n.pk0001.h6 Etiolated Seedling* wre1n.pk162.h10 *These libraries were normalized essentially as described in U.S. Pat. No. 5,482,845, incorporated herein by reference. **Corn developmental stages are explained in the publication "How a corn plant develops" from the Iowa State University Coop. Ext. Service Special Report No. 48 reprinted June 1993. ***Chemicals used included chloramphenicol, cyclohexamide, aurintricarboxylic acid, all of which are commercially available from Calbiochem-Novabiochem Corp. (1-800-628-8470)
[0087] 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.TM. XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. 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) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
[0088] 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.
[0089] 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. 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.
[0090] 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).
[0091] 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.
Example 2
Identification of cDNA Clones
[0092] 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). 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.
[0093] 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.
Example 3
Characterization of cDNA Clones Encoding Polypeptides Involved in Post-Transcriptional Gene Silencing
[0094] 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 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 sequences ("Contig"), or sequences encoding an entire protein, or functionally active polypeptide, derived from an FIS or a contig ("CGS"):
TABLE-US-00003 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
[0095] FIGS. 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-US-00004 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
[0096] 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) 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
[0097] 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 SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI 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.degree. C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue.TM.; 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.TM. 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.
[0098] 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.degree. 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.
[0099] 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) Nature 313:810-812) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
[0100] The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 .mu.m in diameter) are coated with DNA using the following technique. Ten .mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M solution) and spermidine free base (20 .mu.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 .mu.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 .mu.L of ethanol. An aliquot (5 .mu.L) of the DNA-coated gold particles can be placed in the center of a Kapton.TM. flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic.TM. 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.
[0101] 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.
[0102] 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.
[0103] 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).
[0104] 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) Proc Natl Acad Sci USA 97:11650-11654).
Example 5
Expression of Recombinant DNA Constructs in Dicot Cells
[0105] A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the .beta. 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 NcoI (which includes the ATG translation initiation codon), SmaI, KpnI and XbaI. The entire cassette is flanked by HindIII sites.
[0106] 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.
[0107] 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.degree. 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.
[0108] After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.
[0109] Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26.degree. 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.
[0110] 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. Pat. No. 4,945,050). A DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be used for these transformations.
[0111] 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.
[0112] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.L spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (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 .mu.L 70% ethanol and resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five .mu.L of the DNA-coated gold particles are then loaded on each macro carrier disk.
[0113] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60.times.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.
[0114] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
Example 6
Expression of Recombinant DNA Constructs in Microbial Cells
[0115] 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 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.
[0116] 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 .mu.g/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase.TM. (Epicentre Technologies, Madison, Wis.) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 .mu.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.degree. 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 .mu.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.
[0117] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25.degree. C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-.beta.-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25.degree.. Cells are then harvested by centrifugation and re-suspended in 50 .mu.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 .mu.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.
Example 7
Expression of Recombinant DNA Constructs in Yeast Cells
[0118] 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. 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).
[0119] 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.degree. 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.degree. C. with good aeration to an OD.sub.600 of 1.6 (24-30 h). Fifty mL of 20% galactose will be added, and the culture allowed to grow overnight at 30.degree. 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.degree. C. for another 24 hours.
[0120] 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.
Example 8
Expression of Recombinant DNA Constructs in Insect Cells
[0121] 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 E. coli DH5.alpha., 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.
[0122] Spodoptera frugiperda cells (Sf-9) are propagated in ExCell.RTM. 401 media (JRH Biosciences, Lenexa, Kans.) supplemented with 3.0% fetal bovine serum. Lipofectin (50 .mu.L at 0.1 mg/mL, Gibco/BRL) is added to a 50 .mu.L aliquot of the transfer vector containing the toxin gene (500 ng) and linearized polyhedrin-negative AcNPV (2.5 .mu.g, Baculogold.RTM. 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 .mu.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.
Sequence CWU
1
1
5612042DNAZea mays 1ccacgcgtcc gatgaaatct gtcgtggagt acttcaagga aatgtatggt
ttcaccattc 60agcatcctca tcttccttgc cttcaggttg gaaaccaaaa gaaggcgaac
tatttaccaa 120tggaggcctg caagatcgtt gaaggccaga gatacacgaa gaggttgaat
gaaaaacaga 180tcacatcgtt gctaaaggtt acatgccaaa ggcctcgaga acaagagatg
gatattttac 240agacagttca tcaaaatgga tatgagcaag atccatatgc gaaggaattt
gggatcaaca 300ttagtgagaa gctaacctat gttgaagccc gagtccttcc tgcaccttgg
ctgaagtatc 360atgacactgg aaaagagaaa gagtgcttac cacaggttgg tcagtggaac
atggtaaaca 420agaaagtgat aaacggatgc aaggtgagcc actgggcatg tataaacttc
tcaaggagtg 480ttccagaagc cacagctcgg ggattttgcc aggaattggc acaaatgtgt
caaatttcgg 540gcatggaatt taacagtgag cccgtgatgc caatatattc agctagacca
gatcaagtag 600tgaaggcact taaaagtgtg tataatattg cactgaacaa actcaagggt
aaagaacttg 660aacttcttct ggctatactc cccgacaaca atggtccgtt atatggtgac
atcaaacgta 720tttgtgaaac tgatttggga ttgatatcac aatgttgctt aaccaagcat
gtttttaaga 780tcagcaaaca gtacttggca aatgtctcac tgaaaattaa tgttaagatg
ggaggaagaa 840acactgtgct cctggacgca ataagttgga gcattccttt ggtcagtgac
atcccaacta 900ttatatttgg tgcagatgta acacaccctg aaaccgggga ggactcaagt
ccatcaatcg 960ctgccgttgt tgcttctcaa gattggccag aagttacaaa gtatgctgga
ttggtttgtg 1020ctcaggcaca ccggcaagag ctcattcagg acctttacaa aacatggcac
gatcctcaga 1080gaggcactgt aacaggcggc atgatcaggg agctgttaat atccttcagg
aaggccactg 1140ggcagaagcc attgagaata atattctaca gggacggtgt tagtgaaggc
cagttctatc 1200aagttctcct ttacgagtta gatgccatcc gtaaggcatg cgcatcccta
gaaccaaatt 1260accagcctcc tgtaacattt gtggtggttc aaaaacgtca tcatacgaga
ctatttacaa 1320acaatcacaa agacagaagt agcatggaca agagtggaaa tattttgcca
ggaactgttg 1380ttgattctaa gatatgccac ccaacagagt ttgatttcta cctctgtagt
catgctggaa 1440tccagggaac aagtaggccc gctcactacc atgtcctctg ggatgagaac
aatttcacag 1500cagacgaaat gcaaacactg acaaacaacc tttgctacac ttatgcccgg
tgcacacgct 1560cggtttctgt tgtccctcct gcatactacg cacacctggc agcattccgg
gcgcggttct 1620acatggaacc agagatgtcg gagaaccaga cgtcgaagag ctccaatggc
acgaacggag 1680gcttggtgaa gcccctgcct gctgtgaagg agaaggtgaa aagggtgatg
ttctactgct 1740gacgaggtga ccgctttaac aaccattcac atgctgtagc taacttggta
gggttcagta 1800ggggattaga ttagctttct ccaggaacga agaggaaacg ggatgcgtat
