Patent application title: METHOD FOR MODULATING PLANT GROWTH, NUCLEIC ACID MOLECULES AND POLYPEPTIDES ENCODED THEREOF USEFUL AS MODULATING AGENT
Inventors:
Dirk Inze (Aalst, BE)
Veronique Boudolf (Gent, BE)
Lieven De Veylder (Gent, BE)
Juan Antonio Torres Acosta (Gent, BE)
Zoltan Magyar (Gent, BE)
Assignees:
CropDesign N.V.
IPC8 Class: AA01H500FI
USPC Class:
800306
Class name: Plant, seedling, plant seed, or plant part, per se higher plant, seedling, plant seed, or plant part (i.e., angiosperms or gymnosperms) brassica
Publication date: 2012-08-23
Patent application number: 20120216320
Abstract:
The invention provides isolated nucleic acids molecules, designated CCP
nucleic acid molecules, which encode novel cell cycle associated
polypeptides. The invention also provides antisense nucleic acid
molecules, recombinant expression vectors containing CCP nucleic acid
molecules, host cells into which the expression vectors have been
introduced, and transgenic plants in which a CCP gene has been introduced
or disrupted. The invention still further provides isolated CCP proteins,
fusion proteins, antigenic peptides and anti-CCP antibodies.
Agricultural, diagnostic, screening, and therapeutic methods utilizing
compositions of the invention are also provided.Claims:
1. An isolated nucleic acid molecule selected from the group consisting
of: (a) a nucleic acid molecule comprising the nucleotide sequence set
forth in SEQ ID NO: 3, 6, 12, 13, 29, 41, 42, or 45; (b) a nucleic acid
molecule which encodes a polypeptide comprising the amino acid sequence
set forth in SEQ ID NO: 69, 72, 78, 79, 95, 108, or 111; (c) a nucleic
acid molecule which encodes a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence set forth in SEQ ID NOs:
69, 72, 78, 79, 95, 108, or 111; (d) a nucleic acid molecule comprising a
nucleotide sequence which is at least 60% identical to the nucleotide
sequence of SEQ ID NO: 3, 6, 12, 13, 29, 41, 42, or 45, or a complement
thereof; (e) a nucleic acid molecule comprising a fragment of at least 50
nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ
ID NO: 3, 6, 12, 13, 29, 41, 42, or 45, or a complement thereof; (f) a
nucleic acid molecule which encodes a polypeptide comprising an amino
acid sequence at least about 60% identical to the amino acid sequence of
SEQ ID NO: 69, 72, 78, 79, 95, 108, or 111; and (g) a nucleic acid
molecule which encodes a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO: 69, 72, 78, 79, 95, 108, or 111, wherein the
fragment comprises at least 15 contiguous amino acid residues of the
amino acid sequence of SEQ ID NO: 69, 72, 78, 79, 95, 108, or 111.
2-4. (canceled)
5. An isolated nucleic acid molecule which hybridizes to the nucleic acid molecule of claim 1, under stringent conditions.
6. An isolated nucleic acid molecule comprising a nucleotide sequence which is complementary to the nucleotide sequence of the nucleic acid molecule of claim 1.
7. An isolated nucleic acid molecule comprising the nucleic acid molecule of claim 1, and a nucleotide sequence encoding a heterologous peptide.
8. A vector comprising the nucleic acid molecule of claim 1.
9. A cell comprising the nucleic acid molecule of claim 1.
10. A host cell transfected with the vector of claim 8.
11. A method of producing a polypeptide comprising culturing the host cell of claim 10 in an appropriate culture medium to, thereby, produce the polypeptide.
12. An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NOs: 69, 72, 78, 79, 95, 108, or 11, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NOs:69, 72, 78, 79, 95, 108, or 111; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NOs:69, 72, 78, 79, 95, 108, or 111, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of SEQ ID NOs:3, 6, 12, 13, 29, 41, 42, or 45 under stringent conditions; c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NOs:3, 6, 12, 13, 29, 41, 42, or 45; and d) a polypeptide comprising an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NOs:69, 72, 78, 79, 95, 108, or 111.
13. The isolated polypeptide of claim 12 comprising the amino acid sequence of SEQ ID NOs:69, 72, 78, 79, 95, 108, or 111.
14. The polypeptide of claim 12, further comprising one or more heterologous amino acid sequences.
15. An antibody which selectively binds to a polypeptide of claim 12.
16. A method for detecting the presence of a polypeptide of claim 12 in a sample comprising: a) contacting the sample with a compound which selectively binds to the polypeptide; and b) determining whether the compound binds to the polypeptide in the sample to thereby detect the presence of a polypeptide of claim 12 in the sample.
17. The method of claim 16, wherein the compound which binds to the polypeptide is an antibody.
18. A kit comprising a compound which selectively binds to a polypeptide of claim 12 and instructions for use.
19. A method for detecting the presence of the nucleic acid molecule of claim 1 in a sample comprising: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample to thereby detect the presence of nucleic acid molecule of claim 1 in the sample.
20. The method of claim 19, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
21. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.
22. A method for identifying a compound which binds to the polypeptide of claim 12 comprising: a) contacting the polypeptide, or a cell expressing the polypeptide with a test compound; and b) determining whether the polypeptide binds to the test compound.
23. The method of claim 22, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detection of test compound/polypeptide binding; b) detection of binding using a competition binding assay; and c) detection of binding using an assay for CCP activity.
24. A method for modulating the activity of a polypeptide of claim 12 comprising contacting the polypeptide or a cell expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
25. A method for identifying a compound which modulates the activity of a polypeptide of claim 12 comprising: a) contacting a polypeptide of claim 12 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
26. A transgenic plant comprising the nucleic acid molecule of claim 1.
27. The transgenic plant of claim 26, wherein the plant is a monocot plant.
28. The transgenic plant of claim 26, wherein the plant is a dicot plant.
29. The transgenic plant of claim 26, wherein the plant is selected from the group consisting of Arabidopsis thaliana, rice, wheat, maize, tomato, oilseed rape, soybean, sunflower, and canola.
30. A method for modulating the growth of a plant, comprising introducing into the plant a CCP modulator in an amount sufficient to modulate the growth of the plant, thereby modulating the growth of the plant.
31. The method of claim 30, wherein the CCP modulator is a small molecule.
32. The method of claim 30, wherein the CCP modulator is capable of modulating CCP polypeptide activity.
33. The method of claim 32, wherein the CCP modulator is an anti-CCP antibody; or wherein the CCP modulator is a CCP polypeptide comprising the amino acid sequence of SEQ ID NOs: 67-132, 205, 211, 215-216 or 220-227, or a fragment thereof.
34. (canceled)
35. The method of claim 30, wherein the CCP modulator is capable of modulating CCP nucleic acid expression.
36. The method of claim 35, wherein the CCP modulator is an antisense CCP nucleic acid molecule; wherein the CCP modulator is a ribozyme; or wherein the CCP modulator comprises the nucleotide sequence of SEQ ID NO: 1-66 or 228-239, or a fragment thereof.
37-38. (canceled)
39. The method of claim 30, wherein the plant is a monocot plant.
40. The method of claim 30, wherein the plant is a dicot plant.
41. The method of claim 30, wherein the plant is selected from the group consisting of Arabidopsis thaliana, rice, wheat, maize, tomato, alfalfa, oilseed rape, soybean, sunflower, and canola.
42. A method for modulating the cell cycle in a plant, comprising introducing into the plant a CCP modulator in an amount sufficient to modulate the cell cycle in the plant, thereby modulating the cell cycle in the plant.
43. The method of claim 42, wherein the plant is a monocot plant.
44. The method of claim 42, wherein the plant is a dicot plant.
45. The method of claim 42, wherein the plant is selected from the group consisting of Arabidopsis thaliana, rice, wheat, maize, tomato, alfalfa, oilseed rape, soybean, sunflower, and canola
Description:
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent application Ser. No. 60/204,045, filed May 12, 2000. The contents of this provisional patent application are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Cell division plays a crucial role during all phases of plant development. The continuation of organogenesis and growth responses to a changing environment require precise spatial, temporal, and developmental regulation of cell division.
[0003] The basic mechanisms controlling the progression through the cell cycle appear to be conserved in all higher eukaryotes, although the temporal and spatial control of cell division can differ largely, between organisms. Plants have unique developmental features which are not found in either animals or fungi. First, due to the presence of a rigid cell wall, plant cells cannot move and consequently organogenesis is dependent on cell division and cell expansion at the site of formation of new organs. Secondly, cell divisions are confined to specialized regions, called meristems. These meristems continuously produce new cells which, as they move away from the meristem, become differentiated. The meristem identity itself can change from a vegetative to a reproductive phase, resulting in the formation of flowers. Thirdly, plant development is largely post-embryonic. During embryogenesis, the main developmental event is the establishment of the root-shoot axis.
[0004] Most plant growth occurs after germination, by iterative development at the meristems. Lastly, as a consequence of the sessile life of plants, development and cell division are, to a large extent, influenced by environmental factors such as light, gravity, wounding, nutrients, and stress conditions. All these features are reflected in a plant-specific regulation of the factors controlling cell division.
[0005] The unparalleled potential of plants for continuous organogenesis and plastic growth also relies on the competent or active state of the cell division apparaturs. The discovery of a common mechanism underlying the regulation of the cell cycle in yeasts and animals has led to efforts to extend these findings to the plant kingdom and is leading to research aimed at converting the gathered knowledge into useful traits introduced in transgenic plants.
[0006] When eukaryotic cells and, thus, also plant cells divide they go through a highly ordered sequence of events collectively termed as the "cell cycle." Briefly, DNA replication or synthesis (S) and mitotic segregation of the chromosomes (M) occur with intervening gap phases (G1 and G2) and the phases follow the sequence G1-S-G2-M. Cell division is completed after cytokinesis, the last step of the M-phase. Cells that have exited the cell cycle and have become quiescent are said to be in the G0 phase. Cells at the G0 stage can be stimulated to reenter the cell cycle at the G1 phase. The transition between the different phases of the cell cycle are basically driven by the sequential activation/inactivation of a kinase (called "cyclin-dependent kinase", "CDC" or "CDK") by different agonists.
[0007] Proteins called cyclins are required for kinase activation. Cyclins are also important for targeting the kinase activity to a given subset of substrate(s). Other factors regulating CDK activity include CDK inhibitors (CKIs or ICKs, KIPs, CIPs, INKs), CDK activating kinase (CAK) and CDK phosphatase (CDC25) (Mironov et al. (1999) Plant Cell 11, 509-522 and Won K. et al. (1996) EMBO J. 15, 4182-4193).
SUMMARY OF THE INVENTION
[0008] The present invention is based, at least in part, on the discovery of novel plant nucleic acid molecules and polypeptides encoded by such nucleic acid molecules, referred to herein as "cell cycle proteins" or "CCP." The CCP nucleic acid and polypeptide molecules of the present invention are useful as modulating agents in regulating cell cycle progression in, for example, plants. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding CCP polypeptides, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of CCP-encoding nucleic acids.
[0009] In one embodiment, a CCP nucleic acid molecule of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) of SEQ ID NO:1-66 or 228-239, or a complement thereof.
[0010] In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239, or a complement thereof. In another preferred embodiment, an isolated nucleic acid molecule of the invention encodes the amino acid sequence of a plant CCP polypeptide.
[0011] Another embodiment of the invention features nucleic acid molecules, preferably CCP nucleic acid molecules, which specifically detect CCP nucleic acid molecules relative to nucleic acid molecules encoding non-CCP polypeptides. For example, in one embodiment, such a nucleic acid molecule is at least 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1-66\or 228-239, or a complement thereof.
[0012] In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a plant CCP polypeptide, wherein the nucleic acid molecule hybridizes to the nucleic acid molecule of SEQ ID NO:1-66 or 228-239 under stringent conditions.
[0013] Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a CCP nucleic acid molecule, e.g., the coding strand of a CCP nucleic acid molecule.
[0014] Another aspect of the invention provides a vector comprising a CCP nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing a CCP polypeptide, by culturing in a suitable medium a host cell of the invention, e.g., a plant host cell such as a host monocot plant cell (e.g., rice, wheat or corn) or a dicot host cell (e.g., Arabidopsis thaliana, oilseed rape, or soybeans) containing a recombinant expression vector, such that the polypeptide is produced.
[0015] Another aspect of this invention features isolated or recombinant CCP polypeptides. In one embodiment, an isolated CCP polypeptides has one or more of the following domains: a "cyclin destruction box", a "cyclin box motif 1", a "cyclin box motif 2", a "CDC2 motif", a "CDK phosphorylation site", a "nuclear localization signal", a "Cy-like box", an "Rb binding domain", a "DEF domain", a "DNA binding domain", a "DCB1 domain", a "DCB2 domain" and/or a "SAP domain".
[0016] In a preferred embodiment, a CCP polypeptide includes at least one or more of the following domains: a "cyclin destruction box", a "cyclin box motif 1", a "cyclin box motif 2", a "CDC2 motif", a "CDK phosphorylation site", a "nuclear localization signal", a "Cy-like box", an "Rb binding domain", a "DEF domain", a "DNA binding domain", a "DCB1 domain", a "DCB2 domain" and/or a "SAP domain", and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:67-132, 205, 211, 215-216, or 220-227.
[0017] In another preferred embodiment, a CCP polypeptide includes at least one or more of the following domains: a "cyclin destruction box", a "cyclin box motif 1", a "cyclin box motif 2", a "CDC2 motif", a "CDK phosphorylation site", a "nuclear localization signal", a "Cy-like box", an "Rb binding domain", a "DEF domain", a "DNA binding domain", a "DCB1 domain", a "DCB2 domain" and/or a SAP domain and has a CCP activity (as described herein).
[0018] In yet another preferred embodiment, a CCP polypeptide includes one or more of the following domains: a "cyclin destruction box", a "cyclin box motif 1", a "cyclin box motif 2", a "CDC2 motif", a "CDK phosphorylation site", a "nuclear localization signal", a "Cy-like box", an "Rb binding domain", a "DEF domain", a "DNA binding domain", a "DCB1 domain", a "DCB2 domain" and/or a SAP domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1-66 or 228-239.
[0019] In another embodiment, the invention features fragments of the polypeptide having the amino acid sequence of SEQ ID NO:67-132, 205, 211, 215-216, or 220-227, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:67-132, 205, 211, 215-216, or 220-227. In another embodiment, a CCP polypeptide has the amino acid sequence of SEQ ID NO:67-132, 205, 211, 215-216, or 220-227.
[0020] In another embodiment, the invention features a CCP protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:1-66 or 228-239, or a complement thereof. This invention further features a CCP polypeptide, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1-66 or 228-239, or a complement thereof.
[0021] In another embodiment the invention provides transgenic plants (e.g., monocot or dicot plants) containing an isolated nucleic acid molecule of the present invention. For example, the invention provides transgenic plants containing a recombinant expression cassette including a plant promoter operably linked to an isolated nucleic acid molecule of the present invention. The present invention also provides transgenic seed from the transgenic plants. In another embodiment the invention provides methods of modulating, in a transgenic plant, the expression of the nucleic acids of the invention.
[0022] The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-CCP polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind polypeptide of the invention, preferably CCP polypeptide. In addition, the CCP polypeptide or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
[0023] In another aspect, the present invention provides a method for detecting the presence of a CCP nucleic acid molecule, polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a CCP nucleic acid molecule, polypeptide such that the presence of a CCP nucleic acid molecule, polypeptide is detected in the biological sample.
[0024] In another aspect, the present invention provides a method for detecting the presence of CCP activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of CCP activity such that the presence of CCP activity is detected in the biological sample.
[0025] In another aspect, the invention provides a method for modulating CCP activity comprising contacting a cell capable of expressing CCP with an agent that modulates CCP activity such that CCP activity in the cell is modulated. In one embodiment, the agent inhibits CCP activity. In another embodiment, the agent stimulates CCP activity. In one embodiment, the agent is an antibody that specifically binds to a CCP polypeptide. In another embodiment, the agent modulates expression of CCP by modulating transcription of a CCP gene or translation of a CCP mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a CCP mRNA or a CCP gene.
[0026] In one embodiment, the methods of the present invention are used to increase crop yield, improve the growth characteristics of a plant (such as growth rate or size of specific tissues or organs in the plant), modify the architecture or morphology of a plant, improve tolerance to environmental stress conditions (such as drought, salt, temperature, nutrient or deprivation), or improve tolerance to plant pathogens (e.g., pathogens that abuse the cell cycle) by modulating CCP activity in a cell. In one embodiment, the CCP activity is modulated by modulating the expression of a CCP nucleic acid molecule. In yet another embodiment, the CCP activity is modulated by modulating the activity of a CCP polypeptide. Modulators of CCP activity include, for example, a CCP nucleic acid or polypeptide.
[0027] The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a CCP polypeptide; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a CCP polypeptide, wherein a wild-type form of the gene encodes a protein with a CCP activity.
[0028] In another aspect the invention provides methods for identifying a compound that binds to or modulates the activity of a CCP polypeptide, by providing an indicator composition comprising a CCP polypeptide having CCP activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on CCP activity in the indicator composition to identify a compound that modulates the activity of a CCP polypeptide. The identified compounds may be used as herbicides or plant growth regulators.
[0029] Other features and advantages of the invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP1. The complete nucleotide sequence (FIG. 1A) corresponds to nucleic acids 1 to 1715 of SEQ ID NO:39. The complete amino acid sequence (FIG. 1B) corresponds to amino acids 1 to 460 of SEQ ID NO:105. Underlined in FIG. 1A and FIG. 1B are the partially characterized nucleotide (SEQ ID NO:1) and predicted partial amino acid (SEQ ID NO:67) sequence, respectively. Further indicated in FIG. 1A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP1 by PCR. The SEQ ID NOs of the primers used can be found in Table III. Indicated in FIG. 1B are the cyclin destruction box (black shaded box) and the cyclin box motifs 1 and 2 (both in gray shaded boxes).
[0031] FIG. 2 depicts the cDNA sequence of the Arabidopsis thaliana CCP2. The complete nucleotide sequence corresponds to nucleic acids 1 to 2195 of SEQ ID NO:40. Underlined is the partially characterized nucleotide (SEQ ID NO:2) sequence. Nucleotide sequence differences between SEQ ID NO:40 and SEQ ID NO:2 are depicted. Indicated are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP2 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0032] FIG. 3 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP2. The complete amino acid sequence corresponds to amino acids 1 to 664 of SEQ ID NO:106. Underlined is the predicted partial amino acid (SEQ ID NO:68) sequence.
[0033] FIG. 4 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP3. The complete nucleotide sequence (FIG. 3A) corresponds to nucleic acids 1 to 1413 of SEQ ID NO:41. The complete amino acid sequence (FIG. 3B) corresponds to amino acids 1 to 450 of SEQ ID NO:69. Underlined in FIG. 3A and FIG. 3B are the partially characterized nucleotide (SEQ ID NO:3) and predicted partial amino acid (SEQ ID NO:69) sequences, respectively. Indicated in FIG. 3A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP3 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:41 and SEQ ID NO:3 are depicted Indicated in FIG. 3B are the cyclin destruction box (black shaded box) and the cyclin box motifs 1 and 2 (both in gray shaded boxes).
[0034] FIG. 5 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP4. The complete nucleotide sequence (FIG. 5A) corresponds to nucleic acids 1 to 672 of SEQ ID NO:4. The complete amino acid sequence (FIG. 5B) corresponds to amino acids 1 to 223 of SEQ ID NO:70. Indicated in FIG. 5A are stop and start codon (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP4 by PCR. SEQ ID NOs of the primers used can be found in Table III. Indicated in FIG. 5B is the CDK phosphorylation site to (black shaded box).
[0035] FIG. 6 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP5. The complete nucleotide sequence (FIG. 6A) corresponds to nucleic acids 1 to 1287 of SEQ ID NO:5. The complete amino acid sequence (FIG. 6B) corresponds to amino acids 1 to 429 of SEQ ID NO:71. Indicated in FIG. 6A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP5 by PCR. SEQ ID NOs of the primers used can be found in Table III. Indicated in FIG. 6B are the cyclin destruction box (black shaded box) and the cyclin box motifs 1 and 2 (both in gray shaded boxes).
[0036] FIG. 7 depicts the cDNA sequence of the Arabidopsis thaliana CCP6. The complete nucleotide sequence corresponds to nucleic acids 1 to 2766 of SEQ ID NO:42. Underlined is the partially characterized nucleotide (SEQ ID NO:6) sequence. Indicated are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP6 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:42 and SEQ ID NO:6 are depicted.
[0037] FIG. 8 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP6. The complete amino acid sequence corresponds to amino acids 1 to 901 of SEQ ID NO:108. Underlined is the predicted partial amino acid (SEQ ID NO:72) sequence.
[0038] FIG. 9 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP7/CCP8. The complete nucleotide sequence (FIG. 9A) corresponds to nucleic acids 1 to 1260 of SEQ ID NO:43. The complete amino acid sequence (FIG. 9B) corresponds to amino acids 1 to 358 of SEQ ID NO:109. Underlined in FIG. 9A and FIG. 9B are the partially characterized nucleotide (SEQ ID NO:7) and predicted partial amino acid (SEQ ID NO:73) sequence, respectively. Italic sequences in FIG. 9A and FIG. 9B correspond to the partially characterized nucleotide (SEQ ID NO:8) and amino acid (SEQ ID NO:74) sequence, respectively, of another clone found independently to interact with an AtE2F protein in a yeast two-hybrid screen. Indicated in FIG. 9A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP7/8 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:43 and SEQ ID NO:7-8 are depicted.
[0039] FIG. 10 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP9. The complete nucleotide sequence (FIG. 10A) corresponds to nucleic acids 1 to 1308 of SEQ ID NO:9. The complete amino acid sequence (FIG. 10B) corresponds to amino acids 1 to 436 of SEQ ID NO:75. Indicated in FIG. 10A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP9 by PCR. SEQ ID NOs of the primers used can be found in Table III. Indicated in FIG. 10B are the cyclin destruction box (black shaded box) and the cyclin box motifs 1 and 2 (both in gray shaded boxes).
[0040] FIG. 11 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP10. The complete nucleotide sequence (FIG. 11A) corresponds to nucleic acids 1 to 1006 of SEQ ID NO:10. The complete amino acid sequence (FIG. 11B) corresponds to amino acids 1 to 254 of SEQ ID NO:76. Indicated in FIG. 11A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP10 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0041] FIG. 12 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP11. The complete nucleotide sequence (FIG. 12A) corresponds to nucleic acids 1 to 653 of SEQ ID NO:44. Indicated in FIG. 12A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP11 by PCR. SEQ ID NOs of the primers used can be found in Table III. However, during prediction of the open reading frame a frame shift was introduced which effected the CCP11 open reading frame. The stop codon indicated in italics in a black shaded box is the putative correct stop codon. The amino acid sequence in FIG. 12B corresponds to amino acids 1 to 86 of SEQ ID NO:77, the protein encoded by the initially identified open reading frame of SEQ ID NO:11. The putative correct complete amino acid sequence in FIG. 12C corresponds to amino acids 1 to 98 of SEQ ID NO:110.
[0042] FIG. 13 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP12/13. The complete nucleotide sequence (FIG. 13A) corresponds to nucleic acids 1 to 1266 of SEQ ID NO:45. The complete amino acid sequence (FIG. 13B) corresponds to amino acids 1 to 385 of SEQ ID NO:111. Double underlined in FIG. 13A and FIG. 13B are the partially characterized 3' nucleotide (SEQ ID NO:12) and C-terminal predicted partial amino acid (SEQ ID NO:78) sequence, respectively. Single underlined in FIG. 13A and FIG. 13B are the partially characterized 5' nucleotide (SEQ ID NO:13) and N-terminal predicted partial amino acid (SEQ ID NO:79) sequences, respectively. Indicated in FIG. 13A are the stop and start codons (both in black shaded boxes) and the primers (grey shaded boxes) used to amplify the coding region of CCP12/13 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:45 and SEQ ID NO:12 are depicted.
[0043] FIG. 14 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP14. The complete nucleotide sequence (FIG. 14A) corresponds to nucleic acids 1 to 1520 of SEQ ID NO:46. The complete amino acid sequence (FIG. 14B) corresponds to amino acids 1 to 465 of SEQ ID NO:112. Underlined in FIG. 14A and FIG. 14B are the partially characterized nucleotide (SEQ ID NO:14) and predicted partial amino acid (SEQ ID NO:80) sequence, respectively. Indicated in FIG. 14A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP14 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0044] FIG. 15 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP15. The complete nucleotide sequence (FIG. 15A) corresponds to nucleic acids 1 to 1142 of SEQ ID NO:47. The complete amino acid sequence (FIG. 1B) corresponds to amino acids 1 to 313 of SEQ ID NO:113. Underlined in FIG. 15A and FIG. 15B are the partially characterized nucleotide (SEQ ID NO:15) and predicted partial amino acid (SEQ ID NO:81) sequence, respectively. Indicated in FIG. 15A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP 15 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:47 and SEQ ID NO:15 are depicted. Indicated in FIG. 15B are the PSTTLRE motif (boxed) characteristic for the subclass of plant PSTTLRE CDC2 kinases.
[0045] Further indicated in FIG. 15B are three CDC2 motifs (black shaded box, grey shaded box and double underlined). Other residues conserved in CDC2s are underscored by `*` (residues in common with Propom domain PD198850), `+` (residues in common with Propom domain PD015684), (residues in common with Propom domain PD063669), and `1` (residues in common with Propom domain PD195780).
[0046] FIG. 16 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP16. The complete nucleotide sequence (FIG. 16A) corresponds to nucleic acids 1 to 1189 of SEQ ID NO:48. The complete amino acid sequence (FIG. 16B) corresponds to amino acids 1 to 292 of SEQ ID NO:114. Indicated in FIG. 16A are the stop and the three possible start codons (all in black shaded boxes) and the primers (grey shaded boxes) used to amplify the coding region of CCP16 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:48 and SEQ ID NO:16 are depicted. Indicated in FIG. 16B are the DNA binding domain (black shaded box), DEF domain (grey shaded box), DCB1 domain (single underlined) and DCB2 domain (double underlined), all domains characteristic for a DP protein.
[0047] FIG. 17 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP17. The complete nucleotide sequence (FIG. 17A) corresponds to nucleic acids 1 to 794 of SEQ ID NO:17. The complete amino acid sequence (FIG. 17B) corresponds to amino acids 1 to 173 of SEQ ID NO:83. Indicated in FIG. 17A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP17 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0048] FIG. 18 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP18. The complete nucleotide sequence (FIG. 18A) corresponds to nucleic acids 1 to 805 of SEQ ID NO:49. The complete amino acid sequence (FIG. 18B) corresponds to amino acids 1 to 165 of SEQ ID NO:115. Underlined in FIG. 15A and FIG. 15B are the partially characterized nucleotide (SEQ ID NO:18) and predicted partial amino acid (SEQ ID NO:84) sequence, respectively. Indicated in FIG. 18A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP18 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0049] FIG. 19 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP19. The complete nucleotide sequence (FIG. 19A) corresponds to nucleic acids 1 to 1152 of SEQ ID NO:19. The complete amino acid sequence (FIG. 1B) corresponds to amino acids 1 to 383 of SEQ ID NO:85. Indicated in to FIG. 19A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP19 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0050] FIG. 20 depicts the cDNA sequence of the Arabidopsis thaliana CCP20/21. The complete nucleotide sequence corresponds to nucleic acids 1 to 1539 of SEQ ID NO:50. Underlined are the partially characterized 5' nucleotide (SEQ ID NO:20) sequence and the partially characterized 3' nucleotide (SEQ ID NO:21). Indicated in FIG. 20 are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP20/21 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NOs:20-21 and SEQ ID NO:50 are depicted.
[0051] FIG. 21 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP20/21. The complete amino acid sequence corresponds to amino acids 1 to 432 of SEQ ID NO:116. Underlined are the partially characterized N-terminal predicted partial amino acid (SEQ ID NO:50) sequence and the partially characterized C-terminal amino predicted partial acid (SEQ ID NO: 87) sequence. Indicated are further differences in amino acid sequence between SEQ ID NO:87 and SEQ ID NO:116.
[0052] FIG. 22 depicts the cDNA sequence of the Arabidopsis thaliana CCP22. The complete nucleotide sequence corresponds to nucleic acids 1 to 1977 of SEQ ID NO:51. Underlined is the partially characterized nucleotide (SEQ ID NO:22). Indicated are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP22 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0053] FIG. 23 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP22. The complete amino acid sequence corresponds to amino acids 1 to 559 of SEQ ID NO:117. Underlined is the predicted partial amino acid (SEQ ID NO:88) sequence.
[0054] FIG. 24 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP23. The complete nucleotide sequence (FIG. 24A) corresponds to nucleic acids 1 to 525 of SEQ ID NO:52. Indicated in FIG. 24A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP23 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NOs:23 and SEQ ID NO:52 are depicted. The amino acid sequence in FIG. 24B corresponds to amino acids 1 to 98 of SEQ ID NO:89. The complete amino acid sequence in FIG. 24C corresponds to amino acids 1 to 86 of SEQ ID NO:118.
[0055] FIG. 25 depicts the cDNA sequence of the Arabidopsis thaliana CCP24. The complete nucleotide sequence corresponds to nucleic acids 1 to 2610 of SEQ ID NO:53. Underlined is the partially characterized nucleotide (SEQ ID NO:24). Indicated are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP24 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0056] FIG. 26 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP24. The complete amino acid sequence corresponds to amino acids 1 to 784 of SEQ ID NO:119. Underlined is the predicted partial amino acid (SEQ ID NO:90) sequence.
[0057] FIG. 27 depicts the cDNA sequence of the Arabidopsis thaliana CCP25. The complete nucleotide sequence corresponds to nucleic acids 1 to 2235 of SEQ ID NO:54. Underlined is the partially characterized nucleotide (SEQ ID NO:25) sequence. Indicated are stop and start codon (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP25 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0058] FIG. 28 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP25. The complete amino acid sequence corresponds to amino acids 1 to 724 of SEQ ID NO:120. Underlined is the predicted partial amino acid (SEQ ID NO:91) sequence.
[0059] FIG. 29 depicts the cDNA sequence of the Arabidopsis thaliana CCP26. The complete nucleotide sequence corresponds to nucleic acids 1 to 4002 of SEQ ID NO:55. Underlined is the partially characterized nucleotide (SEQ ID NO:26) sequence. Indicated are stop and start codon (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP26 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NOs:26 and SEQ ID NO:55 are depicted.
[0060] FIG. 30 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP26. The complete amino acid sequence corresponds to amino acids 1 to 1313 of SEQ ID NO:121. Underlined is the predicted partial amino acid (SEQ ID NO:92) sequence. Amino acid sequence differences between SEQ ID NOs:92 and SEQ ID NO:121 are depicted.
[0061] FIG. 31 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP27. The complete nucleotide sequence (FIG. 31A) corresponds to nucleic acids 1 to 1251 of SEQ ID NO:56. The complete amino acid sequence (FIG. 31B) corresponds to amino acids 1 to 310 of SEQ ID NO:122. Underlined in FIG. 31A and FIG. 31B are the partially characterized nucleotide (SEQ ID NO:27) and predicted partial amino acid (SEQ ID NO:93) sequence, respectively. Indicated in FIG. 31A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP27 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:27 and SEQ ID NO:56 are depicted in FIG. 31A.
[0062] FIG. 32 depicts the cDNA sequence of the Arabidopsis thaliana CCP28. The complete nucleotide sequence corresponds to nucleic acids 1 to 2955 of SEQ ID NO:56. Underlined is the partially characterized nucleotide (SEQ ID NO:28) sequence. Indicated are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP28 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:28 and SEQ ID NO:57 are depicted.
[0063] FIG. 33 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP28. The complete amino acid sequence corresponds to amino acids 1 to 964 of SEQ ID NO:123. Underlined is the predicted partial amino acid (SEQ ID NO:94) sequence.
[0064] FIG. 34 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP29. The complete nucleotide sequence (FIG. 34A) corresponds to nucleic acids 1 to 546 of SEQ ID NO:29. The complete amino acid sequence (FIG. 34B) corresponds to amino acids 1 to 181 of SEQ ID NO:95. Indicated in FIG. 34A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP29 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0065] FIG. 35 depicts the cDNA sequences and predicted amino acid sequences of the Arabidopsis thaliana CCP30. The complete nucleotide sequence (FIG. 35A) corresponds to nucleic acids 1 to 492 of SEQ ID NO:30. Indicated in FIG. 35A are the stop and start codons (both in black shaded boxes), the complete sense primer and part of the antisense primer (grey shaded boxes) used to amplify the coding region of CCP30 by PCR. SEQ ID NOs of the primers used can be found in Table III. However, after sequencing of the PCR product a sequence error in SEQ ID NO:30 was detected (boxed nucleotide `a` in FIG. 35A not present) which caused a frame shift effectuating the CCP30 open reading frame. The putative correct cDNA sequence is given in FIG. 35B (nucleic acids 1 to 865 of SEQ ID NO:58) wherein the three putative start codons are marked by a black shaded box. The originally identified start codon is indicated in bold letters. The stop codon is unaltered. The amino acid sequence in FIG. 35C corresponds to amino acids 1 to 163 of SEQ ID NO:96, the protein encoded by the initially identified open reading frame of SEQ ID NO:30. The putative correct complete amino acid sequence in FIG. 35D corresponds to amino acids 1 to 222 of SEQ ID NO:124 which comprises the longest possible open reading frame. The Met residues corresponding to the three possible start codons in SEQ ID NO:58 (FIG. 35B) are bold faced.
[0066] FIG. 36 depicts the cDNA sequence of the Arabidopsis thaliana CCP31. The complete nucleotide sequence corresponds to nucleic acids 1 to 723 of SEQ ID NO:31. Indicated in FIG. 1A are the stop and start codons (both in black shaded boxes).
[0067] FIG. 37 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP31. The complete amino acid sequence corresponds to amino acids 1 to 148 of SEQ ID NO:125.
[0068] FIG. 38 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP32. The complete nucleotide sequence (FIG. 38A) corresponds to nucleic acids 1 to 426 of SEQ ID NO:60. The complete amino acid sequence (FIG. 38B) corresponds to amino acids 1 to 70 of SEQ ID NO:126. Underlined in FIG. 38A is the partially characterized nucleotide (SEQ ID NO:32) sequence. Indicated in FIG. 38A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP32 by PCR. SEQ ID NOs of the primers used can be found in Table III. FIG. 38C gives the originally erroneously predicted amino acid sequence of CCP32 (amino acids 1 to 38 of SEQ ID NO:98).
[0069] FIG. 39 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP33. The complete nucleotide sequence (FIG. 39A) corresponds to nucleic acids 1 to 1442 of SEQ ID NO:61. The complete amino acid sequence (FIG. 39B) corresponds to amino acids 1 to 385 of SEQ ID NO:127. Indicated in FIG. 39A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP33 by PCR. SEQ ID NOs of the primers used can be found in Table III. Indicated in FIG. 39B are the DNA binding domain (black shaded box), DEF domain (grey shaded box), DCB1 domain (single underlined) and DCB2 domain (double underlined), all domains characteristic for a DP protein.
[0070] FIG. 40 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP34. The complete nucleotide sequence (FIG. 40A) corresponds to nucleic acids 1 to 1506 of SEQ ID NO:62. The complete amino acid sequence (FIG. 40B) corresponds to amino acids 1 to 437 of SEQ ID NO:128. Underlined in FIG. 40A and FIG. 40B are the partially characterized nucleotide (SEQ ID NO:34) and predicted partial amino acid (SEQ ID NO:62) sequence, respectively. Indicated in FIG. 40A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP34 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0071] FIG. 41 depicts the cDNA sequence of the Arabidopsis thaliana CCP35. The complete nucleotide sequence corresponds to nucleic acids 1 to 2631 of SEQ ID NO:63. Underlined is the partially characterized nucleotide (SEQ ID NO:35) sequence. Indicated are the stop and start codons (both in black shaded boxes) and of the primers (grey shaded boxes) used to amplify the coding region of CCP35 by PCR. SEQ ID NOs of the primers used can be found in Table III. Nucleotide sequence differences between SEQ ID NO:33 and SEQ ID NO:63 are depicted.
[0072] FIG. 42 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP35. The complete amino acid sequence corresponds to amino acids 1 to 749 of SEQ ID NO:129. Underlined is the predicted partial amino acid (SEQ ID NO:101) sequence.
[0073] FIG. 43 depicts the cDNA sequence of the Arabidopsis thaliana CCP36. The complete nucleotide sequence corresponds to nucleic acids 1 to 2743 of SEQ ID NO:64. Underlined is the partially characterized nucleotide (SEQ ID NO:36) sequence. Indicated are the stop and start codons (both in black shaded boxes). Nucleotide sequence differences between SEQ ID NO:36 and SEQ ID NO:64 are depicted.
[0074] FIG. 44 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP36. The complete amino acid sequence corresponds to amino acids 1 to 742 of SEQ ID NO:130. Underlined is the predicted partial amino acid (SEQ ID NO:102) sequence.
[0075] FIG. 45 depicts the cDNA sequence of the Arabidopsis thaliana CCP37. The complete nucleotide sequence corresponds to nucleic acids 1 to 2959 of SEQ ID NO:65. Underlined is the partially characterized nucleotide (SEQ ID NO:37) sequence. Indicated are the stop and start codons (both in black shaded boxes) and primers (grey shaded boxes) used to amplify the coding region of CCP45 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0076] FIG. 46 depicts the predicted amino acid sequence of the Arabidopsis thaliana CCP37. The complete amino acid sequence corresponds to amino acids 1 to 911 of SEQ ID NO:131. Underlined is the predicted partial amino acid (SEQ ID NO:103) sequence. Indicated in a black shaded box is a SAP-like domain.
[0077] FIG. 47 depicts the cDNA sequence and predicted amino acid sequence of the Arabidopsis thaliana CCP38. The complete nucleotide sequence (FIG. 47A) corresponds to nucleic acids 1 to 1295 of SEQ ID NO:66. The complete amino acid sequence (FIG. 47B) corresponds to amino acids 1 to 357 of SEQ ID NO:132. Underlined in FIG. 47A and FIG. 47B arc the partially characterized nucleotide (SEQ ID NO:38) and predicted partial amino acid (SEQ ID NO:104) sequence, respectively. Indicated in FIG. 47A are the stop and start codons (both in black shaded boxes) which are part of the primers (grey shaded boxes) used to amplify the coding region of CCP38 by PCR. SEQ ID NOs of the primers used can be found in Table III.
[0078] FIG. 48 depicts phosphorylation of the Arabidopsis thaliana CCP4 by CDKs. The protein CDC2bDN-IC26M (SEQ ID NO:70) contains a consensus CDK phosphorylation site (TPWK, residues 54-57 of SEQ ID NO:263). The corresponding gene (SEQ ID NO:4) was expressed in E. coli and the protein was purified from the crude extracts. The purified protein was subsequently shown to be phosphorylated by CDKs in an in vitro CDK phosphorylation assay. -: no IC26M added; +: IC26M added.
[0079] FIG. 49 schematically represents the domain organization of AtE2Fa and AtE2Fb. The DNA-binding domain (DB), the dimerization domain (DIM), the marked box (MB), and the Rb-binding domain (RB) are indicated by marked boxes, the N-terminal domains are indicated by open boxes. Numbering on the right refers to the amino acid sequence contained in the different AtE2F constructs, which were used in the in vitro binding assays.
[0080] FIG. 50 depicts AtDPa in vitro interactions with AtE2Fa and AtE2Fb. The c-myc-tagged AtDPa (c-myc-AtDPa) was in vitro translated and used as control. The lower migrating proteins observed in the case of c-myc-AtDPa are most probably due to initiation of translation at internal methionine codons (panel A, unnumbered left lane). The c-myc-AtDPa was in vitro co-translated with HA-AtE2Fb (panels A and B, lane 1), HA-AtE2Fa (panels B, lane 2), the C-terminal deleted form of HA-AtE2Fb (panels A and B, lane 3), HA-AtE2Fa 1-420 (panels A and B, lane 4) and the N-terminal truncated form of HA-AtE2Fa 162-485 (panels A and B, lane 5) as indicated. Numbers in the case of the mutant AtE2Fs refer to the amino acid sequence contained in these constructs (see FIG. 49). An aliquot of each sample was analyzed directly by SDS-PAGE and autoradiographed (panel A; total IVT, total in vitro translation). Another aliquot of the same samples was subjected to immunoprecipitation with anti-c-myc monoclonal antibodies (panel B), lanes are indicated by numbering. The position of c-myc-AtDPa proteins are marked by arrows in both panels. Molecular mass markers are indicated at the left.
[0081] FIG. 51 shows AtDPb in vitro interactions with AtE2Fa and AtE2Fb. The c-myc-tagged AtDPb (c-myc-AtDPb, panels A and B, lane 2) and the HA-tagged AtE2Fb (HA-AtE2Fb, panels A and B, lane 1) were in vitro translated and used as controls. The lower migrating proteins observed in the case of c-myc-AtDPb are most probably due to initiation of translation at internal methionine codons (panel A, lane 2). The c-myc-AtDPb was in vitro co-translated with HA-AtE2Fb (panels A and B, lane 3), HA-AtE2Fa (panels A and B lane 4), HA-AtE2Fa 1-420 (panels A and B, lane 5) and the N-terminal truncated form of HA-AtE2Fa 162-485 (panels A and B, lane 6) as indicated. Numbers in the case of the mutant AtE2Fs refer to the amino acid sequence contained in these constructs (see FIG. 49). An aliquot of each sample was analyzed directly by SDS-PAGE and autoradiographed (panel A; total IVT, total in vitro translation). Another aliquot of the same samples was subjected to immunoprecipitation with anti-c-myc monoclonal antibodies (panel B), lanes are indicated by numbering. The c-myc-AtDPb (panels A and B, lanes 2-6; indicated with `y`) co-migrated almost exactly with the mutant HA-AtE2Fa 1-420 (panels A and B, lane 5; indicated with `x`) and HA-AtE2Fa 162-485 (panels A and B, lane 6; indicated with `z`) in the gel system. These polypeptides as well as the position of c-myc-AtDPa and c-myc-AtDPb proteins are marked by arrows marked with `y`, `x` and `z`, respectively (cfr. supra). Molecular mass markers are indicated at the left.
[0082] FIG. 52 schematically represents AtDPa and mutants. The DNA-binding domain (DB) and the dimerization domain (DIM) are indicated by marked boxes, N- and C-terminal regions are indicated by open boxes. Numbering on the right side refers to the amino acid sequence contained in the different AtDP constructs, which were used in the in vitro binding assays.
[0083] FIG. 53 schematically represents AtDPb and mutants. The DNA-binding domain (DB) and the dimerization domain (DIM) are indicated by marked boxes, N- and C-terminal regions are indicated by open boxes. Numbering on the right side refers to the amino acid sequence contained in the different AtDP constructs, which were used in the in vitro binding assays.
[0084] FIG. 54 shows the mapping of regions in AtDPa required for in vitro binding to AtE2Fb. HA-AtE2Fb was co-translated with series of c-myc-AtDPa mutants. An aliquot of each sample was analyzed directly by SDS-PAGE and autoradiographed (panel A). Another aliquot of the same samples was subjected to immunoprecipitation with anti-HA (panel B) or anti-c-myc (panel C) monoclonal antibodies. The c-myc-AtDPa mutants are marked by dots. Positions of the HA-AtE2Fb proteins are indicated by arrows. Molecular mass markers are indicated at the left.
[0085] FIG. 55 shows the mapping of regions in AtDPb required for in vitro binding to AtE2Fb. HA-AtE2Fb was co-translated with series c-myc-AtDPb mutants. An aliquot of each sample was analyzed directly by SDS-PAGE and autoradiographed (panel A). Another aliquot of the same samples was subjected to immunoprecipitation with anti-HA (panel B) or anti-c-myc (panel C) monoclonal antibodies. The c-myc-AtDPb mutants are marked by dots. Positions of the HA-AtE2Fb proteins are indicated by arrows. Molecular mass markers are indicated at the left.
[0086] FIG. 56 shows the mapping of regions in AtDPb required for in vitro binding to AtE2Fb. HA-AtE2Fb was co-translated with c-myc-AtDPb 182-263. Because of the small size of this protein, it was hardly detectable when it was directly analyzed by SDS-PAGE (data not shown). An aliquot of this sample was subjected to immunoprecipitation with anti-c-myc monoclonal antibodies. The c-myc-AtDP mutant is marked by dots. Position of the HA-AtE2Fb protein is indicated by an arrow. Molecular mass markers are indicated at the left.
[0087] FIG. 57 shows organ- and cell cycle-specific expression of AtE2Fa and AtDPa. Tissue-specific expression of AtDPa and AtE2Fa genes. cDNA prepared from the indicated tissues was subjected to semi-quantitative RT-PCR analysis. The Arath; CDKB1; 1 gene was used as a marker for highly proliferating tissues. The actin 2 gene (ACT2) was used as loading control.
[0088] FIG. 58 shows organ- and cell cycle-specific expression of AtE2Fa and AtDPa. Co-regulated cell cycle phase-dependent transcription of AtE2Fa and AtDPa. The cDNA was prepared from partially synchronized Arabidopsis cells harvested at the indicated time point after removal of the cell cycle blocker was subjected to semi-quantitative RT-PCR analysis. Histone H4 and Arath;CDKB1;1 were used as markers for S and G2/M phase, respectively, and ROCS and Arath;CDKA;1 as loading controls.
[0089] FIG. 59 is a photographic representation of Northern blotting analysis of DPa expression in independent Arabidopsis thaliana DPa overexpressing lines (lines 16-27 as indicated) and one untransformed control line (indicated by C).
[0090] FIG. 60 describes the molecules defined in SEQ ID NOs:199-204 and 240-290.
DETAILED DESCRIPTION OF THE INVENTION
[0091] The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as "cell cycle proteins" or "CCP" nucleic acid and polypeptide molecules. The CCP molecules of the present invention were identified based on their ability, as determined using yeast two-hybrid assays (described in detail in Example 1), to interact with proteins involved in the cell cycle, such as plant cyclin dependent kinases (e.g., a dominant negative form of CDC2b, CDC2bAt.N161), cyclin dependent kinase subunits referred herein as "CKS" (such as CKS1At), cyclin dependent kinase inhibitors referred to herein as "CKI" (such as CKI4), PHO80-like proteins referred to herein as "PLP", E2F, and different domains of kinesin-like proteins referred to herein as "KLPNT.
[0092] Because of their ability to interact with (e.g., bind to) the cyclin dependent kinases, the CCP molecules of the present invention may modulate, e.g., upregulate or downregulate, the activity of plant CDKs, such as CDC2a or CDC2b; CKSs, CKIs, PLPs and KLPNTs. Furthermore, because of their ability to interact with (e.g., bind to) the aforementioned proteins which are proteins involved in cell cycle regulation, the CCP molecules of the present invention may also play a role in or function in cell cycle regulation, e.g., plant or animal cell cycle regulation.
[0093] As used herein, the term "cell cycle protein" includes a polypeptide which is involved in controlling or regulating the cell cycle, or part thereof, in a cell, tissue, organ or whole organism. Cell cycle proteins may also be capable of binding to, regulating, or being regulated by cyclin dependent kinases, such as plant cyclin dependent kinases, e.g., CDC2a or CDC2b, or their subunits. The term cell cycle protein also includes peptides, polypeptides, fragments, variant, homologs, alleles or precursors (e.g., pre-proteins or pro-proteins) thereof.
[0094] As used herein, the term "cell cycle" includes the cyclic biochemical and structural events associated with growth, division and proliferation of cells, and in particular with the regulation of the replication of DNA and mitosis. The cell cycle is divided into periods called: G0, Gap1 (G1), DNA synthesis (S), Gap2 (G2), and mitosis (M). Normally these four phases occur sequentially, however, the cell cycle also includes modified cycles wherein one or more phases are absent resulting in modified cell cycle such as endomitosis, acytokinesis, polyploidy, polyteny, and endoreduplication.
[0095] As used herein, the term "plant" includes reference to whole plants, plant organ (e.g., leaves, stems, roots), plant tissue, seeds, and plant cells and progeny thereof. Plant cell, as used herein includes, without limitation, seeds, e.g., seed suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable of transformation techniques, including both monocotyledonous and dicotyledonous plants. Particularly preferred plants are Arabidopsis thaliana, rice, wheat, maize, tomato, alfalfa, oilseed rape, soybean, cotton, sunflower or canola. The term plant also includes monocotyledonous (monocot) plants and dicotyledonous (dicot) plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Buteafrondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incarnata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis untbellata, Rhopalostylis sapida, Rhus natatlensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, amongst others, or the seeds of any plant specifically named above or a tissue, cell or organ culture of any of the above species.
[0096] The cell cycle proteins of the present invention are involved in cell cycle regulation which is largely, but not completely, similar in plants and animals Accordingly, the nucleic acid molecules and polypeptide of the invention, or derivatives thereof, may be used to modulate the cell cycle in a plant or an animal such as by modulating the activity or level or expression of CCP, altering the rate of the cell cycle or phases of the cell cycle, and entry into and out of the various cell cycle phases. In plants, the molecules of the present invention may be used in agriculture to, for example, improve the growth characteristics of plant such as growth rate or size of specific tissues or organs, the architecture or morphology of the plant, increase crop yield, improve tolerance to environmental stress conditions (such as drought, salt, temperature, or nutrient deprivation), improve tolerance to plant pathogens that abuse the cell cycle or as targets to facilitate the identification of inhibitors or activators of CCPs that may be useful as phytopharmaceuticals such as herbicides or plant growth regulators.
[0097] As used herein, the term "cell cycle associated disorders" includes a disorder, disease or condition which is caused or characterized by a misregulation (e.g., downregulation or upregulation), abuse, arrest, or modification of the cell cycle. In plants cell cycle associated disorders include endomitosis, acytokinesis, polyploidy, polyteny, and endoreduplication which may be caused by external factors such as pathogens (nematodes, viruses, fungi, or insects), chemicals, environmental stress (e.g., drought, temperature, nutrients, or UV) resulting in for instance neoplastic tissue (e.g., galls, root knots) or inhibition of cell division/proliferation (e.g., stunted growth). Cell cycle associated disorders in animals include proliferative disorders or differentiative disorders, such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma.
[0098] The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as CCP protein and nucleic acid molecules, which comprise a family of molecules having certain conserved structural and functional features. The term "family" when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of plant, e.g. Arabidopsis, origin, as well as other, distinct proteins of plant, e.g., Arabidopsis, origin or alternatively, can contain homologues of other plants, e.g., rice, or of non-plant origin. Members of a family may also have common functional characteristics.
[0099] In one embodiment of the invention, a CCP protein of the present invention is identified based on the presence of at least one or more of the following domains:
A. Cyclin Destruction Box
[0100] As used herein, the term "Cyclin destruction box" includes a domain of 9-10 amino acid residues in length which typically contains the following consensus pattern:
TABLE-US-00001 (SEQ ID NO: 267) R-X2-L-X2-[I/V]-X1-2-N,
wherein X can be any amino acid, Xn is a stretch of n Xs, Xn-m is a stretch of n to m Xs, and wherein [I/V] means that an Ile or Val residue can occur at that position. SEQ ID NO:267 depicts the minimal consensus sequence of the cyclin destruction box and underlies the ubiquitin-mediated proteolytic destruction of the cyclins bearing this motif (Yarnano et al. (1998), EMBO J. 17: 5670-5678; Renaudin et al. (1998) in Plant Cell Division (Francis, Dudits and Inze, eds.), Portland Press Research Monograph, Portland Press Ltd. London (1998), pp 67-98).
B. Cyclin Box Motif 1
[0101] As used herein, the term "Cyclin box motif 1" includes a domain of 8 amino acid residues in length and which typically contains the following consensus pattern:
TABLE-US-00002 (SEQ ID NO: 268) MRXIL[I/V]DW,
wherein X can be any amino acid and wherein [I/V] means that an Ile or Val residue can occur at that position. This motif forms part of the helix H1 of the first cyclin fold and is the best conserved motif in the cyclinA/B family (Renaudin et al. (1998) in Plant Cell Division (Francis, Dudits and Inze, eds.), Portland Press Research Monograph, Portland Press Ltd. London (1998), pp 67-98).
C. Cyclin Box Motif 2
[0102] As used herein, the term "Cyclin box motif 2" includes a domain of 8 amino acid residues in length and which typically contains the following consensus pattern:
TABLE-US-00003 (SEQ ID NO: 269) KYEE-X3-P,
wherein X can be any amino acid and wherein Xn is a stretch of n Xs. This motif forms part of the helix H3 of the first cyclin fold wherein the 2 acidic residues are part of the CDK binding site (Renaudin et al. (1998) in Plant Cell Division (Francis, Dudits and Inze, eds.), Portland Press Research Monograph, Portland Press Ltd. London (1998), pp 67-98).
D. CDC2 Motifs
[0103] As used herein, the term "CDC2 motifs" includes domains of about 9-12 amino acid residues in length and which typically contain one of the following consensus patterns:
TABLE-US-00004 (SEQ ID NO: 270) GXG-X2-GXVY (SEQ ID NO: 271) HRDXK-X2-NXL (SEQ ID NO: 272) D-X1-2-[W/Y]SXG-X4-E
wherein X can be any amino acid, Xn is a stretch of n Xs, Xn-m is a stretch of n to m Xs, and wherein [W/Y] means that an Trp or Tyr residue can occur at that position.
E. CDK Phosphorylation Site
[0104] As used herein the term "CDK phosphorylation site" includes a domain of about 5-7 amino acids in length and which contains one or more of the following consensus
TABLE-US-00005 (SEQ ID NO: 273) TPX1-2[R/K] (SEQ ID NO: 274) SPX[R/K] (SEQ ID NO: 275) SPX(Hu) (SEQ ID NO: 276) SP(Hu)X
with Hu being a hydrophobic uncharged amino acid (M, I, L, V) and X any amino acid. The foregoing are typically found in cyclin-dependent kinase substrates such as histone kinase, transcription factors such as E2F or transcription regulators like Rb. CDK phosphorylation sites are described in, for example, Tamrakar et al. 2000, Frontiers Biosci 5, d121-137.
[0105] CCP proteins of the present invention comprising a CDK phosphorylation site can be mutated in said CDK phosphorylation site such that said CCP proteins are no longer able to be phosphorylated on the CDK phosphorylation site. Mutations of a CDK phosphorylation site include all mutations of the ser or thr residue in any of SEQ ID NOs:273-276 into a non-phosphorylatable amino acid residue, e.g., an ala or glu residue. Mutation of one or more CDK phosphorylation site(s) in a CCP protein of the invention is expected to modulate modifications of the CCP protein by CDKs and, thus, to modulate the biological or biochemical function of the CCP protein.
F. E Nuclear Localisation Signal (NLS)
[0106] As used herein the term "nuclear localization signal" or "NLS" includes a domain conferring to a protein comprising the NLS domain the ability to be imported into the nucleus and to, for example, accumulate within the nucleus. NLS domains include one or more of the following concensus patterns:
TABLE-US-00006 (SEQ ID NO: 277) PKKKRKV (SEQ ID NO: 278) KRX10KKKK (SEQ ID NO: 279) KRPRP (SEQ ID NO: 280) PAAKRVKLD
[0107] NLS domains have been found in the SV40 T antigen, in nucleoplasmin (bipartite NLS), in a Adeno EIA, and in c-Myc. NLS domains are described in, for example, Laskey et al. (1998) Biochem. Soc. Trans. 26, 561-567.
G. Cy-Like Boxes
[0108] As used herein, the term "Cy-like box" includes a domain of 3-6 amino acid residues in length with has the consensus motif R-X-X-F (SEQ ID NO:281) with X being any amino acid and one of two Xs preferably being a hydrophobic residue.
H. Rb Binding Domain
[0109] As used herein, the term "Rb binding domain" includes a domain which when present in a protein confers to the protein the ability to bind the Rb protein. Rb binding domains include one or more of the following concensus patterns:
TABLE-US-00007 (SEQ ID NO: 282) LXCXE (SEQ ID NO: 283) LXSXE (SEQ ID NO: 284) DYX7EX3DLFD (SEQ ID NO: 285) DYX6DX4DMWE
Rb binding domains have been found in D-cyclins, in protein phosphatase 1, in human E2F-1, and in plant E2F. Rb binding domains are described in, for example, Rubin et al. (1998) Frontiers Eiosci 3, d1209-1219; Phelps et al. (1992) J. Virol. 66, 2418-2427, and Cress et al. (1993) Mol. Cell. Biol. 13, 6314-6325.
I. DEF Domain
[0110] As used herein the term "DEF domain" includes a protein domain which is required for the formation of heterodimers between DP proteins and E2F proteins. DEF domains comprise the following concensus pattern:
TABLE-US-00008 (SEQ ID NO: 286) [D/N/-][Q/E]KNIR[R/G]RV[Y/D]DALNV[L/F]MA[M/I/L/-] [N/D][V/I]I[S/A][K/R][D/E]KKEI[K/Q/R/-]W[R/K/I]GLP
J. DNA Binding Domain
[0111] As used herein the term "DNA binding domain" includes a domain which is involved in the binding of DP proteins and/or DP-E2F heterodimers to DNA. DNA binding domains include the following concensus pattern:
TABLE-US-00009 (SEQ ID NO: 287) [G/N][K/R]GLR[H/Q]FS[M/V][K/M][I/V]X.sub.(0-17)C[E/Q]K[V/L][Q/E/-][S/-]XK[- G/K]- [R/I/-]TT[S/-]Y[N/K]EVADE[L/I][V/I][A/S][E/D]F
DNA binding domains are described in, for example, Hao et al. (1995) J. Cell Sci. 108, 2945-2954; Bandara et al. (1993) EMBO J. 12, 4317-4324; and Girling et al. (1994) Mol. Biol. Cell 5, 1081-4092.
K. DCB1 Domain:
[0112] As used herein the term "DCB1 domain" includes a protein domain which is conserved among DP proteins and has the following consensus patterns:
TABLE-US-00010 (SEQ ID NO: 288) [R/S][I/V]X[Q/K]KX3[L/S]XE (SEQ ID NO: 289) [R/S][I/V]X[Q/K]KX3[L/S]XE[L/M]X2-3[Q/H]X4-5NL[V/I/M][Q/E]R- N
DCB1 domains are described in, for example, Hao et al. (1995) J. Cell Sci. 108, 2945-2954; Bandara et al. (1993) EMBO J. 12, 4317-4324; and Girling et al. (1994) Mol. Biol. Cell 5, 1081-1092.
L. DCB2 Domain:
[0113] As used herein the term "DCB2 domain" includes a protein domain which is conserved among DP proteins and has the following consensus patern:
TABLE-US-00011 (SEQ ID NO: 290) [L/I]PFI[L/I][V/L]XTX3-4[T/V]VX12-14FX3-4F[E/S][Hu]HDDX.sub- .2[V/I]L[R/K]XM
DCB2 domains are described in, for example, Hao et al. (1995) J. Cell Sci. 108, 2945-2954; Bandara et al. (1993) EMBO J. 12, 4317-4324; and Girling et al. (1994) Mol. Biol. Cell 5, 1081-1092.
M. SAP Domain:
[0114] As used herein the term SAP motif includes a protein domain of about 35 amino acid residues which is found in a variety of nuclear proteins involved in transcription, DNA repair, DNA processing or apoptotic chromatin degradation. It was named after SAF-A/B, Acinus and PIAS, three proteins known to contain it. The SAP motif reveals a bipartite distribution of strongly conserved hydrophobic, polar and bulky amino acids separated by a region that contains a glycine. The SAP domain has been proposed to be a DNA-binding motif (Aravind and Koonin (2000) Trends Biochem. Sci. 25:112-114).
[0115] Isolated CCP proteins of the present invention have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:67-132, 205, 211, 215-216, or 220-227 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:1-66 or 228-239. As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.
[0116] As used interchangeably herein, an "CCP activity", "biological activity of CCP" or "functional activity of CCP", refers to an activity exerted by a CCP protein, polypeptide or nucleic acid molecule on a CCP responsive cell or tissue, or on a CCP protein substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a CCP activity is a direct activity, such as an association with a CCP-target molecule. As used herein, a "target molecule" or "binding partner" is a molecule with which a CCP protein binds or interacts in nature, such that CCP-mediated function is achieved. A CCP target molecule can be a non-CCP molecule or a CCP protein or polypeptide of the present invention, e.g., a plant cyclin dependent kinase, such as CDC2b. In an exemplary embodiment, a CCP target molecule is a CCP ligand. Alternatively, a CCP activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the CCP protein with a CCP ligand. The biological activities of CCP are described herein. For example, the CCP proteins of the present invention can have one or more of the following activities: (1) they may interact with a non-CCP protein molecule, e.g., a CCP ligand; (2) they may modulate a CCP-dependent signal transduction pathway; (3) they may modulate the activity of a plant cyclin dependent kinase, such as CDC2a, CDC2b, or CDC2c, and (4) they may modulate the cell cycle.
[0117] Accordingly, another embodiment of the invention features isolated CCP proteins and polypeptides having a CCP activity. Preferred proteins are CCP proteins having at least one or more of the following domains: a "cyclin destruction box", a "cyclin box motif 1", a "cyclin box motif 2", a "CDC2 motif", a "CDK phosphorylation site", a "nuclear localization signal", a "Cy-like box", an "Rb binding domain", a "DEF domain", a "DNA binding domain", a "DCB1 domain", a "DCB2 domain" and/or a SAP domain, and, preferably, a CCP activity.
[0118] Additional preferred proteins have at least one or more of the following domains: a "cyclin destruction box", a "cyclin box motif 1", a "cyclin box motif 2", a "CDC2 motif", a "CDK phosphorylation site", a "nuclear localization signal", a "Cy-like box", an "Rb binding domain", a "DEF domain", a "DNA binding domain", a "DCB1 domain", a "DCB2 domain" and/or a SAP domain and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1-66 or 228-239.
[0119] The sequences of the present invention are summarized below, in Table I.
TABLE-US-00012 TABLE I SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: CCP Clone Homolog/ partial full-length partial full-length Molecule Name Bait function motif DNA DNA Protein Protein CCP1 CDC2bD CDC2bAt. Novel CYCB2; 3 cyclin box 1 39 67 105 N-IC19 N161 motifs 1 and 2; cyclin destruction box CCP2 CDC2bD CDC2bAt. ARR2 2 40 68 106 N-IC20 N161 CCP3 CDC2bD CDC2bAt. novel A-type cyclin box 3 41 69 107 N-IC21 N161 cyclin motifs 1 and 2; cyclin destruction box CCP4 CDC2bD CDC2bAt. CDK 4 4 70 70 N-IC26M N161 phosphorylation site CCP5 CDC2bD CDC2bAt. Arath cyclin box 5 5 71 71 N-IC39 N161 CYCB2; 1 motifs 1 and 2; cyclin destruction box CCP6 CDC2bD CDC2bAt. 6 42 72 108 N-IC57 N161 CCP7 CDC2bD CDC2bAt. AJH2-COP9 7 43 73 109 N-IC62 N161 CCP8 E2F3ca55 E2F3 N- 8 43 74 109 terminal CCP9 CDC2bD CDC2bAt. Arath cyclin box 9 9 75 75 N-IC9 N161 CYCA2; 2 motifs 1 and 2; cyclin destruction box CCP10 CKSBC001 CKS1At 10 10 76 76 CCP11 CKSBC011 CKS1At gibberellin- 11 44 77 110 regulated protein GASA1 precursor CCP12 CKSBC9 CKS1At 12 45 78 111 8-7 (Cterm) CCP13 CKSBC9 CKS1At 13 45 79 111 8-7 (Nterm) CCP14 CKSBC1 CKS1At 14 46 80 112 03-19 (Cterm) CCP15 CKSBC1 CKS1At PSTTLRE-type CDC2 15 47 81 113 99-20 CDK motifs CCP16 E2F5BB E2F5 DPa DNA-binding 16 48 82 114 C1 dimerisation domain; DEF domain domain; DCB1 and DCB2 domain CCP17 FL67BC4- CKI4 17 17 83 83 2 CCP18 FL67BC12- CKI4 RNA 18 49 84 115 17 polymerase B transcription factor 3 CCP19 JUT1 PLP1 19 19 85 85 CCP20 JUT2 PLP1 20 50 86 116 CCP21 JUT3 PLP1 21 50 87 116 CCP22 JUT6 PLP1 Submergence 22 51 88 117 induced protein2 or Oryza sativa CCP23 kbp1 KLPNT1 HSF1 23 52 89 118 36-508aa (motor domain) KLPNT2 (TH65) 73-186 aa (neck domain) CCP24 kbp3 KLPNT1 24 53 90 119 (427- 867aa) stalk domain CCP25 kbp6 KLPNT2 25 54 91 120 (TH65) 73-186 aa neck domain CCP26 kbp9 KLPNT2 AtKLPNT1 26 55 92 121 (TH65) 73-186 aa neck domain CCP27 kbp11 KLPNT2 27 56 93 122 (TH65) 73-186 aa neck domain CCP28 kbp12 KLPNT2 28 57 94 123 (TH65) 73-186 aa neck domain CCP29 kbp13 KLPNT2 29 29 95 95 (TH65) 73-186 aa neck domain CCP30 kbp15 KLPNT2 Centromere/ 30 58 96 124 (TH65) microtubule 73-186 aa binding neck protein CBF5 domain from yeast CCP31 kbp20 KLPNT2 VU91C 31 59 97 125 (TH65) calmodulin 73-608 aa from yeast stalk domain CCP32 E2F5BB E2F5 32 60 98 126 C16 dimerization CCP33 DPb / DNA-binding 33 61 99 127 domain; DEF domain; DCB1 and DCB2 domain CCP34 E2F3ca1 E2F3 N- 34 62 100 128 terminal CCP35 E2F3ca2 E2F3 N- 35 63 101 129 terminal CCP36 E2F3ca9 E2F3 N- 36 64 102 130 terminal CCP37 E2F3ca12 E2F3 N- SAP 37 65 103 131 terminal domain CCP38 E2F3ca50 E2F3 N- 38 66 104 132 terminal
[0120] Detailed studies of interactions between AtDPs (a and b forms, SEQ ID NO:114 and SEQ ID NO:127, respectively) and AtE2Fs (a and b forms; GenBank accession numbers AJ294534 and AJ294533, respectively) revealed that the regions of AtDPa and AtDPb involved in the binding of AtE2Fb are different.
[0121] Binding of AtDPa to AtE2Fb requires at least the AtDPa dimerization domain and the whole (or possibly part of) the C-terminal domain of AtDPa. The N-terminal domain and the DNA-binding domain of AtDPa do not seem to contribute to the interaction of AtDPa with AtE2Fb (Examples 11, 12, Table 5, FIG. 54).
[0122] Binding of AtDPb to AtE2Fb, however, only requires an intact AtDPb dimerization domain. Neither the region including the N-terminal and DNA-binding domains of AtDPb, nor the C-terminal region of AtDPb seem to contribute to the interaction of AtDPb with AtE2Fb (Examples 11, 12, Table 5, FIG. 55). These observations indicate that modulating the formation of specific E2F/DP-complexes may be useful in modulating cell cycle traversal and the regulation thereof.
[0123] AtDPa and AtDPb, respectively, do not form homodimers but both interact with either AtE2Fa or AtE2Fb (Example 12, Table 5). In reciprocal experiments it was shown that the N-terminal domain of AtE2Fa is not required for binding AtDPa or AtDPb. Likewise, the Rb-binding domains of AtE2Fa and AtE2Fb, respectively, do not seem to contribute to the binding to either AtDPa or AtDPb. The region of AtE2Fa encompassing the dimerization domain and the marked box is sufficient for binding to AtDPa and AtDPb (Examples 11, 12, FIG. 50, FIG. 51, Table 5). The dimerization domain of AtE2Fs appears to be sufficient for binding to AtDPs.
[0124] Accordingly, it is shown herein for the first time (for plant DPs and plant E2Fs) that the minimal DP and E2F proteins or corresponding coding DNA Sequences that can be used in modifying E2F/DP-related processes, e.g., regulation of gene expression by E2F/DP, include:
[0125] (A) Plant DP dimerization domain with or without (part of) the C-terminal DP domain. These domains include the proteins AtDPa143-292 and AtDPa143-213 (numbering indicates the amino acids included in said fragment relative to the full-length AtDPa protein) set forth in SEQ ID NO:221 and SEQ ID NO:222, respectively. The coding sequences corresponding to the foregoing amino acid sequences are set forth in SEQ ID NO:232 and SEQ ID NO:233, respectively. Also included are the corresponding regions of the AtDPb protein characterized by AtDPb182-385 and AtDPb 182-263 (parts of the full-length AtDPb protein). The foregoing regions of AtDPb are set forth in SEQ ID NO:216 and SEQ ID NO:215, respectively, and the coding sequences corresponding thereto are set forth in SEQ ID NO:231 and SEQ ID NO:230, respectively. The AtDPb1-263 domain (SEQ ID NO:223) and the corresponding AtDPa1-214 domain (SEQ ID NO:220) encoded by the nucleic acid sequences SEQ ID NO:234 and SEQ ID NO:239, respectively, can also be used. Further included are nucleic acid sequences hybridizing to SEQ ID NOs:229-234 or SEQ ID NO:239 or encoding a protein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to SEQ ID NOs:211, 215-216 and 220-223.
[0126] (B) Plant E2F dimerization domain with or without (part of) the marked box. These domains include the proteins AtE2Fa232-282, AtE2Fa232-352 and AtE2Fa226-356 set forth in SEQ ID NO:224, SEQ ID NO:225 and SEQ ID NO:205, respectively. The corresponding coding DNA sequences are set forth in SEQ ID NO:235, SEQ ID NO:236 and SEQ ID NO:228, respectively. Also included are the corresponding regions of the AtE2Fb protein characterized by AtE2Fb194-243 and AtE2Fb194-311 set forth in SEQ ID NO:226 and SEQ ID NO:227, respectively. The corresponding coding DNA sequences are set forth in SEQ ID NO:237 and SEQ ID NO:238, respectively. Further included are nucleic acid sequences hybridizing to SEQ ID NO:228 or SEQ ID NOs:235-238 or encoding a' protein at least 70%, 75%, 80%, 85%, 90%, 95%, 98% identical to SEQ ID NO:205 or SEQ ID NOs:224-227.
[0127] (C) Full-length plant DP and plant E2F proteins or corresponding DNA sequences may also be used to modify said E2F/DP-related processes. Furthermore, plant DP and plant E2F proteins or corresponding DNA sequences, or parts thereof, can be used either separately or in combination to modify said E2F/DP-related processes. This is underscored by the demonstration that AtDPs and AtE2Fs are co-expressed in actively dividing cells and in at least some plant tissues (Example 13 and FIGS. 57 and 58).