ttggatcatg 1860aacaatcaat ctgttagcga tcgctgtaaa atactcggaa atgcctgtat
aatagttctt 1920gttggttcag atgcatgcat ccaatgttcc agtgtactat gaaaaggggg
tgtagaagaa 1980accttctggt gttttctagg ttgaaaaaaa aaaaaaaaaa aaaaaacaaa
aaaaaaaaaa 2040aa
20422577PRTZea mays 2Pro 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
32827DNAGlycine max 3ttctagaaca gtaaacaggt ctatcatagc agaactagtg
aggctgtata aagagtctga 60cttggggatg agacttccag catatgatgg cagaaaaagt
ttgtacactg cagggcagct 120tccctttgct tggagagagt ttaagattaa gcttatagat
gaagaggatg gagttaatgg 180ccctaaaagg gaaagagagt acagggtggt gatcaagttc
gttgctcggg ctaacttgta 240tcacttggga cagtttctag ctggtaggcg tgctgatgca
ccgcaagagg cacttcaaat 300tcttgacatt gtattaagag agctgtcaac taagaggtat
tgccctattg ggaggtcctt 360cttttcacct gatattagaa caccgcaacg gcttggagag
ggattagaat catggtgtgg 420attttaccag agtataaggc ctacacaaat gggcctttcc
cttaatattg atatggcgtc 480tgctgcgttt attgagcctc ttccagtagt ggaatttgtt
ggccagctat tagcaaaaga 540tgtgctgtca aggccattgt cagatgctga tcgcattaag
attaagaaag cccttagagg 600agttaaagtt gaagtaacac acagaggaag tgtgagaaga
aaatatcgtg tttctggatt 660gacttctcaa ccaaccagag aacttgtgtt tcctgttgat
gagaactcaa ctatgaaatc 720agtagttgaa tacttccaag agatgtatgg tttcactatt
caatatactc accttccttg 780ccttcaagta ggaaaccaaa agaaggctaa ctatttacct
atggaggcct gcaaaattgt 840tgaggggcaa cgttatacaa aaagattgaa tgagaagcaa
attacagctc tgttgaaagt 900tacttgccag agacctcgcg atcgggaaaa tgacatttta
cggaccgttc aacataatgc 960ttatgatcaa gatccttatg caaaggaatt tggaattaaa
atcagtgaaa agctagcttc 1020tgttgaagca cgaattcttc cggccccttg gcttaaatat
cacgaaagtg ggaaagagaa 1080gaactgttta ccccaagttg gtcagtggaa tatgatgaac
aagaaaatga ttaatggaat 1140gactgttagc cggtgggcat gcataaattt ttcaaggagc
gtgcaagata gtgttgctcg 1200cactttttgt aatgaacttg ctcaaatgtg tcaagtatct
ggcatggaat ttaatccaga 1260gtctgttatt cccatctaca atgccaaacc tgaacaggtg
gaaaaagctt tgaaacatgt 1320ttaccatgtg tcagggagca aaattaaagg aaaggaattg
gagcttttgt tagcaatatt 1380gccagacaat aacgggtctc tctatggtga tctcaagcga
atttgtgaaa ctgaccttgg 1440tttaatttca caatgctgtc tgacaaagca tgtcttcaaa
atcactaaac agtacttggc 1500taatgtgtct ctgaagatca atgtgaagat gggaggtaga
aacactgtac ttcttgatgc 1560tgtaagcagc agaataccat tggttagtga catgccaacc
ataattttcg gagcagatgt 1620aacccaccct gaaaatggag aagaattgag cccttcaata
gcagctgtag tcgcatccca 1680ggactggccc gaagtgacaa aatatgccgg tttagtatgt
gctcaagctc ataggcagga 1740acttatacaa gatttgtaca aaacttggca agaccctgtt
cgtggcacag ttagtggtgg 1800catgatccga gatttactgg tttccttcag aaaggcaaca
ggacaaaagc cactacgaat 1860tatattttac agggatggtg taagtgaagg acaattttac
caagttttac tttatgagtt 1920agatgcaatt cggaaggcat gtgcttcctt agaaccaaac
taccagcctc cagtaacttt 1980catagttgtg caaaaaagac atcatacccg gttatttgca
aacaactaca gggacagaag 2040cagtacagat cggagtggga atatattgcc tgggactgtt
gttgatacca aaatctgcca 2100tccaacagaa tttgattttt atctctgcag ccatgctggc
atccagggta ctagtcggcc 2160agctcattat catgtcctgt gggatgaaaa caacttcaca
cctgatggaa ttcagtctct 2220gacaaacaac ctttgttata catatgccag gtgtacacgc
tcagtatcag ttgttcctcc 2280agcatattat gcacatttag cagcgtttcg agcacgtttc
tatatggaac cagatatgca 2340agacaatggc tctgcaggtg acggtaatgg tcatggtgcc
aaagcaacac gagcagctgg 2400tgattatagt gtcaagccat tgccagactt gaaagaaaat
gtgaagagag tcatgtttta 2460ctgttagact gcttagtggc ttggccttgg tagaatgata
gatatatggg gcaagcatca 2520acatgataag caagttttca aatcatggag tgcaatgttc
acctcacatt actttgtaca 2580ttagtcgtgt aggttttgct gtggtagatc catgattaca
gttcttgagc catagtttag 2640aatgaatttc tacaagcatt attaggtttt atatagatgc
caaatttagc attgtaaaaa 2700atattctctg tcaatctttg tagaaaattt tgccataagg
cctttacaga tgctggagta 2760gaaatttcct tcatctttgc aaggagggga agttttttcc
tagtaaaaaa aaaaaaaaaa 2820aaaaaaa
28274821PRTGlycine max 4Ser 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 51501DNAGlycine max 5gttttgccaa cagttagttc aaatatgcca
aatctcaggc atggaattta gtcaagaccc 60tgtgattcca atatattcag caaaacctga
tctggtaaag aaagccttga agtatgtaca 120ttctgctgta cttgataaac ttggtgggaa
agaactagag ttgttgattg ccattcttcc 180agacaacaat ggctctctgt atggcgatct
caaaagaatc tgtgaaaccg atctggggtt 240gatttctcag tgctgtctta caaaacacgt
attcaagatc aataggcagt atttggcaaa 300tgtggcacta aagatcaatg tcaagatggg
aggaaggaac acagtacttt tggatgccct 360aagttggagg atcccattgg ttagtgacat
tccaacaata atttttggag cagatgtaac 420acatccagaa tctggagagg acccttgtcc
atccattgct gctgttgtag cctcccagga 480ctggccggaa gtaacaaagt acgcaggatt
ggtatgcgct cagcctcatc gtgaggaact 540cattcaagat ctttttaaat gttggaagga
tcctcatcat ggtatagttt atggtggcat 600gatcagagag ctgttactct cttttaagaa
ggcaaccgga caaaaaccat tgaggataat 660attttacagg gatggggtaa gtgaaggaca
gttctaccag gttttgttgt atgagcttga 720tgccatccgt aaggcttgtg catctttgga
acctagttac caacctccgg taacatttgt 780tgtggttcaa aagcgacatc acactagact
cttctcaaac aatcatgacg acagaaatag 840cactgataag agtgggaata tcttacctgg
tactgtggtg gattctaaga tctgtcatcc 900tacggaattc gacttctatt tatgcagtca
tgcgggaatt cagggtacaa gtagaccagc 960tcattatcat gttctgtggg acgagaacaa
tttcactgct gatgagatcc aatctctgac 1020caacaacttg tgctacacct atgcaagatg
tacacgatca gtttctgtag tgcctcctgc 1080gtactatgct catttggcag cttacagagc
tcgattctac atggaaccta atgtccatga 1140aattgctaaa tctcgaggtg caaggtcaaa
agatgagtca gttcggccac tacctgctct 1200gaaagagaag gtgaagaatg taatgtttta
ttgttgaatg agacaaaata gagagacatc 1260taagtagaga aacagcagca tatgtaggaa
aaggaaatta aattagcaga gctcagaaag 1320ctcaatatgt acaacctaac gtgttcataa
ttcataattc tccgcatgga aaattttgac 1380aaagtctagg ttgtttttca gtatttctag
tgcttaggga aggtaataac ttatgtagaa 1440attatttgtg tatcggtttt cgagcttcaa
gacaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500a
15016411PRTGlycine max 6Phe 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 73096DNAZea mays 7gtttcggtgg
ggttcttgcc gctgcggttg ttcgtgcggc gcggatttag ggagggttct 60gaggcgaggg
cttttgcccc cctcgagcga tttgcagctt tgggtccgat acagtgctca 120tcaaggctca
ctaaatggag tctcacaatg gcgaggccaa tgacttgcct ccaccacctc 180ctctgattgc
tggtgttgaa ccacttaaag ctgatgaaac aaagatgcca ttgaaaccta 240ggagtctggt
ccagagaaat ggatttggca gaaaggggca gccaataaag ctgataacaa 300atcacttcaa
agtttctctt gtgaatgctg aagaattttt ctaccattac tatgtcaatt 360tgaagtatga
agatgataca ccggttgatc gcaaagggtc aggaaggaaa gtgattgaaa 420aactgcagca
aacttatgct gctgaacttg caaataaaga ttttgcctat gatggtgaga 480agagcctgtt
cacaattggt gctcttcctc aagttaaaaa tgagtttact gtcgtggttg 540aagatttttc
aactggaaag actcctgcaa acggcagtcc aggaaatgac agtcctcccg 600gaagtgacag
gaaaagggtc agaaggcctt acaatacaaa gacctataag gtcgagctct 660cttttgcagc
aaaaattcct atgagtgcaa tctcacaggc cttaagaggt caggaatcag 720agcacactca
ggaagcaatt cgagtgattg acattattct gaggcagcac tcagctaagc 780agggttgcct
attagtaagg caatcattct tccacaacaa tccttccaat tttgttgacc 840tgggtggtgg
tgtagtgggc tgtagaggtt ttcattctag ttttcgagca acccagagtg 900gactttcact
caatatcgat gtgtcgacta caatgatagt gaaacctggt cctgtcattg 960attttctgct
tgacaatcag aaagttggtg attcaagcat gattgattgg gctaagggca 1020agcgtgcact
gaagaacttg aggataaaaa taagtccagc gaaccaagaa cagaagattg 1080ttggtctcag
cgaaagaact tgtcgtgagc aattattcac actgaaacat aaaaatggta 1140acaatggtga
ctctgaagag atcactgttt atgattactt cgtaaagcag cgtggcatag 1200tgctgcaata
ctctggtgat cttccttgca tcaatgtggg aaaactaaag cggccaacat 1260attttccaat
tgagttatgc agtcttgtgc ctttacaaag atacactaaa gctttgaaca 1320cacttcagag
gtcatcactc gtggagaaat ctaggcagaa accgcaggaa aggatgtctg 1380ttttatctga
tgtgctgcaa agaagcaact atgatgcaga gcccatgttg aaggcatgcg 1440ggattacaat
tgctagaaat ttcacagaag ttgatggtag ggtattgcag ccacctaagc 1500ttaaagctgg
gaatggtgaa gacattttta cacgcaatgg tagatggaac ttcaacaata 1560agaggctcat
tagagcttgt agtgtcgaga aatgggcggt ggtaaacttt tctgcacgat 1620gcaatgtcag
ggatcttgtc cgggatctca tcaagtgtgg aggcatgaag ggcattatgg 1680ttgatgctcc
ttttgctgta tttgatgaga atccttcaat gagacggtca cctgctataa 1740gaagggttga
agacatgttt gaacaagtga aaactaagct tcctggagca ccaaagtttc 1800ttttgtgtgt
tctagctgaa aggaagaatt ctgatattta tgggccttgg aagaagaaat 1860gccttgctga
atttgggatc gttacacaat gtgtggcacc aactagagtg aacgaccagt 1920atcttacaaa
tgtcctactt aagataaatg caaagctggg tggcatgaat tcgttgctcc 1980aaattgaaac
atccccagca attcctcttg tatccaaggt cccaactata atcttgggaa 2040tggatgtgtc
acacggttct cctggacatt ctgatgtacc atctattgct gctgttgtta 2100gttctcgtga
atggcctctt atctcgaaat acagagcttc tgtccgcacc caatcaccta 2160aaatggaaat
gattgactca ttgtttaagc cacgggaagc tgaagatgat ggtctgatcc 2220gggagtgtct
gattgacttc tacaccagtt ctgggaagag aaagcctgac caagttatca 2280tattcaggga
cggtgttagc gaaagtcagt ttaatcaggt gctgaacatt gagttgcaac 2340aaatcatcga
ggcttgcaaa tttcttgatg agaaatggaa tcccaagttc acgttgatta 2400ttgcccagaa
gaatcatcac actaaatttt tcattcctgg aaagccagat aatgtcccac 2460caggaactgt
ggtggacaac aaagtctgcc atccaaagaa cttcgatttc tacatgtgtg 2520cgcatgctgg
aatgatcggg actacgaggc caactcacta ccacatcctg catgatgaga 2580taggcttcag
tcctgatgat ctgcaggagc tggtgcattc gctctcttat gtgtaccaaa 2640ggagcacaac
agccatatca gtcgttgctc ccatctgcta cgcacatctg gcagctgctc 2700aggttggcca
gttcataaag ttcgatgaga tgtcggagac gtcctccagt catggcgggc 2760atacttcggc
gggcagcgtt ccggtccagg agctgccgcg cctgcatgag aaagtgagga 2820gctcgatgtt
cttttgctga gccgtggttt tacttttttg gtggatggtg aacccctcta 2880gttatgtcgg
tagacgctct tggatgacgc tctagttgtg gtccaggaag gctcgagctg 2940gtacgatgtt
aaatgttagt tttttaagcg tcgctgcggc tatgttggtg cctcaggaag 3000acttggaacc
tggttaggat gtcgttaaat ctacccctta tcgttcctgg ttaaaaaaaa 3060aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaa 30968901PRTZea
mays 8Met 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 92446DNAZea mays 9gcacgagatc aaatttgctg ctcgcgctga tctccaccat