[0128] Various aspects of the invention are described in further detail in the following subsections:
I. Isolated Nucleic Acid Molecules
[0129] One aspect of the invention pertains to isolated nucleic acid molecules that encode CCP proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify CCP-encoding nucleic acids (e.g., CCP mRNA) and fragments for use as PCR primers for the amplification or mutation of CCP nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
[0130] An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated CCP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
[0131] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1-66 or 228-239, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of the nucleic acid sequence of SEQ ID NO:1-66 or 228-239, as a hybridization probe, CCP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0132] Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:1-66 or 228-239 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:1-66 or 228-239, respectively.
[0133] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
[0134] Furthermore, oligonucleotides corresponding to CCP nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
[0135] In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239.
[0136] In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239, respectively, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239, respectively, thereby forming a stable duplex.
[0137] In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1-66 or 228-239, or a portion of any of these nucleotide sequences.
[0138] Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:1-66 or 228-239, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a CCP protein. The nucleotide sequence determined from the cloning of the CCP gene allows for the generation of probes and primers designed for use in identifying and/or cloning other CCP family members, as well as CCP homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:1-66 or 228-239, or of a naturally occurring allelic variant or mutant of SEQ ID NO:1-66 or 228-239. In an exemplary embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:1-66 or 228-239.
[0139] Probes based on the CCP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which misexpress a CCP protein, such as by measuring a level of a CCP-encoding nucleic acid in a sample of cells from a subject e.g., detecting CCP mRNA levels or determining whether a genomic CCP gene has been mutated or deleted.
[0140] A nucleic acid fragment encoding a "biologically active portion of a CCP protein" can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:1-66 or 228-239, which encodes a polypeptide having a CCP biological activity (the biological activities of the CCP proteins are described herein), expressing the encoded portion of the CCP protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the CCP protein.
[0141] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239, due to the degeneracy of the genetic code and, thus, encode the same CCP proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:1-66 or 228-239. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a CCP protein.
[0142] In addition to the CCP nucleotide sequences shown in SEQ ID NO:1-66 or 228-239, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the CCP proteins may exist within a population (e.g., an Arabidopsis or rice plant population). Such genetic polymorphism in the CCP genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which include an open reading frame encoding an CCP protein, preferably a plant CCP protein, and can further include non-coding regulatory sequences, and introns. Such natural allelic variations include both functional and non-functional CCP proteins and can typically result in 1-5% variance in the nucleotide sequence of a CCP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in CCP genes that are the result of natural allelic variation and that do not alter the functional activity of a CCP protein are intended to be within the scope of the invention. Differences in preferred codon usage are illustrated below for Agrobacterium tumefaciens (a bacterium), Arabidopsis thaliana, Medicago sativa (two dicotyledonous plants) and Oryza sativa (a monocotyledonous plant). These examples were extracted from http://www.kazusa.or.jp/codon. For example, the codon GGC (for glycine) is the most frequently used codon in A. tumefaciens (36.2%), is the second most frequently used codon in O. sativa but is used at much lower frequencies in A. thaliana and M. sativa (9% o and 8.4%, respectively). Of the four possible codons encoding glycine the GGC codon is most preferably used in A. tumefaciens and O. sativa. However, in A. thaliana the GGA (and GGU) codon is most preferably used, whereas in M. sativa the GGU (and GGA) codon is most preferably used.
[0143] Moreover, nucleic acid molecules encoding other CCP family members and, thus, which have a nucleotide sequence which differs from the CCP sequences of SEQ ID NO:1-66 or 228-239 are intended to be within the scope of the invention. For example, another CCP cDNA can be identified based on the nucleotide sequence of the plant CCP molecules described herein. Moreover, nucleic acid molecules encoding CCP proteins from different species, and thus which have a nucleotide sequence which differs from the CCP sequences of SEQ ID NO:1-66 or 228-239 are intended to be within the scope of the invention. For example, a human CCP cDNA can be identified based on the nucleotide sequence of a plant CCP.
[0144] Nucleic acid molecules corresponding to natural allelic variants and homologues of the CCP cDNAs of the invention can be isolated based on their homology to the CCP nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof; as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
[0145] Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1-66 or 228-239. In other embodiment, the nucleic acid is at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 30%, 40%, 50%, or 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., more preferably at 60° C., and even more preferably at 65° C. Ranges intermediate to the above-recited values, e.g., at 60-65° C. or at 55-60° C. are also intended to be encompassed by the present invention. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1-66 or 228-239 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
[0146] In addition to naturally-occurring allelic variants of the CCP sequences that may exist in nature, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:1-66 or 228-239, thereby leading to changes in the amino acid sequence of the encoded CCP proteins, without altering the functional ability of the CCP proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of a CCP protein. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of CCP without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the CCP proteins of the present invention, are predicted to be particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the CCP proteins of the present invention and other CCP family members are not likely to be amenable to alteration.
[0147] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding CCP proteins that contain changes in amino acid residues that are not essential for activity.
[0148] An isolated nucleic acid molecule encoding a CCP protein homologous to the CCP proteins of the present invention can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:1-66 or 228-239, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:1-66 or 228-239 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a CCP protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a CCP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CCP biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1-66 or 228-239, the encoded protein can be expressed recombinantly and the activity of the protein can be determined. Another alternative embodiment comprises targeted in vivo gene correction or modification which can be achieved by chimeric RNA/DNA oligonucleotides (e.g., Yoon et al. (1996), Proc. Natl. Acad. Sci. USA 93, 2071-2076; Arntzen et al. (1999) WO99/07865).
[0149] In a preferred embodiment, a mutant CCP protein can be assayed for the ability to: (1) regulate transmission of signals from cellular receptors, e.g. hormone receptors; (2) control cell cycle checkpoints, e.g. entry of cells into mitosis; (3) modulate the cell cycle; (4) modulate cell death, e.g., apoptosis; (5) modulate cytoskeleton function, e.g. actin bundling; (6) phosphorylate a substrate; (7) create dominant negative or dominant positive effects in transgenic plants; (8) interact with other cell cycle control proteins in, e.g. a yeast two hybrid assay; (9) modulate CDK activity (e.g., cyclin-CDK activity); (10) regulate cyclin-CDK complex assembly; (11) regulate the commitment of cells to divide, e.g., by integrating mitogenic and antimitogenic signals; (12) regulate cell cycle progression; (13) regulate DNA replication and/or DNA repair; (14) modulate gene transcription, e.g., regulate E2F/DP-dependent transcription of genes; (15) regulate cyclin degradation; (16) modulate cell cycle withdrawal and/or cell differentiation; (17) control organ (e.g., plant organ) and/or organism (e.g., plant organism) size; (18) control organ (e.g., plant organ) and/or organism (e.g., plant organism) growth or growth rate; and (19) regulate endoreduplication.
[0150] In addition to the nucleic acid molecules encoding CCP proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire CCP coding strand, or only to a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding CCP. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding CCP. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
[0151] Given the coding strand sequences encoding CCP disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of CCP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of CCP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CCP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). Preferably, production of antisense nucleic acids in plants occurs by means of a stably integrated transgene comprising a promoter operative in plants, an antisense oligonucleotide, and a terminator.
[0152] Other known nucleotide modifications include methylation, cyclization and `caps` and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine. Modifications of nucleotides include modifications generated by the addition to nucleotides of acridine, amine, biotin, cascade blue, cholesterol, Cy3®, Cy5®, Cy5.5® Dabcyl, digoxigenin, dinitrophenyl, Edam, 6-FAM, fluorescein, 3'-glyceryl, HEX, IRD-700, IRD-800, JOE, phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA-S®, SE, BODIPY®, Marina Blue®, Pacific Blue®, Oregon Green®, Rhodamine Green®, Rhodamine Red®, Rhodol Green® and Texas Red®. Polynucleotide backbone modifications include methylphosphonate, 2'-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2'-OMeRNA. Base modifications include 2-amino-dA, 2-aminopurine, 3'-(ddA), 3' dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N6-Me-dA, abasic site (dSpacer), biotin dT, T-OMe-5Me-C, 2'-OMe-propynyl-C, 3'-(5-Me-dC), 3'-(ddC), 5-Br-dC, 5-I -dC, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA, convertible dC, convertible dG, convertible dT, convertible dU, 7-deaza-dG, 8-Br-dG, 8-oxo-dG, O6-Me-dG, S6-DNP-dG, 4-methyl-indole, 5-nitroindole, 2'-OMe-inosine, 2'-dI, O6-phenyl-dI, 4-methyl-indole, 2'-deoxynebularine, 5-nitroindole, 2-aminopurine, dP(purine analogue), dK(pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT, biotin-dT, carboxy-dT, O4-Me-dT, O4-triazol dT, 2'-OMe-propynyl-U, 5-Br-dU, 2'-dU, 5-F-dU, 5-I-dU, O4-triazol dU.
[0153] The antisense nucleic acid molecules of the invention are typically introduced into a plant or administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CCP protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of introduction or administration of antisense nucleic acid molecules of the invention include transformation in a plant or direct injection at a tissue site in a subject. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a constitutive promoter or a strong pol II or pol III promoter are preferred.
[0154] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
[0155] In another embodiment, the antisense nucleic acid molecule further comprises a sense nucleic acid molecule complementary to the antisense nucleic acid molecule. Gene silencing methods based on such nucleic acid molecules are well known to the skilled artisan (e.g., Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).
[0156] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CCP mRNA transcripts to thereby inhibit translation of CCP mRNA. A ribozyme having specificity for a CCP-encoding nucleic acid can be designed based upon the nucleotide sequence of a CCP cDNA disclosed herein (i.e., SEQ ID NO:1-66 or 228-239). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CCP-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, CCP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.
[0157] The use of ribozymes for gene silencing in plants is known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al. (1997) WO 97/13865 and Scott et al. (1997) WO/97/38116).
[0158] Alternatively, CCP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the CCP (e.g., the CCP promoter and/or enhancers) to form triple helical structures that prevent transcription of the CCP gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.
[0159] In yet another embodiment, the CCP nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
[0160] PNAs of CCP nucleic acid molecules can be used for increasing crop yield in plants or in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of CCP nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as `artificial restriction enzymes` when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
[0161] In another embodiment, PNAs of CCP can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of CCP nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P. J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
[0162] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
II. Isolated CCP Proteins and Anti-CCP Antibodies
[0163] One aspect of the invention pertains to isolated CCP proteins (e.g., the amino acid sequences set forth in SEQ ID NO:67-132, 205, 211, 215-216, or 220-227) and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-CCP antibodies. In one embodiment, native CCP proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, CCP proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a CCP protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
[0164] An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the CCP protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of CCP protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of CCP protein having less than about 30% (by dry weight) of non-CCP protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-CCP protein, still more preferably less than about 10% of non-CCP protein, and most preferably less than about 5% non-CCP protein. When the CCP protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
[0165] The language "substantially free of chemical precursors or other chemicals" includes preparations of CCP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of CCP protein having less than about 30% (by dry weight) of chemical precursors or non-CCP chemicals, more preferably less than about 20% chemical precursors or non-CCP chemicals, still more preferably less than about 10% chemical precursors or non-CCP chemicals, and most preferably less than about 5% chemical precursors or non-CCP chemicals.
[0166] Biologically active portions of a CCP protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the CCP protein, which include less amino acids than the full length CCP proteins, and exhibit at least one activity of a CCP protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the CCP protein. A biologically active portion of a CCP protein can be a polypeptide which is, for example, at least 10, 25, 50, 100 or more amino acids in length.
[0167] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0168] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limiting example of parameters to be used in conjunction with the GAP program include a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0169] In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or version 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
[0170] The nucleic acid and polypeptide sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to Kinase and Phosphatase nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=100, wordlength=3, and a Blosum62 matrix to obtain amino acid sequences homologous to Kinase and Phosphatase polypeptide molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0171] The invention also provides CCP chimeric or fusion proteins. As used herein, a CCP "chimeric protein" or "fusion protein" comprises a CCP polypeptide operatively linked to a non-CCP polypeptide. An "CCP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to CCP, whereas a "non-CCP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the CCP protein, e.g., a protein which is different from the CCP protein and which is derived from the same or a different organism. Within a CCP fusion protein the CCP polypeptide can correspond to all or a portion of a CCP protein. In a preferred embodiment, a CCP fusion protein comprises at least one biologically active portion of a CCP protein. In another preferred embodiment, a CCP fusion protein comprises at least two biologically active portions of a CCP protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the CCP polypeptide and the non-CCP polypeptide are fused in-frame to each other. The non-CCP polypeptide can be fused to the N-terminus or C-terminus of the CCP polypeptide or can be inserted within the CCP polypeptide. The non-CCP polypeptide can, for example, be (histidine)6-tag, glutathione S-transferase, protein A, maltose-binding protein, dihydrofolate reductase, Tag•100 epitope (EETARFQPGYRS; SEQ ID NO:199), c-myc epitope (EQKLISEEDL; SEQ ID NO:200), FLAG®-epitope (DYKDDDK; SEQ ID NO:201), lacZ, CMP (calmodulin-binding peptide), HA epitope (YPYDVPDYA; SEQ ID NO:202), protein C epitope (EDQVDPRLIDGK; SEQ ID NO:203) or VSV epitope (YTDIEMNRLGK; SEQ ID NO:204).
[0172] For example, in one embodiment, the fusion protein is a GST-CCP fusion protein in which the CCP sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant CCP.
[0173] In another embodiment, the fusion protein is a CCP protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., plant or mammalian host cells), expression and/or secretion of CCP can be increased through use of a heterologous signal sequence.
[0174] The CCP fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a plant or a subject in vivo. The CCP fusion proteins can be used to affect the bioavailability of a CCP substrate. Use of CCP fusion proteins may be useful agriculturally for the increase of crop yields or therapeutically for the treatment of cellular growth related disorders, e.g., cancer. Moreover, the CCP-fusion proteins of the invention can be used as immunogens to produce anti-CCP antibodies in a subject, to purify CCP ligands and in screening assays to identify molecules which inhibit the interaction of CCP with a CCP substrate, e.g., a kinase such as CDC2b.
[0175] Preferably, a CCP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A CCP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CCP protein.
[0176] The present invention also pertains to variants of the CCP proteins which function as either CCP agonists (mimetics) or as CCP antagonists. Variants of the CCP proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a CCP protein. An agonist of the CCP proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a CCP protein. An antagonist of a CCP protein can inhibit one or more of the activities of the naturally occurring form of the CCP protein by, for example, competitively modulating a cellular activity of a CCP protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the CCP protein.
[0177] In one embodiment, variants of a CCP protein which function as either CCP agonists (mimetics) or as CCP antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a CCP protein for CCP protein agonist or antagonist activity. In one embodiment, a variegated library of CCP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of CCP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential CCP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of CCP sequences therein. There are a variety of methods which can be used to produce libraries of potential CCP variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential CCP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
[0178] In addition, libraries of fragments of a CCP protein coding sequence can be used to generate a variegated population of CCP fragments for screening and subsequent selection of variants of a CCP protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CCP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the CCP protein.
[0179] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of CCP proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify CCP variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
[0180] In one embodiment, cell based assays can be exploited to analyze a variegated CCP library. For example, a library of expression vectors can be transfected into a cell line which ordinarily synthesizes and secretes CCP. The transfected cells are then cultured such that CCP and a particular mutant CCP are secreted and the effect of expression of the mutant on CCP activity in cell supernatants can be detected, e.g., by any of a number of enzymatic assays. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of CCP activity, and the individual clones further characterized.
[0181] An isolated CCP protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind CCP using standard techniques for polyclonal and monoclonal antibody preparation. A full-length CCP protein can be used or, alternatively, the invention provides antigenic peptide fragments of CCP for use as immunogens. The antigenic peptide of CCP comprises at least 8 amino acid residues and encompasses an epitope of CCP such that an antibody raised against the peptide forms a specific immune complex with CCP. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
[0182] Preferred epitopes encompassed by the antigenic peptide are regions of CCP that are located on the surface of the protein, e.g., hydrophilic regions.
[0183] A CCP immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed CCP protein or a chemically synthesized CCP polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic CCP preparation induces a polyclonal anti-CCP antibody response.
[0184] Accordingly, another aspect of the invention pertains to anti-CCP antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as CCP. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind CCP. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of CCP. A monoclonal antibody composition thus typically displays a single binding affinity for a particular CCP protein with which it immunoreacts.
[0185] Polyclonal anti-CCP antibodies can be prepared as described above by immunizing a suitable subject with a CCP immunogen. The anti-CCP antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized CCP. If desired, the antibody molecules directed against CCP can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-CCP antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol 127:539-46; Brown et al. (1980) J. Biol. Chem. 0.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a CCP immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds CCP.
[0186] Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-CCP monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind CCP, e.g., using a standard ELISA assay.
[0187] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-CCP antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with CCP to thereby isolate immunoglobulin library members that bind CCP. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP® Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Rang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:13704372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
[0188] Additionally, recombinant anti-CCP antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al, (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.
[0189] An anti-CCP antibody (e.g., monoclonal antibody) can be used to isolate CCP by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-CCP antibody can facilitate the purification of natural CCP from cells and of recombinantly produced CCP expressed in host cells. Moreover, an anti-CCP antibody can be used to detect CCP protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the CCP protein. These antibodies can also be used, for example, for the immunoprecipitation and immunolocalization of proteins according to the invention as well as for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention.
[0190] Anti-CCP antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
III. Computer Readable Means
[0191] The CCP nucleotide sequences of the invention (e.g., SEQ ID NO:1-66 or 228-239) or amino acid sequences of the invention (e.g., SEQ ID NO:67-132, 205, 211, 215-216, or 220-227) are also provided in a variety of mediums to facilitate use thereof. As used herein, "provided" refers to a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a nucleotide or amino acid sequences of the present invention. Such a manufacture provides the nucleotide or amino acid sequences, or a subset thereof (e.g., a subset of open reading frames (ORI's)) in a form which allows a skilled artisan to examine the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exist in nature or in purified form.
[0192] In one application of this embodiment, a nucleotide or amino acid sequence of the present invention can be recorded on computer readable media. As used herein "computer readable media" includes any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such a CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. The skilled artisan will readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention.
[0193] As used herein "recorded" refers to a process of storing information on computer readable medium. The skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising the nucleotide or amino acid sequence information of the present invention.
[0194] A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase Oracle, or the like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
[0195] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identity fragments or regions of the sequences of the invention which match a particular target sequence or target motif As used herein, a "target sequence" can be any DNA or amino acid sequence of six or more nucleotide or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or form about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be shorter length.
[0196] As used herein, "a target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
[0197] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software of conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPatter (EMBL), BLASTN and BASTX (NCBIA).
[0198] For example, software which implements the BLAST (Altschul et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system can be used to identify open reading frames (ORFs) of the sequences of the invention which contain homology to ORFs or proteins from other libraries. Such ORFs are protein encoding fragments and are useful in producing commercially important proteins such as enzyme used in various reactions and in the production of commercially useful metabolites.
IV. Recombinant Expression Vectors and Host Cells
[0199] Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a CCP protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
[0200] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, e.g., a plant cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CCP proteins, mutant forms of CCP proteins, fusion proteins, and the like).
[0201] The vectors of the invention comprise a selectable and/or scorable marker. Selectable marker genes useful for the selection of transformed plant cells, callus, plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable genes have been described, namely trpB, which allow cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
[0202] Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, the marker is a gene encoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or B-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a vector of the invention.
[0203] A "plant promoter" is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected. For example, copper-responsive, glucocorticoid-responsive or dexamethasone-responsive regulatory elements may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible, glucocorticoid-inducible, or dexamethasone-inducible expression respectively, on said nucleic acid molecule. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, endosperm, embryos, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue preferred." Promoters which initiate transcription only in certain tissue are referred to as "tissue specific." A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter which is active under most environmental conditions.
[0204] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
[0205] A host cell can be any prokaryotic or eukaryotic cell. For example, a CCP protein can be expressed in plant cells, bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
[0206] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
[0207] Means for introducing a recombinant expression vector of this invention into plant tissue or cells include, but are not limited to; transformation using CaCl2 and variations thereof, in particular the method described by Hanahan (J. Mol. Biol. 166, 557-560, 1983), direct DNA uptake into protoplasts (Krens et al, Nature 296: 72-74, 1982; Paszkowski et al, EMBO J. 3:2717-2722, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, Plant Cell Reports 9: 335-339, 1990) microparticle bombardment, electroporation (Fromm et al., Proc. Natl. Acad. Sci. (USA) 82:5824-5828, 1985), microinjection of DNA (Crossway et al., Mol. Gen. Genet. 202:179-185, 1986), microparticle bombardment of tissue explants or cells (Christou et al, Plant Physiol 87: 671-674, 1988; Sanford, Particulate Science and Technology 5: 27-37, 1987), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et al. (EMBO J. 4:277-284, 1985), Herrera-Estrella et al. (Nature 303: 209-213, 1983a; EMBO J. 2: 987-995, 1983b; In: Plant Genetic Engineering, Cambridge University Press, N.Y., pp 63-93, 1985), or in planta method using Agrobacterium tumefaciens such as that described by Bechtold et al., (C. R. Acad. Sci. (Paris, Sciences de la vie/Life Sciences)316: 1194-1199, 1993), Clough et al (Plant J. 16: 735-743, 1998), Trieu et al. (Plant J. 22:531-541, 2000) or Kloti (WO01/12828, 2001). Methods for transformation of monocotyledonous plants are well known in the art and include Agrobacterium-mediated transformation (Cheng et al. (1997) WO 97/48814; Hansen (1998) WO 98/54961; Hiei et al. (1994) WO 94/00977; Hiei et al. (1998) WO 98/17813; Rikiishi et al. (1999) WO 99/04618; Saito et al. (1995) WO 95/06722), microprojectile bombardment (Adams et al. (1999) U.S. Pat. No. 5,969,213; Bowen et al. (1998) U.S. Pat. No. 5,736,369; Chang et al. (1994) WO 94/13822; Lundquist et al. (1999) U.S. Pat. No. 5,874,265/U.S. Pat. No. 5,990,390; Vasil and Vasil (1995) U.S. Pat. No. 5,405,765; Walker et al. (1999) U.S. Pat. No. 5,955,362), DNA uptake (Eval et al. (1993) WO 93/181,168), microinjection of Agrobacterium cells (von Holt 1994 DE 4309203), sonication (Finer et al. (1997) U.S. Pat. No. 5,693,512) and flower-dip or in planta-transformation (Kloti, WO01/12828, 2001).
[0208] The vector DNA may further comprise a selectable marker gene to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct. Suitable selectable marker genes contemplated herein include the ampicillin resistance (Ampr), tetracycline resistance gene Tcr), bacterial kanamycin resistance gene (Kang, phosphinothricin resistance gene, neomycin phosphotransferase gene (nptII), hygromycin resistance gene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), and luciferase gene.
[0209] For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat. No. 4,945,050). When using ballistic transformation procedures, the gene construct may incorporate a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
[0210] A whole plant may be regenerated from the transformed or transfected cell, in accordance with procedures well known in the art. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a gene construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
[0211] The term "organogenesis", as used herein, includes a process by which shoots and roots are developed sequentially from meristematic centres.
[0212] The term "embryogenesis", as used herein, includes a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
[0213] Preferably, the plant is produced according to the methods of the invention by transfecting or transforming the plant with a genetic sequence, or by introducing to the plant a protein, by any art-recognized means, such as microprojectile bombardment, microinjection, Agrobacterium-mediated transformation (including in planta transformation), protoplast fusion, or electroporation, amongst others. Most preferably the plant is produced by Agrobacterium-mediated transformation. Agrobacterium-mediated transformation or agrolistic transformation of plants, yeast, moulds or filamentous fungi is based on the transfer of part of the transformation vector sequences, called the T-DNA, to the nucleus and on integration of said T-DNA in the genome of said eukaryote.
[0214] The term "Agrobacterium" as used herein, includes a member of the Agrobacteriaceae, more preferably Agrobacterium or Rhizobacterium and most preferably Agrobacterium tumefaciens.
[0215] The term "T-DNA", or "transferred DNA", as used herien, includes the transformation vector flanked by T-DNA borders which is, after activation of the Agrobacterium vir genes, nicked at the T-DNA borders and is transferred as a single stranded DNA to the nucleus of an eukaryotic cell.
[0216] As used herein, the terms "T-DNA borders", "T-DNA border region", or "border region" include either right T-DNA borders (RB) or left T-DNA borders (LB), which comprise a core sequence flanked by a border inner region as part of the T-DNA flanking the border and/or a border outer region as part of the vector backbone flanking the border. The core sequences comprise 22 bp in case of octopine-type vectors and 25 bp in case of nopaline-type vectors. The core sequences in the right border region and left border region form imperfect repeats.
[0217] As used herein, the term "T-DNA transformation vector" or "T-DNA vector" includes any vector encompassing a T-DNA sequence flanked by a right and left T-DNA border consisting of at least the right and left border core sequences, respectively, and used for transformation of any eukaryotic cell.
[0218] As used herein, the term "T-DNA vector backbone sequence" or "T-DNA vector backbone sequences" includes all DNA of a T-DNA containing vector that lies outside of the T-DNA borders and, more specifically, outside the nicking sites of the border core imperfect repeats.
[0219] The present invention includes optimized T-DNA vectors such that vector backbone integration in the genome of a eukaryotic cell is minimized or absent. The term "optimized T-DNA vector" as used herein includes a T-DNA vector designed either to decrease or abolish transfer of vector backbone sequences to the genome of a eukaryotic cell. Such T-DNA vectors are known to the one of skill in the art and include those described by Hanson et al. (1999) and by Stuiver et al. (1999--WO9901563).
[0220] The current invention clearly considers the inclusion of a DNA sequence encoding a CCP, homologue, analogue, derivative or immunologically active fragment thereof as defined supra, in any T-DNA vector comprising binary transformation vectors, super-binary transformation vectors, co-integrate transformation vectors, R1-derived transformation vectors as well as in T-DNA carrying vectors used in agrolistic transformation.
[0221] As used herein, the term "binary transformation vector" includes a T-DNA transformation vector comprising: a T-DNA region comprising at least one gene of interest and/or at least one selectable marker active in the eukaryotic cell to be transformed; and a vector backbone region comprising at least origins of replication active in E. coli and Agrobacterium and markers for selection in E. coli and Agrobacterium. Alternatively, replication of the binary transformation vector in Agrobacterium is dependent on the presence of a separate helper plasmid. The binary vector pGreen and the helper plasmid pSoup form an example of such a system (Hellens et al. (2000), Plant Mol. Biol. 42, 819-832; http://www.pgreen.ac.uk).
[0222] The T-DNA borders of a binary transformation vector can be derived from octopine-type or nopaline-type Ti plasmids or from both. The T-DNA of a binary vector is only transferred to a eukaryotic cell in conjunction with a helper plasmid. As used herein, the term "helper plasmid" includes a plasmid that is stably maintained in Agrobacterium and is at least carrying the set of vir genes necessary for enabling transfer of the T-DNA. The set of vir genes can be derived from either octopine-type or nopaline-type Ti plasmids or from both.
[0223] As used herein, the term "super-binary transformation vector" includes a binary transformation vector additionally carrying in the vector backbone region a vir region of the Ti plasmid pTiBo542 of the super-virulent A. tumefaciens strain A281 (EP0604662, EP0687730). Super-binary transformation vectors are used in conjunction with a helper plasmid.
[0224] As used herein, the term "co-integrate transformation vector" includes a T-DNA vector at least comprising: a T-DNA region comprising at least one gene of interest and/or at least one selectable marker active in plants; and a vector backbone region comprising at least origins of replication active in Escherichia coli and Agrobacterium, and markers for selection in E. coli and Agrobacterium, and a set of vir genes necessary for enabling transfer of the T-DNA. The T-DNA borders and the set of vir genes of the T-DNA vector can be derived from either octopine-type or nopaline-type Ti plasmids or from both.
[0225] The term "Ri-derived plant transformation vector" includes a binary transformation vector in which the T-DNA borders are derived from a Ti plasmid and the binary transformation vector being used in conjunction with a `helper` Ri-plasmid carrying the necessary set of vir genes.
[0226] The terms "agrolistics", "agrolistic transformation" or "agrolistic transfer" include a transformation method combining features of Agrobacterium-mediated transformation and of biolistic DNA delivery. As such, a T-DNA containing target plasmid is co-delivered with DNA/RNA enabling in planta production of VirD1 and VirD2 with or without VirE2 (Hansen and Chilton 1996; Hansen et al. 1997; Hansen and Chilton 1997--WO9712046).
[0227] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a CCP protein. Accordingly, the invention further provides methods for producing a CCP protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a CCP protein has been introduced) in a suitable medium such that a CCP protein is produced. In another embodiment, the method further comprises isolating a CCP protein from the medium or the host cell.
[0228] The host cells of the invention can also be used to produce transgenic plant or non-human transgenic animals in which exogenous CCP sequences have been introduced into their genome or homologous recombinant plants or animals in which endogenous CCP sequences have been altered. Such plants and animals are useful for studying the function and/or activity of a CCP and for identifying and/or evaluating modulators of CCP activity.
Trangenic Plants
[0229] As used herein, "transgenic plant" includes a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heteroglogous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses as asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring event such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
[0230] A transgenic plant of the invention can be created by introducing a CCP-encoding nucleic acid into the plant by placing it under the control of regulatory elements which ensure the expression in plant cells. These regulatory elements may be heterologous or homologous with respect to the nucleic acid molecule to be expressed as well with respect to the plant species to be transformed. In general, such regulatory elements comprise a promoter active in plant cells. These promoters can be used to modulate (e.g. increase or decrease) CCP content and/or composition in a desired tissue. To obtain expression in all tissues of a transgenic plant, preferably constitutive promoters are used, such as the 35 S promoter of CaMV (Odell, Nature 313 (1985), 810-812) or promoters from such genes as rice actin (McElroy et al. (1990) Plant Cell 2:163-171) maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. 231:276-285) or promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689). In order to achieve expression in specific tissues of a transgenic plant it is possible to use tissue specific promoters (see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251 or Table II, below).
TABLE-US-00013 TABLE II EXPRESSION GENE SOURCE PATTERN REFERENCE α-amylase (Amy32b) aleurone Lanahan, M. B., e t al., Plant Cell 4: 203- 211, 1992; Skriver, K., et al. Proc. Natl. Acad. Sci. (USA) 88: 7266-7270, 1991 cathepsin β-like gene aleurone Cejudo, F. J., et al. Plant Molecular Biology 20: 849-856, 1992. Agrobacterium rhizogenes rolB cambium Nilsson et al., Physiol. Plant. 100: 456-462, 1997 PRP genes cell wall http://salus.medium.edu/mmg/tierney/html barley Itr1 promoter endosperm synthetic promoter endosperm Vicente-Carbajosa et al., Plant J. 13: 629- 640, 1998. AtPRP4 flowers http://salus.medium.edu/mmg/tierney/html chalene synthase (chsA) flowers Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990. LAT52 anther Twell et al Mol. Gen Genet. 217: 240-245 (1989) apetala-3 flowers chitinase fruit (berries, grapes, etc) Thomas et al. CSIRO Plant Industry, Urrbrae, South Australia, Australia; http://winetitles.com.au/gwrdc/csh95-1.html rbcs-3A green tissue (eg leaf) Lam, E. et al., The Plant Cell 2: 857-866, 1990.; Tucker et al., Plant Physiol. 113: 1303-1308, 1992. leaf-specific genes leaf Baszczynski, et al., Nucl. Acid Res. 16: 4732, 1988. AtPRP4 leaf http://salus.medium.edu/mmg/tierney/html Pinus cab-6 leaf Yamamoto et al., Plant Cell Physiol. 35: 773-778, 1994. SAM22 senescent leaf Crowell, et al., Plant Mol. Biol. 18: 459- 466, 1992. R. japonicum nif gene nodule U.S. Pat. No. 4,803,165 B. japonicum nifH gene nodule U.S. Pat. No. 5,008,194 GmENOD40 nodule Yang, et al., The Plant J. 3: 573-585. PEP carboxylase (PEPC) nodule Pathirana, et al., Plant Mol. Biol. 20: 437- 450, 1992. leghaemoglobin (Lb) nodule Gordon, et al., J. Exp. Bot. 44: 1453-1465, 1993. Tungro bacilliform virus gene phloem Bhattacharyya-Pakrasi, et al, The Plant J. 4: 71-79, 1992. sucrose-binding protein gene plasma membrane Grimes, et al., The Plant Cell 4: 1561- 1574, 1992. pollen-specific genes pollen; microspore Albani, et al., Plant Mol. Biol. 15: 605, 1990; Albani, et al., Plant Mol. Biol. 16: 501, 1991) Zm13 pollen Guerrero et al Mol. Gen. Genet. 224: 161- 168 (1993) apg gene microspore Twell et al Sex. Plant Reprod. 6: 217-224 (1993) maize pollen-specific gene pollen Hamilton, et al., Plant Mol. Biol. 18: 211- 218, 1992. sunflower pollen-expressed gene pollen Baltz, et al., The Plant J. 2: 713-721, 1992. B. napus pollen-specific gene pollen; anther; tapetum Arnoldo, et al., J. Cell. Biochem., Abstract No. Y101, 204, 1992. root-expressible genes roots Tingey, et al., EMBO J. 6: 1, 1987. tobacco auxin-inducible gene root tip Van der Zaal, et al., Plant Mol. Biol. 16, 983, 1991. β-tubulin root Oppenheimer, et al., Gene 63: 87, 1988. tobacco root-specific genes root Conkling, et al., Plant Physiol. 93: 1203, 1990. B. napus G1-3b gene root U.S. Pat. No. 5,401,836 SbPRP1 roots Suzuki et al., Plant Mol. Biol. 21: 109- 119, 1993. AtPRP1; AtPRP3 roots; root hairs http://salus.medium.edu/mmg/tierney/html RD2 gene root cortex http://www2.cnsu.edu/ncsu/research TobRB7 gene root vasculature http://www2.cnsu.edu/ncsu/research AtPRP4 leaves; flowers; lateral http://salus.medium.edu/mmg/tierney/html root primordia seed-specific genes seed Simon, et al., Plant Mol. Biol. 5: 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin seed Pearson, et al., Plant Mol. Biol. 18: 235- 245, 1992. legumin seed Ellis, et al., Plant Mol. Biol. 10: 203-214, 1988. glutelin (rice) seed Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987. zein seed Matzke et al Plant Mol Biol, 14(3): 323- 32 1990 napA seed Stalberg, et al, Planta 199: 515-519, 1996. sunflower oleosin seed (embryo and dry Cummins, et al., Plant Mol. Biol. 19: seed) 873-876, 1992 LEAFY shoot meristem Weigel et al., Cell 69: 843-859, 1992. Arabidopsis thaliana knat1 shoot meristem Accession number AJ131822 Malus domestica kn1 shoot meristem Accession number Z71981 CLAVATA1 shoot meristem Accession number AF049870 stigma-specific genes stigma Nasrallah, et al., Proc. Natl. Acad. Sci. USA 85: 5551, 1988; Trick, et al., Plant Mol. Biol. 15: 203, 1990. class I patatin gene tuber Liu et al., Plant Mol. Biol. 153: 386-395, 1991. blz2 endosperm EP99106056.7 PCNA rice meristem Kosugi et al, Nucleic Acids Research 19: 1571-1576, 1991; Kosugi S. and Ohashi Y, Plant Cell 9: 1607-1619, 1997.
The promoters listed in the foregoing table are provided for the purposes of exemplification only and the present invention is not to be limited by the list provided therein. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention. The promoters listed may also be modified to provide specificity of expression as required.
[0231] Known are also promoters which are specifically active in tubers of potatoes or in seeds of different plants species, such as maize, Vicia, wheat, barley and the like. Inducible promoters may be used in order to be able to exactly control expression under certain environmental or developmental conditions such as pathogens, anaerobia, or light. Examples of inducible promoters include the promoters of genes encoding heat shock proteins or microspore-specific regulatory elements (WO96/16182). Furthermore, the chemically inducible Tet-system may be employed (Gatz, Mol. Gen. Genet. 227 (1991); 229-237). Further suitable promoters are known to the person skilled in the art and are described, e.g., in Ward (Plant Mol. Biol. 22 (1993), 361-366). The regulatory elements may further comprise transcriptional and/or translational enhancers functional in plants cells. Furthermore, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A tail to the transcript which may improve its stability.
[0232] In the case that a nucleic acid molecule according to the invention is expressed in the sense orientation, the coding sequence can be modified such that the protein is located in any desired compartment of the plant cell, e.g., the nucleus, endoplasmatic reticulum, the vacuole, the mitochondria, the plastids, the apoplast, or the cytoplasm.
[0233] Methods for the introduction of foreign DNA into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, biolistic methods like particle bombardment, pollen-mediated transformation, plant RNA virus-mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus and other methods known in the art. The vectors used in the method of the invention may contain further functional elements, for example "left border"- and "right border"-sequences of the T-DNA of Agrobacterium which allow for stably integration into the plant genome. Furthermore, methods and vectors are known to the person skilled in the art which permit the generation of marker free transgenic plants, i.e., the selectable or scorable marker gene is lost at a certain stage of plant development or plant breeding. This can be achieved by, for example, cotransformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161; Peng, Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems which utilize enzymes capable of promoting homologous recombination in plants (see, e.g., WO97/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361); Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet. 230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378). Methods for the preparation of appropriate vectors are described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
[0234] Suitable strains of Agrobacterium tumefaciens and vectors, as well as transformation of Agrobacteria, and appropriate growth and selection media are described in, for example, GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12 (1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976; Koncz, Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example, if a phenotype conferred by said strain is desired.
[0235] Methods for the transformation using biolistic methods are known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant Science 1, 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, N.Y. (1995).
[0236] The transformation of most dicotyledonous plants may be performed using the methods described above or using transformation via biolistic methods as, e.g., described above as well as protoplast transformation, electroporation of partially permeabilized cells, or introduction of DNA using glass fibers.
[0237] In general, the plants which are modified according to the invention may be derived from any desired plant species. They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture interest, such as crop plants (e.g., maize, rice, barley, wheat, rye, oats), potatoes, oil producing plants (e.g., oilseed rape, sunflower, pea nut, soy bean), cotton, sugar beet, sugar cane, leguminous plants (e.g., beans, peas), or wood producing plants, preferably trees.
[0238] The present invention also relates to a transgenic plant cell which contains (preferably stably integrated into its genome) a nucleic acid molecule of the present invention linked to regulatory elements which allow expression of the nucleic acid molecule in plant cells. The presence and expression of the nucleic acid molecule in the transgenic plant cells leads to the synthesis of a CCP protein and may lead to physiological and phenotypic changes in plants containing such cells.
[0239] Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced with a polynucleotide of the present invention.
[0240] Plant cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillilan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
[0241] Transformed plant cells, calli or explant can be cultured on regeneration medium in the dark for several weeks, generally about 1 to 3 weeks to allow the somatic embryos to mature. Preferred regeneration media include media containing MS salts, such as PHI-E and PHI-F media. The plant cells, calli or explant are then typically cultured on rooting medium in a light/dark cycle until shoots and roots develop. Methods for plant regeneration are known in the art and preferred methods are provided by Kamo et al., (Bot. Gaz. 146(3):324-334, 1985), West et al., (The Plant Cell 5:1361-1369. 1993), and Duncan et al. (Planta 165:322-332, 1985).
[0242] Small plantlets can then be transferred to tubes containing rooting medium and allowed to grow and develop more roots for approximately another week. The plants can then be transplanted to soil mixture in pots in the greenhouse.
[0243] The regeneration of plants containing the foreign gene introduced by Agrobacterium from leaft explants can be achieved as described by Horsch et al., Science, 227:1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al., Proc. Natl. Acad. Sci, U.S.A. 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.
[0244] Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys., 38:467-486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, from example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissback, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting ht transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis. (1988).
[0245] One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
[0246] In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype, (e.g., altered cell cycle content or composition).
[0247] Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
[0248] Transgenic plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
[0249] A preferred embodiment of the invention is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered cell division relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.
[0250] The present invention also relates to transgenic plants and plant tissue comprising transgenic plant cells according to the invention. Due to the (over)expression of a CCP molecule, e.g., at developmental stages and/or in plant tissue in which they do not naturally occur, these transgenic plants may show various physiological, developmental and/or morphological modifications in comparison to wild-type plants.
[0251] Therefore, part of this invention is the use of the CCP molecules to modulate the cell cycle and/or plant cell division and/or growth in plant cells, plant tissues, plant organs and/or whole plants. To the scope of the invention also belongs a method for influencing the activity of CDKs such as CDC2a, or CDC2b, CKSs, CKIs, PLPs and KLPNTs in a plant cell by transforming the plant cell with a nucleic acid molecule according to the invention and/or manipulation of the expression of the molecule.
[0252] Furthermore, the invention also relates to a transgenic plant cell which contains (preferably stably integrated into its genome) a nucleic acid molecule of the invention or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis of a CCP. In a preferred embodiment, the reduction is achieved by an anti-sense, sense, ribozyme, co-suppression and/or dominant mutant effect. The reduction of the synthesis of a protein according to the invention in the transgenic plant cells can result in an alteration in, e.g., cell division. In transgenic plants comprising such cells this can lead to various physiological, developmental and/or morphological changes.
[0253] In yet another aspect, the invention relates to harvestable parts and to propagation material of the transgenic plants of the invention which either contain transgenic plant cells expressing a nucleic acid molecule according to the invention or which contain cells which show a reduced level of the described protein. Harvestable parts can be in principle any useful parts of a plant, for example, flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots etc. Propagation material includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks, and the like.
Transgenic Animals
[0254] As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous CCP gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
[0255] A transgenic animal of the invention can be created by introducing a CCP-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The CCP cDNA sequence of SEQ ID NO:1-66 or 228-239 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a human CCP gene, such as a mouse or rat CCP gene, can be used as a transgene. Alternatively, a CCP gene homologue, such as another CCP family member, can be isolated based on hybridization to the CCP cDNA sequences of SEQ ID NO:1-66 or 228-239 (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a CCP transgene to direct expression of a CCP protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of a CCP transgene in its genome and/or expression of CCP mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a CCP protein can further be bred to other transgenic animals carrying other transgenes.
V. Agricultural, Phytopharmaceutical and Pharmaceutical Compositions
[0256] The CCP nucleic acid molecules, CCP proteins, and anti-CCP antibodies (also referred to herein as "active compounds") of the invention can be incorporated into compositions useful in agriculture and in plant cell and tissue culture. Plant protection compositions can be prepared by conventional means commonly used for the application of, for example, herbicides and pesticides. For example, certain additives known to those skilled in the art stabilizers or substances which facilitate the uptake by the plant cell, plant tissue or plant may be used.
[0257] The CCP nucleic acid molecules, CCP proteins, and anti-CCP antibodies (also referred to herein as "active compounds") of the invention can also be incorporated into pharmaceutical compositions suitable for administration into animals. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[0258] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a plant or subject by, for example, injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The agricultural or pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the agricultural or pharmaceutical preparation can include one or more cells which produce the gene delivery system.
[0259] The agricultural and pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
VI. Uses and Methods of the Invention
[0260] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) agricultural uses (e.g., to increase plant yield and to develop phytopharmaceuticals); b) screening assays; c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials); d) methods of treatment (e.g., phytotherapeutic, therapeutic and prophylactic); e) transcriptomics; f) proteomics; g) metabolomics; h) ligandomics; and i) pharmacogenetics or pharmacogenomics. The isolated nucleic acid molecules of the invention can be used, for example, to express CCP protein (e.g., via a recombinant expression vector in a host cell or in gene therapy applications), to detect CCP mRNA (e.g., in a biological sample) or a genetic alteration in a CCP gene, and to modulate CCP activity, as described further below. The CCP proteins can be used to treat disorders characterized by insufficient or excessive production of a CCP substrate or production of CCP inhibitors. In addition, the CCP proteins can be used to screen for naturally occurring CCP substrates, to screen for drugs or compounds which modulate CCP activity, as well as to treat disorders characterized by insufficient or excessive production of CCP protein or production of CCP protein forms which have decreased or aberrant activity compared to CCP wild type protein. Moreover, the anti-CCP antibodies of the invention can be used to detect and isolate CCP proteins, regulate the bioavailability of CCP proteins, and modulate CCP activity.
[0261] A. Agricultural Uses:
[0262] In another embodiment of the invention, a method is provided for modifying cell fate and/or plant development and/or plant morphology and/or biochemistry and/or physiology comprising the modification of expression in particular cells, tissues or organs of a plant, of a genetic sequence encoding a CCP, e.g., a CCP operably connected with a plant-operable promoter sequence.
[0263] Modulation of the expression in a plant of a CCP or a homologue, analogue or derivative thereof as defined in the present invention can produce a range of desirable phenotypes in plants, such as, for example, the modification of one or more morphological, biochemical, or physiological characteristics including: (i) modification of the length of the G1 and/or the S and/or the G2 and/or the M phase of the cell cycle of a plant; (ii) modification of the G1/S and/or S/G2 and/or G2/M and/or M/G1 phase transition of a plant cell; (iii) modification of the initiation, promotion, stimulation or enhancement of cell division; (iv) modification of the initiation, promotion, stimulation or enhancement of DNA replication; (v) modification of the initiation, promotion, stimulation or enhancement of seed set and/or seed size and/or seed development; (vi) modification of the initiation, promotion, stimulation or enhancement of tuber formation; (vii) modification of the initiation, promotion, stimulation or enhancement of fruit formation; (viii) modification of the initiation, promotion, stimulation or enhancement of leaf formation; (ix) modification of the initiation, promotion, stimulation or enhancement of shoot initiation and/or development; (x) modification of the initiation, promotion, stimulation or enhancement of root initiation and/or development; (xi) modification of the initiation, promotion, stimulation or enhancement of lateral root initiation and/or development; (xii) modification of the initiation, promotion, stimulation or enhancement of nodule formation and/or nodule function; (xiii) modification of the initiation, promotion, stimulation or enhancement of the bushiness of the plant; (xiv) modification of the initiation, promotion, stimulation or enhancement of dwarfism in the plant; (xv) modification of the initiation, promotion, stimulation or enhancement of senescence; (xvi) modification of stem thickness and/or strength characteristics and/or wind-resistance of the stem and/or stem length; (xvii) modification of tolerance and/or resistance to biotic stresses such as pathogen infection; and (xviii) modification of tolerance and/or resistance to abiotic stresses such as drought stress or salt stress.
[0264] Methods to effect expression of a CCP or a homologue, analogue or derivative thereof as defined in the present invention in a plant cell, tissue or organ, include either the introduction of the protein directly to a cell, tissue or organ such as by microinjection of ballistic means or, alternatively, introduction of an isolated nucleic acid molecule encoding the protein into the cell, tissue or organ in an expressible format. Methods to effect expression of a CCP or a homologue, analogue or derivative thereof as defined in the current invention in whole plants include regeneration of whole plants from the transformed cells in which an isolated nucleic acid molecule encoding the protein was introduced in an expressible format.
[0265] The present invention clearly extends to any plant produced by the inventive method described herein, and any and all plant parts and propagules thereof. The present invention extends further to encompass the progeny derived from a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by the inventive method, the only requirement being that the progeny exhibits the same genotypic and/or phenotypic characteristic(s) as those characteristic(s) that (have) been produced in the parent by the performance of the inventive method.
[0266] By "cell fate and/or plant development and/or plant morphology and/or biochemistry and/or physiology" is meant that one or more developmental and/or morphological and/or biochemical and/or physiological characteristics of a plant is altered by the performance of one or more steps pertaining to the invention described herein. "Cell fate" includes the cell-type or cellular characteristics of a particular cell that are produced during plant development or a cellular process therefor, in particular during the cell cycle or as a consequence of a cell cycle process.
[0267] The term "plant development" or the term "plant developmental characteristic" or similar terms shall, when used herein, be taken to mean any cellular process of a plant that is involved in determining the developmental fate of a plant cell, in particular the specific tissue or organ type into which a progenitor cell will develop. Cellular processes relevant to plant development will be known to those skilled in the art. Such processes include, for example, morphogenesis, photomorphogenesis, shoot development, root development, vegetative development, reproductive development, stem elongation, flowering, and regulatory mechanisms involved in determining cell fate, in particular a process or regulatory process involving the cell cycle.
[0268] The term "plant morphology" or the term "plant morphological characteristic" or similar term will, when used herein, be understood by those skilled in the art to include the external appearance of a plant, including any one or more structural features or combination of structural features thereof. Such structural features include the shape, size, number, position, color, texture, arrangement, and patternation of any cell, tissue or organ or groups of cells, tissues or organs of a plant, including the root, stem, leaf, shoot, petiole, trichome, flower, petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre, fruit, cambium, wood, heartwood, parenchyma, aerenchyma, sieve element, phloem or vascular tissue.
[0269] The term "plant biochemistry" or the term "plant biochemical characteristic" or similar term will, when used herein, be understood by those skilled in the art to include the metabolic and catalytic processes of a plant, including primary and secondary metabolism and the products thereof, including any small molecules, macromolecules or chemical compounds, such as but not limited to starches, sugars, proteins, peptides, enzymes, hormones, growth factors, nucleic acid molecules, celluloses, hemicelluloses, calloses, lectins, fibres, pigments such as anthocyanins, vitamins, minerals, micronutrients, or macronutrients, that are produced by plants.
[0270] The term "plant physiology" or the term "plant physiological characteristic" or similar term will, when used herein, be understood to include the functional processes of a plant, including developmental processes such as growth, expansion and differentiation, sexual development, sexual reproduction, seed set, seed development, grain filling, asexual reproduction, cell division, dormancy, germination, light adaptation, photosynthesis, leaf expansion, fibre production, secondary growth or wood production, amongst others; responses of a plant to externally-applied factors such as metals, chemicals, hormones, growth factors, environment and environmental stress factors (e.g., anoxia, hypoxia, high temperature, low temperature, dehydration, light, daylength, flooding, salt, heavy metals, amongst others), including adaptive responses of plants to said externally-applied factors.
[0271] The CCP molecules of the present invention are useful in agriculture. The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used to modulate the protein levels or activity of a protein involved in the cell cycle, e.g., proteins involved in the G1/S and/or the G2/M transition in the cell cycle due to environmental conditions, including abiotic stress such as cold, nutrient deprivation, heat, drought, salt stress, or biotic stress such as a pathogen attack.
[0272] Thus, the CCP molecules of the present invention may be used to modulate, e.g., enhance, crop yields; modulate, e.g., attenuate, stress, e.g. heat or nutrient deprivation; modulate tolerance to pests and diseases; modulate plant architecture; modulate plant quality traits; or modulate plant reproduction and seed development.
[0273] The CCP molecules of the present invention may also be used to modulate endoreduplication in storage cells, storage tissues, and/or storage organs of plants or parts thereof. The term "endoreduplication" includes recurrent DNA replication without consequent mitosis and cytokinesis. Preferred target storage organs and parts thereof for the modulation of endoreduplication are, for example, seeds (such as from cereals, oilseed crops), roots (such as in sugar beet), tubers (such as in potatoes) and fruits (such as in vegetables and fruit species). Increased endoreduplication in storage organs, and parts thereof, correlates with enhanced storage capacity and, thus, with improved yield. In another embodiment of the invention, the endoreduplication of a whole plant is modulated.
[0274] B. Screening Assays:
[0275] The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to CCP proteins, have a stimulatory or inhibitory effect on, for example, CCP expression or CCP activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a CCP substrate.
[0276] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a CCP protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a CCP protein or polypeptide or biologically active portion thereof, e.g., modulate the ability of CCP to interact with its cognate ligand. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the `one-bead one-compound` library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).
[0277] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckeimann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.
[0278] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).
[0279] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a CCP target molecule (e.g., a plant cyclin dependent kinase) with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the CCP target molecule. Determining the ability of the test compound to modulate the activity of a CCP target molecule can be accomplished, for example, by determining the ability of the CCP protein to bind to or interact with the CCP target molecule, or by determining the ability of the target molecule, e.g., the plant cyclin dependent kinase, to phosphorylate a protein.
[0280] The ability of the target molecule, e.g., the plant cyclin dependent kinase, to phosphorylate a protein can be determined by, for example, an in vitro kinase assay. Briefly, a protein can be incubated with the target molecule, e.g., the plant cyclin dependent kinase, and radioactive ATP, e.g., [γ-32P] ATP, in a buffer containing MgCl2 and MnCl2, e.g., 10 mM MgCl2 and 5 mM MnCl2. Following the incubation, the immunoprecipitated protein can be separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to a membrane, e.g., a PVDF membrane, and autoradiographed. The appearance of detectable bands on the autoradiograph indicates that the protein has been phosphorylated. Phosphoaminoacid analysis of the phosphorylated substrate can also be performed in order to determine which residues on the protein are phosphorylated. Briefly, the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis. The products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to ninhydrin-stained phosphoaminoacid standards.
[0281] Determining the ability of the CCP protein to bind to or interact with a CCP target molecule can be accomplished by determining direct binding. Determining the ability of the CCP protein to bind to or interact with a CCP target molecule can be accomplished, for example, by coupling the CCP protein with a radioisotope or enzymatic label such that binding of the CCP protein to a CCP target molecule can be determined by detecting the labeled CCP protein in a complex. For example, CCP molecules, e.g., CCP proteins, can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, CCP molecules can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
[0282] It is also within the scope of this invention to determine the ability of a compound to modulate the interaction between CCP and its target molecule, without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of CCP with its target molecule without the labeling of either CCP or the target molecule. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between compound and receptor.
[0283] In a preferred embodiment, determining the ability of the CCP protein to bind to or interact with a CCP target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase), or detecting a target-regulated cellular response.
[0284] In yet another embodiment, an assay of the present invention is a cell-free assay in which a CCP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the CCP protein or biologically active portion thereof is determined. Binding of the test compound to the CCP protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the CCP protein or biologically active portion thereof with a known compound which binds CCP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a CCP protein, wherein determining the ability of the test compound to interact with a CCP protein comprises determining the ability of the test compound to preferentially bind to CCP or biologically active portion thereof as compared to the known compound.
[0285] In another embodiment, the assay is a cell-free assay in which a CCP protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the CCP protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a CCP protein can be accomplished, for example, by determining the ability of the CCP protein to bind to a CCP target molecule by one of the methods described above for determining direct binding. Determining the ability of the CCP protein to bind to a CCP target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S, and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
[0286] In an alternative embodiment, determining the ability of the test compound to modulate the activity of a CCP protein can be accomplished by determining the ability of the CCP protein to further modulate the activity of a CCP target molecule (e.g., a CCP mediated signal transduction pathway component). For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined as previously described.
[0287] In yet another embodiment, the cell-free assay involves contacting a CCP protein or biologically active portion thereof with a known compound which binds the CCP protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the CCP protein, wherein determining the ability of the test compound to interact with the CCP protein comprises determining the ability of the CCP protein to preferentially bind to or modulate the activity of a CCP target molecule.
[0288] The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of proteins (e.g., CCP proteins or biologically active portions thereof). In the case of cell-free assays in which a membrane-bound form a protein is used it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.
[0289] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either CCP or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a CCP protein, or interaction of a CCP protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/CCP fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or CCP protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of CCP binding or activity determined using standard techniques.
[0290] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a CCP protein or a CCP target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated CCP protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with CCP protein or target molecules but which do not interfere with binding of the CCP protein to its target molecule can be derivatized to the wells of the plate, and unbound target or CCP protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the CCP protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the CCP protein or target molecule.
[0291] In another embodiment, modulators of CCP expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of CCP mRNA or protein in the cell is determined. The level of expression of CCP mRNA or protein in the presence of the candidate compound is compared to the level of expression of CCP mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of CCP expression based on this comparison. For example, when expression of CCP mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of CCP mRNA or protein expression. Alternatively, when expression of CCP mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of CCP mRNA or protein expression. The level of CCP mRNA or protein expression in the cells can be determined by methods described herein for detecting CCP mRNA or protein.
[0292] In yet another aspect of the invention, the CCP proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993). J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with CCP ("CCP-binding proteins" or "CCP-bp") and are involved in CCP activity. Such CCP-binding proteins are also likely to be involved in the propagation of signals by the CCP proteins or CCP targets as, for example, downstream elements of a CCP-mediated signaling pathway. Alternatively, such CCP-binding proteins are likely to be CCP inhibitors. Alternatively, a mammalian two-hybrid system can be used which includes e.g. a chimeric green fluorescent protein encoding reporter gene (Shioda et al. 2000, Proc. Natl. Acad. Sci. USA 97, 5520-5224). Yet another alternative consists of a bacterial two-hybrid system using e.g. HIS as reporter gene (Joung et al. 2000, Proc. Natl. Acad. Sci. USA 97, 7382-7387).
[0293] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a CCP protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a CCP-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the CCP protein.
[0294] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate plant or animal model. For example, an agent identified as described herein (e.g., a CCP modulating agent, an antisense CCP nucleic acid molecule, a CCP-specific antibody, or a CCP-binding partner) can be used in a plant or animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in a plant or animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for the agricultural and therapeutic uses described herein.
[0295] C. Detection Assays
[0296] Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; identify an individual from a minute biological sample (tissue typing); and aid in forensic identification of a biological sample. Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the CCP nucleotide sequences, described herein, can be used to map the location of the CCP genes on a chromosome. The mapping of the CCP sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
[0297] Briefly, CCP genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the CCP nucleotide sequences. Computer analysis of the CCP sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of cell hybrids containing individual plant or human chromosomes. Only those hybrids containing the plant or human gene corresponding to the CCP sequences will yield an amplified fragment.
[0298] Other mapping strategies which can similarly be used to map a CCP sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
[0299] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).
[0300] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
[0301] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.
[0302] Moreover, differences in the DNA sequences between plants affected and unaffected with a disease associated with the CCP gene, can be determined. If a mutation is observed in some or all of the affected plants but not in any unaffected plants, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected plants generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several plants can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
[0303] D. Predictive Medicine:
[0304] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining CCP protein and/or nucleic acid expression as well as CCP activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant CCP expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with CCP protein, nucleic acid expression or activity. For example, mutations in a CCP gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with CCP protein, nucleic acid expression or activity.
[0305] Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of CCP in clinical trials.
[0306] E. Methods of Treatment:
[0307] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant CCP expression or activity. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomies. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype".) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the CCP molecules of the present invention or CCP modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
[0308] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing are incorporated herein by reference.
EXAMPLES
Example 1
Identification of Plant CCP Polypeptides Using the Two Hybrid System with CDC2B as a Bait
[0309] A two-hybrid screening was performed using as bait a fusion between the GAL4 DNA-binding domain and one of the following: CDC2bAt.N161 (GenBank accession number D10851; residue Asp161 converted into Asn161); CKS1At (GenBank accession number AJ000016); E2Fa (=E2F5) (GenBank accession number AJ294534) dimerization domain (226-356aa; SEQ ID NO:205); CKI4 (SEQ ID NO:264); PLP1 (GenBank accession number T01601); KLPNT1 (GenBank accession number AB011479; protein ID number BAB11568) motor domain (36-508 aa); KLPNT1 (GenBank accession number AB011479; protein ID number BAB11568) stalk domain (427-867 aa); KLPNT2=TH65 (GenBank accession number AJ001729) neck domain (3-186 aa); KLPNT2=TH65 (GenBank accession number AJ001729) stalk domain (73-608 aa); E2Fb (=E2F3) (GenBank accession number AJ294533) N-terminal domain (1-385 aa; SEQ ID NO:206), respectively
[0310] CDC2bAt.N161 is a dominant negative form of the CDC2bAt protein. The D161 residue in CDC2bAt is crucial for ATP binding and, thus, the mutation of this residue results in an inactive kinase. The interactions between this mutated CDK and its substrates and regulatory proteins are also more stabilised as a result of this mutation.
[0311] In yeast the PHO genes are part of a complex regulatory network linking phosphate availability with the expression of phosphatases. When phosphate levels are high the PHO80/PHO85 cyclin/CDK complex phosphorylates a transcription factor. This transcription factor of phosphatase genes thereby becomes inactive. The S. cerevisiae PHO85 protein can interact with the G1 specific cyclins PCL1 and PCL2 (close homologues to the PHO80). In a yeast strain deficient for the G1 cyclins CLN1 and CLN2, PHO80 is required for G1 progression. This result suggests that PHO85 is involved in a regulatory pathway that links the nutrient status of the cell with cell division activity. The five PLP of A. thaliana show similarity to the yeast cyclin-like PHO80 gene.
[0312] Kinesins use the cytoskeleton to move around vesicles, organelles, chromosomes and the like in the cell. They can also be involved in spindle formation. Kinesins consist of three functional unrelated domains: the motor domain (involved in microtubule binding; contains the ATPase domain), the stalk region (involved in homo- or heterodimirisation of the kinesins), and the tail (involved in the interaction with the `substrates` of the kinesin). Two hybrid screens were performed using different parts of two-kinesin-related proteins (KLPNT1 and KLPNT2 (being more than 80% identical to KLPNT1). Other information obtained by the two hybrid approach is the dimerization of the kinesins: the KLPNT1 and KLPNT2 interact (stalks and stalks-tail) with and between themselves.
[0313] Vectors and strains used were provided with the Matchmaker Two-Hybrid System (Clontech, Palo Alto, Calif.). The bait was constructed by inserting the CDC2bAt.N161 (GenBank accession number D10851; residue Asp161 converted into Asn161); CKS1At (GenBank accession number AJ000016); E2Fa (=E2F5) (GenBank accession number AJ294534) dimerization domain (226-356aa; SEQ ID NO:205); CKI4 (SEQ ID NO:264); PLP1 (GenBank accession number T01601); KLPNT1 (GenBank accession number AB011479; protein ID number BAB11568) motor domain (36-508 aa); KLPNT1 (GenBank accession number AB011479; protein ID number BAB11568) stalk domain (427-867 aa); KLPNT2=TH65 (GenBank accession number AJ001729) neck domain (3-186 aa); KLPNT2=TH65 (GenBank accession number AJ001729) stalk domain (73-608 aa); E2Fb (=E2F3) (GenBank accession number AJ294533) N-terminal domain (1-385 aa; SEQ ID NO:206), respectively, into the pGBT9 vector. Bait vectors where constructed by introducing the PCR fragment created from the corresponding cDNA using primers to incorporate EcoRI and BamH1 restriction enzyme sites. The PCR fragment was cut with EcoRI and BamH1 and cloned into the EcoRI and BamH1 sites of pGBT9, resulting in the desired plasmid. The GAL4 activation domain cDNA fusion library was constructed as described in De Veylder et al 1999, 208(4) p453-62 from mRNA of Arabidopsis thaliana cell suspensions harvested at various growing stages: early exponential, exponential, early stationary, and stationary phase.
[0314] For the screening a 1-liter culture of the Saccharomyces cerevisiae strain HF7c (MATaura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3,112 gal4-542 gal80-538 LYS2::GAL1.sub.UAS-GAL1.sub.TATA-HIS3 URA3::GAL417mers(3x)-CyC1.sub.TATA-LacZ) was sequentially transformed with the bait plasmid and 20 μg DNA of the library using the lithium acetate method (Geitz et al. (1992) supra). To estimate the number of independent cotransformants, 1/1000 of the transformation mix was plated on Leu- and Trp-medium. The rest of the transformation mix was plated on medium to select for histidine prototrophy (Tip-, Leu-, His-). After 5 days of growth at 30° C., the colonies larger than 2 mm were streaked on histidine-lacking medium. At total for each screening at least 107 independent cotransformants were screened for there ability to grow on histidine free medium. Of the His.sup.+ colonies the activation domain plasmids were isolated as described (Hoffman and Winston, 1987, Gene 57, 267-272). The hybriZAP® inserts were PCR amplified and the PCR fragments were digested with AluI and fractionized on a 2% agarose gel. Plasmid DNA of which the inserts gave rise to different restriction patterns were electroporated into Escherichia coli XL1-Blue, and the DNA sequence of the inserts was determined. Extracted DNA was also used to retransform HF7c to test the specificity of the interaction.
[0315] Using the foregoing technique, 61 cDNAs were identified, their sequences were determined and found to contain open reading frames termed CCP1 through CCP61 (FIGS. 1-61).
Example 2
Extension of CCP Encoding Polynucleotides to Full Length or to Recover Regulatory Elements
[0316] The CCP encoding nucleic acid sequences (SEQ ID NO:1-66 or 228-239) are used to design oligonucleotide primers for extending a partial nucleotide sequence to full length or for obtaining 5' sequences from genomic or cDNA libraries. One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF). Primers allow the extension of the known CCP encoding sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest. The initial primers are designed from the cDNA using OLIGO® 4.06 Primer Analysis Software (National Biosciences), or another appropriate program, to be preferably 22-30 nucleotides in length, to have a GC content of preferably 50% or more, and to anneal to the target sequence at temperatures preferably about 68°-72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations is avoided. The original, selected cDNA libraries, prepared from mRNA isolated from actively dividing cells or a plant genomic library are used to extend the sequence; the latter is most useful to obtain 5' upstream regions. If more extension is necessary or desired, additional sets of primers are designed to further extend the known region.
[0317] Sense XLF primers can also be designed based on publicly available genomic sequences. GENEMARK.hmm (hidden morkov model) version 2.2a software (default parameters) can e.g. be used to predict open reading frames. The 5' end of the predicted open reading frame is then subsequently used to design the sense XLF primer. Said XLF primer and the appropriate XLR primer are then used in an RT-PCR (reverse transcription-polymerase chain reaction) reaction to amplify the predicted cDNA. The resulting PCR product is cloned in a suitable vector and subjected to DNA sequence analysis to verify the prediction.
[0318] Primers used to amplify coding regions of the CCPs of the invention are designed such that the PCR product can be cloned in the pDONR201 vector (Gateway® cloning system, Invitrogen). Thus, a sense primer has the attB1 site (SEQ ID NO:246) at its 5' end. For current purposes, the attB1 site is followed by a consensus Kozak sequence (SEQ ID NO:247; Kozak (1989)J Cell Biol 108:229-241; Lutck et al. (1987) EMBO J. 6:43-48). The 3' end of the sense primer comprises the gene-specific parts as indicated in FIGS. 1-46. An antisense primer has at the 5' end the attB2 site (SEQ ID NO:248) followed by the inverse complement of the gene/coding region of interest as indicated in FIGS. 1-46. Primers used for CCP amplification by PCR are given with their SEQ ID NOs in Table 3. The sequence of cloned CCP PCR products was or is determined using the sense primer prm1024 (SEQ ID NO:265) and the antisense primer prm1025 (SEQ ID NO:266).
TABLE-US-00014 TABLE III sense antisense primer primer CCP PCR primers SEQ ID SEQ ID Molecule sense + antisense NO: NO: CCP1 prm0733 + prm0734 133 134 CCP2 prm0663 + prm0664 135 136 CCP3 prm0705 + prm0706 137 138 CCP4 prm0659 + prm0660 139 140 CCP5 prm0749 + prm0750 141 142 CCP6 prm0707 + prm0708 143 144 CCP7/8 prm0657 + prm0658 145 146 CCP9 prm0582 + prm0583 147 148 CCP10 prm0671 + prm0672 149 150 CCP11 prm0729 + prm0730 151 152 CCP12 + prm1676 + prm1677 153 154 CCP13 CCP14 prm0701 + prm0702 155 156 CCP15 prm0445 + prm0446 157 158 CCP16 prm0321 + prm0322 159 160 CCP17 prm0632 + prm0633 161 162 CCP18 prm0488 + prm0489 163 164 CCP19 prm0661 + prm0662 165 166 CCP20 + prm0709 + prm0710 167 168 CCP21 CCP22 prm0711 + prm0712 169 170 CCP23 prm0819 + prm0820 171 172 CCP24 prm0739 + prm0740 173 174 CCP25 prm0741 + prm0742 175 176 CCP26 prm0703 + prm0704 177 178 CCP27 prm0817 + prm0818 179 180 CCP28 prm0713 + prm0714 181 182 CCP29 / / / CCP30 prm0480 + prm0481 183 184 CCP31 prm0737 + prm0738 185 186 CCP32 prm1493 + prm1494 187 188 CCP33 prm0319 + prm0320 189 190 CCP34 prm1377 + prm1378 191 192 CCP35 prm1381 + prm1382 193 194 CCP36 / / / CCP37 prm1379 + prm1380 195 196 CCP38 prm1383 + prm1384 197 198
[0319] By following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix, high fidelity amplification is obtained. Beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, PCR is performed suing the Peltier Thermal Cycle (PTC200; MJ Research, Watertown Mass.) and the following parameters:
TABLE-US-00015 Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 min Step 4 94° for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 min Step 7 Repeat steps 4-6 for 15 additional cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step 8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)
[0320] A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were selected and cut out of the gel. Further purification involves using a commercial gel extraction method such as QIAQuick® (QIAGEN Inc). After recovery of the DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning. After ethanol precipitation, the products are redissolved in 13 μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4 polynucleotide kinase are added, and the mixture is incubated at room temperature for 2-3 hours or overnight at 16° C. Competent E. coli cells (in 40 μl of appropriate media) are transformed with 3 μl of ligation mixture and cultured in 80 μl of SOC medium (Sambrook, supra). After incubation for one hour at 37° C., the whole transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook, supra) containing 2×Carb. The following day, several colonies are randomly picked from each plate and cultured in 150 p. 1 of liquid LB/2×Carb medium placed in an individual well of an appropriate, commerically-available, sterile 96-well microtiter plate. The following day, 5 μl of each overnight culture is transferred into a non-sterile 96-well plate and after dilution 1:10 with water, 5 μl of each sample is transferred into a PCR array. For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×) containing 4 units of 4Tth DNA polymerase, a vector primer and both of the gene specific primers used for the extension reaction are added to each well. Amplification is performed using the following conditions:
TABLE-US-00016 Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (and holding)
Aliquots of the PCR reactions are run on agarose gels together with molecular weight markers. The sizes of the PCR products are compared to the original partial cDNAs, and appropriate clones are selected, ligated into plasmid and sequenced.