ttggctatgt ttcttgctgg 60gaggcagcca gatgcccctc aagaggctct tcaagtactt
gacatcgtgc tacgtgaaat 120gcctactgcc aagtattgtc ctgttggtag atcattttat
tctcccaagt tagggagacc 180tcagcaactt ggtgaaggtt tggaaacttg gcgtggtttc
taccaaagca taaggcccac 240acagatgggt ctttctctga atattgatat gtcctctact
gcattttttg aggccctccc 300tgtaattgat tttgtttctc agcttcttaa tagagatatc
tcagttagac cattgtctga 360ttctgatcgc gtgaagatta aaaaagccct acgaggtgtg
aaagtggagg tcacacaccg 420tggaaacatg cgtaggaaat atcggatatc tggccttact
ccacaagcaa caagggagtt 480atcattccct attgatgatc gtggtactgt taagactgtg
gtgcaatact tcctggagac 540ttatggtttc agtattcagc acaccacttt accttgtttg
caagtgggca atcagcaaag 600accaaattat ctgcctatgg aggtctgtaa gatagttgag
gggcagcgct actcaaaacg 660acttaatgat aaacagatca ctgctctact gaaggtgact
tgccaacgtc cccaagcgcg 720tgagaaggac atcttggaga ctgtgtatca caatgcctac
tccaaggatc cttatgccca 780ggaatttggt ataacgattg atgagcgtct tgcatcggtt
gaagctcgtg ttctgcctcc 840cccaaggctg aaataccatg atagtggcag agaaagggat
gtattgccaa aagttggcca 900gtggaacatg atgaataaga aaatggtcaa tggtggtaga
gttagcagct gggcatgcat 960taacttctca cggaatgtgc aagatggtgc tgccgggggt
ttctgtcatg aattggcttt 1020gatgtgccaa gtatcaggaa tggattttgt acttgaacct
gtgctgtcac cttgctatgc 1080aaggcctgaa cttgttgaaa gagcactaaa gggacgctat
caagatgcga tgaacatact 1140cgggcctcag ggccgagaac tcgacttgct gattgttata
ctgcctgaca ataatggttc 1200tctttacggg gatgtcaaaa ggatctgtga gactaatctt
ggattggtct cccaatgctg 1260tctgactaaa catgttttca aggtgaacaa gcagcagtat
cttgcaaatg ttgccctgaa 1320aataaatgtg aaggttgggg gaaggaatac tgtgcttgtt
gatgctttgg caaggagaat 1380cccccttgtc agtgacatag cgactattat ctttggtgct
gatgtgaccc atccccatcc 1440tggggaagat tctagtcctt ccattgcagc tgtggttgct
tctcaagact ggcctgaggt 1500tacaaagtat gcaggattgg tgagtgctca agcccatcgt
caagaattga tacaggatct 1560tttcaaggta tggcaagatc ccgaaagggg gactgtctct
ggtggcatga tcagggagct 1620tctcatatct ttctggaggg caactggaca gaaaccaaag
aggatcatat tctacaggga 1680tggcgtcagt gagggacaat tctaccaagt tctgttgtat
gaacttgatg ccattagaaa 1740ggcctgtgcg tcattggagt ctgactacca gcctccagtt
acttttgtcg tggtccagaa 1800gcgtcatcac accaggttgt ttgctaataa tcacaatgat
aatcgtgctg tcgataaaag 1860cgggaacata ctgcctggca ccgtggtgga ctcgaagatc
tgccatccaa ctgagtttga 1920tttctacctg tgcagccatg ctggcattca gggaacaagc
cgccctgccc attaccatgt 1980tctgtgggat gagaacaact ttacggctga tgggttgcaa
actctcacca acaacttgtg 2040ttacacgtat gctaggtgca cacgctcagt atcgattgtt
cctcctgcat actatgctca 2100cctggcagcc ttccgagctc ggttctacat ggagccagat
acgagtgaca gtggatctat 2160ggcaagccgt ggccctccac caggggggcg caacaccaag
gctgccggtg ttgggaatgt 2220tgctgtgagg ccattacctg ccctcaagga aaacgtgaag
cgggtcatgt tctactgcta 2280agactgatgc tgttaaggca gagctacctt ttattattac
agtatatcgt gaagactaga 2340gtattttttt ccacgtactt gatgatgctg agctaccttt
taaaaaaaaa aaaaaaaaaa 2400aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaa 244610757PRTZea mays 10Ile 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 113808DNAZea mays 11ctgctttctc cagtgagccg cacccgcact
accgctgacg ctaattaacc acaagcgacc 60gtcgccttcc cccacctcct cccttcctca
aaaaaaggcg gaggcgggag tggtggtggt 120gctcgtgggc acgcagcgga gcaccctgta
cagcagcagc gctgcggcag tagagagtgc 180cattggtgga gctggtaact agccctcccc
cctccgttcc cgtcccgcgc gcagccgtct 240gccgagcctg ctcagtgccc atcatggtga
ggaagaagag aactggccct ggtggctctg 300gagaaacttc tggagagtct tcaggagcct
ctggacaagg ttcctcacag cagcctgagc 360gaactcaaca acctggggga ggacgtggct
gggtgcctca acagggtggc catggtggtg 420ggcaacacca gggtcgtgat cgacattatc
agggacgtgg aggaccaggg ccacatcacc 480ttggtagtgg ggcacctgag tatcacccgc
gtgaatacca gggacgtggt ggtgaatatc 540agggacatgg tggtgagtac cagggacggg
gtggtgacta ccagggacgt ggtggtggcc 600gctccagagg tggaatgcca cagccatact
atggtgggca taggggaggt aatgttggac 660gcaatgttcc tccaggtccg tccaggacag
ttcccgagct gcaccaagcc ccatatgtcc 720agtatccagc cccggtggtt tcgccctccc
catcgggacc tggctcatcc tcacagccta 780tggcagaggt gagctctgga caagtccagc
aacagtttca gcaacttgcc gatcgtggtc 840agagttccac gagccaagaa attcaagtgg
caccagcatc aagcaaatcg gttcgattcc 900cgttacggcc cggcaagggc acttatgggg
acaggtgcat tgtgaaggca aatcattttt 960ttgctgagct tcctgacaaa gaccttcacc
aatatgatgt atctataaca cctgaggtta 1020cttcacgtgg cgtcaatcgt gctgtcatgg
gtgagcttgt aacaatatat agacaatccc 1080atttgggtgg gcgtctacct gcatacgatg
gaagaaagag cctgtatact gctggaccat 1140tgccatttac ttctatggca tttgaaatta
ccttgcaaga tgaggaagat agtcttggcg 1200gtcgccaagg tggacatagg cgtgagagag
tatttagggt ggtgatcaaa tttgcagccc 1260gtgctgatct ccaccatctg gctatgtttc
tagctggaag gcaagcagat gcccctcagg 1320aagctcttca agtgcttgac attgtactac
gtgaattgcc taccgcgagg tattctcctg 1380tcggtaggtc attttactct cccaacttag
ggagacgtca aaaacttggt gagggattgg 1440aaagttggcg tggtttttac caaagcataa
ggccgacaca gatgggcctt tcactgaata 1500ttgatatgtc ctctactgca tttatcgagc
ctctccctgt gatcgatttt gttgctcagc 1560ttcttaacag agatatctca gttaggccat
tgtctgattc tgatcgcgtg aagattaaaa 1620aagccctaag aggtgtgaag gttgaggtga
ctcacagggg aaacatgcgc agaaaatatc 1680gcatttctgg cctcacctca caagcaacaa
gagagctatc attccctgtt gatgatcgtg 1740gtactgtgaa gactgtggtg caatacttca
tggagactta tggttttagt atccagcaca 1800ccactttacc atgcttgcaa gtgggtaatc
aacaaagacc aaattatctg cctatggagg 1860tttgcaagat agttgaagga cagcgttact
caaagcgact caatgagaaa caaatcactg 1920ctctactgaa agtgacctgc cagcgccctc
aagagcgcga gctggacatc ttacagactg 1980tgcatcacaa tgcatactat gaagacccct
atgcactgga atttggtata agaattgatg 2040aacgtcttgc tgcagttgaa gctcgtgttc
tgccaccacc aagacttaaa taccatgata 2100gtggccgaga gaaggatgtt ttgcccagag
ttggccaatg gaacatgatg aataagaaaa 2160tggttaatgg tggcagagtg agcaactggg
catgtattaa cttctctcgg aatgtgcaag 2220atagtgccgc taggggtttc tctcatgagt
tggcagtcat gtgccaaata tcaggaatgg 2280attttgctct tgagcctgtg ctgcctccag
tgactgcaag gccagaacat gttgagagag 2340cgttaaaggc acgttatcaa gatgcaatga
acatactgag gccacaggga agggaacttg 2400atctgctgat cgtaatactg cctgacaaca
atggttctct ttatggggat ctcaaaagga 2460tctgtgagac tgaactcgga ttggtctccc
agtgttgtct gactaaacat gtttttaaga 2520tgagcaagca gtaccttgca aatgttgcac
tcaaaataaa tgttaaggtt gggggaagga 2580atactgtact tttagatgct ttgtcaagga
gaatccccct tgtcagtgac agaccgacca 2640taatatttgg tgctgatgtt acccatccac
atcctggaga agattccagt ccttccattg 2700cagccgttgt tgcttcgcaa gactggcccg
aggtcacgaa atacgctgga ctagtgagtg 2760cgcaagccca tcgccaggag ctgatacagg
atcttttcaa agtatggcag gacccgcaga 2820gaaggacggt aactggcggc atgataaagg
aacttctcat ttctttcaag agggcaactg 2880gacagaagcc ccagaggatc atattctaca
gggatggtgt cagtgaggga cagttctatc 2940aagtattgct gtacgaactt gatgccatta
gaaaggcctg tgcgtccctg gagcccaact 3000accagcctcc agttactttt gtcgtggtac
agaagcgcca tcacactagg ctgtttgcga 3060acaaccacag tgatcagcgc acagtcgata
gaagcggaaa catactgcct ggcaccgtgg 3120tcgattcgaa gatttgccat cctactgagt
ttgacttcta cctgtgtagc catgctggca 3180ttcagggaac gagccgccct gctcactacc
atgtcctgtg ggacgagaac aagttcacag 3240ctgacgagct gcagaccctg acgaacaacc
tgtgctacac gtacgctagg tgcacccgct 3300ccgtgtccat cgtgcccccg gcgtactacg
ctcatctggc agccttccga gctcgcttct 3360acatggagcc agacacctct gacagcgggt
cactggccag cggtgcccgt ggccccccac 3420ccggtgcggc acgcagcagc acgagagggg
ccgggagtgt cgaggtcagg cccctacctg 3480ctctcaagga gaacgtgaag cgtgtcatgt
tttactgctg agacgctggt gggctgcctt 3540cgccaaggaa aatgccctgg agcattccca
tgtacccgca ctgtttcggt gatacagtac 3600tatctaacgc cgattttgcg cgttaagact
tccagtgatc tgggaaattt cttgtacgac 3660tgttgtagtg ttgtgtattc gtaatgtgat
gacgcggcag ttcttctagg agcttagtgc 3720cgtgtaaaat atctgttgta agttgtaacc
tgtcaccctc tagtgttatg tcatgatgaa 3780ccaaattaaa aaaaaaaaaa aaaaaaaa
3808121085PRTZea mays 12Met 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
133714DNAZea maysunsure(1789)n = A, C, G or T 13gccttccccc cctcccctcc
tcaaaaaagg cggagaggtg gtggtgctcg tgggcacgca 60gtggagcacc cagtacagca
gcagcgctgc ggcagtggag ttaggagctt agcactccgc 120ctccgttccc atcccgcgcg
cagccgtcgg ccgagcctgc tcagtgccca tcatggtgag 180gaagaagaga actggccctg
gcggctctgg agaaacttct ggagagtctt caggagcttc 240tggacaaggt tcctcacagc
ggcctgaacg gactcaacaa cctggggcag gacgtggctg 300ggtgcctcag cagggtggcc
gtggtggcgg gcaacaccag ggtcgtggtg gacattatca 360aggccgtgga gggccaggtc
cacatcaccc tggtggactg cctgagtatc accagcgtga 420ataccaggga cgaggtggtg
agtaccaggg acagtaccag gggcgtggtg gtgcccgctc 480cagaggtgga atttcacagc
catactatgg tgggcatagg ggaggtagtg ttggacgaaa 540tgttcctcca ggtccatcca
gaacagttcc cgagctgcac caagccccat acgtccagta 600tcaagccccg gtgatttcac
catccccatc gggacctggc tcatcctcac agcctatggc 660agaggtgagc tctggacaag
tccagcaaca gtttgagcaa cttgccattc atggtcagag 720ttccatgagt caagaagttc
aagtggcacc agcatcaagc aaatcggttc gattcccatt 780acgccccggc aagggcactt
atggggacag gtgcattgtg aaggcgaatc atttttttgc 840tgagcttcct gacaaagacc
ttcaccaata tgatgtaact ataacacctg aagttacttc 900acgtggcgtt aatcgtgctg
tcatgggaga gcttgtaaca ctatatagac aatcccattt 960gggcgggcgt ctacctgcgt
acgatggaag aaagagcctt tataccgctg gaccattgcc 1020ttttacttct atgacatttg
aaattacctt gcaagatgag gaagatagtg ttggcggtgg 1080ccagggcgga caaaggcgcg
agagagtatt tagggtggtg atcaaatttg cggcccgtgc 1140tgatctccat catctggcta
tgtttctagc tggaaggcaa gcagacgctc ctcaagaagc 1200tcttcaagtg cttgacattg
tactacgtga attgcctact gcgaggtatt ctcctgttgg 1260taggtcattt tattctccca
acttagggag acgtcagcaa cttggtgagg gtttggaaag 1320ttggcgcggt ttttaccaaa
gcataaggcc gacacagatg ggcctttcac tgaatattga 1380tatgtcctct actgcattta
tcgagcctct ccctgtgatt gattttgttg ctcagcttct 1440taatagagat atttcagtta
ggccattgtc tgattctgat cgcgtgaaga tcaaaaaagc 1500cttaagaggt gtgaaggttg
aggtcactca caggggaaac atgcgcagaa agtatcgcat 1560ttctggcctc acctcacaag
caacaagaga gctatcattc cctgttgatg atcgtggtac 1620tgtgaagact gtggtccaat