Example 3
Expression of Recombinant CCP Proteins in Transgenic Plants
[0321] In this example, the CCP molecules of the present invention were expressed in a 35S expression vector in transgenic plants. The CCP molecules of this invention were cloned using standard cloning procedures between a suitable promoter, e.g. the CaMV35S promoter or any promoter from e.g. Table II, and a suitable terminator, e.g., the NOS 3' untranslated region. The resulting recombinant gene is subsequently cloned in a suitable binary vector and the resulting plant transformation vector is then transferred to Agrobacterium tumefaciens. Arabidopsis thaliana is transformed with this Agrobacterium applying the in planta flower-dip transformation method (Clough and Bent, Plant J. 16:735-743, 1998). Transgenic plant lines are selected on a growth medium containing the suitable selection agent (e.g., kanamycin or Basta) or on the basis of scoring the expression of a screenable marker (e.g., luciferase, green fluorescent protein).
[0322] For tissue-specific expression, the CCP gene can also be expressed under control of the minimal 35S promoter containing UAS elements. These UAS elements are sites for transcriptional activation by the GAL4-VP16 fusion protein. The GAL4-VP16 fusion protein in turn is expressed under control of a tissue-specific promoter. The UAS-CCP construct and the GAL4-VP16 construct are combined by co-transformation of both constructs, subsequent transformation of single constructs or by sexual cross of lines that contain the single constructs. The advantage of this two-component system is that a wide array of tissue-specific expression patterns can be generated for a specific transgene, by simply crossing selected parent lines expressing the UAS-CCP construct with various tissue-specific GAL4-VP16 lines. A tissue-specific promoter/CCP combination that gives a desired phenotype can subsequently be recloned in a single expression vector, to avoid stacking of transgene constructs in commercial lines.
[0323] Primary transformants are characterized by Northern and Western blotting using 1-4 week old plantlets. Expression levels were compared with those of non-transformed (control) plants.
Example 4
Downregulation of Target CCP Genes in Transgenic Plants
[0324] Plant genes can be specifically downregulated by antisense and co-suppression technologies. These technologies are based on the synthesis of antisense transcripts, complementary to the mRNA of a given CCP gene. There are several methods described in literature, that increase the efficiency of this downregulation, for example to express the sense strand with introduced inverted repeats, rather than the antisense strand. The constructs for downregulation of target genes are made similarly as those for expression of recombinant proteins, i.e., they are fused to promoter sequences and transcription termination sequences (see example 3). Promoters used for this purpose are constitutive promoters as well as tissue-specific promoters.
Example 5
Agrobacterium-Mediated Rice Transformation
[0325] Mature dry seeds of the rice japonica cultivars Nipponbare or Taipei 309 are dehusked, sterilised and germinated on a medium containing 2,4-D (2,4-dichlorophenoxyacetic acid). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli are excised and propagated on the same medium. Selected embryogenic calluses are then co-cultivated with Agrobacterium. Widely used Agrobacterium strains such as LBA4404 or C58 harbouring binary T-DNA vectors can be used. The hpt gene in combination with hygromycin is suitable as a selectable marker system but other systems can be used. Co-cultivated callus is grown on 2,4-D-containing medium for 4 to 5 weeks in the dark in the presence of a suitable concentration of the selective agent. During this period, rapidly growing resistant callus islands develop. After transfer of this material to a medium with a reduced concentration of 2,4-D and incubation in the light, the embryogenic potential is released and shoots develop in the next four to five weeks. Shoots are excised from the callus and incubated for one week on an auxin-containing medium from which they can be transferred to the soil. Hardened shoots are grown under high humidity and short days in a phytotron. Seeds can be harvested three to five months after transplanting. The method yields single locus transformants at a rate of over 50% (Aldemita and Hodges (1996) Planta 199:612-617; Chan et al. (1993) Plant Mol. Biol. 22: 491-506; Hiei et al. (1994) Plant J. 6:271-282).
Example 6
Expression of Recombinant CCP Proteins in Bacterial Cells
[0326] In this example, the CCP molecules of the present invention are expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, CCP molecules are fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-CCP fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.
Example 7
Expression of Recombinant CCP Proteins in COS Cells
[0327] To express the CCP gene of the present invention in COS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire CCP protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
[0328] To construct the plasmid, the CCP DNA sequence is amplified by PCR using two primers. The 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the CCP coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the CCP coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the Kinase and/or Phosphatase gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5a, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
[0329] COS cells are subsequently transfected with the CCP-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of the CCP polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly, the cells are labelled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
[0330] Alternatively, DNA containing the Kinase and/or Phosphatase coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the CCP polypeptide is detected by radiolabelling and immunoprecipitation using a CCP specific monoclonal antibody.
Example 8
In Vitro Phosphorylation of CDC2DN-IC26M by Plant CDKs
[0331] The CDC2bDN-IC26M coding region (SEQ ID NO:4) was amplified by PCR with Pfu polymerase (Stratagene, La Jolla, Calif.). The PCR product was subcloned into pET19b (Novagen, Madison, Wis.), to obtain CDC2bDN-IC26 MpET19b. The CDC2bDN-IC26M gene is located downstream of a T1lac promoter, in frame with a sequence encoding a 10-histidine tag followed by an enterokinase recognition site. Escherichia coli BL21(DE3) cells (Novagen) containing the CDC2bDN-IC26 MpET19b plasmid were grown at 37° C. in M9 medium (Sambrook and Russel, Molecular Cloning, A Laboratory Manual, 3rd Edition, CSHL Press, CSH New York, 2001), supplemented with 100 μg/ml of ampicillin, to obtain a cell density corresponding to an A600 of 0.6. Subsequently, expression of the CDC2bDN-IC26M gene was induced by addition of 0.4 mM isopropyl β-D-thiogalactoside, and culture was continued for 4 h at 30° C.
[0332] Cells were collected in lysis buffer containing 50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl, 0.1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and were lysed on ice by sonication. The extract was clarified by centrifugation for 20 minutes at 20,000×g. The crude extract was loaded at 4° C. on a nickel-nitrilotriacetic acid-agarose affinity resin (Qiagen), and protein fractionation was performed according to the manufacturer's instructions. The fractions containing the CDC2bDN-IC26M fusion protein were pooled.
[0333] CDC2bDN-IC26M kinase assays were performed with CDK complexes purified from total plant (Arabidopsis seedlings) protein extracts by p13.sup.suc1-Sepharose affinity binding according to Azzi et al. (Eur. J. Biochem. 203: 353-360). Briefly, p13.sup.suc1 was purified from an overproducing E. coli strain by chromatography in Sephacryl S2000, and conjugated to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's instructions. Total plant protein extracts (300 μg) were incubated with 50 μl 50% (v/v) p13.sup.suc1-Sepharose beads for 2 h at 4° C. The washed beads were combined with 30 μl kinase buffer containing ˜1 mg/ml CDC2bDN-IC26M, 150 mM ATP and 1 μCi of [-32P]ATP (Amersham). After 20 minutes of incubation at 30° C., samples were analysed by SDS-PAGE and autoradiographed.
[0334] As shown in FIG. 48, the purified CDC2bDN-IC26M protein is phosphorylated by CDKs in vitro.
Example 9
PCR Amplification of AtDPb
[0335] Based on available sequence data of putative plant DP-related partial clones from the databank (soybean DP(AI939068), tomato DP(AW217514), and cotton DP (AI731675)), three oligonucleotides, corresponding to the most conserved part of the DNA-binding and E2F heterodimerization domains (MKVCEKV, SEQ ID NO:240; LNVLMAMD, SEQ ID NO:241 and FNSTPFEL, SEQ ID NO:242), were synthesized and designated A (ATAGAATTCATGAAAGTTTGTGAAAAGGTG, SEQ ID NO:243), B (ATAGAATTCCTGAATGTTCTCATGGCAATGGAT, SEQ ID NO:244) and C (ATAGGATCCCAGCTCAAAAGGAGTGCTATTGAA, SEQ ID NO:245), respectively.
[0336] PCR was performed on an Arabidopsis/yeast two-hybrid suspension culture cDNA library. The PCR products were purified, digested with EcoRI and BamHI, and ligated into pCR-XL-TOPO vector (Invitrogen). The cloned inserts were sequenced by double-stranded dideoxy sequencing.
Example 10
Construction of AtDP and AtE2F Mutants, In Vitro Transcription-Translation System and Immunoprecipitation
[0337] Influenza hemagglutinin (HA)-tagged versions of the wild-type and mutant AtE2Fa and AtE2Fb were constructed by cloning into the pSK plasmid (Stratagene) containing the HA-tag (SEQ ID NO:202). The AtE2F mutants, namely AtE2Fa 1-420 (SEQ ID NO:217), AtE2Fa 162-485 (SEQ ID NO:218), and AtE2Fb 1-385 (SEQ ID NO:206), were obtained by PCR and cloned into the EcoRI and BamHI sites of HA-pSK. The c-myc (SEQ ID NO:200)-tagged versions of wild-type and AtDP mutants (AtDPa 1-292, SEQ ID NO:114; AtDPa 121-292, SEQ ID NO:211; AtDPa 1-142, SEQ ID NO:208; AtDPa 172-292, SEQ ID NO:213; AtDPa 121-213, SEQ ID NO:212; and AtDPb 1-385, SEQ ID NO:127; AtDPb 182-385, SEQ ID NO:216; AtDPb 1-263, SEQ ID NO:223; AtDPb 1-193, SEQ ID NO:214; and AtDPb 182-263, SEQ ID NO:215) were generated by PCR and cloned into the EcoRI and PstI sites of the pBluescript plasmid (Stratagene) containing a double c-myc tag. All cloning steps were carried out according to standard procedures, and the reading frames were verified by direct sequencing.
[0338] In vitro transcription and translation experiments were performed using the TNT T7-coupled wheat germ extract kit (Promega) primed with appropriate plasmids for 90 min at 30° C. For immunoprecipitation, 10 μl of the total in vitro translated extract (50 μl) was diluted at 1:5 in Nonidet P40 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P40, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin/aprotinin/pepstatin) and incubated for 2 h at 4° C. with anti-c-myc (9E10; BabCo) or anti-HA (16B12; BabCo) antibodies. Protein-A-Sepharose (40 μl 25% (v/v)) was added and incubated for 1 h at 4° C., then the beads were washed four times with Nonidet P40 buffer. Immune complexes were eluted with 10 μl 2 U sodium dodecyl sulfate (SDS) sample buffer and analyzed by 10% or 15% SDS-PAGE and by autoradiography.
[0339] An overview of the AtDP and AtE2F fragments and their SEQ ID NOs is given in Table 4.
TABLE-US-00017 TABLE IV SEQ ID NO SEQ ID NO amino acid DNA CCP or partial CCP sequence sequence AtE2Fa 226-356 205 228 AtE2Fb 1-385 206 AtE2Fb 1-127 207 AtDPa 1-142 208 AtDPa 42-142 209 AtDPa 42-292 210 AtDPa 121-292 211 229 AtDPa 121-213 212 AtDPa 172-292 213 AtDPb 1-193 214 AtDPb 182-263 215 230 AtDPb 182-385 216 231 AtE2Fa 1-420 217 AtE2Fa 162-485 218 AtE2Fa 1-38 219 AtDPa 1-214 220 239 AtDPa 143-292 221 232 AtDPa 143-213 222 233 AtDPb 1-263 223 234 AtE2Fa 232-282 224 235 AtE2Fa 232-352 225 236 AtE2Fb 194-243 226 237 AtE2Fb 194-311 227 238
Example 11
In Vitro Interaction Between AtDPs, AtE2Fs and Mutants Thereof Illustrated by Immunoprecipitation Experiments
[0340] The AtDPa and AtDPb can efficiently interact in vitro with AtE2Fa and AtE2Fb. As a first step in comparing the biochemical properties of AtDPa and AtDPb, the ability of these molecules to heterodimerize with AtE2Fa and AtE2Fb was tested. For this purpose, the coupled in vitro transcription-translation system was used in which the c-myc-tagged AtDPa or AtDPb was co-expressed with the HA-tagged AtE2Fa or AtE2Fb. One part of each sample was resolved by SDS-PAGE (FIGS. 50 and 51, panels A), while another part was subjected to immunoprecipitation with monoclonal anti-c-myc antibodies (FIGS. 50 and 51, panels B). In the absence of DP proteins, no AtE2F2a or AtE2F2b was precipitated by the anti-c-myc antibodies (FIG. 51, panel B, lane 1). However, both HA-AtE2F proteins co-precipitated reproducibly with c-myc-tagged AtDPa (FIG. 50, panel B, lanes 1 and 2) and AtDPb (FIG. 51, panel B, lanes 3 and 4). Identical results were obtained in a reciprocal experiment with anti-HA monoclonal antibodies. These data revealed that both Arabidopsis DP-related proteins interacted in vitro with the different Arabidopsis E2F-related proteins.
[0341] The conserved dimerization domain of the AtE2Fs seemed to be important for the interaction with the AtDPs, because mutational analysis showed that deletion neither of the N-terminal extension nor the C-terminal part of AtE2Fa and AtE2Fb impaired the interaction with the DPs (FIGS. 50 and 51, panels B). Similar results were obtained by two-hybrid analysis (see Table 5 of Example 12). To test whether the structural requirements for heterodimerization of the AtDPs were similar to those of their animal homologs, several deletion mutants of AtDPa and AtDPb were constructed (for a schematic illustration, see FIGS. 52 and 53), tagged with the c-myc epitope (FIGS. 54 and 55, panels A). The interactions between the mutant AtDPs and AtE2Fb were analyzed in immunoprecipitation experiments with the specific anti-HA or anti-c-myc antibodies (Figures A6 and A7, panels B and C, respectively). As shown in FIGS. 54 and 55, mutant AtDP proteins with deleted DNA-binding domain could bind sufficiently to the co-translated HA-AtE2Fb proteins (FIG. 54, panel C, lane 2; and FIG. 55, panel C, lane 2). No detectable interaction was found between the AtE2Fb protein and mutant DP proteins containing the complete DNA-binding domain, but lacking the putative dimerization domain (FIG. 54, panel C, lane 3; FIG. 55, panel C, lane 4). Thus, the N-terminal part of both AtDP proteins, including the conserved DNA-binding domain, was not sufficient for the in vitro interaction to occur. In contrast, a mutant form of AtDPb (amino acids 1-263; SEQ ID NO:223) could bind to AtE2Fb (FIG. 55, panel C, lane 3), indicating that the region of AtDPb between amino acids 182 and 263 was required for interaction with AtE2Fb.
[0342] To confirm this hypothesis, a deletion mutant of AtDPb (182-263, SEQ ID NO:215) was constructed and, as expected, it could bind to AtE2Fb (FIG. 56). The requirement for the homologous dimerization domain of AtDPa for the interaction with AtE2Fb was supported by a binding assay in which the mutant AtDPa 172-292 (SEQ ID NO:213), with the N-terminal part of the dimerization domain deleted, failed to bind to AtE2Fb (FIG. 54, panels B and C, lanes 4). However, when the E2F-binding activity of the predicted dimerization domain of the AtDPa (amino acid positions 121-213, SEQ ID NO:212) was tested, no interaction could be detected between this region and the AtE2Fb protein (FIG. 54, panel B, lane 5). These data indicate that other carboxyl-terminal regions of AtDPa are required for the stable interaction with AtE2Fb.
Example 12
Yeast Two-Hybrid Experiments for Showing Interaction Between DP and E2F Mutants
[0343] For library screening, vectors and strains (HF7c) were provided with the Matchmaker two-hybrid system (Clontech). The dimerization and DNA-binding domains of the AtE2Fa (amino acids 226-356; SEQ ID NO:205) were amplified by polymerase chain reaction (PCR) and subcloned in-frame with the GAL4 DNA-binding domain of pGBT9 (Clontech) to create the bait plasmid pGBTE2Fa226-356. Screens were performed as described previously (De Veylder et al. 1999; Planta 208, 453-462). A second library screening was performed with the AtE2Fb construct (pGBTE2Fb-Rb) lacking the Rb-binding domain (amino acids 1-385; SEQ ID NO:206). Plasmids from interacting clones were isolated and sequenced.
[0344] For the yeast two-hybrid interaction experiments, a number of yeast two-hybrid prey (in pAD-GAL424) plasmids were created by PCR amplification of fragments from the AtDPa (DPa 1-292, SEQ ID NO:114; DPa 1-142, SEQ ID NO:208; DPa 42-142, SEQ ID NO:209; DPa 42-292, SEQ ID NO:210; DPa 121-292, SEQ ID NO:211; DPa 121-213, SEQ ID NO:212; and DPa 172-292, SEQ ID NO:213) and AtDPb (DPb 1-385, SEQ ID NO:127; DPb 1-193, SEQ ID NO:214; DPb 182-263, SEQ ID NO:215; and DPb 182-385, SEQ ID NO:216) genes and confirmed by sequencing. Different combinations between bait (pGBTE2Fa226-356, pGBTE2Fb-Rb, or pGBTE2Fb 1-127, SEQ ID NO:207) and prey constructs were transformed into yeast cells and assayed for their ability to grow on His.sup.- minimal media after 3 days of incubation at 30° C. Bait plasmids co-transformed with empty pAD-GAL424 and prey plasmids co-transformed with empty pGBT9 were assessed along as controls for the specificity of the interaction.
[0345] An overview of the AtDP and AtE2F fragments and their SEQ ID NOs is given in Table 4.
[0346] The results obtained were confirmed by two-hybrid interaction analysis. pGBTE2Fa226-356 and pGBTE2Fb-Rb were co-transformed in an appropriate yeast reporter stain with a plasmid producing the full-length AtDPa or AtDPb protein fused to the GAL4 transactivation domain. The specific reconstitution of GAL4-dependent gene expression measured as the ability to grow in the absence of histidine confirms the interaction between the two DP and E2F proteins (Table 5).
TABLE-US-00018 TABLE V AtDPs and AtE2Fs interaction in yeast two-hybrid assays. Preys DPa DPa DPa DPa DPa DPa DPa DPb DPb DPb DPb E2Fa pAD- Baits 1-292 1-142 42-142 42-292 121-292 121-213 172-292 1-385 1-193 182-263 182-385 226-356 GAL424 pGBT + - - + + - - + - + + - - E2Fa 226-356 pGBT + - - + + - - + - + + - - E2Fb- Rb pGBT - NT NT NT NT NT NT - NT NT NT - - E2Fb 1-127 pGBT - NT NT NT NT NT NT - NT NT NT + - DPa 1-292 pGBT NT NT NT NT NT NT NT - NT NT NT + - DPb 1-385 pBGT9 - - - - - - - - - - - - - Different combinations between AtE2Fs bait and AtDPs prey constructs were tested for growth on His.sup.- minimal media. -, no interaction; +, positive interaction; NT, not tested.
Example 13
RNA Isolation and Reverse Transcription-(RT)-PCR analysis of AtDP and AtE2F Expression
[0347] A. thaliana (L.) Heynh. cell suspension cultures were maintained as described previously (Glab et al. 1994, FEBS Lett. 17, 207-211). The cells were partially synchronized by the consecutive addition of aphidicolin (5 μg/ml) and propyzamide (1.54 μg/ml). The aphidicolin block was left for 24 hours. The cells were washed for 1 hour in B5 medium before the addition of propyzamide. Samples were taken at the end of the 24 hour aphidicolin block, at the end of a 1 hour washing step, and at 1, 2, 3, and 4 hours after the addition of propyzamide to the culture medium. Total RNA was isolated from the Arabidopsis cell suspension culture according to Magyar et al. (1997), Plant Cell 9, 223-235, and with the Triazol reagent (Gibco/BRL) from different organs. Semi-quantitative RT-PCR amplification was carried out on reverse-transcribed mRNA, ensuring that the amount of amplified product stayed in linear proportion to the initial template present in the reaction. 10 μl from the PCR was transferred onto Hybond-N/membrane, hybridized to fluorescein-labeled gene-specific probes (Gene-Images random prime labeling module; Amersham Pharmacia Bio-tech), detected with the CDP-Star detection module (Amersham), and visualized by short exposure to Kodak X-OMAT autoradiography film.
[0348] The following primer pairs (forward and reverse) were used for the amplification: 5'-ATAGAATTCATGTCCGGTGTCGTACGA-3' (SEQ ID NO:249, EcoRI site underlined) and 5'-ATAGGATCCCACCTCCAATCTTTCTGCAGC-3' (SEQ ID NO:250, BamHI site underlined) for AtE2Fa (GenBank accession number AJ294533); 5'-ATAGAATTCGAGAAGAAAGGGCAAT CAAGA-3' (SEQ ID NO:251, EcoRI site underlined) and 5'-ATACTGCAGAGAAATCTCGATTTCGACTAC-3' (SEQ ID NO:252, PstI site underlined) for AtDPa (GenBank accession number AJ294531); 5'-GCCACTCTCATAGGGTTCTC CATCG-3' (SEQ ID NO:253) and 5'-GGCATGCCTCCAAGATCCTTGAAGT-3' (SEQ ID NO:254) for Arath;CDKA;1 (Genbank accession number X57839); 5'-GGGTCTTGGTCGTTTTACTGTT-3' (SEQ ID NO:255) and 5'-CCAAGACGATGACAACAGATACAGC-3' (SEQ ID NO:256) for Arath;CDKB1;1 (Genbank accession number X57840); 5'-ATAAACTAAATCTTCGCTGAA-3' (SEQ ID NO:257) and 5'-CAAACGCGGATCTGAAAAACT-3' (SEQ ID NO:258) for histone 114 (Genbank accession number M17132); 5'-TCTCTCTTCCAAATCTCC-3' (SEQ ID NO:259) and 5'-AAGTCTCT CACTTTCTCACT-3' (SEQ ID NO:260) for R005 (AtCYP1, GenBank accession number U072676) (Chou and Gasser 1997, Plant Mol. Biol. 35, 873-892); 5'-CTAAGCTCTCAAGATCAAAGGCTTA-3' (SEQ ID NO:261) and 5'-TTAACATTG CAAAGAGTTTCAAGGT-3' (SEQ ID NO:262) for actin 2 gene (GenBank accession number U41998) (An et al. 1996, Plant J. 10, 107-121).
Example 14
The AtDPa and the AtE2Fa Genes are Co-Expressed in a Cell Cycle Phase-Dependent Manner
[0349] The identification of the AtDPa in a yeast two-hybrid screen as a gene encoding an AtE2Fa-associating protein indicated that it might act cooperatively in the plant cells as a functional heterodimer. To strengthen this hypothesis, we investigated whether both genes were co-regulated at the transcriptional level. Tissue-specific expression analysis revealed that both genes were clearly up-regulated in flowers and were very strongly transcribed in actively dividing cell suspension cultures (FIG. 57). Expression in these tissues could be a sign for the correlation between the actual proliferation activity of a given tissue and the transcript accumulation, as can be seen from the Arath;CDKB1;1 gene. AtDPa transcripts were also detectable in leaf and, to a lesser extent, in root and stem tissues, whereas AtE2Fa transcripts were virtually undetectable in roots and stem with only slight levels of expression in leaf tissues. Cell cycle phase-dependent gene transcription was studied using an Arabidopsis cell suspension that was partially synchronized by the sequential treatment with aphidicolin and propyzamide. The Arabidopsis histone H4 and the Arath;CDKB1;1 gene were included to monitor the cell cycle progression (FIG. 58) (Chaubet et al. 1996, Plant J. 10, 425-435; Segers et al. 1996, Plant J. 10, 601-612). Bearing in mind the partial synchronization of the culture, it can be observed that histone H4 transcript levels peaked immediately after the removal of the inhibitor and decrease gradually thereafter (FIG. 58). The opposite expression pattern could be observed for the Arath;CDKB1;1 gene, illustrating that cells entered the G2-M phases with partial synchrony. Within this experimental setting, the AtDPa and the AtE2Fa genes show a very similar expression pattern. Both exhibit higher transcript accumulation before the peak of histone H4 gene expression and quickly decay in the following samples (FIG. 58). The similarity in the expression patterns of Arabidopsis AtDPa and AtE2Fa supports the possibility that they act cooperatively as a heterodimer during the S phase.
Example 15
Transformation of Arabidopsis Thaliana with CaMV35S::DPa
[0350] Arabidopsis plants were transformed (using the in planta flower dip method; Clough and Bent, Plant J. 16:735-743, 1998) with a construct containing the DPa gene under the control of the CaMV 35S promoter. The lines were molecularly analysed by northern blotting. As can be seen in FIG. 59, all lines showed increased DPa levels in comparison with the untransformed control. Generally, two classes of lines were observed: weakly expressing (e.g., 16) and strongly expressing (e.g., 23) lines (see FIG. 59). The plants are subsequently analyzed for phenotypic alterations as described herein.
EQUIVALENTS
[0351] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence CWU
1
29011255DNAArabidopsis thaliana 1ccacatatcc gtgatgagga aactaagaaa
ccagactcag tttcaagtga agaaccagag 60acgattatca ttgatgtgga tgaaagtgat
aaagaaggag gtgactctaa tgagccaatg 120tttgtacaac atactgaagc aatgctggag
gagattgaac agatggagaa ggagattgaa 180atggaagatg cagacaaaga agaagagcct
gtgatcgata ttgatgcctg tgataagaat 240aatcctttgg ctgcggttga atatatccat
gatatgcata ccttctacaa gaattttgag 300aaacttagtt gcgtgcctcc taactatatg
gacaatcaac aagatcttaa tgagagaatg 360agaggaatcc tcattgactg gttaattgag
gtgcactaca agtttgaact gatggaggaa 420actctttatc tcacaatcaa tgtcatcgac
agattccttg cggttcatca aatcgtgagg 480aaaaagcttc agcttgttgg tgttactgct
ttgttgcttg catgtaaata tgaagaagtt 540tcagttccag tggtagatga tctcatcttg
atctctgaca aagcttactc tagaagagaa 600gtgctagata tggagaagct aatggccaac
accttgcaat tcaatttctc tctaccaact 660ccatatgttt tcatgaaacg atttctcaaa