acttcatgga gacttatggt tttagcatcc agcacaccac 1680tttaccgtgc ttgcaagtgg
gcaatcaaca aagaccaaat tatctgccta tggaggtttg 1740caagatagtt gaaggacagc
gttactcaaa gcgactcaat gagaaacana tcactgcttt 1800actgaaagtg acctgccagc
gccctcaaga gcgtgagctg gacattttac agactgtgca 1860tcacaatgcg tactatgaag
acccgtatgc acaggaattt ggtataagaa ttgatgaacg 1920ccttgctgca gttgaagctc
gtgttctgcc accaccaagg cttaaatacc atgatagtgg 1980ccgagagaag gatgttttgc
ccagagttgg ccaatggaac atgatgaata agaaaatggt 2040aaatggtggc agagtcagca
actgggcatg tattaacttc tctcggaatg tgcaagatag 2100tgccgctagg ggtttctgtc
atgaactggc aatcatgtgc caaatatcag gaatggattt 2160ttcccttgag cctgtgctgc
ctccagtgac tgcaaggcca gaacatgttg aaagagcgtt 2220gaaggcacgt tatcaagatg
caatgaacat actgaggcca caggggaggg aacttgatct 2280gctgattgta atactgcctg
acattaatgg ttccttatat ggggatctca aaaggatctg 2340tgagactgat ctcggattgg
tctcccagtg ttgtctgact aaacatgttt ttaagatgag 2400caagcagtat cttgcaaatg
ttgcactcaa aataaatgtt aaggttggtg gaaggaatac 2460tgtacttgta gatgctttga
caaggagaat cccccttgtc agtgacagac cgaccataat 2520atttggtgct gatgttaccc
atccacatcc tggagaagat tccagtcctt ccattgcagc 2580tgtggttgct tcgcaagact
ggcctgaggt caccaaatat gctggactag tgagtgccca 2640agcccatcgc caggagctga
tacaggatct tttcaaagta tggcaagatc cacagagaag 2700gacagtaact ggtggcatga
taaaggaact tctcatttct ttcaagagag caactggaca 2760gaagccccag aggatcatat
tctacaggga tggtgtcagt gagggacagt tctatcaagt 2820attgttgtat gaacttgatg
ccatcagaaa ggcatgtgca tccttggagc ccaactacca 2880gcctccagtt acttttgtcg
tggtgcagaa acgacatcac actaggctgt ttgctaataa 2940ccacaacgat cagcgtacag
ttgatagaag cggaaacata ctgcctggca ccgtggttga 3000ttcgaagatt tgccatccta
ctgaatttga tttctacctg tgtagccatg ctggcattca 3060gggaacaagc cgccctgctc
attaccatgt cctgtgggac gagaacaagt tcacagctga 3120tgagctgcag actctgacaa
acaacctatg ctacacgtac gctaggtgca cccgctccgt 3180gtcaattgtg cccccggcat
actatgctca tctggcagcc ttccgagctc gcttctacat 3240ggagccagat acctctgaca
gtggctcaat ggccagtggt gcccgtggcc ctccaccagg 3300tgcggcacgc agcatgagag
gagcggggag tgttgcggtc aggcccctac ctgctctcaa 3360ggaaaacgtg aagcgtgtca
tgttttactg ctgagatgct gagctacctt caccaagaaa 3420atatcctgac ttgttccatg
tacccgcact gtttcggtga tactatctga caccgaattt 3480atgcattaag tcttccagtg
gtctggagat tttaagtaac gcctgttttt attcgtgagt 3540tgtaacgctg cagttcgagg
agcttcagtg ctgtatgatg tgtaaactat ttgttgtaag 3600ttgtaaccaa ttgttgtaag
ttgtaaccag ccactatgtt ataatcctgt ttgtttcagc 3660taaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 3714141073PRTZea
maysUNSURE(539)Xaa = ANY AMINO ACID 14Met 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 153072DNAZea maysunsure(411)n = A, C, G
or T 15ctccaaatac gtaacaaact tctacgtgag cgccagggca gttgcgccca tgggaaggca
60tccgcctgtc gatgaggcga tggacttcaa cggcaacgga cgggacgagg caaacccgag
120cggctctgag gcggggaacc acaacgagca ccgcggcgac gacccctcgc gcgttggcca
180gagcctgccc gccgatatcc gccaaaatgg gcagccaacc ctcggggagg agatcaccgc
240gccgctgtgg gaggagttcg aggcgctcgg catccacgtc cgccgctccg agcccgtgtt
300cccgccgcgc ccagggtacg gcgccgcggg gacgccgtac gtcgtcaggg ccaacctctt
360cctcggtcgc ctcgtcgacg aggccctgca tcagtacaac gtaaccattt ngcccgagcc
420gacgcccaag gccgcgtaca gagagatcat gacgaagctg ttgtccgaga accagcacac
480ggatttcgac ggccgcttct ccgtgtacga tgatggtgac tcgctcttca cagccggtgc
540gctgccgttc gacaccaagg agttcgaggt ccccctctct gcaggcggcg acgaaaagat
600ggacaggaag tacaaggtga tgatcaacca tgccgcaacg attagtctgc tacagctgag
660gatgctgtta gcgggctatc ccacggacat ccccgcgcag gcgctcgtgg tcctcgacac
720cgtgctgcgt gacgtcttca acgaacgcaa tgacatggaa tgcgtcgtga ttgacaaaaa
780ggatcgcaca ctgggtgttg acgcatggaa ggggctctat ctgagcatca ggccaacaca
840aaactgcttg tctctgattg cagacgtgtc ctcatctgta ttcgttcaac ccctgctatt
900gattgaattc gttcagaaga tcctaaagat agatgccgtg gataggaact tgactaaacc
960tgagtatgac aagctcttga aggccctcag gggtgtgagg attcaagtca cacacagaga
1020taatagacgc cgagtatggt caaagaaaaa agataataga cgccaactct ctacgtacag
1080agttgctggc ttgtcagtga atcctactaa tgatttgagt tttgaatcaa aggttggagt
1140cacaacgact gtgattgatt acttcagaga aatatacggc ctggaactga aatacaaata
1200tctcccatgc gtcaatgctg gcagcgagca ggatccaatc tattttccta tagaggtttg
1260caagatagct cccaagcagt gttaccagaa gaagctggaa ggtagtcagt tttctactcc
1320aaggaagtca gcctggatcc atcctgaagc cgagcaatcc tgtcctcaga ttgttgagca
1380gaggcagtac aaacaaacca aacgtgcaaa tgaatttgac ttagaatttg atggcaatct
1440tacaacagtt gctgctagag ttctgctgcc tccaaatctt aagtatgatg attctgtatc
1500acagaaaaca tggtttccac tggatgggta ctggaatatg aaagacaaga aagtaataaa
1560tggtgccaag atcagaaact gggcatgtct taatttttgt gaagatttat ccaaggaaga
1620tattaagaag ttttgcttta agctggctga aatgtctcgt attactggac tggactttgc
1680cgatttgaag ctcccaatat tcactgcacg tccagatcga gttgaagatg gtattcgtag
1740gtgctatcag gaagcgaaga acaagctaag ggatcagaag attgatttac tgcttgctat
1800actaccagat aaaaaagaca gtttatatgg aaatattaaa aggatctgtg agacagatat
1860tggtcttgtg tcacagtgtt gtcgaaggtc aagagtctta gtgaataata atcagatatt
1920ggcaaatatt gctattaaga tcaatgccaa ggttggagga agaatctcag tattcgatga
1980cgtacagaag agtttaccgg ttgtttcaaa taagccaaca attatatttg gtgctcatgt
2040ttctcaccct tctgttgtag atggttctac tggcccttct attgcttctg tcgttgcatc
2100ccaagactgg catgaggtgt ctaagtataa tggtgttgtt cgtgcacaag gtcacactga
2160agagatcggt ggccttgaag acattgtcaa ggagctcctt catgcatttg caaacgagtc
2220caaggagaag ctccagcagc tgatattcta cagggatggc ataagtgagg gtcaattcaa
2280tcgaattttg gagaaagaaa tcccagcgat agaaaaggct tggaacgcac tgtatgacaa
2340tgagaagcca caaatcacct tcgttgttgt gcagaagagg cataaactga ggctgttccc
2400cgtggacgac aactataaga tccgttctgc taagaagaaa attgttgagc ctggcacagt
2460ggttgatagt gagatctgtc acccagcaga atttgatttc ttcctttgca gccaatctgg
2520tggtatcaaa ggcccaaggc gtcctgtgag gtaccttgta ctgcgagatg ataacaactt
2580cacggcagat gaactgcagg ctctcacaaa taacctgtgc tacacttatt caggcggcaa
2640tcgttcgttg tcggtcgctc ctcccgcata ctacgcccaa aagctcgcac atcgggcccg
2700cgtctacctc gccaaaggct cggacaataa tgcagcagct gctaatggtg gtcggaagca
2760aattccagag ataaagaatg agctgaaggg gtccatgttc tactgctagt cctttgcctg
2820ctgaacggac gatgcattgt tctatagtga aagacttgag tgtgctctga gtctctgact
2880gacatctgga gaaggatggc atctgcaata gtcgccgtgt tctttttagt acactagaat
2940aaatggatgt tttttgtgga cgcccatgtt gaactagttt tcttttccag taagtacttc
3000agaatgagtg agataaatat atcattcagc gtctggtggt ctggcattgg aaaaaaaaaa
3060aaaaaaaaaa ag
307216919PRTZea maysUNSURE(121)Xaa = ANY AMINO ACID 16Met 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 17400DNAZea
maysunsure(286)n = A, C, G or T 17caagaaggca caagggtgtc agttgtgcat
tactttaaac aacgatataa ctactactta 60caatacactc actggccatg ccttcaagct
ggccgtgttg acaagcagat ctatttacct 120atagaggttt gcagcatagt tcagggacaa
cgctactcca gtaagctgaa tgagaatcaa 180gtcaggaaca tcctgcagtt tacctgcgag
cgaccagcag ataggcaaac tagaactttt 240gaggtattca agaattacaa atctgatgga
tcaacttatg caaaanaatt tggccttacg 300tttgatggat caacttacgn ntnggatgct
cgagttgctc ccagtccaag gcttaaatac 360catgatccga naaaaaaagt ttnggcaacc
tccatcggaa 40018126PRTZea maysUNSURE(96)Xaa =
ANY AMINO ACID 18Gln 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
19550DNAZea maysunsure(479)n = A, C, G or T 19cggacgcgtg ggcaagattg
tagaagggca gagatactct aagaagctta atgacagaca 60agtgacgaac atacttagag
caacttgtaa acgtccccag gagagagaga agagcatacg 120tgatatggtt ctgcataaca
agtatgcaga tgataagttt gctcaggagt ttggcatcga 180agttagcagt gatctagtga
ctgttccagc ccgtgtgctg cctccacccc tgttgaaata 240tcatgactct ggtagggaga
aaacttgtgc accaagtgtt ggacaatgga acatgatcaa 300taagaaaatg atcaatggtg
gaactattga taactggact tgtttgaact tttcacgcat 360gcgccctgat gaagtacaaa
ggttctgtat ggatctgact catatgtgca atgccactgg 420aatggttgtc aatccacgcc
catttattga aatccggtct gctgctccta accatatana 480naatgctttg ananatgttc
acaagaaaac cncccaaata cttgcccaca aacatgggaa 540atcnactcca
55020163PRTZea
maysUNSURE(160)Xaa = ANY AMINO ACID 20Gly 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 213009DNAZea mays 21ctcgcctcgt ccgtcctcct
gcctacttcc ttgcttttgg taggtgctgc ttgttttatc 60ttgaaatggg ctctcatgat
ggcgaggatg aagagttgcc acccccccct ccggtgccac 120cagatgtgat tcccattaaa
gctgaagatg ctgtgggtga atcaccagca aaccatatat 180taaagccaaa gagattactg
atggacaggc ctggtatagg aagaaaaggg cagccgaccc 240agctctattc aaatcacttt
aaagtcgctg tgaagagtac agaagacgtc ttctttcact 300actatgtaaa cctgaagtat
gaggatgatc gacccgttga tggtaaaggg atcggcagaa 360aggtgattga taaactgcag
cagacatatc gtgcagagct ttctaacaag gactttgcat 420atgatggaga aaagagcctg
tttacagttg gtggtcttcc acaaaaaaag aatgagttca 480ccgttgtctt ggaggacgta
tctactggaa agactgctgc caatgggagc cctggaggta 540atgacagtcc tggaggtggt
gataggaaga gagtgaggag gccataccag acgaaaactt 600tcaaagtgga gataaatttt
gcagcagagg ttcctatgag tgctattggt caagtcatta 660gaggcgaaga atctgagaac
tccctggagg cgcttcgtgt tcttgatatc atactgaggc 720agcattccgc agaacaaggc
tgccttttgg ttaagcaatc atttttctac aacaaccctt 780catgctttgt tgacttgggt
ggtggtgtga tgggttgtcg tggatttcat tcaagcttcc 840gtggcacaca gagtggactt
tccctcaatg ttgatgtctc aacaacaatg atcgtgaaac 900ctggccctgt tattgatttt
cttctttcta accagaatgt taatgatcct agcagaattg 960attggcaaaa ggccaagcgt
gctctcaagg gcttgaggat tagaaccact cctgcaaatt 1020cagaattcaa gatttttggt
ctcagcgaga ggatctgcaa agaacaaacg tttccgctga 1080ggcagagaaa tggtagcaac
ggagattgtg ataccattga aataactgtc tatgactact 1140atgcaaagaa aggaatcgat
ctaaagtatt ctggtgattt cccctgtata aatacaggga 1200aggcaaagcg cccaacatat
tttccaatcg agctatgctc gcttgttccg cttcaaagat 1260acaccaaagc tttgtctacg
ctacaaaggt catcccttgt ggagaagtct agacagaagc 1320ctgaagaaag gatgaccgtt
ctaaatgatg cactgcaacg cagtaactac gattctgacc 1380ccatgttgag ggcatgtggt
gtttcagttg ctccaaaatt tacccaagtt gaaggaagga 1440tccttcaagc cccaaagctg
aaagccggca atggtgatga tatcttttca cgaaatggac 1500ggtggaattt cactaatagg
aagttttatg aaacctgctc tgtgaataag tgggcggtcg 1560ttaatttctc tgcacgttgt
gatgttcgga atcttatccg tgacctgatg aggaatgcat 1620ctgcaaaggg aattcaaatg
gaggaacctt ttgatgtgtt tgaagagagt ccctctatga 1680ggcgtgcacc tgtgtcaaga
agggtggatg atatgtttgg gcagataaaa tcaaaacttc 1740ctggagctcc taggttcctc
ttgtgccttc tccctgagag gaaaaattgt gaaatctatg 1800gtccttggaa gagaaagtgc
ctggccgagt ttggtattgt cacacagtgt ctagctccat 1860taagagtcaa tgatccgtac
ctgcttaatt tgctgatgaa gatcaatgca aagcttggtg 1920gtctgaactc gttgctgcaa
gttgaagcat cttcgtcaat accacatgtg tcgcaagtac 1980ccaccatcat cttaggtatg
gatgtttcac atggtcatcc aggacaagat agaccttcgg 2040ttgcagcggt ggttagttct
cgtcaatggc ctcttatctc tagatataga gcatcagtgc 2100acacccaatc tgccagacta
gaaatgatgt cctcgttgtt taagccgcgg ggtactgatg 2160atgatggcct catccgggaa
tcactgatcg acttccacac tagctctgga aagcgaaaac 2220cagaacacat aattattttc
agggatggag tcagtgaaag tcagtttacc caggtcatca 2280acattgagct ggatcagatc
atcgaggcat gtaagtttct ggatgagaag tggtcaccca 2340agttcactgt gattgttgct
caaaagaacc accacaccaa gttctttcag acggcatcac 2400cagacaatgt tcttcctgga
actgtggtgg atagtaaagt ttgccatcct aagaacttcg 2460acttctacat gtgtgcacat
gctgggatga ttggaacaac aaggccgacc cactatcatg 2520ttctgcacga cgagataggt
ttcagtgccg acgagatgca ggagtttgtt cattcgctct 2580cttacgtgta ccagaggagc
acgacagcca tctcagttgt tgctccagtg tgctacgccc 2640acctcgctgc agcccaggtg
agcacgttcc tgagattgga ggagatgtca gacgcgtcct 2700ccagccaggg aggagggcat
acctcggctg gcagtgctcc tgtgccggag ctgcctcgcc 2760tgcatgacaa agtcaggagc
tccatgttct tctgctagct gatgtgcgtg cgcatcagga 2820tcgagctcca tgttttgtgt
tagtaaggcc tagttagtaa ggctgtagaa agaatgttta 2880atgtttgcat gctaaagtcc
aaacaatcaa aaccactact atatctacca gagcactgat 2940cgatcaaaca acaagagtca
gcatcaatca atcaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000aaaaaaaaa
300922910PRTZea mays 22Met
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
232429DNAOryza sativa 23aaaccattca agttcttgat gttgtcctta gggagtcacc
atcttggaat tatgtcacag 60tgtccagatc cttcttctct acccagtttg gtcaccgggg
tgacattggt gagggacttg 120agtgttggag aggttactat cagagcctgc gcccaacaca
gatgggcctt tcgctgaata 180tagatatatc tgcaacgtcc ttttttaagc ctgtgacagt
gatccaattt gtggaggagt 240tcctgaacat acgtgacacc tcaagacctt tgtcagaccg
ggatcgtgtg aagataaaga 300aagcattacg tggggttcgc attgaaacaa accaccaaga
ggaccaaatc agaagataca 360agataacagg gattaccccc attcctatga gccagctgat
atttcctgtt gatgataatg 420ggacaaggaa gactgttgtt cagtacttct gggataggta
caattacaga ctgaagtacg 480cttcttggcc ctgcctacag tctggcagtg attctcgccc
tgtatactta cctatggagg 540tgtgcaagat tgtagaaggg cagaggtact ccaagaagct
taatgacaaa caagtgacca 600acatccttag agcaacctgt caacgccccc agcagaggga
acagagcatt catgagatgg 660ttctccacaa caagtataca gaggataggt ttgctcagga
gttcggtatc aaggtctgca 720atgacctagt ctctgttcca gcccgtgtgc tgcctccacc
catgttgaag tatcatgatt 780ctggaaggga gaaaacttgt gcacccagtg ttggacagtg
gaacatgatt aacaagaaaa 840tgatcaatgg aggaactgtg gataactgga catgtctgag
tttttcacga atgcgtcctg 900aggaggtaca aaggttctgt ggtgacctga ttcagatgtg
caatgccact ggaatgtctt 960tcaatccaag accagtcgtg gatgtccggt caacaaatcc
taacaatata gagaatgctc 1020tgagggatgt tcacaggaga acatcagaac tgctagccag
agagggaaag ggaggcctgc 1080agcttttaat tgtaattctg cctgaagtta gtggttctta
tgggaaaatt aaaagggtct 1140gtgagactga ccttggcatt gtatctcaat gttgtttgcc
aaggcatgcc agcaggccga 1200acaagcaata tttggaaaat gttgcactca aaatcaatgt
caaggtcgga gggcgcaaca 1260ctgttcttga gcgagccttt atccgcaatg gcataccatt
tgtgtcagaa gtcccaacaa 1320tcatctttgg cgctgatgtc acacaccctc cacctggaga
ggactctgca tcatctattg 1380ctgcggttgt ggcatctatg gattggcctg aaatcaccaa
ataccgaggt ctggtctctg 1440ctcaaccaca tagacaggag ataatagaag atctctttag
tgttggtaaa gatccagtga 1500aggttgtaaa tggtgggatg atcagggagt tgcttatcgc
attccgcaag aagactggca 1560gaaggcctga gaggataatc ttctatagag atggtgtaag
tgaaggtcag ttcagccatg 1620tgcttcttca tgaaatggat gccatcagaa aggcttgtgc
atctttggag gagggatatc 1680taccacctgt cacatttgta gtagttcaga aaaggcatca
cacaaggctt ttcccagagg 1740ttcatgggag gcgagacatg actgacaaga gcggaaacat
ccttcctgga actgtcgtgg 1800accgtcagat ttgccatcct acagagttcg atttctacct
gtgtagccat gctggcatac 1860agggtactag caggccaact cattaccatg tcctttacga
tgagaaccat tttacagccg 1920atgcacttca gtccctgacc aacaatcttt gctataccta
tgcgcgatgc acccgggcag 1980tgtctgtggt cccaccggcc tactatgctc atcttgctgc
attccgcgct cgctactacg 2040tggaaggaga gagttcggat ggtggctcga cccctggcag
cagcgggcag gctgtggcgc 2100gagagggccc tgtggaggtg cgccagcttc ccaagatcaa
ggagaacgtc aaggacgtca 2160tgttctactg ctgaggagat tgttggcaag gagagcccaa
tattctggta gttttttggt 2220tggtagactt gtttgtgtcc ttggtttgga gctggttgct
tgtagttcca tttgctgttt 2280ccgagtagcc ggattgtgac tgagcttttg tggtctttaa
ggccttaact ctgcttgaga 2340caatgcaagt cttttaaatt tccctgtggc taaaaaagaa
gaaaaacaag aaaaaaaaaa 2400aaaaaaaaaa aaaaaaaaaa aaaaaaaaa
242924723PRTOryza sativa 24Thr 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 251619DNAOryza sativa 25cttacattct
ggaagggtga gagtatgcac gccagaggat ggggcgtgga acatgaaaga 60caagaaagta
gttaacggtg ctacaattaa aagctgggca tgtgtcaact tgtgcgaggg 120tttggataat
cgtgttgttg aagcattctg ccttcaattg gtcagaacgt ccaaaataac 180tggactggac
tttgcgaatg tgagccttcc aatattgaaa gctgatcctc ataatgttaa 240aactgatctt
cctatgcgct atcaggaagc atgcagctgg tcgagggata acaagattga 300cctcctactt
gttgtaatga cagatgataa aaataatgcc agcttatatg gtgacgttaa 360aagaatctgt
gaaacagaaa tcggtgtatt gtcacagtgt tgtcgagcga agcaagtcta 420caaggagagg
aatgttcagt actgcgcaaa tgttgctctt aagatcaatg ccaaggctgg 480aggaaggaac
tcggtatttc ttaatgtaga agcaagttta ccggttgttt caaagagccc 540aactattata
tttggtgctg atgttaccca tcctgggtcc tttgatgaaa gtaccccttc 600cattgcttcg
gttgttgctt ccgcagactg gcctgaggtg accaagtata attctgttgt 660tcgtatgcaa
gcttctcgta aggagattat acaagatctt gatagcattg ttagggaact 720tctcaatgca
ttcaaaaggg actccaagat ggagccgaag cagctcattt tctacaggga 780cggcgtaagc
gagggtcagt tccagcaagt tgtagagagc gaaataccgg agatagaaaa 840ggcttggaag
tctctgtatg ctggcaagcc acgaattacc ttcatagtgg tgcagaagag 900gcatcataca
aggctgttcc ccaacaatta caatgatcca cgcggcatgg atgggactgg 960aaatgttcgt
ccaggcacag tagttgatac agtgatctgt caccctcgag agtttgattt 1020cttcctgtgc
agccaagccg ggatcaaagg gacaagccgt cctagccatt accatgtgct 1080gcgcgacgac
aacaacttca ccgcagatca gcttcagtct gtcacaaaca acctgtgcta 1140cttatataca
agctgcactc gctcggtgtc tattccacct cctgtttact acgctcataa 1200gctcgcattc
cgcgctcgtt tctacctcac ccaagttccc gtcgccggtg gagatccagg 1260tgctgctaag
ttccagtggg tacttccaga gattaaggaa gaggtgaaaa agtccatgtt 1320cttttgctag
tcgtccttgt gcccccctga aactgaagcc tggagccagc cggcaagctc 1380tggaaatgct
ctgaataatc aaacttggaa gaataagcac ctgcccaggt tgccattcgt 1440ttccatgtgg
catggaggat ggcatcctga aaaggatatt gtcatgtttg tgtggttttt 1500aaacgacatt
gaagtttatc tccggtgtta ctatctcagc actttggatg ttttattttg 1560ttatgtctga
agatatagac acaaaacttc atttttgttt caaaaaaaaa aaaaaaaaa
161926442PRTOryza sativa 26Leu 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 273549DNAOryza sativa 27gttggacaac
gggtactact cccatcaagc tttagccatg atgagaaaga aaaaaactga 60accccgtaat
gctggggaaa gttctggaac tcaacaagcc actggagctc ctggacgggg 120tccttcacag
cgacctgaga gagctcaaca gcatggaggt ggtggttggc agcctgccaa 180tcctcaatat
gctcaacaag ctggtcgtgg tggtggacaa caccagggac gtggtggacg 240ttaccagggt
cgtggagggc caacatcaca tcaaccaggt ggtggtccgg ttgaatatca 300agcacatgag
tactatggcc gtggtgtcca acggcaagga ggaatgccac aacacaggag 360tggcagtggt
ggacatggag ttcctgccag tccatcaaga acagttcccg agctgcacca 420agcctcacaa
gaccagtacc aagctacggt ggttgcacca tcaccatcaa gaactggccc 480atcttcgctg
cctgttgagg ccagcagcga agaagtccaa catcagtttc aggaacttgc 540catccagggt
caaagcccca ctagccaggc cattcaacca gcaccaccat cgagcaaatc 600agtgagattt
ccaatgcgcc ctggcaaggg tacatttggt gataggtgca tcgtgaaagc 660caaccatttc
tttgctgaat tgcctgacaa agaccttcac cagtatgatg tgtctataac 720tcctgaggtt
ccttcacgtg gtgtcaatcg tgctgtcatt ggagaaattg taacacaata 780taggcagtct
catttgggtg gccgtcttcc agtctatgat ggaaggaaga gcttatacac 840agctggtcca
ttaccattta cttctaggac ctttgacgtt attctgcagg atgaggaaga 900gagccttgct
gttgggcaag gtgcacagag gcgtgagaga ccatttaagg tcgtgatcaa 960atttgctgca
cgcgctgatc tccaccattt agccatgttt ttagctggaa ggcaagcgga 1020tgctcctcaa
gaagctcttc aagttcttga cattgttcta cgtgaattgc ctactgcaag 1080gtactctcca
gttgcaaggt cattttattc gcctaactta ggaaggcgcc aacaacttgg 1140cgagggcctg
gaaagttggc gtggttttta ccaaagcata cgacccacgc agatgggact 1200ttctctgaat
attgatatgt catcgacagc attcattgag cctctacctg tgattgactt 1260tgttgcacag
cttttgaaca gagacatctc agttagacca ttatctgatg ctgatcgtgt 1320gaagatcaag
aaggccctaa ggggtgtaaa ggttgaggtc acacatagag gcaatatgcg 1380caggaagtat
cgcatttctg gccttacctc gcaagcaaca cgagagttgt cttttcccat 1440tgataatcat
ggtactgtga agacggtggt gcaatacttc caggagacat atggatttaa 1500cattaagcac
acaactttgc cttgcttgca agtgggcaat caacaaaggc caaattatct 1560accaatggag
gtctgtaaga ttgtggaggg acagcgttac tcaaaaagac taaatgagaa 1620gcagataact
gctcttctta aagtgacctg ccagcgccct caagagcgtg agctggacat 1680tttgcagact
gtgcatcaca atgcatacca tcaggatcca tatgcacagg agtttggcat 1740aaggatcgat
gagcgacttg catctgttga agctcgtgtt ctaccacccc cctggcttaa 1800gtaccacgat
agtggcagag agaaggatgt cttgccaaga attggccaat ggaatatgat 1860gaataagaaa
atggtcaatg gtggtagagt taacaactgg acatgcatca atttttctcg 1920tcatgtccaa
gataatgctg ctaggagttt ctgtcgcgag cttgctatta tgtgccaaat 1980atctgggatg
gacttctcaa ttgatcctgt ggttcctcta gtgactgcaa gacctgaaca 2040tgtggaaaga
gcgctcaagg cacgctatca agaggccatg aatatactga aaccacaggg 2100cggggagctt