gctgcccaat ctgacaagaa gcttgagatt 720ttatcattct ttatgatcga gctttgcctt
gtggagtatg agatgctaga gtatcttcca 780tctaagctgg cggcctcagc aatctacact
gctcagtgta cacttaaggg atttgaagaa 840tggagcaaaa cctgtgagtt tcacacaggc
tacaacgaaa aacagctact ggcatgtgcg 900agaaagatgg ttgctttcca tcacaaggca
ggaacaggga agctcacagg agttcacaga 960aagtacaaca catctaagtt ctgtcatgct
gcaagaactg aaccagctgg gtttctgatt 1020taatattaat aagaatctaa tatgacttaa
ctcgagtttt tctttagaac aaaaagagtg 1080tgagagaaag agagatagta gagcaagttg
cccaaaatgg gagaagaatg gatctttaga 1140tatcatggca agtagcccaa aaagagtgta
ttcttctctt tctaaggtct ttagatcttt 1200cttcacttga gagagaataa aaagaatctt
ctgaaaaaaa aaaaaaaaaa aaaaa 12552471DNAArabidopsis thaliana
2cccgattcgg gtactgctgc tggtgggtca aactccgacc cgtttcctgc gaatcttcga
60gttcttgtcg ttgatgatga tccaacttgt ctcatgatct tagagaggat gcttatgact
120tgtctctaca gagtaactaa atgtaacaga gcagagagcg cattgtctct gcttcggaag
180aacaagaatg gttttgatat tgtcattagt gatgttcata tgcctgacat ggatggtttc
240aagctccttg aacacgttgg tttagagatg gatttacctg ttatcatgat gtctgcggat
300gattcgaaga gcgttgtgtt gaaaggagtg actcacggtg cagttgatta cctcatcaaa
360ccggtacgta ttgaggcttt gaagaatata tggcaacatg tggtgcggaa gaagcgtaac
420cgagtggaat ggttctgaac attctggagg aagtattgaa gatactggcg g
47131351DNAArabidopsis thaliana 3atggggaagg aaaatgctgt gtctcggcca
ttcactcgtt cccttgcctc tgctttgcgc 60gcttcagaag tgacttctac tacacagaat
caacagagag taaacacaaa aagaccagcc 120ttggaggata caagagccac tggacccaac
aagaggaaga agcgagcggt tctaggggag 180atcacaaatg ttaactccaa tacagctata
cttgaggcca aaaacagcaa gcagataaag 240aaaggacgcg gtcatggatt ggcgagtaca
tcccagttgg caacttctgt tacttcagaa 300gtcacagatc ttcagtccag gaccgatgca
aaagttgaag ttgcatcaaa tacagcagga 360aacctttctg tttctaaagg cacagataac
acagctgata actgtattga gatatggaat 420tctagattgc ctccaagacc tcttgggaga
tcagcttcta cagctgagaa aagtgctgtt 480attggtagtt caactgtacc ggatatccca
aaatttgtag acatcgattc agatgacaag 540gatcctttac tgtgctgcct ctatgcccct
gaaatccact acaatttgcg tgtttcagag 600cttaaacgca gaccacttcc ggactttatg
gagagaatac agaaggatgt cacccagtcc 660atgcggggaa ttctggttga ttggcttgtg
gaggtctctg aagaatacac acttgcatct 720gacactctct acctcacagt gtatctcata
gactggttcc tccatggaaa ctacgtgcaa 780agacagcaac ttcaactgct cggcatcact
tgcatgctaa ttgcctcgaa gtatgaggaa 840atctctgctc cacgcattga ggagttttgc
ttcattacgg ataacaccta cacaagagat 900caggtcctgg aaatggagaa ccaagtactt
aagcatttta gctttcaaat atacactccc 960actccaaaaa cgttccttag gagatttctc
agagcagctc aagcctctcg cctgagccca 1020agccttgaag tcgagtttct agccagctat
ctaacagagt tgacattaat agactaccat 1080ttcttaaagt ttcttccttc cgttgttgct
gcttcagcgg gttttctcgc caagtggaca 1140atggaccaat caaaccaccc atggaatcca
acacttgagc attacacaac gtacaaagca 1200tcggatctga aagcatctgt tcatgcctta
caagatctgc agcttaacac caaaggttgc 1260cccttgagcg ctatacgcat gaagtatagg
caagagaaat acaaatctgt ggcggttctc 1320acgtctccaa agctacttga cacgctattc t
13514672DNAArabidopsis thaliana
4atggggaaga agtgtgattt atgtaacggt gttgcaagaa tgtattgcga gtcagatcaa
60gctagtttat gttgggattg cgacggtaaa gttcacggcg ctaatttctt ggtagctaaa
120cacacgcgtt gtcttctctg tagcgcttgt cagtctctta cgccgtggaa agctactggg
180cttcgtcttg gcccaacttt ctccgtctgc gagtcatgcg tcgctcttaa aaacgccggc
240ggtggccgtg gaaacagagt tttatcggag aatcgtggtc aggaggaggt taatagtttc
300gagtccgaag aagatcggat tagagaagat cacggtgacg gtgacgacgc ggagtcttac
360gatgatgatg aggaagaaga tgaggatgaa gagtacagcg acgatgagga tgaggatgat
420gatgaggatg gtgatgatga ggaagcggag aatcaagttg tgccgtggtc tgcggcggcg
480caagttcctc cggtgatgag ttcttcatct tctgacggag gaagcggagg ttcagtgacg
540aagaggacga gggctagaga gaattcagat cttctctgct ccgatgatga gatcggaagc
600tcttcagctc aagggtcaaa ctattctcgg ccgttgaagc gatcggcgtt taaatcaacg
660gttgttgttt aa
67251287DNAArabidopsis thaliana 5atggttaact catgcgagaa caaaatcttc
gttaaaccca cttcaacgac gattcttcaa 60gatgaaacaa gaagtagaaa attcggacaa
gagatgaaga gggagaagag aagagtgttg 120cgtgtgatta accagaatct cgctggtgca
agagtttatc cttgtgttgt caacaagaaa 180ggaagcttat tgtctaataa gcaagaagaa
gaagaaggat gtcaaaagaa gaagtttgat 240tctttgcgtc cttcagttac aagatctgga
gttgaggaag agactaacaa gaagctgaag 300ccctcagttc caagtgctaa cgacttcggt
gattgtatat ttattgatga ggaggaagct 360acattggacc ttccaatgcc aatgtcgctt
gagaaaccat acattgaagc tgatccaatg 420gaagaagttg agatggagga tgtaacagtg
gaagaaccga tcgtggatat cgatgtctta 480gactcgaaga actcgcttgc ggctgttgaa
tatgttcaag atctttacgc attttacaga 540acaatggaga gatttagttg tgttccagta
gactatatga tgcaacaaat cgacttaaac 600gagaagatga gagcaatact aatcgactgg
ttaatcgagg tacatgacaa gtttgatctg 660atgaacgaga cactgtttct gacagtgaat
ctgatagata gattcttgtc caagcaaaat 720gttatgagaa agaagcttca gcttgtaggg
ttagtagctt tgctgttagc ttgtaagtat 780gaggaggttt cggttcctgt tgtcgaagat
ttagtactca tttcggacaa agcgtatacg 840aggaacgatg ttctagagat ggagaaaaca
atgttgagta ctttgcaatt caatatctcg 900ttaccgacac aatacccgtt cttgaaaaga
ttcctcaagg cagctcaagc agacaagaag 960tgtgaggtct tggcgtcgtt cttgatcgag
cttgcccttg tggagtacga gatgcttcgg 1020tttccaccat cattactagc tgccacatct
gtgtacactg ctcaatgtac acttgatggt 1080tccaggaaat ggaacagtac atgtgaattc
cattgtcatt actctgaaga ccagctcatg 1140gaatgttcac ggaagctggt gagtctgcat
cagagggcgg cgacaggaaa cttaacagga 1200gtatatagga agtacagcac aagcaaattt
ggttacatag caaaatgtga agctgcacac 1260tttctagtgt ctgagtctca tcattct
128761078DNAArabidopsis thaliana
6actaagcagg aggccaaagc tgctttcaag tctcttttgg aatctgtaaa tgttcattcc
60gactggacat gggaacagac attgaaagag attgttcacg ataaaagata tggtgctttg
120aggacactcg gcgagcggaa acaagcgttt aacgagtatc ttggccaaag gaaaaaagtg
180gaagctgagg aaagacgaag gaggcagaag aaagctcggg aagaatttgt caagatgcta
240gaggagtgtg aagaactttc atcatccctg aaatggagca aagcaatgag tttgttcgaa
300aatgatcagc gttttaaagc tgttgaccgt cctagggatc gtgaagatct ttttgacaat
360tacattgtgg aacttgagag gaaggaaaga gaaaaggcag cggaggaaca tcggcagtat
420atggcagact atcggaagtt tcttgaaacc tgtgactata tcaaagctgg tacacaatgg
480cgcaaaattc aagatagact ggaggatgat gacagatgct catgtcttga aaagatagat
540cgtctgattg gttttgagga atacattctt gacctagaga aggaagaaga agagctgaag
600agagtagaga aagaacatgt aaggcgggcc gagagaaaaa accgtgatgc atttcgtaca
660ctattggaag aacatgttgc tgcaggcatc cttacagcca agacgtactg gttggattat
720tgcattgagt taaaagactt gccccaatac caagctgttg catctaatac atctggttca
780actccgaaag acttgtttga agatgtcaca gaagaattag agaagcagta tcatgaggat
840aagagctatg tgaaggatgc tatgaagtca agaaagattt ccatggtctc ctcgtggctg
900tttgaagatt ttaaatctgc tatttcagaa gatctcagta ctcaacagat atcagacata
960aatttaaagc ttatatatga tgacttggtt gggagagtga aggaaaaaga agaaaaagag
1020gccagaaagc ttcagcgtct ggctgaagaa tttaccaatc tgttgcacac tttcaagg
10787511DNAArabidopsis thaliana 7caagagaaac cgtgggagaa tgatcctcac
tactttaaac gagtcaagat ctcagcgctc 60gctcttctta agatggtggt tcacgctcgc
tctggtggta caattgaaat aatgggtctt 120atgcaaggta agaccgatgg tgatactatc
attgttatgg atgcttttgc tttaccagtg 180gaaggtactg agacaagggt taatgctcag
gatgatgctt atgagtacat ggttgagtat 240tcacagacca acaagctcgc ggggccggct
ggagaatgtt gttggatggt atcactctca 300ccctggatat ggatgctggc tctccggtat
tgatgtttct acgcagaggc ttaaccaaca 360gcatcaggag ccatttttag ctgttgttat
tgatcccaca aggactgttt cagctggtaa 420ggttgagatt ggtgctttca gaacatactc
taaaggatat aaagccctcc agatgaacct 480gtttctgagt atcaaaacta ttcctttaaa t
51181155DNAArabidopsis thaliana
8agtagactca cctgattcaa cctccgacaa catcttctac tacgacgata cttcacagac
60taggttccag caagagaaac cgtgggagaa tgatcctcac tactttaaac gagtcaagat
120ctcagcgctc gctcttctta agatggtggt tcacgctcgc tctggtggta caattgaaat
180aatgggtctt atgcaaggta agaccgatgg tgatactatc attgttatgg atgcttttgc
240tttaccagtg gaaggtactg agacaagggt taatgctcag gatgatgctt atgagtacat
300ggttgagtat tcacagacca acaagctcgc ggggcggctg gagaatgttg ttggatggta
360tcactctcac cctggatatg gatgctggct ctccggtatt gatgtttcta cgcagaggct
420taaccaacag catcaggagc catttttagc tgttgttatt gatcccacaa ggactgtttc
480agctggtaag gttgagattg gtgctttcag aacatactct aaaggatata agcctccaga
540tgaacctgtt tctgagtatc aaactattcc tttaaataag attgaggact ttggtgttca
600ctgcaaacag tactattcat tagatgtcac ttatttcaag tcatctcttg attctcacct
660tctggatcta ctatggaaca agtactgggt gaacactctt tcttcttctc cactgctggg
720taatggagac tatgttgctg gacaaatatc agacttagct gagaagcttg agcaagccga
780gagtcatctg gttcagtctc gctttggagg agttgtgcca tcatcccttc ataagaaaaa
840agaggatgag tctcaactaa ctaagataac tcgggatagc gcaaagataa ctgtggaaca
900ggtccatgga ctaatgtcgc aggtcataaa agatgaatta ttcaactcaa tgcgtcagtc
960caacaacaaa tctcccactg actcgtcgga tccagaccct atgattacat attgaagttg
1020ctcttctttt ggtttctagt tttggattga cccatcattt gttgtccttt catttatttt
1080ctgttgtgta aagaattata atgctaatca gaataataca gaagaagatt ttggttaaaa
1140aaaaaaaaaa aaaaa
115591308DNAArabidopsis thaliana 9atgtattgct cttcttcgat gcatccaaat
gcaaacaaag aaaatatctc tacttcagat 60gtacaggaga gttttgtacg aataacgaga
tcacgagcta aaaaagccat gggaagagga 120gtatcaatac ctccaacaaa accttctttt
aaacagcaaa agagacgtgc agtacttaag 180gatgtgagta atacctctgc agatattatt
tattcagaac ttcgaaaggg aggcaacatc 240aaggcaaaca gaaaatgtct aaaagagcct
aaaaaagcag caaaggaagg tgctaacagt 300gccatggata ttctggtaga tatgcataca
gaaaaatcaa aattagcaga agatttgtcc 360aagatcagga tggctgaagc ccaagatgtc
tctctttcaa actttaaaga tgaagaaatt 420actgagcaac aagaagatgg atcaggtgtc
atggagttac ttcaagttgt agatattgat 480tccaacgtcg aagatccaca gtgttgcagc
ttgtatgctg ctgatatata tgacaacata 540catgttgcag agcttcaaca acgacccttg
gctaattata tggagcttgt gcagcgagat 600atcgacccag acatgagaaa gattctgatt
gactggcttg tagaagtttc tgacgactac 660aagctggttc cagatacgct ttaccttaca
gtgaatctta tcgaccggtt tctgtccaac 720agttacattg aaaggcaaag actccagctc
cttggtgtct cttgcatgct tatagcttca 780aaatatgaag agctttccgc accaggggtg
gaggagtttt gcttcattac ggccaacaca 840tacacaagac gagaagtgct gagcatggag
attcaaattc taaattttgt gcactttaga 900ttatcggttc ctaccaccaa aacatttctg
aggcggttca ttaaagcagc tcaagcttcg 960tacaaggtgc ctttcattga actggagtat
ttagcaaact atctcgccga attgacactg 1020gtggaatata gtttcctaag gttcctgcca
tcactaattg ctgcttcagc tgttttccta 1080gcccgatgga cactcgacca aactgaccat
ccttggaacc ctactctgca acactacacc 1140agatatgagg tagctgagct gaagaacaca
gttctcgcca tggaggactt gcagctcaac 1200accagtggct gtactctcgc tgccacccgt
gagaaataca accaaccaaa gtttaagagc 1260gtggcaaagc tgacatctcc caaacgagtc
acattactat tctcaaga 1308101006DNAArabidopsis thaliana
10agacttcaca ttttaccatt atttgctctg agctcagtag gagagttcaa gaaacaatgg
60caaagatgca attatcaatc tttatcgctg tcgttgcgct tatcgtctgc tctgcatctg
120ctaaaaccgc aagccctcca gctccagtgc tgccaccgac accagctcca gcaccagccc
180cggaaaatgt gaatctcacc gagcttttaa gtgtagctgg tccgttccac acattcctcg
240actaccttct ctcgactgga gtcattgaga ctttccaaaa ccaagctaac aacactgagg
300aaggcatcac aatctttgtc cctaaagatg atgctttcaa agctcagaag aatcctcctt
360tgtcaaatct cacaaaggat cagcttaagc agcttgttct cttccatgct ctgcctcatt
420actattcgct ttcggaattc aagaacttga gccaatctgg tccagtgagc acctttgctg
480gtggtcaata ctccttgaaa ttcactgatg tttctggcac ggttaggatt gattctttat
540ggaccaggac taaagtcagc agcagtgttt tctccactga ccctgttgcg gtttaccaag
600tgaaccgcgt gcttctaccc gaagcaatct ttggtactga tgtccctcca atgcctgctc
660cagctcctgc tcctatcgtt agtgctcctt cggattctcc ttcagttgct gattctgaag
720gagcttcttc accaaagtcc tcacacaaga actccggaca aaagctgcta cttgcaccaa
780tctccatggt tatttccggt ttggtggcat tgttcttgtg atcagatggt tttgcagatt
840gagttatgtt tttaagttac aatgtgaaag attgtattac atcatttgaa ttgtcttttt
900gatttttgaa acccattttt tattatacat ttttatcatt attattgttt gtcattacga
960ttgttgtgaa ttgaaattgt tcctccaaaa aaaaaaaaaa aaaaaa
100611643DNAArabidopsis thaliana 11atttatcatt acagtctgat ttgagctaag
ttctctcatc ataaactctc cttggagaat 60catggctatt tcaaaagctc ttatcgcttc
tcttctcata tctcttcttg ttctccaact 120cgtccaggct gatgtcgaaa actcacagaa
gaaaaatggt tacgcaaaga agatcgattg 180tgggagtgcg tgtgtagcac ggtgcaggct
ttcgaggagg ccgaggctgt gtcacagagc 240gtgcgggact tgctgctaca ggtgcaactg
tgtgcctccg ggtacgtacg gaaactacga 300caagtgccag tgctacgcta gcctcaccac
ccacggtgga cgccgcaagt gcccataaga 360agaaacaaag ctcttaattg ctgcggataa
tgggacgatg tcgttttgtt agtatttact 420ttggcgtata tatgtggatc gaataataaa
cgagaacgta cgttgtcgtt gtgagtgtga 480gtactgtatt attaatggtt ctatttgttt
ttacttgcaa gttttcttgt tttgaatttg 540tttttttcat atttgtatat cgattcgtgc
attattgtat tatttcaatt tgtaataaga 600ttatgttacc tttgagtggt tgtttaaaaa
aaaaaaaaaa aaa 64312484DNAArabidopsis thaliana
12aaggaagaag caggaatgta ttggggatac aaagtacgat atgcatcaca attaagttca
60gtattcaagg aatgcccttt cgagggtggt tacgattatt tgattggtac ctcggagcac
120ggcctggtaa ttagttcatc tgagctgaaa ataccaacat ttaggcacct attgattgca
180tttggtggac ttgctgggct tgaagaaagt attgaagatg ataatcagta taaggggaaa
240aacgttcgag atgtgtttaa tgtatacttg aatacttgtc cacatcaagg tagccgaacc
300attcgagcag aggaagcgat gtttatatca cttcagtact tccaggaacc catcagcagg
360gcagtgagaa gactttaagc ttcgataaaa agagtcaaag aagctatttt gttctcatag
420atctgaggtt tgtctgaaaa agagtgatgt aatgtaactg ttttagaaaa aaaaaaaaaa
480aaaa
48413688DNAArabidopsis thaliana 13agatggggaa gaagaacaag agaagtcaag
acgagtctga gctcgaattg gagccagagc 60taacgaaaat aatcgatgga gactctaaaa
agaagaaaaa taagaataag aagaagagaa 120gccatgaaga tacggagata gaaccggagc
aaaagatgag tctcgacgga gactcgaggg 180aggagaagat aaagaagaag aggaagaaca
agaaccaaga ggaggagcca gagcttgtga 240cggagaaaac gaaagtccaa gaggaggaaa
agggaaatgt agaagagggt agagccactg 300ttagcatagc catagctggt tcaatcatcc
acaacactca atcacttgag ctcgccacac 360gcgtaatctc tctttctctc tatctctccc
ttcgtttctc tgtttttcca ttcccagata 420atttaaagtc cccttcttcc atttctaaca
tttctcagct cgccggccaa attgctcgtg 480cagctacaat tttccgaatc gacgagatcg
tagtgttcga caataagagc agctcagaaa 540tcgaatcagc tgctacgaat gcttctgata
gcaatgaaag tggtgcctcc tttctcgttc 600gtatcttgaa gtatctagag acaccacaat
atttgaggaa atctctcttc cccaagcaaa 660atgatcttag atatgtgggt atgttgcc
68814461DNAArabidopsis
thalianamisc_feature(396)..(396)n = a, c, g or t 14gtcagtgctg tctggcatgg
actgtatcct ggatacatta tattctttgt gcaatcagca 60ttgatgatcg atggttcgaa
agctatttac cggtggcaac aagcaatacc tccgaaaatg 120gcaatgctga gaaatgtttt
ggttctcatc aatttcctct acacagtagt ggttctcaat 180tactcatccg tcggtttcat
ggttttaagc ttgcacgaaa cactagtcgc cttcaagagt 240gtatattaca ttggaacagt
tatacctatc gctgtgcttc ttctcagcta cttagttcct 300gtgaagcctg ttagaccaaa
gaccagaaaa gaagaataat gttgtctttt taaaaaatca 360acaacatttt ggttcttttc
tttttttcca cttggnccgt tttatgtaaa acaagagaaa 420tcaagatttg aggttttatt
cttaaaaaaa aaaaaaaaaa a 46115862DNAArabidopsis
thalianamisc_feature(292)..(292)n = a, c, g, or t 15ggtttttgaa tacatggaca
ctgatgtcaa gaaattcatc agaagtttcc gtagcactgg 60caagaacatt ccaacccaaa
ctatcaagag cttgatgtat caactatgca aaggtatggc 120attctgccat ggtcacggga
tattgcacag agatctcaag cctcacaatc tcttgatgga 180tcccaagaca atgaggctca
aaatagcaga tcttggttta gccagagcct tcactctgcc 240aatgaagaag tatacccatg
agatattaac tctttggtat agagctccag angnttcttc 300ttggtgccac ccattactct
acagctgtgg atatgtggnc tgttggctgc atatttgctg 360aacttgtgac caaccaagca
atctttcagg gagactctga gctccaacag ctcctccata 420ttttcaagtt gttgggacac
ccaatgaaga aatgtggcca ggagtgagca cactcaagaa 480ctggcatgaa tacccacagt
ggaaaccatc gactctatct ctgctgttcc aaacctcgac 540gaggctggag ttgatcttct
atctaaaatg ctgcagtacg agccagcgaa acgaatatca 600gcaaagatgg ctatggagca
tccttacttt gatgatctgc cagaaaagtc ctctctctaa 660ggatttaaaa tcttcagtta
gtatctttcc aagttttatg gtttttctag ttttgcttct 720ttcaagcata tctctagtgt
gctgcttccc cctctatgaa tcatcctttc tttagcataa 780tatatcactt ctgattgttg
tttctttcta ttcgaatatt tggattaacg gctttaatgt 840tcttaaaaaa aaaaaaaaaa
aa 862161114DNAArabidopsis
thaliana 16acccaaaaga aggatgagta tggagatgga gttgtttgtc actccagaga
agcagaggca 60acatccttca gtgagcgttg agaaaactcc agtgagaagg aaattgattg
ttgatgatga 120ttctgaaatt ggatcagaga agaaagggca atcaagaact tctggaggcg
ggcttcgtca 180attcagtgtt atggtttgtc agaagttgga agccaagaag ataactactt
acaaggaggt 240tgcagacgaa attatttcag attttgccac aattaagcaa aacgcagaga
agcctttgaa 300tgaaaatgag tacaatgaga agaacataag gcggagagtc tacgatgcgc
tcaatgtgtt 360catggcgttg gatattattg caagggataa aaaggaaatc cggtggaaag
gacttcctat 420tacctgcaaa aaggatgtgg aagaagtcaa gatggatcgt aataaagtta
tgagcagtgt 480gcaaaagaag gctgcttttc ttaaagagtt gagagaaaag gtctcaagtc
ttgagagtct 540tatgtcgaga aatcaagaga tggttgtgaa gactcaaggc ccagcagaag
gatttacctt 600accattcatt ctacttgaga caaaccctca cgcagtagtc gaaatcgaga
tttctgaaga 660tatgcaactt gtacacctcg acttcaatag cacacctttc tcggtccatg
atgatgctta 720cattttgaaa ctgatgcaag aacagaagca ggaacagaac agagtatctt
cttcttcatc 780tacacatcac caatctcaac atagctccgc tcattcttca tccagttctt
gcattgcttc 840tggaacctca ggcccggttt gctggaactc gggatccatt gatactcgct
gaccgagctt 900ctattcccaa attcttcaag aagaagaagt aatgatctaa ttggtatact
aaaaaattat 960acatctggtt tagtgttcaa ttgagagaga ctgtaaaatc aattcatagg
ccaacaaatg 1020tttgtttatc caattttcct ttttattcga acttgatgcg atatttcaac
ggaaacagaa 1080actattgttt taaaccaaaa aaaaaaaaaa aaaa
111417794DNAArabidopsis thaliana 17aagatgcaac cgacagagac
gtcgcagccg gcgccgtcgg atcaaggccg ccggcttaag 60gatcagttat cggagagtat
gagcttcagt agccaaatga agaaggaaga cgatgagttg 120tcgatgaaag ctttgtcggc
gttcaaggcc aaagaagagg agatcgagaa gaagaagatg 180gagatcagag aaagagttca
agctcagctt ggtcgtgttg aagatgagtc caagcgtctc 240gctatgattc gcgaggaact
tgaaggtttt gctgatccca tgaggaagga agttactatg 300gtgaggaaga agattgattc
tctcgacaaa gaattaaagc cattggggaa tacagttcag 360aaaaaggaaa cagagtacaa
ggatgctctt gaagcattca atgaaaagaa caaggagaag 420gtggagctga tcaccaagct
acaggagttg gagggagaaa gcgagaaatt caggttcaag 480aagctggagg agctaagcaa
gaacattgat ctaaccaaac cttagtgttg gacgagcaga 540gtcgctggga tttggctatt
caaagttcta aaaaagtcac tttttagagt attttcattg 600ttcttttatg attctagtaa
tatatataat ttataaaata aaaagtaaga agatatgtgt 660ttgaactaga tgttgcaaag
aaaatgtaac aaagttacga tggcactaca ttatcgacgt 720gattggcaga attgtaatag
taatgtaaag aaactatgtt tgttccggaa aaaaaaaaaa 780aaaaaaaaaa aaaa
79418448DNAArabidopsis
thaliana 18cagaaacaag ctccaggtgc aggtgatgtc ccagcaacaa tccaagaaga
ggacgatgat 60gatgatgtcc cagatcttgt agtgggagag actttcgaga cccctgctac
tgaagaggct 120cccaaagctg ctgcttctta gaggaggagg aagaagaagg agaagagctc
acctgcaaaa 180cccatcataa aaatgtttgt cgctcgacct cttctgagca ctgtcagatt
cttgttttct 240ctaatgcttg cgaacagaaa gacttggttt tattatcact tgatgctttt
tggtccgaac 300agcaattttc cttttattaa ggttagatcg ctttttgttt accacctgtt
caaatgagta 360ctactatgtc ctgtcgcttc atacacttct tgcaacacag tcctttgttt
tgagtcaaaa 420aaaaaaaaaa aaaaaaaaaa aaaaaaaa
448191152DNAArabidopsis thaliana 19atggaggacg acgacgagat
tcagtcaatt ccatctccgg gagattcttc cctttcacca 60caagctcctc cttctccgcc
gattttgcca acaaacgacg tgacggtggc cgtcgtgaag 120aaaccacaac cggggctttc
ttctcaatct ccgtccatga acgctttagc gttagtggtt 180catactcctt ctgtaaccgg
tggtggtggt agcggaaaca gaaacggacg aggaggagga 240ggaggaagcg gtggtggtgg
aggaggaaga gatgattgtt ggagcgaaga agctacaaag 300gttctaatcg aagcttgggg
agatcgattc tctgaaccag gtaaaggaac tttgaagcaa 360caacattgga aagaagtagc
tgagattgtg aacaagagtc gtcaatgcaa ataccctaaa 420actgatattc agtgtaagaa
cagaattgat acggtgaaga agaagtataa gcaagagaaa 480gctaagattg cttctggtga
tggacctagt aaatgggttt tcttcaagaa gcttgagagt 540ttgattggtg gtactacaac
attcattgct tcttcaaaag cttcagagaa ggctcctatg 600ggaggagctc ttgggaatag
ccgttcgagt atgtttaaac ggcaaactaa aggtaatcag 660attgtgcagc aacaacaaga
gaagagaggc tctgattcga tgcggtggca ttttaggaaa 720cgtagtgctt ctgagactga
gtctgagtct gatcctgaac ctgaggcttc tcctgaggaa 780tctgctgaga gtctcccacc
tttgcaaccg attcaaccgc tttcgtttca tatgccaaag 840cggttgaagg tggataagag
tggaggtgga gggagtggag ttggagatgt ggcgagggcg 900atacttggat ttacggaagc
ttatgagaag gcggaaactg ctaagcttaa gttaatggcg 960gaactggaaa aggagaggat
gaaatttgct aaagagatgg agttgcagag aatgcagttc 1020ttgaaaactc aattggagat
aacacagaac aatcaagaag aggaagagag gagcaggcag 1080cgaggagaaa ggaggatcgt
tgatgatgat gatgatcgca atggcaagaa taacggcaat 1140gtaagtagct ga
115220409DNAArabidopsis
thalianamisc_feature(201)..(201)n = a, c, g, or t 20cctccttctc cacgcttctt
cttcttcttc ctcaatctct cttacgattc cttcaaatca 60ttcttccatg gccaccgtat
cttcttcctc ctggccaaac cccaacccta atcccgattc 120cacgtctgcc tcagattccg
attctacttt tccctctcac cgcgatcgcg tagacgaacc 180cgactctctc gattccttct
nctccatgag tcttaactcc gacgaaccta atcagacttc 240taatcaatcg cctctttctc
cccctacgcc caatttaccg gtgatgcctc ctccgttcgt 300gctttatctt tcctttaacc
aagatcatgc ttgcttcgcc tgtnggcact gaccgtggct 360ttacngatnc ttaattgcga
tccctttcgc gagattttcc ggcgggatt 40921758DNAArabidopsis
thaliana 21gtcaggctca tgattccaga atagcttgct tcgctctcac gcaggatggc
catttgttgg 60ccactgctag ctctaagggt actctggttc ggatcttcaa tactgttgat
ggtaccttgc 120gtcaagaggt aaggaggggt gcggatagag cagagatcta cagtttggcc
ttctcttcaa 180atgctcagtg gttagctgtc tcaagtgaca aaggaacggt ccatgtcttt
ggtctcaaag 240tcaactccgg atctcaagtg aaagactcat cccgaattgc acctgatgct
actccctcat 300ccccatcgtc gtctctgtct ttattcaaag gagtgttacc gaggtatttc
agctcggagt 360ggtcggtggc tcagttcagg ttggttgaag gaactcagta catagccgcc
tttggccatc 420aaaagaacac cgttgttatt cttggcatgg atgggagctt ctacagatgc
cagtttgatc 480cggtgaacgg cggtgaaatg tctcagcttg agtaccacaa ctgtctgaaa
ccgccttcag 540ttttctaaaa gctttactac ttatactctt ttgttccttc tctctcttta
tatctctctg 600caacttaagc ggtgagatat ggtgtatagt tttgtgtata taataatgat
gggtcgtcct 660ataatttgta aaacctttta tcgctacccg ggtcgactct agagccctat
agtgagtcgt 720attactgcag agatctatga atcgtagata ctgaaaaa
75822624DNAArabidopsis thaliana 22atggactttt gtgaggtatg
cccggaaaag cttccaaact atgaagtgaa agtgaagagc 60tttttcgaag aacatttaca
cactgatgag gagatccgtt actgcgttgc aggaactggt 120tactttgatg tgagagatcg
taatgaagct tggattaggg tattggtaaa gaagggaggt 180atgatagtct tacctgctgg
gatctatcat cgcttcactg tggactctga caactatatc 240aaggcaatgc ggctattcgt
gggtgaaccg gtatggacac catacaatcg cccacacgac 300catcttcctg caaggaaaga
atatgtcgat aacttcatga tcaatgcctc ggcttagaga 360gcttcctctc tctatatctg
gctttctgaa acaaggatct ataaacaagg cctacaataa 420agaaagcttt cctgtcaagt
attggatatt tatatgtatt cctgtgtaga atgatggctt 480ttggtatgct tgagttgttg
taaacttagt tacactctct gatatgtctc tctttaccat 540ctttgtcgta tcccatatac
gaaaagatta cattgggatt catattgtct tacgttcgtt 600cctatgtgca atatgttgag
tttt 62423495DNAArabidopsis
thaliana 23ccagttttcc gatcactcgc aagaaaaccc taaaaatgga tggtcatgat
tctgaggata 60ctaagcagag cactgctgat atgactgctt ttgtccaaaa tcttctccag
cagatgcaaa 120ccaggttcca gacaatgtcg gactccatca tcacaaagat tgatgacatg
ggaggcagaa 180tcaatgagct ggagcaaagc atcaatgatc taagagccga gatgggagta
gaaggcactc 240ctcctccagc ctccaaatca ggcgatgaac ccaaaacacc ggctagttcc
tcttaaaaag 300gaatgtggtg ttcattgaca tgtccgaagg aaaaagaaaa actatgaaat
atgttaagag 360cagtattact tttaaaattc ctgtttaaga aacgagtttg ttgtttatta
aagttcatca 420aatagattga tgatgtggtg cattacatta ttctccacct atgaattgca
tttctatttt 480ggtctaaaaa aaaaa
49524580DNAArabidopsis thaliana 24cgcgcaggta cgagcaaaaa
tgctcaaaga agttgccacg gagaagcaaa ccgccgtgga 60cactcatttc gcaaccgcta
aaaagcttgc tcaagaagga gacgcgttgt tcgttaaaat 120cttcgcaatc aagaaactgt
tggcgaaact tgaagcagag aaagaatctg ttgatggaaa 180gtttaaggag actgtgaaag
aactttctca tcttctggct gatgcttctg aggcttacga 240agagtatcat ggcgcggtga
ggaaggcgaa agacgagcaa gcggctgagg aatttgcgaa 300agaggcgacg caaagtgcag
agatcatttg ggttaagttt cttagttctc tttagagaac 360aattgagatt cttggttgtg
ttaagagcaa atctagagct cttgttggtt cttgttatgt 420attttgtgat gatgttctgt
ttcagagttt gtgtgttggt tgtatcagga gaaagaggct 480gggagataga gagaaagaga
gtctctgcga aaactaataa tgttttttca gatatctaaa 540taataagctt tttacaaaaa
aaaaaaaaaa aaaaaaaaaa 58025656DNAArabidopsis
thaliana 25cggccgcgtc gacgcttgag agattcctct ggctaaaccc agatggagtt
tggatctttt 60cttgtgtcct tagggacatc ttttgttatc ttcgtcattc tcatgcttct
cttcacctgg 120ctttctcgca aatctggaaa tgctcccatt tattacccga atcggatcct
taaagggctg 180gagccatggg aaggcacctc cttgactcga aacccttttg cttggatgcg
tgaagctttg 240acttcctctg aacaagatgt cgttaactta tccggcgtcg atactgctgt
ccactttgtc 300ttcttgagca ctgttctggg gatatttgct tgttccagtc ttcttctcct
accaactcta 360ctgcctctag ccgctacaga caacaacata aagaacacaa agaatgcgac
agataccaca 420agcaaaggaa cttttagcca acttgataat ctatcaatgg ctaacatcac
aaaaaaaagt 480tcgaggctgt gggcgttcct aggagctgtt tactggatat ctttggtcac
atatttcttc 540ttgtggaaag cttataagca tgtctcttca ttgagagctc aagctctgat
gtctgctgat 600gtaaaacccg agcaattcgc tattcttgtt agggatatgc ctgcaccacc
tgacgg 65626985DNAArabidopsis thaliana 26gttcacactc cggctggtga
actgcaaaga cagattaggt catggcttgc agaaagtttt 60gagtttctct ctgttacagc
agatgatgtt tcaggagtaa ccactggcca attagagctt 120ctttccacag caattatgga
tggctggatg gctggagtag gagctccggt gcctcctcac 180acagacgctt taggacagct
tgtgtctgag tatgcaaagc gagtctacac ttctcagatg 240cagcatctaa aggatattgc
cggtactttg gcttcggaag aggcagaaga tgctggtcaa 300gtcgcgaagc ttcgatcagc
tctcgagtct gttgaccaca aaagaagaaa gattttgcaa 360caaatgagaa gtgatgcagc
tttgtttacc ttggaagaag gcagttcccc tgttcaaaat 420ccatctacag cagccgaaga
ctcgagatta gcctccctca tttctctgga tgccatactg 480aagcaagtca aggaaataac
aagacaagcc tctgtccacg ttttgagtaa aagcaagaaa 540aaggcattgc ttgagtctct
tgatgaactt aacgaacgaa tgccttctct gcttgatgtt 600gatcatccat gtgcacagag
agaaattgat acggctcacc agttggtcga gacaattcca 660gaacaagagg acaatcttca
agacgaaaag agaccttcaa tagattcaat atcttcgact 720gaaaccgatg tgtctcaatg
gaatgttttg caattcaaca caggaggctc ttcagctcca 780ttcatcataa aatgcggagc
taactccaac tcagagctcg tgatcaaagc ggatgcccgt 840attcaagaac ctaaaggagg
cgaaatagtg agagttgtgc caagaccttc ggttttagaa 900aacatgagct tagaggaaat
gaaacaagtg tttggtcagt tgcccgaagc tctaagttca 960ctggccttag ctagaacagc
tgatg 98527527DNAArabidopsis
thalianamisc_feature(512)..(512)n = a, c, g, or t 27acttatgaga ggttaccgat
tgaggaagaa caacagcaag agcagccgct tcaactagaa 60gatgggaaga agcagaaaga
agagaatgat gataacgaga gtgggaataa cggaaacgaa 120ggatcgatgc agccgccgat
gtataatatg cctcctaatt ttatcccaaa tggtcatcaa 180atggctcaac acgacgtgta
ttggggtggt cctccgcctc gtgctcctcc ttcgtattga 240ttaagttaga taggcggtgg
ttggtgcgtt ctttttactg gaatgattat attttccatt 300aggatgggta ggcttttgtt
attaaagcta tcaagtttct ttttttttac ggataattcg 360gatgacaatt agctagtgtt
tgtttgtttg ttttgtggcc ggcttttctg cttgactatt 420ttgatcgcgg atagctttgt
atgaaagtga attgattgta gaatcgtctt ttgaattttg 480atgttggaaa aaaccaagca