gacctgctga ttgcaatatt gcctgacaat aatggttctc tttatggcga 2160tctcaaaagg
atatgtgaga ctgatcttgg attggtctcg caatgctgtc ttacgaagca 2220tgtttttaag
atgagcaaac agtatttagc aaacgttgcc cttaaaatca atgttaaggt 2280gggaggaaga
aatacagtac ttgttgatgc tttgacaagg aggattcccc ttgtcagtga 2340cagaccaact
atcatatttg gtgcggatgt tactcatcct catcctggag aagattccag 2400tccttccatt
gcagctgtgg ttgcttctca agactggcct gaagtcacta agtatgctgg 2460attggtgagt
gcccaagccc atcgtcaaga attgatacaa gatcttttca aagtatggca 2520agacccgcat
agaggaactg ttactggtgg catgatcaag gagcttctca tttctttcaa 2580gagggctact
ggacagaaac ctcagaggat aatattttac agggatggtg tcagcgaggg 2640gcagttttat
caagttttgt tgtatgagct tgatgccatt agaaaggctt gtgcatccct 2700ggaacccaac
tatcagcctc cagttacctt tgtggtggtc cagaagcggc atcacacaag 2760gttgtttgct
aataatcaca acgaccagcg tactgttgat agaagtggaa acattctgcc 2820tggaactgtt
gttgactcaa agatttgcca tccaaccgag tttgatttct acctgtgtag 2880ccatgctggc
atacagggaa caagccgtcc tgctcattat catgttctgt gggatgagaa 2940caaatttact
gcagacgagt tgcaaaccct cacgaacaac ttgtgctaca cgtatgcaag 3000gtgcactcgc
tctgtatcaa ttgtgcctcc tgcgtactat gctcatctgg cagccttccg 3060agctcgcttt
tacatggagc cagagacatc tgacagtgga tcaatggcga gtggagctgc 3120aacgagccgt
ggccttccac caggtgtgcg cagcgccagg gttgctggaa atgtagccgt 3180caggcctcta
cctgctctca aggaaaacgt gaagcgtgtc atgttttact gctaagagct 3240tgggctgtac
cccgtatgcg ccaaggaatg tagtactatg ttatgttatt ttagcacttg 3300cactctgtcg
ttgatcccgt taaaacgggt atgctaccat aagctgttgg actattctgg 3360gtattgtagt
actacttgtt ttgtatttgt gtttgtgacg ctgcagagcg tgaacaacgc 3420aagtctggta
cttgtatcgt tgtgtttgtg ggaacctaaa tcttgttgga cctttgttgt 3480gcttgaagaa
ccaagttaaa taatcctgtc agtataggga tttaattgca aaaaaaaaaa 3540aaaaaaaaa
3549281065PRTOryza
sativa 28Met 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 29772DNAOryza sativa 29gttctaaccg ccggccgccg ccctccccgc acgacgccga
cgccgccctc ctcgcccaac 60gccggctcag ccccttctcc tccccgcccg acgccgcccc
ttctcctccc cgtcccacgc 120cgaccccgcc cgacgccggc gccactctgc ttgtccccgg
ccggcgccga gcctgctcct 180ccccgcccga cgccggcgcc gccgacgctg ctctgctcct
ccccgaccgg cgccgacctg 240ctcctcccag cccggagccc gacgccggca catctcatcc
agatgtccga taacatggct 300gccaaaattg gtgaaattgt ccaagtacat aatgataatc
ctgtaaagag agtacctatt 360gcacgaccta gctttggccg tgaaggaaag caaatcaagc
tgctctcaaa ccacttcact 420gtgaagctta gtggaattga tgcggttttc taccaataca
gtgtttccat caaatctgag 480gatgataagg tgattgatgg aaagggtatt ggccgaaagg
tcatggataa agtgctgcaa 540acatacagct ctgagcttgc tgggaaggaa tttgcgtatg
atggtgaaaa atgtctattt 600actgtggggc ctcttccaca gaacaacttt gagttcactg
ttatcttgga ggaaacatct 660tcaagagctg ctggtgggag tctagggcat ggaagcccta
atcaaggtga catcaaaaaa 720aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gaaaaaaaaa
aaaaaaaaaa aa 77230238PRTOryza sativa 30Val 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 311238DNAOryza sativa
31tcacaatgct gttgcacgaa gcaggtgttc aaaatgaaca aacaaattct tgcaaatctt
60gctctgaaga taaatgtcaa ggttgggggc aggaacactg tgctggtgga tgctgtgtca
120aggcgtattc ctctggtaac cgacagacct acaattatat ttggtgctga tgttacccat
180cctcatcctg gagaggacag cagtccctca attgctgctg ttgtagcctc ccaagattgg
240cctgaggtga caaagtatgc tgggttggtt tctgctcaag cccaccgaca agagctgata
300gaagatctat ataaaatctg gcaggatcca cagagaggaa cagttagtgg tggcatgatc
360cgtgagctgc ttatatcctt caaaagatca actggtgaga agccccagcg aataatattt
420tacagggatg gcgttagtga aggccaattt taccaagttc tactttatga attgaatgca
480atccgaaaag catgtgcctc cctggagaca aattaccaac caaaggtgac tttcattgtg
540gttcagaaac gtcaccacac aagattattt gcacataatc acaacgatca gaactcagtt
600gacaggagcg ggaacatact ccctggtacg gttgtagatt caaagatctg tcatccaact
660gagtttgact tctacctgtg tagccatgct ggcattaagg gtactagtcg tccagctcat
720tatcatgtct tgtgggatga aaacaacttc acagctgatg cattgcagat tcttaccaac
780aacctttgct acacctatgc aaggtgcact cgctctgtat caattgttcc acctgcttat
840tatgctcatc tggctgcctt ccgtgctcgt ttctatatgg aaccagatac atctgacagc
900agctctgtcg ttagtgggcc tggtgtacgt gggccacttt ctggctcatc aacatcacgt
960actcgggccc ctggtggtgc agctgttaag ccacttcctg ctctgaagga tagtgtgaag
1020agggtcatgt tctactgctg aagctagggc ctacatagct aaagctcttc gtttcttggc
1080aacctgccta tgatggttgt aattatgtgt caaaaaatcc cataataatc tgccagctgc
1140tatcttctcc attgtactat gctggtcatg tttgccaaag ttaccctata tgtatgtata
1200ttatgctatt gtttttttaa aaaaaaaaaa aaaaaaaa
123832346PRTOryza sativa 32Ser 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
33551DNAOryza sativaunsure(23)n = A, C, G or T 33ttgccatggc ctaccatacg
acnaaccaga ttacgctcat atggccatgg aggccagtgc 60aagaattggc caatggaata
tgatgaataa gaaaatggtc aatggtggta gagttaacaa 120ctggacatgc atcaattttt
ctcgtcatgt ccaagataat gctgctagga gtttctgtcg 180cgagcttgct attatgtgcc
aaatatctgg gatggacttc tcaattgatc ctgtggttcc 240tctagtgact gcaagacctg
aacatgtgga aagagcgctc aaggcacgct atcaagaggc 300catgaatata ctgaaaccac
agggcgggga gcttgacctg ctgattgcaa tattgcctga 360caataatggt tctctttatg
gcgatctcaa aaggatatgt gagactgatc ttggattggt 420ctcgcaatgc tgtcttacga
agcatgtttt taagatgagc aaacagtatt taacaaacgt 480tgcccttaaa atcaatgtta
aggngggaag gaaaaaatac aagtactttg ttggatgcct 540ttgacnaagg g
55134169PRTOryza
sativaUNSURE(8)Xaa = ANY AMINO ACID 34Cys 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
35966DNAGlycine max 35cttcggaagt tgagggatta cctcagtgga
agcgtgcttt cgatccctag ggatgttttg 60cacggcttgg atttggtggt gaaggaaaat
ccttcgaagc agtgtgtttc cttggggcgg 120tgcttcttcc ccatgaaccc tcctttgagg
aagaaagatc ttaaccatgg cataattgcg 180attggagggt ttcagcagag tcttaagtct
acttctcagg gattgtcctt gtgcctggac 240tattcggttt tgtcctttcg gaagaagctg
ttggtgttgg attttctgca cgagcatatt 300agggacttca atttaaggga gtttgggcgg
ttcaggagac aagttgagca tgtacttatt 360gggttgaagg ttaatgttaa acaccggaag
acaaagcaga agtacactat tactaggttg 420acacccaagg ttacgagaca tatcacattc
cctattttgg atcccgaggg ccggaatccc 480ccaaaggaag ctactctggt tggttacttt
ctagagaagt atggtgtgaa cattgaatac 540aaggacattc ctgccttgga ttttggaggc
aacaagacga attttgtgcc tatggagttt 600tgtgagttgg ttgaggggca gagatatccc
aaagagaatt tggacaaata tgctgccaag 660gacttaaaag acatgtcagt ggctcctcca
agggtgaggc aaagtacaat acaagcaatg 720gtaaactcag aggacggacc gtgcggaggt
ggtgttatta aaaattttgg aatgagtgtc 780aacacttcca tgacaaatgt gacaggacgt
gtaattcagc ctccacaatt gaagctaggt 840aatccaaatg gccagactgt tagtatgaca
cttgaagtag agaaatgtca gtggaatcta 900gtgggacgat caatggtgga aggcaagcca
gttgagtgtt ggggcattct tgattttacc 960tcgtgc
96636322PRTGlycine max 36Leu 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
373613DNAGlycine max 37ttcttgcaag catctcattt ctctctttct ctctttctct
ctctttggga gaaaacccac 60tcttcttttc tctctcttgc acacatatac acactcctct
tttttattcc cttcttcact 120ccactgccca gcttcgccct gtccatcgct caccgtttgc
agtagcttct ctacttttca 180ctttctccct gagatcatgg tcagaaagag aagaactgaa
ctacccagtg ggggtgaaag 240ctctgaggct caacgccctg ctgaaaggag tgcaccaccc
caacaacagg ctgctgctgc 300tgccccagga ggggctggac cccaaggagg cagaggttgg
ggtccccaag gaggacgagg 360aggctatggt gggggccgca gtcgtgggat gccccaacag
caatatggtg cccctcctga 420atatcaaggt aggggaaggg gagggccttc tcagcaagga
ggccgtggag ggtatggcgg 480tggccgaagt ggtggtggta tgggcagtgg ccgtggcgta
ggtccttcat atggtggccc 540atccaggcca ccggcacccg agctgcacca agcaacctca
gttcaattct atcaaactgg 600ggtgagttct cagcctgcat tatctgaggc cagttcatca
ctgccgccgc cggaacctgt 660tgatttggaa cagtcaatgg cgcagatggt gcttcattct
gaagctgctc cttctccgcc 720tcctgcaagt aaatcatcaa tgaggttccc tcttcgacca
ggaaagggta gctatggcac 780caaatgtgtt gtcaaggcta atcatttctt tgccgagttg
cccaacaaag atctgcatca 840atatgatgta acaattactc ctgaagtgac atcaagagga
gtgaaccgtg ctgttatgga 900gcagttggtg aggctgtatc gggaatctca cttgggtaag
agacttcctg cttacgatgg 960gcgcaagagc ctctatactg ctggaccact tccttttatg
tcaaaggagt tcagaattgt 1020tcttgctgat gatgatgaag gagctggagg ccagaggagg
gacagggaat tcaaggttgt 1080gataaaattg gctgcacggg cagatcttca ccatttagga
ctctttttac agggaaggca 1140aactgatgct cctcaagagg ctttgcaggt ccttgacatt
gttctgcgtg aactccctac 1200tacaaggtat tgtcctgtag gaagatcatt ttattcacct
gatttgggta gaagacagcc 1260tttaggtgag ggattggaaa gctggcgtgg tttctaccag
agtattcggc ctacacagat 1320ggggctatcc ctgaacattg atatgtcttc cactgcattt
attgagccat tgccggtaat 1380tgacttcgta aatcaactgc tgaacagaga tgtatctgcc
cggccattat ctgatgctga 1440tcgtgttaag atcaagaaag ctcttcgagg tatcaaagtt
gaagtaacac atcgtggaaa 1500catgagaagg aaatatcgta tctctggtct gacttcacag
gcaaccagag aattgacatt 1560cccagtagat gaaaggggaa ccatgaaatc tgttgtggag
tacttctatg agacatatgg 1620gtttgtcatt caacatactc agtggccttg tctgcaagtt
ggcaatacac agagacctaa 1680ctatttgcca atggaggttt gcaagatagt ggaaggtcaa
aggtactcaa aaaggcttaa 1740tgagaggcaa atcaccgctt tgctgaaagt tacatgccag
cgtcctgttg agagggagcg 1800tgatatcatg cagacagtac accacaatgc ataccatgaa
gatccttatg ccaaagaatt 1860tgggatcaag atcagtgaga agcttgctca agttgaagct
cgcatccttc ctgctccatg 1920gctcaaatat cacgatacgg gcagagaaaa ggattgtctt
cctcaagttg ggcaatggaa 1980tatgatgaat aagaaaatgg ttaatggggg aacagttaac
aactggttct gcataaactt 2040ttcgaggaat gttcaagata gtgttgcccg cggtttttgc
tatgaacttg ctcagatgtg 2100ttatatatct ggaatggcat ttacacctga gccagtagtt
cccccagtca gtgctcgccc 2160tgatcaagtg gaaaaggttc ttaaaactcg gtatcacgat
gccaagaata aactgcaagg 2220aaaagagctt gatttactca ttgttatctt gccggataat
aatggatcac tatatggtga 2280cctcaaacgt atttgtgaga cagatctagg acttgtttca
caatgttgct taactaagca 2340tgtcttcaaa atgagcaagc agtaccttgc aaatgttgct
ttgaaaatta atgtcaaagt 2400tggagggaga aacactgtac tggttgatgc gctctcacga
cgcattccct tggtcagtga 2460cagacctaca attatttttg gagctgatgt gactcatcca
catcctggag aggattcaag 2520tccatcaatt gcagcagttg tggcttcgca agactatcct
gaaattacaa agtatgctgg 2580tttagtttgt gcccaagctc ataggcagga actcatccag
gatcttttca aacaatggca 2640agatccagtc agaggaacag tgactggtgg aatgatcaag
gaacttctta tatcttttag 2700gagagctaca ggacaaaagc cacaacgcat catattttat
agggatggtg ttagtgaggg 2760tcaattttat caggttctac tgtttgagct tgatgctatt
cgaaaggcat gtgcatccct 2820ggaacccaac tatcagcctc ctgtgacttt tgtggtggtt
caaaagcgtc accacacaag 2880gctctttgcc agcaaccatc acgataagag ttcttttgac
aggagtggca acatattgcc 2940tggtactgtt gttgactcca aaatctgcca tcccaccgaa
tttgactttt atctctgcag 3000ccatgctgga atacagggta caagccgtcc tgctcactac
catgtgttgt gggatgaaaa 3060caattttact gctgatgcct tgcaaacact caccaataat
ctttgctaca catatgctcg 3120gtgcacccga tctgtttcaa ttgtgcctcc tgcatactat
gctcaccttg ctgcattccg 3180tgcaaggttt tacatggaac ccgagacttc ggatagtggc
tctatgacaa gtggtgctgt 3240tgcaggccgt gggatgggtg gcggcggtgg tggtggtgta
gggcgtagca cccgggcacc 3300tggtgctaat gctgctgtga gaccattgcc tgcactcaaa
gagaacgtta agagagttat 3360gttttattgt taagaagata tgatatgcat gccaaagatt
acttttagca accttgtttt 3420gtggaggagt gctttttccc ttgctgcttt caaactatct
ccagtggtgt ggtctgtgtc 3480attagtattg agttttttga aactatttaa ggtgtgtggt
gtgttgaata aggttgtcca 3540gtgtggagtg gagtgtttta tctttgctat gagggtctga
tatttgatgc aaaaaaaaaa 3600aaaaaaaaaa aaa
3613381058PRTGlycine max 38Met 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
393239DNAGlycine max 39ttctaaactc actctctcac tttctcactc cctcactccc
tccgttgacg tttttgtttt 60ctttttctct gtgttctgaa gaagttttag ggtttcgttt
tgtttctctc ttcggccact 120tcaggctatg gattcatttg agccagatgg aaatgggaag
gagtcactgc caccaccacc 180tcctgttgtt ccctctgata ttgtacctct caaagcagag
gaggtgctct gtacccctac 240cgagcataat aagaaaaagg cttcccgact tccaatagcc
agatctggtc tgggatcaaa 300aggaaataaa atacaattac taaccaatca cttcaaagtt
aatgttgcta aaaatgatgg 360gcatttcttc cattatagtg tggcttttac ttatgaagat
ggacgccctg tagaaggtaa 420gggtgtaggg agaaagataa tagatagggt gcaggagaca
tatcattctg acttaaatgg 480taaggacttt gcatatgatg gggagaaaag tctgtttact
gttggctctc ttcctcaaaa 540caagcttgag tttgaagttg ttcttgagga tgtcacctct
aacaggaata atggcaattg 600cagccctgat ggtctagggg acaatgagag tgacagaaag
aggatgcgac gtccttatcg 660ttcgaagtca ttcaaagtag agataagctt tgctgcaaaa
attccaatgc aggccattgc 720cagtgcctta cgcgggcaag agactgagaa ttttcaagaa
gccatcagag ttcttgatat 780cattttgagg cagcatgctg ctaagcaagg ctgcttactt
gtacgccaat cctttttcca 840caataatcca aataattttg ctgatgtagg aggtggtgtc
ctaggctgta gaggattcca 900ctcaagcttt agaactacac agagtggcct gtctcttaac
atagatgtgt caactacaat 960gataatttct cctgggcctg tggtggattt cttaatttcc
aatcaaaatg tgagagatcc 1020ttttcaactt gactgggcta aggccaaaag gaccctaaaa
aatctgagga ttaaaactag 1080cccatccaat caagaattca aaatttctgg gctcagtgaa
ctcccatgca gagagcagac 1140ttttactttg aaaggtaaag gtggggggga tggtgaagat
ggtaatgagg aaatcactgt 1200atatgattat tttgttaagg ttcgtaagat agatctccga
tactctgctg accttccatg 1260tatcaatgtt ggcaagccta aacgaccaac atttttcccc
attgaggttt gtgaattggt 1320atcattgcaa cgatatacaa aagctctgtc cacgcttcaa
agggcttcat tagtggagaa 1380gtcgaggcag aagccacaag agaggatgaa aattttgtct
gatgcactga gaacaagcaa 1440ctatggtgct gaacctatgc tccggaattg tggaatttct
ataagcactg gcttcactga 1500agtggagggc cgggtgttgc ctgcaccaag gttgaagttt
ggcaatggtg aggatctcaa 1560tcctaggaat gggagatgga atgtcagcag agtgaaattt
gtggaaccat caaagataga 1620aagatgggct gttgctaact tttctgcacg ctgtgatgta
cgaggacttg tacgggacct 1680cattagaatt ggagatatga aaggaattac tatagaacaa
ccatttgacg tgtttgatga 1740gaatccacag tttaggcgtg ccccccctat ggttagagtg
gagaaaatgt tcgagcatat 1800ccaatctaaa cttcctgggg ctcctcagtt ccttctctgt
ttgcttcctg atcggaaaaa 1860ttgtgatatt tatggtccat ggaaaaagaa gaatcttgct
gattttggaa tcataaatca 1920gtgtatgtgt cctttaaggg tcaatgacca gtacctgact
aatgttatgt tgaagatcaa 1980tgccaagctt ggtgggttga attcattgtt aggcgttgaa
cattctcctt ctcttcctgt 2040tgtttccaaa gctcccaccc tcattctggg aatggacgtg
tcacatggct cacctgggca 2100gactgacatt ccttcaattg ctgcggtggt cagctctaga
cactggcctc tgatatcaaa 2160gtatagggca tgtgttcgta cgcaatctgc aaagatggaa
atgattgata atttgttcaa 2220gctagtatct gaaaaggaag atgaaggcat cataagggaa
cttttgcttg atttctatac 2280aacttctggg aggagaaaac cggaaaatat aatcatattc
agggatgggg ttagtgagtc 2340acaattcaat caagttttga atattgaact cgatcgaatc
attgaggctt gcaaatttct 2400cgatgaaaat tgggagccaa aatttgtggt aattgttgct
cagaagaacc accacactag 2460atttttccag cctggctctc ccgacaatgt cccacctgga
actgttatcg acaataaaat 2520ttgtcatccc agaaattatg atttctacct atgtgcacat
gctggaatga taggaactag 2580taggcctacc cattatcatg tgctgcttga tcaggttggt
ttctctccgg atcagctgca 2640ggagcttgtc cattcattat catatgtgta tcagaggagc
actactgcca tttctgttgt 2700tgctccaata tgctatgcgc acttggctgc tactcagttg
gggcagttca tgaaatttga 2760ggacaaatct gaaacatctt caagccatgg tggattgagc
ggtgcaagtg ctgttcccgt 2820ccctcagttg cctcccttgc aagagaatgt ccgcaacaca
atgttctttt gttgaagcct 2880taatgctctg ccctgtctcc tcaagtggtg aaaatgctgt
acataaaact atgtttctaa 2940tcttgcaagt tatgcggacg aagtttatat tgtggtagac
ttggtcttct tagccatatt 3000tagtctttct agcacaagcc ttttcaaatg ttcggggacc
ttaccttact ttttgtagca 3060agactctctt tagcgcaacg tctttttgta gcaaggcttg
atcttcagca cgtcttttgt 3120taggcccccc cttttttaag gtttaattgc actttttacc
tcgaatgctg taatttatag 3180tgaattttac ttatctccaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaa 323940915PRTGlycine max 40Met 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 413151DNATriticum aestivum 41gttgttcgag
gagaggggag ggggagagac gagaagggga acggaaaaga aagccaagcc 60ctctctcgcg
gaggccaacg gcgaggcttc ctcccttgcg ccctcgcaga tcagttcagc 120ggttcggctc
ctcgggacca ttgttggttc gctgaaatgg agtcacacgg agaggacctg 180ccaccaccac
caccactccc gccaaatgca gagccgataa aagctgagtc ggctgatgac 240ttgccaccac
caccacccct gctgcctatc aaacctgaag aagcaaagaa gatctcaaag 300cctaagaggg
ccctgatcgc tcgtcctggt tttggcaaga ggggaaatcc tatacagctt 360gtgacaaatc
atttcaaagt ctcgttgaag acgacagacg agttcttcca tcattactat 420gtaaatctga
agtatgaaga tgacaggcct gttgatggaa aaggtgttgg tagaaaagtc 480attgataagc
ttgctcagac ttatccatcg gaactagccc ataaagactt tgcctatgat 540ggtgaaaaga
gtctttttac cattggtgcc ctcccacaaa ttaacaatga gtttgttgtg 600gttcttgaag
atgtttccag tggaaagact cctgcaaatg gcagccctgg aaacgacagt 660ccagacaaga
agagagtgaa aaggccatat caaactaaaa ccttcaaggt ggagttgagc 720tttgctgcta
gaatccccat gagtgctatt gcaatggcac tcaaaggcca ggaatcagag 780cacacgcaag
aagccattcg ggttattgat atcatattaa gacagcactc tgccaaacag 840ggctgcctgt
tagtccgcca gtcatttttt cacaacaatc cttcaaactt tgtggacttg 900ggtgggggtg
tgatgggctg ccgaggtttc cactcaagct ttcgagccac acagagcggg 960ctttctctta
atattgatgt ttctacaaca atgattgtga aacctggccc tgttgtcgat 1020tttctgctgg
ccaaccagaa ggttgaccac cctaataaaa ttgattgggc taaggccaag 1080cgtgcactta
agaatttaag gataaaaaca agcccagcaa atacagaata caagattgtt 1140ggtttgagtg
agaggaattg ttatgaacaa atgttttccc tcaagcaaag gaatggtggg 1200aatggtgacc
ctgaagcaat agaaatatct gtttatgatt actttgtgaa gaaccgtggc 1260attgagctga
ggtactctgg tgatttccct tgtataaatg ttgggaaacc taggcggcca 1320acatattttc
ccattgagct ctgccagctg gtccctttac aaaggtatac caaatctttg 1380agtaccctac
aaagatcatc tcttgttgag aagtccaggc agaagcctca agagaggatg 1440tcagttttgt
ctgatgtact gaaacgcagc agctatgata cagaacccat gttgaaggca 1500tgtggaattt
cgatagctca gggctttaca caggtggctg gtagggtact gcaggccccc 1560aagctcaaag
ctggaaatgg tgaagatatt ttcacaagga atggacgttg gaatttcaac 1620aacaagaggc
ttgctagagc ttgtgtggtg gacagatggg cagttgtaaa cttttcggct 1680aggtgtaaca
ccatgaacct tgtcaatgac ctcatcaagt gtgggggcat gaagggcatt 1740acagtagaaa
aacctcatat tgtaattgaa gagaatggtt caatgagacg tgcacctgct 1800ccaaaaaggg
ttgaggatat gtttgagcaa gtgaagtcta agcttcctgg ggctccgaag 1860tttctcttgt
gtattcttgc tgagaggaag aactcagatg tttatggtcc atggaagcga 1920aaatgccttg
ctgactttgg gattgtcact caatgtgtgg ccccaacaag ggtcaatgac 1980caatatctga
caaatgttct gctgaagatc aatgcaaaac ttggtggaat gaactcacta 2040ctacaaattg
aaatgtcccc aagtatacct cttgtatcaa aggtcccaac tctcatcttg 2100ggaatggatg
tgtcccatgg atcccctgga cagtctgata taccgtccat tgcagcagtt 2160gttggttctc
gggaatggcc tcttgtctcg aaatataggg cttcagtgcg ctcgcagtca 2220ccaaagctcg
aaatgataga ttcattgttc aagccacaag gaactgatga tgatggcctt 2280gttcgggagt
gtctcattga cttctacacc agttctggaa aaaggaaacc agatcagatc 2340atcatcttca
gggatggtgt tagtgagagc cagtttaatc aggtgctgaa cattgaattg 2400gatcaaataa
ttgaggcctg caagttcttg gatgaaaatt ggaaccccaa gttcacgctg 2460attgttgccc
agaaaaatca ccacaccaaa ttcttcatac ctggatctcc tgacaatgtc 2520cctccaggca
ctgttgtaga taatgcagtc tgccatccaa ggaattatga cttctacatg 2580tgcgctcatg
ctggaatgat tgggactaca aggccaacac actaccatat cctgcatgat 2640gagatacact
ttgctgcgga tgacctgcag gatcttgtgc actcgctctc atatgtgtac 2700caaaggagca
cgacagccat atcagttgtt tctccaatct gctatgcaca tcttgcggct 2760gctcaggtgg
cgcagttcat aaagtttgat gagatgtctg agacgtcgtc gagccagggc 2820ggtggccaca
cctctgccgg cagcgctcca gtgcaggagc tgcctcgcct ccatgagaaa 2880gtccgcagca
gcatgttctt ctgctgagcc agccagccag ccgcacttgc gcgttccaac 2940ttttggtgat
gcgcttggtt atctagtact agtagtatgt agtagtggcc tgtgatggcc 3000tgttggactc
ctgggatgtt gtgttcctaa gctggttgct gcacttggtg cctcagaacc 3060tttgaatcct
gtcagggtgc tgcagttgaa cctttactat cgaaccatct aatttgttgc 3120tttcaaaaaa
aaaaaaaaaa aaaaaaaaaa a
315142916PRTTriticum aestivum 42Met 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 43791DNATriticum aestivum 43ctcgtgccga attcggcacg agaacttcct
caaatacgca acgcatgccg cctgaaatat 60cctgcaaccg acaccaaacg agggctcccc
aggatcacaa ttgttgtctg tggtaaacgc 120caccacactc gattctaccc taaaaacagc
ggtgacgctg ataaatcatc gaatttgatg 180gctggaactg ttgtcgatcg tggcgttaca
gagactcgaa actgggactt ttacctacaa 240gcccatgcat gtcttcaggg aacagcccgt
gcctgtcatt actatgtgat aatagacgaa 300attttccggt ccaataaggt taagggtggt