atggtgtgtg gnctttgcaa tggaagc 52728610DNAArabidopsis
thalianamisc_feature(482)..(482)n = a, c, g, or t 28atcaaaagct agagtcttgg
ccattcctga tgatctagca aatgtgtcat gcggtgtgga 60acagattgaa gaactgaaag
gattgaacct tgttgagaaa gatggtggtt catcttcttc 120tgacggggct aggaacacta
atcctgaaac tagaaggtac agtggttcct tgggtgtaga 180ggatggagcc tatactaatg
agatgctcca gtccatagag atggttactg atgtgctgga 240ctctcttgtg aggagggtta
cagtagcaga atctgagtct gctgtcaaaa ggagagggca 300cttttgggag aggaaagaaa
tcagtaggaa agactatcca aatcgaaaat ttgtccgtga 360agttagaaga gatggaacga
tttgcttatg ggactaatag tgttctaaac gaaatgcggg 420aaaggattga ggaattagtt
gaagagcgat gaggcagagg gaaaaagctg tggaaaacga 480anaggagttg tgtnntgtga
agagagagtc gagtcnttaa aagctcctca gtactttacc 540atgtcgagaa cactctttcn
ccggagncat tcaaaccatg aggagtnttt gacggtggca 600ctaaacnccg
61029546DNAArabidopsis
thaliana 29atgaccaata tcgccatggc tgatgctctc aaatctcttg agattgttga
tggtcttgat 60gaatacatga atcaatctga atccagtgct ccgcattctc caaccagtgt
agcaaagctg 120ccaccaagca ctgcaactag aacaactcga cggaagacca caacaaaagc
tgagcctcag 180ccatcatctc agttggtgtc ccgttcttgt cgttcgacga gcaagtctct
tgctggagat 240atggaccagg aaaacataaa caagaatgtt gctcaagaaa tgaagactag
caatgtcaag 300tttgaagcca atgtgctcaa aactccagca gcaggaagca caaggaaaac
ttcagcagca 360acttcttgca ctaagaagga tgaattggtc cagtcggtct acagcactag
gagatcaacc 420aggctgttag agaaatgtat ggccgatctg agtttgaaga ctaaagaaac
tgtggataat 480aaacctgcca agaatgaaga tacagaacag aaagtatctg cacaggagaa
gaatctaact 540ggttag
54630492DNAArabidopsis thaliana 30atgctgatgc tgtgtgggtt
cacggtcttg gatatgctaa agcaccacga ccttgggaag 60atccgagcac ccttgcatcc
tctcagaaag aagatgcaga ttcagcacgc ttaccagcag 120atacatcagg ggtcaaaact
gttgaagatg gaccggatga tgttgagagg gaccaaaaga 180aggataggcg tgaggaaagg
aaacctgcaa agagagagaa ggaagaaaga catgataggc 240gtgaaaaacg cgaaaggcat
gagaagcgaa gcgctcgtga ttcagatgat agaaagaagc 300acaagaaaga gaagaaggag
aaaaaaagaa ggcatgactc tgattctgat tgaagcgaat 360tgtcccagga tggaacattt
tgctcttcag aggaagagtg gtcggctagg taccaaaatc 420cagctaccac ttctgcaaga
tttaaatctg ttgcttattt catttacgaa tcgtggagta 480aagtgttgtt ga
49231723DNAArabidopsis
thalianamisc_feature(559)..(559)n = a, c, g, or t 31gcaaaagaga gaaacatctg
acccggaatc tgacctgaaa acccggaaga atcgaaaaat 60ggggaaagat ggtctgagcg
acgatcaggt ctcgtcgatg aaggaagcct tcatgctctt 120cgacaccgat ggcgacggca
aaatcgcacc gtcagagctc gggatcctca tgcgatctct 180cggcggaaac ccgacccaag
cccagctgaa atccataatc gcatccgaga atctctcttc 240accgtttgat ttcaacagat
tcctcgatct catggcgaaa catctgaaga cggaaccttt 300cgatcgccag ctccgtgacg
cattcaaagt gctcgataag gaaggtaccg ggttcgttgc 360tgtggcggat ctgaggcata
ttctgaccag tatcggagag aagctggagc ctaatgagtt 420cgatgagtgg atcaaggagg
tggatgttgg atccgatgga aagatccggt atgaagattt 480catagcaagg atggttgcta
agtgagatct aatcttttat gttttgaaag ttgaaatttt 540taagaagaga ttcttttgng
gttttttcac ttggttggtt tgatttcgag cgaatcctaa 600ctaggggttg gtttatcatt
gnggaatttg cttactaact ttggcttctt catggttggg 660tttcaatttt taatggnaaa
tggtggctgg gggaattcct aaaaaaaaaa aaaaaaaaaa 720aaa
72332344DNAArabidopsis
thaliana 32cgcggagtct cctttcgatc aagagaaatg cgtccgattt ttgcaatctc
tcagagaatg 60cgttctatca aagaaagtaa agaagttctc gataccgagt caagatcacg
actctgaggg 120agcagcttca gctacaaaga gaccttcata acgttctttg ttccgatttt
cttttatcgt 180ttgagttgta atcatgtaat tgattttaat gtcatgcctt ggattcataa
gctgggtcat 240gccttgtttc ccctttgttg tcttgtatgt tgaatattgc aaactctaaa
gagcatattt 300ataagaagaa ataaaagttt ctacaaaaaa aaaaaaaaaa aaaa
344331131DNAArabidopsis thaliana 33atgacaacta ctgggtctaa
ttctaatcac aaccaccatg aaagcaataa taacaacaat 60aaccctagta ctaggtcttg
gggcacggcg gtttcaggtc aatctgtgtc tactagcggc 120agtatgggct ctccgtcgag
ccggagtgag caaaccatca ccgttgttac atctactagc 180gacactactt ttcaacgcct
gaataatttg gacattcaag gtgatgatgc tggttctcaa 240ggagcttctg gtgttaagaa
gaagaagagg ggacagcgtg cggctggtcc agataagact 300ggaagaggac tacgtcaatt
tagtatgaaa gtttgtgaaa aggtggaaag caaaggaagg 360acaacttaca atgaggttgc
agacgagctt gttgctgaat ttgcacttcc aaataacgat 420ggaacatccc ctgatcagca
acagtatgat gagaaaaaca taagacgaag agtatatgat 480gctttaaacg tcctcatggc
tatggatata atatccaagg ataaaaaaga aattcaatgg 540agaggtcttc ctcggacaag
cttaagcgac attgaagaat taaagaacga acgactctca 600cttaggaaca gaattgagaa
gaaaactgca tattcccaag aactggaaga acaaagaaat 660gagcacttat atagctcagg
aaatgctccc agtggcggtg ttgctcttcc ttttatcctt 720gtccagactc gtcctcacgc
aacagtagaa gtggagatat cagaagatat gcagctcgtg 780cattttgatt tcaacagcac
tccatttgag ctccacgacg acaattttgt cctcaagact 840atgaagtttt gtgatcaacc
gccgcaacaa ccaaacggtc ggaacaacag ccagctggtt 900tgtcacaatt tcacgccaga
aaaccctaac aaaggcccca gcacaggtcc aacaccgcag 960ctggatatgt acgagactca
tcttcaatcg caacaacatc agcagcattc tcagctacaa 1020atcattccta tgcctgagac
taacaacgtt acttccagcg ctgatactgc tccagtgaaa 1080tccccgtctc ttccagggat
aatgaactcc agcatgaagc cggagaattg a 113134631DNAArabidopsis
thaliana 34agagtatctg aagaaagggt caccaataag cgcgctcaaa agtttcatct
cgtctctctc 60tgaacctcct caagacatca tggacgcact cttcaatgct ctctttgatg
gtgtgggaaa 120gggattcgcc aaagaagtga ctaagaagaa gaattactta gcggctgctg
caacaatgca 180agaggatgga tcacagatgc atctgctcaa ttcgattggg acattctgtg
gaaagaatgg 240aaacgaagaa gctttgaaag aggtggctct ggttcttaaa gcattgtacg
accaagacat 300cattgaggaa gaggtagtgt tggattggta cgaaaagggt ctcaccggag
ctgacaaaag 360ctcgccggtt tggaagaatg ttaagccttt tgtggagtgg cttcagagcg
ctgagtctga 420gtccgaagag gaggattgag tcactttttt cttccctcct aacttttctt
tgcggcattt 480cttataatac ttcgtcagtt ttcagaattc ttaaatcttt ttgctgtgtt
cttataaaga 540aacatcatct attaaagttg tcttcgtttg gatttggttt tgacgacttt
gggaaatatt 600tatgtttaag aaaaaaaaaa aaaaaaaaaa a
631351333DNAArabidopsis thaliana 35gctggaggta gagaggaatg
cgtctgctgt tgctgccagt gaaacaatgg cgatgatcaa 60taggttgcat gaggagaaag
ctgcgatgca gatggaagcg ttgcagtatc agagaatgat 120ggaggagcaa gctgagtttg
atcaagaagc tttgcagttg ttgaatgagc ttatggtgaa 180tagagagaag gagaatgctg
agcttgagaa ggagctagag gtgtatagaa agagaatgga 240ggagtatgaa gctaaagaga
aaatggggat gttgaggagg agattgagag attcctctgt 300tgattcgtat agaaataatg
gcgattctga tgagaatagc aatggagagt tacagtttaa 360gaacgttgaa ggggttacgg
attggaaata tagagagaat gagatggaga atacgccggt 420ggatgttgta cttcgtcttg
atgagtgttt agatgattat gatggagaga ggctttcgat 480tcttgggaga ttgaagtttc
ttgaagagaa actcacagat cttaataacg aagaggacga 540cgaggaggag gctaaaacgt
ttgagagtaa tggtagcatc aatggaaatg agcatattca 600tggcaaagaa acaaacggga
agcacagagt tatccagtca aaaagattac ttcccctgtt 660tgatgcggtc gatggagaga
tggaaaacgg gttaagtaac ggaaaccatc acgaaaacgg 720gtttgatgat tcggagaagg
gtgagaatgt gacgatagaa gaagaagtgg atgagcttta 780cgagaggtta gaagctctag
aggcagatag agagttctta agacattgtg ttggttcatt 840gaaaaaagga gacaaaggtg
tacatctcct ccatgagatt ctgcaacatc ttcgtgatct 900aaggaatatc gatcttactc
gcgtcagaga aaacggagac atgagtttat gagtttgatt 960ttgagttttg ggtttgagtc
cactctttgc atagtgaccc aaagaacaag aaaaatcata 1020caggtatgga agtgacatgt
tgcttgtgag gcaaggaaca acgacaaggt ttcagatgaa 1080gaagaaaacg ttctcagaat
aaaagtattt taagtatata ctctgaggaa aagtgtcaga 1140tcagaatgtt cgtctttctt
cgttcatttt cattattata agttttgttt tttatattga 1200agatttattt agagagaggg
aagtgtcagt ataatttcac ttttatattt tatatttggg 1260agttgtcttt atgagtggtg
gtaatagaaa aaggtagaat gatgagtgaa gaaaaaaaaa 1320aaaaaaaaaa aaa
1333362289DNAArabidopsis
thaliana 36cttatgcaaa ctctcagcag attctgatgc caatgtccaa agtgctgctc
atcttttgga 60tcgccttgtt aaggatattg tgacggaaag tgatcagttc agtattgagg
aattcatacc 120tcttttaaaa gagcgaatga acgttctcaa cccttacgtc cggcaatttc
tggttggatg 180gatcactgtt cttgatagtg ttccagacat tgacatgctt gggtttctgc
cagactttct 240cgatgggtta ttcaatatgt tgagcgactc tagtcatgaa atacgacagc
aagctgattc 300agctctttca gagtttcttc aagagataaa aaattcacca tctgtagatt
atggtcgcat 360ggctgaaata ctggtgcaga gggctgcttc tcctgatgaa ttcactcgat
taacagccat 420cacgtggata aacgagttcg taaaacttgg gggagaccag ctcgtgcgtt
attatgctga 480cattcttggg gctatcttgc cttgcatatc tgacaaagaa gagaaaatca
gggtggttgc 540tcgtgaaacc aatgaagaac ttcgttcaat ccatgttgaa ccctcagatg
gttttgatgt 600tggcgcaatt ctctctgttg caaggaggca gctatcaagt gagtttgagg
ctactcggat 660tgaagcattg aattggatat caacactttt aaacaagcat cgtactgagg
tcttgtgctt 720cctgaatgac atatttgaca cccttctaaa gcactatctg attcttctga
tgacgtggtg 780ctcttggttc tggaggttca tgctggtgta gcaaaagatc cacaacactt
tcgccagctc 840atcgtatttc ttgtccacaa tttccgagct gataattctc ttttggaaag
gcgcggtgcc 900cttattgtcc gaagaatgtg tgtacttttg gatgccgaaa gagtctaccg
agagctctct 960acaattcttg agggagaaga taatcttgac tttgcttcta ccatggttca
ggcattgaat 1020ttgattttgc ttacttcccc ggagttatcg aaactgagag aactattaaa
aggttcactc 1080gtcaatcgcg aagggaaaga acttttcgtt gccttgtata cttcatggtg
ccattcaccc 1140atgggcaatt ataagcctct gcttattagc tcaggcttac caagcatgcg
agtgtcgtaa 1200tccaatcctt ggtaaaagaa gacattaacg tccaaatttc ttaggccagc
ttgataaaat 1260tgatccggct tctggaaact ccaatcttta cttaccttag attgcagctt
ctggaaccag 1320gaaggtacac atggttgctg aaaacacttt atggtcttct tatgttactt
cctcagcaaa 1380gtgcggcgtt caagatactt aggacaagac tcaaaactgt gccaacgtac
tcattcagta 1440ctggaaacca aataggcaga gcaacttcag gagttccttt ctctcagtat
aagcatcaaa 1500acgaggacgg tgacttagaa gacgataaca tcaacagttc tcaccaagga
atcaattttg 1560ctgtgcggct acaacagttc gaaaacgtac agaatctaca tcgtggccag
gcaaggacta 1620gagtgaacta ctcatatcac tcttcctctt cttctacatc aaaggaggtg
aggagatctg 1680aagaacaaca acagcagcag cagcaacaac aacagcaaca acaacaacaa
caacgaccac 1740caccttcttc gacatcatca tcagttgcag ataacaatag acctccatca
agaacttcaa 1800gaaaaggccc tggtcaatta cagctttaac ctacctggta atcataaata
ataaataata 1860ttccatcccc gacaatcatc atcttcatct tctttgtgtg gacaccaccg
atcccttttg 1920tctcctgtaa aattgtatat ctctcttttt tagtaactct tcaagtttcg
acggaacttg 1980tggaaaagct acggtcgtgt ccatcatctc tttctctctg tcgggttttt
tttatttacg 2040agagattctt cttcagtccc tcagtctacc tttatattgt ttttttgggg
gtttctcgtt 2100tctttgaatt tgtttcattg tttggagctt tttatatttt taccttatgt
ggagatgtaa 2160gaaaaagaag tgatcatgtg gttttgtgtt gtttttttat aactggaaaa
ccacatgagt 2220ttgtagaggt cacttattgg atattttatg tcaaatgatg ctccttttta
caaaaaaaaa 2280aaaaaaaaa
2289371094DNAArabidopsis thaliana 37cttgattgaa acttcagttg
aatccaagga aacgactgaa tcagtggtta caggtgaatc 60ggagaaagcg attgaagata
tttcaaaaga agctgacaat gaggaggatg atgatgagga 120ggaacaagaa ggagatgagg
atgatgatga aaatgaagag gaagaagtgg ttgttccaga 180aactgagaat cgagcagaag
gagaagattt agtgaagaat aaggcagctg acgcgaagaa 240gcatcttcaa atgattggag
tccaactctt gaaagaatcc gatgaagcaa acagaacaaa 300gaaacgtggg aagagggcat
ctcgtatgac acttgaggat gatgcagatg aggattggtt 360ccctgaggaa ccatttgaag
cattcaaaga aatgagggaa agaaaagtgt tcgatgtggc 420tgacatgtat acaatagcag
acgtttgggg ttggacatgg gagaaggatt ttaagaacaa 480aactccaagg aaatggtcac
aagagtggga agtcgagttg gcaattgtgc tcatgacaaa 540ggtgattgaa ttgggtggaa
ttccaacgat tggtgattgt gcagtgatat tacgagctgc 600tttaagagct cccatgcctt
cagccttctt gaagatcttg cagacgacac acagtcttgg 660ctactcattt ggcagcccgt
tgtacgatga gatcatcaca ttgtgtttgg accttggaga 720acttgatgca gccatcgcca
tagttgcaga tatggaaacc acagggatca ctgtccctga 780tcaaaccctt gacaaggtca
tatctgctag acaatctaat gagagtccgc ggtctgagcc 840tgaagagcca gcatcaacag
taagctctta gttatcatat cctcttctgc ttgttgtgaa 900gtctctataa gaaacagaaa
tcggtagaag gagctgaatc tgtcttagtt atgaaagttt 960tgttcattat aagtacaagt
catgtagttc cgagtgtaga acagttttta ctagtgttgc 1020accaggtccc tccagtctga
tacttaattc tttagtgttg gatctttcta tataagaaaa 1080aaaaaaaaaa aaaa
1094381204DNAArabidopsis
thaliana 38aaccgattca gcctccgaca gtatattcca ctacgacgac gcttcacaag
ccaaaatcca 60gcaggagaag ccatgggcct ccgatcctaa ctacttcaag cgcgttcaca
tctcagccct 120tgctcttctc aagatggtgg ttcacgctcg ctccggtggc acaatcgaga
tcatgggtct 180tatgcagggt aaaaccgagg gtgatacaat catcgttatg gatgcttttg
ctttgcctgt 240tgaaggtact gagactaggg ttaatgctca gtctgatgcc tatgagtata
tggttgaata 300ctctcagacc agcaagctgg ctgggaggtt ggagaacgtt gttggatggt
atcactctca 360ccctgggtat ggatgttggc tctcgggtat tgatgtttcg acacagatgc
ttaaccaaca 420gtatcaggag ccattcttag ctgttgttat tgatccaaca aggactgttt
cggctggtaa 480ggttgagatt ggggcattca gaacatatcc agagggacat aagatctcgg
atgatcatgt 540ttctgagtat cagactatcc ctcttaacaa gattgaggac tttggtgtac
attgcaaaca 600gtactactca ttggacatca cttatttcaa gtcatctctc gatagtcacc
ttctggatct 660cctttggaac aagtactggg tgaacactct ttcttcttcc ccactgttgg
gcaatggaga 720ctatgttgcc gggcaaatat cagacttggc tgagaagctc gagcaagcgg
agagtcagct 780cgctaactcc cggtatggag gaattgcgcc agccggtcac caaaggagga
aagaggatga 840gcctcaactc gcgaagataa ctcgggatag tgcaaagata actgtcgagc
aggtccatgg 900actaatgtca caggttatca aagacatctt gttcaattcc gctcgtcagt
ccaagaagtc 960tgctgacgac tcatcagatc cagagcccat gattacatcg tgaagttggt
ctattctttt 1020gttttttggc tgcggaaatt gactatcggt ttgacccggt ttatgaggca
atgcccattg 1080ttccctatat ctctagtgta gtatctgctt cagacaaaga tctttgggtt
attaaatgac 1140attaacataa atcgatcatt atgtttttgc gttaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1200aaaa
1204391715DNAArabidopsis thaliana 39cttttaagtt gggggatgtt
tcgattttga aatttgattt cttcaagaga agagatttaa 60tgaaaataaa taacttccgc
agataacgaa gaagaagaaa atggttagat cagatgaaaa 120tagccttgga ttaatcggat
caatgagtct ccaaggtacc ctaaatcgat cgattttgtt 180attaaaaatc aaaactttcg
ttctctttga tttttccccc aaattgattt tgaatttact 240tgatgtaggg ggaggagtag
taggaaagat caagacgacg gcaacaacag gaccgacaag 300aagagcacta agtactatta
acaagaacat cactgaagcg ccgtcttacc cttatgctgt 360caacaagaga tcagtttctg
aaagagatgg catttgtaat aaaccacctg tgcatcgacc 420agttactagg aagtttgctg
ctcagttagc agatcataag ccacatatcc gtgatgagga 480aactaagaaa ccagactcag
tttcaagtga agaaccagag acgattatca ttgatgtgga 540tgaaagtgat aaagaaggag
gtgactctaa tgagccaatg tttgtacaac atactgaagc 600aatgctggag gagattgaac
agatggagaa ggagattgaa atggaagatg cagacaaaga 660agaagagcct gtgatcgata
ttgatgcctg tgataagaat aatcctttgg ctgcggttga 720atatatccat gatatgcata
ccttctacaa gaattttgag aaacttagtt gcgtgcctcc 780taactatatg gacaatcaac
aagatcttaa tgagagaatg agaggaatcc tcattgactg 840gttaattgag gtgcactaca
agtttgaact gatggaggaa actctttatc tcacaatcaa 900tgtcatcgac agattccttg
cggttcatca aatcgtgagg aaaaagcttc agcttgttgg 960tgttactgct ttgttgcttg
catgtaaata tgaagaagtt tcagttccag tggtagatga 1020tctcatcttg atctctgaca
aagcttactc tagaagagaa gtgctagata tggagaagct 1080aatggccaac accttgcaat
tcaatttctc tctaccaact ccatatgttt tcatgaaacg 1140atttctcaaa gctgcccaat
ctgacaagaa gcttgagatt ttatcattct ttatgatcga 1200gctttgcctt gtggagtatg
agatgctaga gtatcttcca tctaagctgg cggcctcagc 1260aatctacact gctcagtgta
cacttaaggg atttgaagaa tggagcaaaa cctgtgagtt 1320tcacacaggc tacaacgaaa
aacagctact ggcatgtgcg agaaagatgg ttgctttcca 1380tcacaaggca ggaacaggga
agctcacagg agttcacaga aagtacaaca catctaagtt 1440ctgtcatgct gcaagaactg
aaccagctgg gtttctgatt taatattaat aagaatctaa 1500tatgacttaa ctcgagtttt
tctttagaac aaaaagagtg tgagagaaag agagatagta 1560gagcaagttg cccaaaatgg
gagaagaatg gatctttaga tatcatggca agtagcccaa 1620aaagagtgta ttcttctctt
tctaaggtct ttagatcttt cttcacttga gagagaataa 1680aaagaatctt ctgaaaaaaa
aaaaaaaaaa aaaaa 1715402195DNAArabidopsis
thaliana 40aacccacgtc aattcttttt caaaggcata tattctctct gtttcaaact
ttgtgtctct 60tcttctcctt ctctgatcgt tcgttttctg gacgagagag atggtaaatc
cgggtcacgg 120aagaggaccc gattcgggta ctgctgctgg tgggtcaaac tccgacccgt
ttcctgcgaa 180tcttcgagtt cttgtcgttg atgatgatcc aacttgtctc atgatcttag
agaggatgct 240tatgacttgt ctctacagag taactaaatg taacagagca gagagcgcat
tgtctctgct 300tcggaagaac aagaatggtt ttgatattgt cattagtgat gttcatatgc
ctgacatgga 360tggtttcaag ctccttgaac acgttggttt agagatggat ttacctgtta
tcatgatgtc 420tgcggatgat tcgaagagcg ttgtgttgaa aggagtgact cacggtgcag
ttgattacct 480catcaaaccg gtacgtattg aggctttgaa gaatatatgg caacatgtgg
tgcggaagaa 540gcgtaacgag tggaatgttt ctgaacattc tggaggaagt attgaagata
ctggcggtga 600cagggacagg cagcagcagc atagggagga tgctgataac aactcgtctt
cagttaatga 660agggaacggg aggagctcga ggaagcggaa ggaagaggaa gtagatgatc
aaggggatga 720taaggaagac tcatcgagtt taaagaaacc acgcgtggtt tggtctgttg
aattgcatca 780gcagtttgtt gctgctgtga atcagctagg cgttgacaaa gctgttccta
agaagatctt 840agagatgatg aatgtacccg ggctaacgcg agaaaacgta gccagtcacc
tccagaagta 900tcggatatat ctgagacggc ttggaggagt atcgcaacac caaggaaata
tgaaccattc 960gtttatgact ggtcaagatc agagttttgg acctctttct tcgttgaatg
gatttgatct 1020tcaatcttta gctgttactg gtcagctccc tcctcagagc cttgcacagc
ttcaagcagc 1080tggtcttggc cggcctacac tcgctaaacc agggatgtcg gtttctcccc
ttgtagatca 1140gagaagcatc ttcaactttg aaaacccaaa aataagattt ggagacggac
atggtcagac 1200gatgaacaat ggaaatttgc ttcatggtgt cccaacgggt agtcacatgc
gtctgcgtcc 1260tggacagaat gttcagagca gcggaatgat gttgccagta gcagaccagc
tacctcgagg 1320aggaccatcg atgctaccat ccctcgggca acagccgata ttgtcaagca
gcgtttcaag 1380aagaagcgat ctcactggtg cgctggcggt tagaaacagt atccccgaga
ccaacagcag 1440agtgttacca actactcact cggtcttcaa taacttcccc gcggatctac
ctcgcagcag 1500cttcccgttg gcaagtgccc cagggatttc agttccagta tcagtttctt
accaagaaga 1560ggtcaacagc tcggatgcaa aaggaggttc atcagctgct actgctggat
ttggtaaccc 1620aagctacgac atatttaacg attttccgca gcaccaacag cacaacaaga
acatcagcaa 1680taaactaaac gattgggatc tgcggaatat gggattggtc ttcagttcca
atcaggacgc 1740agcaactgca accgcaaccg cagcattttc cacttcggaa gcatactctt
cgtcttctac 1800gcagagaaaa agacgggaaa cggacgcaac agttgtgggt gagcatgggc
agaacctgca 1860gtcaccgagc cggaatctgt atcatctgaa ccacgttttt atggacggtg
gttcagtcag 1920agtgaagtca gaaagagtgg cggagacagt gacttgtcct ccagcaaata
cattgtttca 1980cgagcagtat aatcaagaag atctgatgag cgcatttctc aaacaggaag
gcatcccatc 2040cgtagataac gagttcgaat ttgacggata ctccatcgat aatatccagg
tctgactaca 2100gaaactcaga ctagactgca agattctttg tttttcttct ccctccttcg
aggtacaaag 2160ctcaaaacat ggcaataacc gaagggaaag ataga
2195411413DNAArabidopsis thaliana 41aggctgtgtt ttatcgtggg
atttttaaac atggggaagg aaaatgctgt gtctcggcca 60ttcactcgtt cccttgcctc
tgctttgcgc gcttcagaag tgacttctac tacacagaat 120caacagagag taaacacaaa
aagaccagcc ttggaggata caagagccac tggacccaac 180aagaggaaga agcgagcggt
tctaggggag atcacaaatg ttaactccaa tacagctata 240cttgaggcca aaaacagcaa
gcagataaag aaaggacgcg gtcatggatt ggcgagtaca 300tcccagttgg caacttctgt
tacttcagaa gtcacagatc ttcagtccag gaccgatgca 360aaagttgaag ttgcatcaaa
tacagcagga aacctttctg tttctaaagg cacagataac 420acagctgata actgtattga
gatatggaat tctagattgc ctccaagacc tcttgggaga 480tcagcttcta cagctgagaa
aagtgctgtt attggtagtt caactgtacc ggatatccca 540aaatttgtag acatcgattc
agatgacaag gatcctttac tgtgctgcct ctatgcccct 600gaaatccact acaatttgcg
tgtttcagag cttaaacgca gaccacttcc ggactttatg 660gagagaatac agaaggatgt
cacccagtcc atgcggggaa ttctggttga ttggcttgtg 720gaggtctctg aagaatacac
acttgcatct gacactctct acctcacagt gtatctcata 780gactggttcc tccatggaaa
ctacgtgcaa agacagcaac ttcaactgct cggcatcact 840tgcatgctaa ttgcctcgaa
gtatgaggaa atctctgctc cacgcattga ggagttttgc 900ttcattacgg ataacaccta
cacaagagat caggtcctgg aaatggagaa ccaagtactt 960aagcatttta gctttcaaat
atacactccc actccaaaaa cgttccttag gagatttctc 1020agagcagctc aagcctctcg
cctgagccca agccttgaag tcgagtttct agccagctat 1080ctaacagagt tgacattaat
agactaccat ttcttaaagt ttcttccttc cgttgttgct 1140gcttcagcgg gttttctcgc
caagtggaca atggaccaat caaaccaccc atggaatcca 1200acacttgagc attacacaac
gtacaaagca tcggatctga aagcatctgt tcatgcctta 1260caagatctgc agcttaacac
caaaggttgc cccttgagcg ctatacgcat gaagtatagg 1320caagagaaat acaaatctgt
ggcggttctc acgtctccaa agctacttga cacgctattc 1380tgaaggtttc aactcctaac
cgataatagt ttt 1413422766DNAArabidopsis
thaliana 42atttgagagg aagctttatt ttgtgtgtag atggcgaata atcctccgca
gtcttctggt 60acccagggtc agcattttgt tcctgcagct tcacaacctt ttcaccctta
tggacatgta 120cctccaaatg ttcaaagtca gcctccacag tattctcagc cgatacagca
gcagcagctc 180tttccagtga gaccaggtca gcctgtgcat attacatcat cctcacaggc
tgtatcagtt 240ccgtatattc aaacgaacaa gattctcact tctggatcta ctcaaccaca
gccaaatgca 300cctccaatga cgggctttgc tacatctgga cctccatttt cttctccata
tacttttgta 360ccatcatctt atcctcagca acaaccaaca tccttggtcc aaccaaattc
tcagatgcat 420gtagctggcg tccctccagc agcaaacact tggcctgttc ctgttaatca
aagcacatca 480cttgtttccc ctgtgcagca gactgggcaa caaacaccgg tcgcagtttc
cacagaccca 540ggaaacttga ctccgcaatc tgcatctgac tggcaggagc atacatctgc
tgatgggaga 600aaggctgatg catccactgt atggaaggaa tttacaacac ctgaaggaaa
gaaatattat 660tataacaagg ttacaaagga gtctaagtgg acaattccgg aagatttaaa
gttagctcgg 720gaacaagccc aactagctag tgaaaaaacg tccctttcgg aagctggatc
tacccctcta 780tcccaccatg ctgcatcctc gtctgatcta gcagttagca ctgtgacttc
tgttgttccc 840agcacatctt cagcacttac tggacattct tcaagcccta ttcaagcggg
tttggctgta 900cctgtcaccc gtcctccctc tgttgctcct gttactccaa catctggtgc
aattagtgac 960actgaggcta ctacaatgta ctatttttcc ttgggaagtt ttgctgagaa
taaggaaatg 1020tctgtgaatg gaaaagccaa tttgtcacct gctggtgaca aagcaaatgt
cgaggaacct 1080atggtatatg ctactaagca ggaggccaaa gctgctttca agtctctttt
ggaatctgta 1140aatgttcatt ccgactggac atgggaacag acattgaaag agattgttca
cgataaaaga 1200tatggtgctt tgaggacact cggcgagcgg aaacaagcgt ttaacgagta
tcttggccaa 1260aggaaaaaag tggaagctga ggaaagacga aggaggcaga agaaagctcg
ggaagaattt 1320gtcaagatgc tagaggagtg tgaagaactt tcatcatccc tgaaatggag
caaagcaatg 1380agtttgttcg aaaatgatca gcgttttaaa gctgttgacc gtcctaggga
tcgtgaagat 1440ctttttgaca attacattgt ggaacttgag aggaaggaaa gagaaaaggc
agcggaggaa 1500catcggcagt atatggcaga ctatcggaag tttcttgaaa cctgtgacta
tatcaaagct 1560ggtacacaat ggcgcaaaat tcaagataga ctggaggatg atgacagatg
ctcatgtctt 1620gaaaagatag atcgtctgat tggttttgag gaatacattc ttgacctaga
gaaggaagaa 1680gaagagctga agagagtaga gaaagaacat gtaaggcggg ccgagagaaa
aaaccgtgat 1740gcatttcgta cactattgga agaacatgtt gctgcaggca tccttacagc
caagacgtac 1800tggttggatt attgcattga gttaaaagac ttgccccaat accaagctgt
tgcatctaat 1860acatctggtt caactccgaa agacttgttt gaagatgtca cagaagaatt
agagaagcag 1920tatcatgagg ataagagcta tgtgaaggat gctatgaagt caagaaaggc
aaattttaaa 1980tctgctattt cagaagatct cagtactcaa cagatatcag acataaattt
aaagcttata 2040tatgatgact tggttgggag agtgaaggaa aaagaagaaa aagaggccag
aaagcttcag 2100cgtctggctg aagaatttac caatctgttg cacactttca aggaaatcac
cgtagcttca 2160aattgggaag atagcaaaca actagtagaa gaaagtcaag agtacagatc
gattggagat 2220gaaagtgtta gccaagggtt atttgaggaa tacataacga gtttacaaga
aaaggcaaag 2280gagaaggagc gtaagcgtga cgaggaaaag gttagaaaag agaaggaaag
ggacgagaaa 2340gagaaacgga aagacaagga taaggagaga agggaaaagg aaagagaacg
tgaaaaagag 2400aagggaaaag agaggagtaa acgggaagaa tcagatggtg agactgctat
ggatgtgagc 2460gaaggtcata aagacgagaa aagaaaggga aaagatcgtg acagaaaaca
tcgaagacgg 2520catcacaaca attctgatga agatgttagt tctgataggg atgacagaga
tgagtcgaag 2580aaatcatccc gtaaacatgg taatgatcgc aaaaaatcaa gaaagcacgc
aaactcgcct 2640gaatcggaga gtgaaaaccg gcataaaaga cagaaaaaag agagtagtcg
ccgaagtggt 2700aatgacgagc tagaggatgg agaagttggg gagtgatagt gaaattcgac
attaatctga 2760aacctt
2766431260DNAArabidopsis thaliana 43tgaaacctag atttctgcaa
ctgaattcct aattcgaaaa agaatggagg gttcgtcgtc 60gacgatagca aggaagacat
gggaactaga gaacagcatt ctaacagtag actcacctga 120ttcaacctcc gacaacatct
tctactacga cgatacttca cagactaggt tccagcaaga 180gaaaccgtgg gagaatgatc
ctcactactt taaacgagtc aagatctcag cgctcgctct 240tcttaagatg gtggttcacg
ctcgctctgg tggtacaatt gaaataatgg gtcttatgca 300aggtaagacc gatggtgata
ctatcattgt tatggatgct tttgctttac cagtggaagg 360tactgagaca agggttaatg
ctcaggatga tgcttatgag tacatggttg agtattcaca 420gaccaacaag ctcgcggggc
ggctggagaa tgttgttgga tggtatcact ctcaccctgg 480atatggatgc tggctctccg
gtattgatgt ttctacgcag aggcttaacc aacagcatca 540ggagccattt ttagctgttg
ttattgatcc cacaaggact gtttcagctg gtaaggttga 600gattggtgct ttcagaacat
actctaaagg atataagcct ccagatgaac ctgtttctga 660gtatcaaact attcctttaa
ataagattga ggactttggt gttcactgca aacagtacta 720ttcattagat gtcacttatt
tcaagtcatc tcttgattct caccttctgg atctactatg 780gaacaagtac tgggtgaaca
ctctttcttc ttctccactg ctgggtaatg gagactatgt 840tgctggacaa atatcagact
tagctgagaa gcttgagcaa gccgagagtc atctggttca 900gtctcgcttt ggaggagttg
tgccatcatc ccttcataag aaaaaagagg atgagtctca 960actaactaag ataactcggg
atagcgcaaa gataactgtg gaacaggtcc atggactaat 1020gtcgcaggtc ataaaagatg
aattattcaa ctcaatgcgt cagtccaaca acaaatctcc 1080cactgactcg tcggatccag
accctatgat tacatattga agttgctctt cttttggttt 1140ctagttttgg attgacccat
catttgttgt cctttcattt attttctgtt gtgtaaagaa 1200ttataatgct aatcagaata
atacagaaga agattttggt taaaaaaaaa aaaaaaaaaa 126044653DNAArabidopsis
thaliana 44cttaaactac atttatcatt acagtctgat ttgagctaag ttctctcatc
ataaactctc 60cttggagaat catggctatt tcaaaagctc ttatcgcttc tcttctcata
tctcttcttg 120ttctccaact cgtccaggct gatgtcgaaa actcacagaa gaaaaatggt
tacgcaaaga 180agatcgattg tgggagtgcg tgtgtagcac ggtgcaggct ttcgaggagg
ccgaggctgt 240gtcacagagc gtgcgggact tgctgctaca ggtgcaactg tgtgcctccg
ggtacgtacg 300gaaactacga caagtgccag tgctacgcta gcctcaccac ccacggtgga
cgccgcaagt 360gcccataaga agaaacaaag ctcttaattg ctgcggataa tgggacgatg
tcgttttgtt 420agtatttact ttggcgtata tatgtggatc gaataataaa cgagaacgta
cgttgtcgtt 480gtgagtgtga gtactgtatt attaatggtt ctatttgttt ttacttgcaa
gttttcttgt 540tttgaatttg tttttttcat atttgtatat cgattcgtgc attattgtat
tatttcaatt 600tgtaataaga ttatgttacc tttgagtggt tgtttaaaaa aaaaaaaaaa
aaa 653451266DNAArabidopsis thalianamisc_feature(757)..(761)n =
a, c, g, or t 45agatggggaa gaagaacaag agaagtcaag acgagtctga gctcgaattg
gagccagagc 60taacgaaaat aatcgatgga gactctaaaa agaagaaaaa taagaataag
aagaagagaa 120gccatgaaga tacggagata gaaccggagc aaaagatgag tctcgacgga
gactcgaggg 180aggagaagat aaagaagaag aggaagaaca agaaccaaga ggaggagcca
gagcttgtga 240cggagaaaac gaaagtccaa gaggaggaaa agggaaatgt agaagagggt
agagccactg 300ttagcatagc catagctggt tcaatcatcc acaacactca atcacttgag
ctcgccacac 360gcgtaatctc tctttctctc tatctctccc ttcgtttctc tgtttttcca
ttcccagata 420atttaaagtc cccttcttcc atttctaaca tttctcagct cgccggccaa
attgctcgtg 480cagctacaat tttccgaatc gacgagatcg tagtgttcga caataagagc
agctcagaaa 540tcgaatcagc tgctacgaat gcttctgata gcaatgaaag tggtgcctcc
tttctcgttc 600gtatcttgaa gtatctagag acaccacaat atttgaggaa atctctcttc
cccaagcaaa 660atgatcttag atatgtgggt atgttgccgg gtatgttgcc acctcttgat
gctcctcacc 720atctgcgtaa gcacgagtgg gaacaatacc gtgaagnnnn nattgttcca