cacaaaaatc acgctgatgc ccttgaggaa 360ttgacaaaca atatgagtca tctgtttgga
cgagcaacaa aagccgtcag tctttgtcct 420cctgcttact atgctgattt actctgcaca
agggtacgct gctacttatc tgaagttttc 480gacccaagtg aggcccagag tgtgatgagt
ggcggcacca accaaacgat cgaggacatt 540gttattccgc cgagtatgag ggattccatg
tactacatct aagctcattg catgagaatg 600agaatcatta aaccataacc ttcggtgtta
gttacagaat tagctgtgtc aagtcattat 660agacgaaata ccatttctgt attgtagact
ttgcgttccg aaatatttta tgcacacgca 720aatgtatagc caaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780aaaaaaaaaa a
79144193PRTTriticum aestivum 44Leu 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
45327DNATriticum aestivum 45tgcgttctga catccattcg aggcccctct
cagacgccga acgtgttaag atcaagaagg 60cactgagagg agtaaaggtg gaagttactc
atcgtggcaa catgcgaagg aagtaccgaa 120tatctggtct gacaacccag gcaactcgag
agctaacttt tcctgttgat gaagggggta 180cagtaaagtc agtcgtacaa tactttcagg
agacatatgg ctttgccatc cagcacacgt 240acctgccttg cctccaagtt ggcaatcagc
agcgtccaaa ttacttgggg gatcctctag 300aggcgaccgg caggcataca agcttgg
3274695PRTTriticum aestivum 46Arg 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 47571DNATriticum aestivumunsure(234)n = A, C, G or T
47attagtgtta ccaaaagtcg gcaagtggga catgtggtgc aagaaaatgg tcaatggagg
60agtagttaac acctgggcat gcattaactt tgcttgggaa gtcacagatg ctcatgctct
120gaatttttgt gatgagttgg tgctgatgtg caatgtatcc gggatggact tcaggcctga
180acctgtgctc cctgtaacag cttatgaccc taaatccgta gcacggtcac tcanaganac
240accataaang tntcatgaac atacctggnc cacngcgcca aanactcgac ctgctgattc
300naatattgct gacaagtant ggcacccttt atggtgacat caggagaata ttngggacag
360atattgggag tggtctctca nngttgtctt gcaaaacatg tttttaancc caaaaaacat
420atttnncaat gttgccctta aaataatgnt aangcnggag ganaaancgg tcntttangc
480ttgaaaggaa cccccctatg ggaaaaaacg cnncnatttg ggcgnantag cntcaaaccn
540gcaagggttc caccctccat gnnngtgtgg t
5714877PRTTriticum aestivum 48Leu 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 491565DNATriticum
aestivum 49actcgaatat gaggaccctc acactgtaat tgaagagagc ccgtcactga
gacgagctcc 60ggtggcacga agagtggagg agatgtttgc ccagataaag gccaagctac
ctggagcacc 120cttgtttctt ttgtgcctcc tccctgagag gaagaactgc gaagtttacg
gtccttggaa 180gaagaagtgt cttgctgatt tcggcatagt cacccaatgt ctagctccgc
aaagagtcaa 240tgaccagtac ttgagtaatc tgctactcaa gataaatgct aagctcggtg
gactcaacac 300actgcttcaa attgaagcag cccgtgcaat acccattgtg gggaaggtgc
ctactatcat 360cctgggcatg gatgtctcgc atggtcaacc tggccaatcc gacaggcctt
ccattgctgc 420ggtggtgagt tctcgtgagt ggcctctcat ctctaaatac agagcaacag
tgcacactca 480gtcacccaaa caggaggtga tggcttccct gtttaagcca cggggagctg
aagatgatgg 540ccttattcgg gaatctctta ttgacttgta cactagctct gggaagcgaa
agccagacca 600agttattatt ttcagggatg gagttagcga aagccagttt actcaggtga
taaacattga 660gcttgagcag atcattgagg catgcaagtg ccttgacgac aagtgggagc
ccaagttcac 720ggtcattgtt gctcagaaaa accatcatac caggtttttc cagacaaact
cgccagaaaa 780tgttcctcct ggcactgtgg tggataaaca agtgtgccat cccaagaact
ttgacttcta 840catgtgcgcg catgctggga tgattggcac gtcgaggcca acgcattacc
atgttctgca 900tgatgagatc ggcttcagtg gggatgagct ccaggagttt gtgcactcgc
tctcctatgt 960gtaccagagg agcacgacgg cgatatcagt agctgctccg atagcgtacg
cgcatctggc 1020ggcggcgcag gtgggcacct tcatgaagtt tgaggacatg tcggacacgt
cgtcgagcca 1080gggagggggc cacacgtctg cgggcagcgc cccggtgccg gagctgcctc
ggctgcacga 1140gaaagtgagg agctccatgt tcttctgctg atctgatgct gctcttgaac
ttgatcgatg 1200ccgctttctg tcagtggagg ttgaaccgtg cgtctgtata aataaaacct
actagtacct 1260atctatctat gtactatcta gatggcacct ggaactttag ctgttatcca
gggtgcccgt 1320aagtcggtcc gttgtgtcgg gtgccgctgg gaacgttccc atggatgtta
ccgtttgtgg 1380tgttggcgtt gttgaaccaa ccaacctgac cctagcttaa ccttgcttgg
attggatgat 1440gtgctagcta gctagagcta gagctagagt tagaccatgc atggctgatg
gtatgtattg 1500tgggatcata tctatctatc tccatcctga cttggtgata aaaaaaaaaa
aaaaaaaaaa 1560aaaaa
156550389PRTTriticum aestivum 50Leu 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
51541DNATriticum aestivumunsure(33)n = A, C, G or T 51gattccatgt
cctttgggat ggaacaattt acngcggatg tttacgatct cagaacaatt 60tgtgtacact
acgcaaggtg cacccgttct gtatngattg tgcctccggc atactatgct 120cacctcgcgg
cttttcgagc tcggttctac atggaaccgg atacctccga tggtggctcg 180gtcgcgagcg
gtgccacgac aagccgtgcc cctcctggtg cacgcggcgg cagtagagct 240gcagggaatg
ttgctgttaa gcctctgcct gagctcaagg aaaacgtgaa gcgtgtcatg 300ttttactgct
gataagttgg ggcaacgcct ccggggtccg ggctatctat tccccgtgat 360cccaactgaa
gtgcctgctg atttaccaat cctttctttg cggcagaaaa tcaatcatca 420gtcatcacat
gagtgtatct atatatgtat cagtgctgcc atgtttcctg tgcaacctga 480acatctcaat
tcctcttttc atctacagat tttcaaatgg cattttccct gttaaaaaaa 540a
54152103PRTTriticum aestivumUNSURE(32)Xaa = ANY AMINO ACID 52Asp 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 3705DNAOryza sativaunsure(3616)n = A, C, G or T 53gagcagcagt
gcggtagtgc aagcgctagt ggaggagttg ggaggaggcc ccctagggtt 60tcccgagacc
gcctcccccc gcgcctgcgc cgccgctcgc cgagcgcgcg ctccgtgccc 120atcatggtga
agaagaaaag aactgggtct ggcagcaccg gtgagagttc tggagaggct 180ccaggagctc
ctggccatgg ttcttcacag cgagctgaga gaggtcctca acagcatggg 240ggaggacgtg
gttgggtgcc tcaacatggt ggccgtggtg gtgggcaata ccagggccgt 300ggtggacatt
atcagggccg tggagggcaa ggttcacacc atccaggtgg agggcctcct 360gagtatcagg
gtcgtggagg gccaggttca catcatccag gtggtgggcc tcctgactat 420cagggccgtg
gaggatcagg ttcacatcac ccaggtggtg ggcctcccga gtatcaaccg 480cgtgactatc
aaggacgtgg tggtccacgc cccagaggtg gaatgccaca gccatactat 540ggcggaccta
gggggagtgg cggacgtagt gttccttcag gttcatcaag aacagttccc 600gagctgcacc
aagccccaca tgtccaatac caagccccga tggtttcacc aaccccatcg 660ggagctggct
catcctctca gcctgcggcg gaggtgagca gtggacaagt ccaacaacag 720tttcagcaac
ttgccacccg tgatcaaagt tcgaccagcc aagccattca aatagcacca 780ccgtcaagca
aatcagttag attcccgttg cgccctggca agggtacata tggggacagg 840tgcattgtga
aggcgaacca tttctttgct gaacttcctg ataaagacct tcaccaatac 900gacgtatcta
ttactcctga ggttacttca cgtggcgtga atcgtgctgt tatgtttgag 960ttagtaacgc
tgtatagata ttcccatttg ggcgggcgtc tacctgccta tgatggaagg 1020aagagtcttt
acacagctgg accattgcca tttgcttcta ggacatttga aattactctt 1080caagatgagg
aagatagtct tggtggtggc caaggcaccc aaaggcgtga gagactattt 1140agggtggtga
tcaagtttgc tgcccgtgct gatcttcacc atttggctat gtttctagct 1200ggaaggcaag
cagatgctcc tcaagaagcc cttcaagtcc ttgacattgt gttacgtgaa 1260ttgcctacca
caaggtactc accagttggt cggtcatttt attctcccaa tttagggaga 1320cgccagcaac
ttggtgaggg tttggaaagt tggcgtggtt tttaccaaag cataaggcct 1380acccagatgg
gtctctcact gaatattgat atgtcatcaa ctgcatttat tgagcctcta 1440cctgtgattg
actttgttgc tcagcttctg aacagagaca tctcagttag accattatct 1500gattctgatc
gtgtgaagat aaagaaagct ctaagaggtg tgaaggttga ggtgacgcat 1560agaggaaaca
tgcgtagaaa atatcgtata tctggactca cttcacaggc aacaagggag 1620ttatcattcc
ctgtcgatga tcgtggtact gtgaagactg tggtgcaata ttttctggag 1680acatatggtt
ttagtattca gcacaccact ttgccttgcc ttcaagtggg caatcagcaa 1740aggcccaatt
atctgcctat ggaggtttgt aagatcgttg agggacagcg ttactcgaag 1800cggcttaacg
agaaacagat tactgcgcta ttgaaagtga cttgccagcg acctcaagag 1860cgtgaactgg
atattttgcg gactgtatct cacaatgcat accatgaaga tcagtatgcg 1920caggaatttg
gcataaaaat tgatgagcgt cttgcatctg ttgaagctcg tgttctgcct 1980cccccaaggc
ttaaatacca tgatagtggg agagaaaagg atgtattgcc gagagttggc 2040cagtggaaca
tgatgaataa gaaaatggtc aatggtggga gagtcaacaa ctgggcatgt 2100attaacttct
ctagaaatgt gcaagatagt gctgccaggg gcttctgtca tgagctggct 2160atcatgtgcc
aaatatctgg aatggatttt gcactggaac ctgtgctgcc cccacttact 2220gctagacctg
aacatgtgga aagagcactg aaggcacgct atcaagatgc aatgaacatg 2280ctcagaccgc
agggcaggga acttgattta ctgattgtaa tactgcctga caataatggt 2340tctctttatg
gggatctcaa aagaatctgt gagactgatc ttggattggt ctcccaatgt 2400tgtttgacaa
aacatgtttt taaaatgagc aagcagtatc ttgcaaatgt tgcccttaaa 2460ataaacgtta
aggtgggggg aaggaatact gtacttgtgg atgctttgac aaggaggatt 2520ccccttgtca
gtgacagacc aactatcata tttggtgcgg atgttactca tcctcatcct 2580ggagaagatt
ccagtccttc cattgcagct gtggttgctt ctcaagactg gcctgaagtc 2640actaagtatg
ctggattggt gagtgcccaa gcccatcgtc aagaattgat acaagatctt 2700ttcaaagtat
ggcaagaccc gcatagagga actgttactg gtggcatgat caaggagctt 2760ctcatttctt
tcaagagggc tactggacag aaacctcaga ggataatatt ttacagggat 2820ggtgtcagcg
aggggcagtt ttatcaagtt ttgttgtatg agcttgatgc cattagaaag 2880gcttgtgcat
ccctggaacc caactatcag cctccagtta cctttgtggt ggtccagaag 2940cggcatcaca
caaggttgtt tgctaataat cacaacgacc agcgtactgt tgatagaagt 3000ggaaacattc
tgcctggaac tgttgttgac tcaaagattt gccatccaac cgagtttgat 3060ttctacctgt
gtagccatgc tggcatacag ggaacaagcc gtcctgctca ttatcatgtt 3120ctgtgggatg
agaacaaatt tactgcagac gagttgcaaa ccctcacgaa caacttgtgc 3180tacacgtatg
caaggtgcac tcgctctgta tcaattgtgc ctcctgcgta ctatgctcat 3240ctggcagcct
tccgagctcg cttttacatg gagccagaga catctgacag tggatcaatg 3300gcgagtggag
ctgcaacgag ccgtggcctt ccaccaggtg tgcgcagcgc cagggttgct 3360ggaaatgtag
ccgtcaggcc tctacctgct ctcaaggaaa acgtgaagcg tgtcatgttt 3420tactgctaag
agcttgggct gtaccccgta tgcgccaagg aatgtagtac tatgttatgt 3480tattttagca
cttgcactct gtcgttgatc ccgttaaaac gggtatgcta ccataagctg 3540ttggactatt
ctgggtattg tagtactact tgttttgtat ttgtgtttgt gacgctgcag 3600agcgtgaaca
acgcanaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660aaaaaaaaaa
aaaccaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa
3705541101PRTOryza sativa 54Met 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 55904PRTOryza sativa 55Met 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
561048PRTArabidopsis thaliana 56Met 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
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