ccctctaagc 780caagggaaga agcaggaatg tattggggat acaaagtacg atatgcatca
caattaagtt 840cagtattcaa ggaatgccct ttcgagggtg gttacgatta tttgattggt
acctcggagc 900acggcctggt aattagttca tctgagctga aaataccaac atttaggcac
ctattgattg 960catttggtgg acttgctggg cttgaagaaa gtattgaaga tgataatcag
tataagggga 1020aaaacgttcg agatgtgttt aatgtatact tgaatacttg tccacatcaa
ggtagccgaa 1080ccattcgagc agaggaagcg atgtttatat cacttcagta cttccaggaa
cccatcagca 1140gggcagtgag aagactttaa gcttcgataa aaagagtcaa agaagctatt
ttgttctcat 1200agatctgagg tttgtctgaa aaagagtgat gtaatgtaac tgttttagaa
aaaaaaaaaa 1260aaaaaa
1266461520DNAArabidopsis thalianamisc_feature(1455)..(1455)n =
a, c, g, or t 46atggaattgc ttgacatgaa ctcaatggct gcctcaatcg gcgtctccgt
cgccgttctc 60cgtttcctcc tctgtttcgt cgcaacgata ccaatctcat ttttatggcg
attcatcccg 120agtcgactcg gtaaacacat atactcagct gcttctggag ctttcctctc
ttatctctcc 180tttggcttct cctcaaatct tcacttcctt gtcccaatga cgattggtta
cgcttcaatg 240gcgatttatc gacccttgtc tggattcatt actttcttcc taggcttcgc
ttatctcatt 300ggctgtcatg tgttttatat gagtggtgat gcttggaaag aaggaggaat
tgattctact 360ggagctttga tggtattaac actgaaagtg atttcgtgtt cgataaacta
caacgatgga 420atgttgaaag aagaaggtct acgtgaggct cagaagaaga accgtttgat
tcagatgcct 480tctcttattg agtactttgg ttattgcctc tgttgtggaa gccatttcgc
tggcccggtt 540ttcgaaatga aagattatct cgaatggact gaagagaaag gaatttgggc
tgtttctgaa 600aaaggaaaga gaccatcgcc ttatggagca atgattcgag ctgtgtttca
agctgcgatt 660tgtatggctc tctatctcta tttagtacct cagtttccgt taactcggtt
cactgaacca 720gtgtaccaag aatggggatt cttgaagaga tttggttacc aatacatggc
gggtttcacg 780gctcgttgga agtattactt tatatggtct atctcagagg cttctattat
tatctctggt 840ttgggtttca gtggttggac tgatgaaact cagacaaagg ctaaatggga
ccgcgctaag 900aatgtcgata ttttgggggt tgagcttgcc aagagtgcgg ttcagattcc
gcttttctgg 960aacatacaag tcagcacatg gctccgtcac tacgtatatg agagaattgt
gaagcccggg 1020aagaaagcgg gtttcttcca attgctagct acgcaaaccg tcagtgctgt
ctggcatgga 1080ctgtatcctg gatacattat attctttgtg caatcagcat tgatgatcga
tggttcgaaa 1140gctatttacc ggtggcaaca agcaatacct ccgaaaatgg caatgctgag
aaatgttttg 1200gttctcatca atttcctcta cacagtagtg gttctcaatt actcatccgt
cggtttcatg 1260gttttaagct tgcacgaaac actagtcgcc ttcaagagtg tatattacat
tggaacagtt 1320atacctatcg ctgtgcttct tctcagctac ttagttcctg tgaagcctgt
tagaccaaag 1380accagaaaag aagaataatg ttgtcttttt aaaaaatcaa caacattttg
gttcttttct 1440ttttttccac ttggnccgtt ttatgtaaaa caagagaaat caagatttga
ggttttattc 1500ttaaaaaaaa aaaaaaaaaa
1520471142DNAArabidopsis thaliana 47ttatataacc tatctacact
ttgatctccg acaattcact ttcccaataa gaaccaactg 60agagagagag agcgccggag
aagaagaatt ttagagagcg atggacgagg gagttatagc 120agtttccgcc atggatgctt
tcgagaagct tgagaaagtt ggtgaaggga catacgggaa 180agtttacaga gccagagaga
aagctaccgg gaaaatcgtc gctctaaaga agacgcgtct 240ccatgaggac gaagaaggcg
ttccttccac cactctccgc gagatctcca ttttgcgaat 300gctcgctcgt gatcctcacg
tcgtcaggtt aatggatgtt aagcaaggac taagcaaaga 360aggcaaaact gtactgtacc
tggtttttga atacatggac actgatgtca agaaattcat 420cagaagtttc cgtagcactg
gcaagaacat tccaacccaa actatcaaga gcttgatgta 480tcaactatgc aaaggtatgg
cattctgcca tggtcacggg atattgcaca gagatctcaa 540gcctcacaat ctcttgatgg
atcccaagac aatgaggctc aaaatagcag atcttggttt 600agccagagcc ttcactctgc
caatgaagaa gtatacccat gagatattaa ctctttggta 660tagagctcca gaggttcttc
ttggtgccac ccattactct acagctgtgg atatgtggtc 720tgttggctgc atatttgctg
aacttgtgac caaccaagca atctttcagg gagactctga 780gctccaacag ctcctccata
ttttcaagtt gtttgggaca cccaatgaag aaatgtggcc 840aggagtgagc acactcaaga
actggcatga atacccacag tggaaaccat cgactctatc 900ctctgctgtt ccaaacctcg
acgaggctgg agttgatctt ctatctaaaa tgctgcagta 960cgagccagcg aaacgaatct
cagcaaagat ggctatggag catccttact ttgatgatct 1020gccagaaaag tcctctctct
aaggatttaa aatcttcagt tagtatcttt ccaagtttta 1080tggtttttct agttttgctt
ctttcaagca tatctctagt gtgctgcttc cccctctatg 1140aa
1142481189DNAArabidopsis
thaliana 48tagtcaacga tggatttgag acatgaacaa ctaattgatt tgatttcgtg
tagctaactt 60tgttaattgg taaattgtgt agagaaggat gagtatggag atggagttgt
ttgtcactcc 120agagaagcag aggcaacatc cttcagtgag cgttgagaaa actccagtga
gaaggaaatt 180gattgttgat gatgattctg aaattggatc agagaagaaa gggcaatcaa
gaacttctgg 240aggcgggctt cgtcaattca gtgttatggt ttgtcagaag ttggaagcca
agaagataac 300tacttacaag gaggttgcag acgaaattat ttcagatttt gccacaatta
agcaaaacgc 360agagaagcct ttgaatgaaa atgagtacaa tgagaagaac ataaggcgga
gagtctacga 420tgcgctcaat gtgttcatgg cgttggatat tattgcaagg gataaaaagg
aaatccggtg 480gaaaggactt cctattacct gcaaaaagga tgtggaagaa gtcaagatgg
atcgtaataa 540agttatgagc agtgtgcaaa agaaggctgc ttttcttaaa gagttgagag
aaaaggtctc 600aagtcttgag agtcttatgt cgagaaatca agagatggtt gtgaagactc
aaggcccagc 660agaaggattt accttaccat tcattctact tgagacaaac cctcacgcag
tagtcgaaat 720cgagatttct gaagatatgc aacttgtaca cctcgacttc aatagcacac
ctttctcggt 780ccatgatgat gcttacattt tgaaactgat gcaagaacag aagcaagaac
agaacagagt 840atcttcttct tcatctacac atcaccaatc tcaacatagc tccgctcatt
cttcatccag 900ttcttgcatt gcttctggaa cctcaggccc ggtttgctgg aactcgggat
ccattgatac 960tcgctgaccg agcttctatt cccaaattct tcaagaagaa gaagtaatga
tctaattggt 1020atactaaaaa attatacatc tggtttagtg ttcaattgag agagactgta
aaatcaattc 1080ataggccaac aaatgtttgt ttatccaatt ttccttttta ttcgaacttg
atgcgatatt 1140tcaacggaaa cagaaactat tgttttaaac caaaaaaaaa aaaaaaaaa
118949805DNAArabidopsis thaliana 49atgaataggg aaaagttgat
gaagatggct aacactgtcc gcactggcgg aaaggggaca 60gtaagaagaa agaagaaggc
tgttcacaag accactacaa ccgatgacaa gaggctccag 120agcactctta agagagttgg
agtcaattcc attcccgcca ttgaagaagt taacattttt 180aaggatgatg tagtcattca
gttcattaac cctaaagttc aagcttcaat tgctgctaac 240acatgggttg tgagtggtac
accacagacg aaaaaattgc aagacattct tcctcagatt 300atcagccaac ttggaccaga
taacttggac aacctgaaga agctagcaga gcaattccag 360aaacaagctc caggtgcagg
tgatgtccca gcaacaatcc aagaagagga cgatgatgat 420gatgtcccag atcttgtagt
gggagagact ttcgagaccc ctgctactga agaggctccc 480aaagctgctg cttcttagag
gaggaggaag aagaaggaga agagctcacc tgcaaaaccc 540atcataaaaa tgtttgtcgc
tcgacctctt ctgagcactg tcagattctt gttttctcta 600atgcttgcga acagaaagac
ttggttttat tatcacttga tgctttttgg tccgaacagc 660aattttcctt ttattaaggt
tagatcgctt tttgtttacc acctgttcaa atgagtacta 720ctatgtcctg tcgcttcata
cacttcttgc aacacagtcc tttgttttga gtcaaaaaaa 780aaaaaaaaaa aaaaaaaaaa
aaaaa 805501539DNAArabidopsis
thaliana 50aagctttact acttatactc ttttgttcct atggccaccg tatcttcttc
ctcctggcca 60aaccccaacc ctaatcccga ttccacgtct gcctcagatt ccgattctac
ttttccctct 120caccgcgatc gcgtagacga acccgactct ctcgattcct tctcctccat
gagtcttaac 180tccgacgaac ctaatcagac ttctaatcaa tcgcctcttt ctccccctac
gcccaattta 240ccggtgatgc ctcctccgtc cgtgcttcat ctttccttta accaagatca
tgcttgcttc 300gctgtcggca ctgaccgtgg cttccggatc cttaattgcg atccctttcg
cgagattttc 360cggcgtgatt tcgatcgtgg cggtggtgtt gcagtcgtgg agatgctttt
cagatgcaat 420atattagccc tagttggtgg cggacctgat cctcaatatc ctcctaataa
ggttatgatt 480tgggatgatc accagggccg atgtatcgga gaactctctt tcaggtccga
tgttagatcc 540gtccggctta ggagggatcg gattattgtc gttcttgagc agaagatttt
tgtctacaat 600ttctctgacc tcaagctgat gcatcagatt gaaaccattg ccaaccctaa
gggtttgtgt 660gctgtttctc agggtgttgg ttctatggtt ttggtatgtc caggtttgca
gaaaggtcaa 720gttcggatcg agcactacgc ttctaaacgg accaaattcg tcatggctca
tgattccaga 780atagcttgct tcgctctcac gcaggatggc catttgttgg ccactgctag
ctctaagggt 840actctggttc ggatcttcaa tactgttgat ggtaccttgc gtcaagagtc
tggcacttct 900gaggatgaaa taggtaagga gggtgcggat agagcagaga tctacagttt
ggccttctct 960tcaaatgctc agtggttagc tgtctcaagt gacaaaggaa cggtccatgt
ctttggtctc 1020aaagtcaact ccggatctca agtgaaagac tcatcccgaa ttgcacctga
tgctactccc 1080tcatccccat cgtcgtctct gtctttattc aaagtgttac cgaggtattt
cagctcggag 1140tggtcggtgg ctcagttcag gttggttgaa ggaactcagt acatagccgc
ctttggccat 1200caaaagaaca ccgttgttat tcttggcatg gatgggagct tctacagatg
ccagtttgat 1260ccggtgaacg gcggtgaaat gtctcagctt gagtaccaca actgtctgaa
accgccttca 1320gttttctaaa agctttacta cttatactct tttgttcctt ctctctcttt
atatctctct 1380gcaacttaag cggtgagata tggtgtatag ttttgtgtat ataataatga
tgggtcgtcc 1440tataatttgt aaaacctttt atcgctaccc gggtcgactc tagagcccta
tagtgagtcg 1500tattactgca gagatctatg aatcgtagat actgaaaaa
1539511977DNAArabidopsis thaliana 51agagcttcct ctctctatat
ctggctttct atggatgtag gagttactac ggcgaagtct 60atacttgaga agcctctgaa
gcttctcact gaagaagaca tttctcagct tactcgcgaa 120gattgccgca aattcctcaa
agagaaaggt ttcttcttct tcctttctcc atttttttcc 180ggtcttattg tcttcgacga
atggcggctg acacgtgtcg aaacaggaat gcgcaggcct 240tcgtggaata aatctcaggc
gatccagcaa gttttatctc ttaaagctct ctatgaacct 300ggagatgatt ccggcgccgg
aatcctccgc aagatccttg tttctcagcc gccaaatccg 360cctcgcgtta caacaacgtt
gattgagcca aggaacgagc tcgaagcttg tggaaggatt 420cctttacagg aagatgatgg
tgcgtgccat agaagggatt ctccaagatc agctgagttt 480tctggtagtt ctggtcagtt
tgttgcggat aaagatagcc acaagactgt ttctgtttcc 540cccagaagcc cagctgaaac
aaatgcggtg gttgggcaaa tgacgatatt ttatagtggc 600aaagtgaatg tatatgatgg
agtaccacct gaaaaggccc ggtctatcat gcattttgca 660gccaatccaa ttgatttgcc
tgaaaatggt atttttgctt ctagtagaat gatttcgaaa 720cccatgagta aagagaagat
ggtggagctt ccccaatatg gacttgaaaa ggcacctgct 780tctcgtgatt ctgatgttga
gggtcaggcg aacagaaaag tatcgttgca aagatatctt 840gaaaagcgga aagacagatt
ttctaagacc aagaaggctc caggagttgc gtcctctagc 900ttggagatgt ttctgaatcg
tcagccacgg atgaacgctg catattcaca aaaccttagt 960ggcacagggc attgcgagtc
acctgaaaat caaacaaaaa gtcccaatat ctcagttgat 1020ctaaacagtg atctaaacag
cgaaggtgcc aaaagaactg gagatggtac tacgggtcaa 1080aaggcgggaa gaacaatttc
atgttcttat aacatgacta agacatcacg aggaacacga 1140tgggtgaagc ggtcaagaga
agaagtgatt caagcttggt atatggatga tagtgaagag 1200gatcagagac ttcctcacca
caaggatcct aaagagtttg tatcgttgga caaacttgca 1260gagctgggag tacttagctg
gagacttgat gctgataact atgaaaccga tgaggatttg 1320aaaaagatcc gtgaatctcg
tggttactct tacatggact tttgtgaggt atgcccggaa 1380aagcttccaa actatgaagt
gaaagtgaag agctttttcg aagaacattt acacactgat 1440gaggagatcc gttactgcgt
tgcaggaact ggttactttg atgtgagaga tcgtaatgaa 1500gcttggatta gggtattggt
aaagaaggga ggtatgatag tcttacctgc tgggatctat 1560catcgcttca ctgtggactc
tgacaactat atcaaggcaa tgcggctatt cgtgggtgaa 1620ccggtatgga caccatacaa
tcgcccacac gaccatcttc ctgcaaggaa agaatatgtc 1680gataacttca tgatcaatgc
ctcggcttag agagcttcct ctctctatat ctggctttct 1740gaaacaagga tctataaaca
aggcctacaa taaagaaagc tttcctgtca agtattggat 1800atttatatgt attcctgtgt
agaatgatgg cttttggtat gcttgagttg ttgtaaactt 1860agttacactc tctgatatgt
ctctctttac catctttgtc gtatcccata tacgaaaaga 1920ttacattggg attcatattg
tcttacgttc gttcctatgt gcaatatgtt gagtttt 197752525DNAArabidopsis
thaliana 52catcgctttt cgctgaaatc aaaatttctc cagttttccg atcagtcgca
agaaaaccct 60aaaaatggat ggtcatgatt ctaaggatac taagcagagc actgctgata
tgactgcttt 120tgtccaaaat cttctccagc agatgcaaac caggttccag acaatgtcgg
actccatcat 180cacaaagatt gatgacatgg gaggcagaat caatgagctg gagcaaagca
tcaatgatct 240aagagccgag atgggagtag aaggcactcc tcctccagcc tccaaatcag
gcgatgaacc 300caaaacaccg gctagttcct cttaaaaagg aatgtggtgt tcattgacat
gtccgaagga 360aaaagaaaaa ctatgaaata tgttaagagc agtattactt ttaaaattcc
tgttttaaga 420aacgagtttg ttgtttatta aagttcatca aatagattga tgatgtggtg
cattacatta 480ttctccacct atgaattgca tttctatttt ggtctaaaaa aaaaa
525532610DNAArabidopsis thaliana 53agaacaattg agattcttgg
ttgtgttaag atggaaatct acaccatgaa aacgaatttt 60cttgtactgg ctttgtcttt
gtgtatcctt ctttcaagct tccatgaggt ttcttgtcag 120gatgatggta gtggtttgag
taatttggat ctaatagaac gtgattatca agatagtgtc 180aatgctcttc aaggcaagga
cgatgaagat cagtctgcaa agatacagag tgaaaaccag 240aataacacta cagtgactga
taagaacact atttctctat ctctatcaga tgaatctgag 300gttggatctg ttagtgatga
aagcgttgga cgttcgagtc tgttggatca aatcaaactt 360gaattcgaag ctcatcacaa
tagtattaac caagctggat ctgatggtgt caaggctgaa 420tccaaggatg atgatgaaga
attatctgct catagacaga aaatgttgga agaaatcgaa 480catgagtttg aagctgcttc
agatagtctg aaacaactaa agactgatga tgtaaacgaa 540ggaaatgatg aagaacattc
tgcaaagagg caaagtttgt tggaagagat cgaacgtgag 600tttgaagctg ctacaaaaga
acttgaacaa ctaaaggtta atgacttcac cggggacaaa 660gatgacgaag aacactctgc
aaagagaaaa agtatgcttg aagctattga acgcgagttt 720gaagctgcta tggaaggcat
tgaagcactt aaggtttctg attccacagg aagcggagat 780gatgaagaac aatctgcaaa
gagactaagt atgcttgaag agatcgaacg ggaatttgaa 840gctgcttcaa aaggtcttga
acaactaagg gctagcgatt caaccgcgga caataacgaa 900gaagaacacg ctgcaaaggg
acaaagtttg ttagaagaga tcgaacgaga gttcgaagct 960gctacagaga gccttaagca
acttcaagtt gatgattcta ctgaagacaa agaacactgt 1020aaagcactct tcttcttatt
atctgctatt ctttctctat ggttatctga atcaggcttt 1080gaatgtattg tagttacagc
tgcaaagagg caaagtctgc tggaagagat tgaacgtgaa 1140tttgaagctg caacaaaaga
tcttaaacaa ctaaatgatt tcactgaagg cagtgctgat 1200gatgaacaat ctgcaaagag
aaacaaaatg ttggaagata tcgaacgcga atttgaagct 1260gctacaatag gtcttgaaca
actaaaggct aatgatttct ctgaaggcaa taataatgaa 1320gaacaatctg caaagagaaa
gagtatgctt gaagagatcg aacgcgagtt cgaagctgct 1380attggaggtc ttaaacagat
caaagttgat gattccagaa atcttgaaga agaatctgct 1440aagagaaaga taattttgga
agagatggaa cgtgaatttg aagaagcaca cagtggtatt 1500aatgcaaagg ctgacaaaga
agaatctgca aagaaacaga gtggctctgc tataccagag 1560gttcttggac taggacagtc
aggtggttgt agctgttcta aacaagacga agattcctcg 1620attgttatac caacaaaata
tagcatagaa gatatcctct ctgaagaatc tgcagtccag 1680ggaacagaga cttctagtct
caccgcgtct ttgactcaac tcgttgagaa tcacaggaaa 1740gaaaaggaat ctctactcgg
acacagagtt ctcacttctc cttctatagc ttcttccaca 1800agcgaatcat ctgctacatc
agagactgta gaaaccctaa gggctaaact gaatgagctt 1860cgcggcttaa ccgctcgtga
gcttgtgaca cgtaaagatt tcggtcagat tctcattacg 1920gctgcgagtt ttgaagagct
aagttcagct ccaatcagtt acatttctag gttagctaaa 1980tacagaaacg tcatcaaaga
aggacttgaa gcttctgaga gagttcacat cgcgcaggta 2040cgagcaaaaa tgctcaaaga
agttgccacg gagaagcaaa ccgccgtgga cactcatttc 2100gcaaccgcta aaaagcttgc
tcaagaagga gacgcgttgt tcgttaaaat cttcgcaatc 2160aagaaactgt tggcgaaact
tgaagcagag aaagaatctg ttgatggaaa gtttaaggag 2220actgtgaaag aactttctca
tcttctggct gatgcttctg aggcttacga agagtatcat 2280ggcgcggtga ggaaggcgaa
agacgagcaa gcggctgagg aatttgcgaa agaggcgacg 2340caaagtgcag agatcatttg
ggttaagttt cttagttctc tttagagaac aattgagatt 2400cttggttgtg ttaagagcaa
atctagagct cttgttggtt cttgttatgt attttgtgat 2460gatgttctgt ttcagagttt
gtgtgttggt tgtatcagga gaaagaggct gggagataga 2520gagaaagaga gtctctgcga
aaactaataa tgttttttca gatatctaaa taataagctt 2580tttacaaaaa aaaaaaaaaa
aaaaaaaaaa 2610542235DNAArabidopsis
thaliana 54aatttgaatc caatccccaa attatctcat atggagtttg gatcttttct
tgtgtcctta 60gggacatctt ttgttatctt cgtcattctc atgcttctct tcacctggct
ttctcgcaaa 120tctggaaatg ctcccattta ttacccgaat cggatcctta aagggctgga
gccatgggaa 180ggcacctcct tgactcgaaa cccttttgct tggatgcgtg aagctttgac
ttcctctgaa 240caagatgtcg ttaacttatc cggcgtcgat actgctgtcc actttgtctt
cttgagcact 300gttctgggga tatttgcttg ttccagtctt cttctcctac caactctact
gcctctagcc 360gctacagaca acaacataaa gaacacaaag aatgcgacag ataccacaag
caaaggaact 420tttagccaac ttgataatct atcaatggct aacatcacaa aaaaaagttc
gaggctgtgg 480gcgttcctag gagctgttta ctggatatct ttggtcacat atttcttctt
gtggaaagct 540tataagcatg tctcttcatt gagagctcaa gctctgatgt ctgctgatgt
aaaacccgag 600caattcgcta ttcttgttag ggatatgcct gcaccacctg acgggcagac
acagaaagag 660tttattgatt cttatttcag agaaatatac cctgagacat tctacagatc
gcttgtcgca 720acagaaaaca gcaaggttaa taaaatatgg gaaaaattgg aaggttacaa
gaagaagctt 780gcgcgagcag aagcaatatt agcagcaact aataaccgtc ccacgaacaa
aaccggcttc 840tgtgggctag tcggtaaaca agtagacagc attgagtatt acactgagct
aatcaacgag 900tctgtagcca aactggaaac agagcagaaa gcggttcttg ctgagaagca
gcaaaccgca 960gcagtggttt tcttcacaac cagggttgct gctgcatcag cagctcagtc
tctgcactgc 1020cagatggttg ataaatggac tgtgaccgaa gctcctgagc cacggcagct
cctatggcag 1080aatctcaaca tcaagctctt cagcagaata atccggcaat acttcatcta
cttctttgtt 1140gcagtgacca ttctgtttta catgatacca atcgcgttcg tctctgccat
caccactctt 1200aagaatcttc agaggattat tccgttcata aagccggttg tggagataac
cgccataaga 1260accgttttgg agtctttcct tcctcagatt gcgctcattg ttttcttggc
catgttgccg 1320aagcttctct tgtttctctc caaagccgag gggattcctt cacagagcca
tgccattagg 1380gctgcttcag ggaagtactt ttacttctcg gtctttaatg tcttcattgg
tgttaccctt 1440gctgggactt tgttcaacac agtgaaggat atcgcgaaaa atcccaaact
cgacatgatt 1500attaaccttt tggctactag cctccctaag agcgcaactt tcttcctgac
ctacgttgct 1560ctcaagttct ttatcggtta tggccttgag ctgtctcgga tcataccttt
gataatcttc 1620cacctgaaaa agaagtatct ctgcaaaacc gaagcggagg tcaaagaagc
ttggtacccg 1680ggagacttaa gctatgcgac tagggttccc ggagacatgc tcatcctcac
aatcacgttc 1740tgctattcag tcattgctcc tcttatcctc atattcggca tcacctactt
tggtttaggc 1800tggctagtcc tcaggaatca ggcgttgaaa gtgtacgttc catcatacga
gagctatgga 1860agaatgtggc cgcatattca ccagcgcata ctagcagcgt tgtttctatt
ccaagtggta 1920atgtttggct acttaggagc caagacattc ttctacacgg cccttgtgat
ccctctcatt 1980atcacctctc tcatcttcgg atatgtgtgc cgccagaaat tctacggagg
gttcgaacac 2040acagctctcg aggtagcttg ccgtgagctg aagcagagtc cagacctaga
ggagattttc 2100agagcataca ttccgcatag cttgagctct cacaaaccag aagaacacga
gttcaaaggc 2160gcaatgtctc gttatcaaga tttcaacgca atagcaggcg tttaaagctt
gagagattcc 2220tctggctaaa cccag
2235554002DNAArabidopsis thaliana 55aacaataaga agaaaaagtt
tcattttctg atggcggagc agaagagtac caatatgtgg 60aactgggagg tgactgggtt
cgaatcgaag aagtcgcctt ctagtgagga aggcgttcat 120cggacaccgt cttctatgct
tcgacggtac tcgatcccga agaactcgct tccaccgcac 180tcgtcggagc ttgcgtctaa
ggttcagagt ttgaaggata aagttcagct tgcaaaggac 240gattatgtgg gattgagaca
ggaagctact gatcttcaag agtactccaa tgcgaagctt 300gaaagggtta cacgttattt
aggtgttctg gctgataaaa gtcgtaaact ggatcaatat 360gcacttgaga ctgaggctag
gatatctcca cttatcaatg agaagaagag actgttcaat 420gacttactga cgaccaaagg
tgcacatctt ccatttccga cgtcattctc tatccttact 480tctattgata ttgatcacac
cagaccctta tttgaagacg agggtccctc tatcattgaa 540tttcctgata actgcactat
acgcgtaaac actagtgatg atactctgtc caatcccaag 600aaggaatttg aatttgatag
agtttatggg cctcaagttg gacaagcttc actgttcagt 660gatgtccaac cttttgtgca
atccgctctg gatggatcga acgtttctat atttgcgtat 720ggccaaactc acgcggggaa
gacatacacc atggttgccc ctcctttccc tttcctctct 780gaaattagat ataggtcttg
tttggattta aatatgatag gcaagttcat ggacgttcat 840agtaagttca tggacgaagg
atctaatcag gaccgtggtt tatatgctcg ttgttttgag 900gaacttatgg acttggccaa
ttctgattca acttccgcat ctcagttcag tttctctgtt 960tcagtgtttg agctttataa
cgaacaggtc agggatttac tctcgggttg tcagagcaat 1020ttgccaaaga tcaatatggg
tttacgcgaa tcggttatag aactttcaca ggaaaaagtt 1080gataatccat cagagttcat
gagagtcctg aactctgcat ttcagaatag agggaatgat 1140aaatcaaagt ctactgtgac
ccatctgatt gtctcgatac acatttgtta tagcaacaca 1200attacgagag aaaatgtaat
tagcaagctt tctttagttg acctggctgg aagtgaaggt 1260ttaactgtgg aggatgacaa
tggagatcat gtaactgatc tgctccatgt aacaaattca 1320atttccgcgc tgggagatgt
tttatcatct ttgacgtcaa aaagagatac cattccttac 1380gagaactcat ttcttacaag
aatacttgca gattcactag gagggagctc caaaacattg 1440atgatcgtca acatttgtcc
aagtgcacgg aacttgtctg aaataatgtc gtgtctcaac 1500tatgctgcta gagctcgaaa
tactgtacca agccttggga atcgagacac aattaagaaa 1560tggagagacg tggcaaatga
tgctcggaag gaggtattgg agaaagagag ggaaaatcag 1620cgtctaaaac aagaggttac
gggtttaaaa caagcactta aagaagcaaa tgaccaatgt 1680gtactgctct ataatgaagt
acagagagcg tggagagttt cattcacact gcaatcagat 1740ttaaagtcag agaatgcgat
ggttgtagac aaacataaaa tagaaaagga gcagaatttt 1800cagttaagaa atcaaatagc
tcaactttta cagttagaac aggaacaaaa gctgcaggcg 1860caacaacaag attccaccat
tcaaaatctc cagtctaaag tgaaagactt agaatcacaa 1920ctaagtaaag ctctgaagtc
tgacatgaca agatcgagag atcccttgga acctcagccc 1980agagcagctg agaacacact
cgattcttct gcagttacca agaaacttga ggaagaattg 2040aaaaaacgtg atgcactgat
tgagaggttg catgaagaaa atgaaaaatt gttcgacaga 2100ttaacagaaa agtcagtggc
tagctcgact caggtatcta gcccctcatc aaaagcttca 2160ccaacagtgc agcctgcaga
tgttgacagg aaaaatagcg cgggcacttt accgtcttca 2220gtggataaaa atgagggcac
gattacatta gtaaaatcca gctctgaatt agtaaaaacc 2280actccagctg gagaatactt
aacagctgca ttgaatgatt ttgatcccga acaatatgaa 2340ggtcttgcag ccatagctga
tggcgcaaac aagcttctga tgctggtctt agcagctgtc 2400ataaaggctg gtgcttccag
agagcatgaa atccttgctg agatcagaga ttctgtcttt 2460tcatttatcc ggaaaatgga
accaaggaga gtaatggata caatgcttgt ttctcgagtc 2520aggatattgt acataaggtc
cttacttgca cgatcacctg agcttcagtc gatcaaggtt 2580tctcctgttg aacgcttttt
ggagaagcca tatactggtc gaactagaag ctccagcggg 2640agtagcagcc caggtagatc
accagttcga tattatgatg agcagattta tggctttaaa 2700gttaatttaa agccagaaaa
gaaaagtaag ttggtatctg tagtttcaag aatccgtgga 2760catgaccagg atactgggag
gcagcaagtg actggaggaa agctgaggga gatacaagat 2820gaagccaaaa gttttgccat
tggaaacaaa cccttagctg ctttatttgt tcacactccg 2880gctggtgaac tgcaaagaca
gattaggtca tggcttgcag aaagttttga gtttctctct 2940gttacagcag atgatgtttc
aggagtaacc actggccaat tagagcttct ttccacagca 3000attatggatg gctggatggc
tggagtagga gctgcggtgc cacctcacac agacgcttta 3060ggacagcttt tgtctgagta
tgcaaaacga gtctacactt ctcagatgca gcatctaaag 3120gatattgccg gtactttggc
ttcggaagag gcagaagatg ctggtcaagt cgcgaagctt 3180cgatcagctc tcgagtctgt
tgaccacaaa agaagaaaga ttttgcaaca aatgagaagt 3240gatgcagctt tgtttacctt
ggaagaaggc agttcccctg ttcaaaatcc atctacagca 3300gccgaagact cgagattagc
ctccctcatt tctctggatg ccatactgaa gcaagtcaag 3360gaaataacaa gacaagcctc
tgtccacgtt ttgagtaaaa gcaagaaaaa ggcattgctt 3420gagtctcttg atgaacttaa
cgaacgaatg ccttctctgc ttgatgttga tcatccatgt 3480gcacagagag aaattgatac
ggctcaccag ttggtcgaga caattccaga acaagaggac 3540aatcttcaag acgaaaagag
accttcaata gattcaatat cttcgactga aaccgatgtg 3600tctcaatgga atgttttgca
attcaacaca ggaggctctt cagctccatt catcataaaa 3660tgcggagcta actccaactc
agagctcgtg atcaaagcgg atgcccgtat tcaagaacct 3720aaaggaggcg aaatagtgag
agttgtgcca agaccttcgg ttttagaaaa catgagctta 3780gaggaaatga aacaagtgtt
tggtcagttg cccgaagctc taagttcact ggccttagct 3840agaacagctg atggcacacg
ggctcgatac tctagactct acagaactct agccatgaag 3900gttccctctc ttagggacct
cgttggagag cttgagaaag gaggagtctt aaaagataca 3960aaatcgacat gataggatta
gggttttttc gtgaatttga aa 4002561251DNAArabidopsis
thaliana 56ttagttagat aggcggtggt tggtgcgttc atggcgaatc cttggtgggt
agggaatgtt 60gcgatcggtg gagttgagag tccagtgacg tcatcagctc cttctttgca
ccacagaaac 120agtaacaaca acaacccacc gactatgact cgttcggatc caagattgga
ccatgacttc 180accaccaaca acagtggaag ccctaatacc cagactcaga gccaagaaga
acagaacagc 240agagacgagc aaccagctgt tgaacccgga tccggatccg ggtctacggg
tcgtcgtcct 300agaggtagac ctcctggttc caagaacaaa ccaaagagtc cagttgttgt
taccaaagaa 360agccctaact ctctccagag ccatgttctt gagattgcta cgggagctga
cgtggcggaa 420agcttaaacg cctttgctcg tagacgcggc cggggcgttt cggtgctgag
cggtagtggt 480ttggttacta atgttactct gcgtcagcct gctgcatccg gtggagttgt
tagtttacgt 540ggtcagtttg agatcttgtc tatgtgtggg gcttttcttc ctacgtctgg
ctctcctgct 600gcagccgctg gtttaaccat ttacttagct ggagctcaag gtcaagttgt
gggaggtgga 660gttgctggcc cgcttattgc ctctggaccc gttattgtga tagctgctac
gttttgcaat 720gccacttatg agaggttacc gattgaggaa gaacaacagc aagagcagcc
gcttcaacta 780gaagatggga agaagcagaa agaagagaat gatgataacg agagtgggaa
taacggaaac 840gaaggatcga tgcagccgcc gatgtataat atgcctccta attttatccc
aaatggtcat 900caaatggctc aacacgacgt gtattggggt ggtcctccgc ctcgtgctcc
tccttcgtat 960tgattagtta gataggcggt ggttggtgcg ttctttttac tggaatgatt
atattttcca 1020ttaggatggt taggcttttg tttattaaag ctatcaagtt tctttttttt
ttacggataa 1080ttcggatgac aattagctag tgtttgtttg tttgttttgt ggcggctttt
ctgacttgac 1140tattttgatc gcggatagct ttgtatgaaa gtgaattgat tgtagaatcg
tcttttgaat 1200tttgatgttg gaaaaaacca agcaatggtg tgtggccttt gcaatggaag c
1251572955DNAArabidopsis thaliana 57aatttgcttt atctttgcat
tgttgttggc atggctctca atctccgtca gaaacagact 60gaatgtgtaa tccggatgtt
gaatctgaac caacctttga atccaagtgg aactgcgaac 120gaagaagttt acaagatctt
gatttacgat aggttttgtc agaacattct atctccattg 180acccatgtca aggatctgcg
taagcatgga gttacactct tctttctcat agacaaagat 240cgacaacctg ttcatgatgt
tcccgctgtc tactttgttc aaccaactga atccaacctc 300cagaggatca tagccgatgc
ttctagatct ctctacgata cctttcatct gaatttctcg 360tcttcgatcc ctcgtaagtt
tcttgaagag ctagcttctg ggactcttaa atctggttct 420gttgagaaag tctcgaaagt
gcatgatcag tatctggagt ttgtgacttt ggaagataac 480ttgttctcgc tggctcagca
atctacctat gttcaaatga atgacccatc agcaggggag 540aaagagatta atgagattat
cgaaagggtc gctagtggtt tgttttgtgt gttggtaacg 600cttggtgtgg ttcctgttat
ccgatgccct agtggtggac ctgcagagat ggtggcgtct 660ttgttggatc agaaactgag
ggatcatctt ttgtccaaga acaatctgtt tactgaaggt 720ggcggtttca tgagctcgtt
tcagcgtccc ctcttgtgca tatttgatag gaactttgag 780ctctcggttg ggattcagca
tgatttcaga taccggcctc tcgttcacga tgttctcggg 840ttaaagctca accaattgaa
agtgcaggga gagaaaggac caccgaaatc gtttgagctg 900gacagttcgg acccattctg
gtcagcaaac agtactctgg agtttccaga tgtcgctgtg 960gagatcgaaa cacagttgaa
caagtacaag agagacgttg aagaggttaa caagaaaacc 1020ggaggtggga gcggcgctga
gtttgatggg acagatctga ttggaaacat ccacaccgag 1080catctcatga acactgtgaa
atcgctcccg gagttaactg agcgaaagaa agtgattgac 1140aaacacacca atatcgcaac
agcgctctta ggacagatca aggagagatc tattgacgct 1200ttcactaaga aagaaagcga
catgatgatg aggggcggaa tcgacagaac tgaacttatg 1260gctgctctga aaggcaaagg
gacaaagatg gacaagctcc ggtttgcaat catgtacctg 1320atctccacag aaaccataaa
ccaatcggaa gttgaagcag tggaggcagc attgaatgaa 1380gctgaggctg atacaagtgc
gtttcagtat gtaaagaaaa tcaaatcgtt aaacgcatct 1440tttgcagcta catcagcgaa
ttcagctagc agaagcaaca ttgtagactg ggccgagaag 1500ctttacggac agtctataag
cgcagtgact gcaggagtca agaatctgtt atctagtgat 1560caacaattgg cagtgactcg
aacagtcgaa gctttaacag aaggaaaacc aaacccggag 1620atcgattctt accgcttcct
ggacccaaga gctccaaagt cgtctagctc cggtggtagc 1680catgtaaaag gaccgttcag
agaagctata gtgttcatga tcggtggagg taactatgtt 1740gagtatggaa gtttgcagga
gttgactcag agacagttaa ccgttaaaaa cgttatttat 1800ggagccactg agattcttaa
cggaggtgag ttggtggagc agcttggact tttgggaaag 1860aagatgggat taggaggtcc
ggtcgcttca acgctgaaga ggctgggaat ggctggtaaa 1920gaggagactg atgtatctgc
acaagggtct ttaaccaggg aggccactga gatatggagg 1980agtgagttgg aatctcgccg
gtttcaggta gatagtttag aagctgaact tgtggatgtc 2040aaggcttacc ttgagtttgg
ctcagaagaa gatgccagaa aggagttagg agttctttcg 2100ggtagggtca gatcgactgc
aactatgttg cgttatttga gatcaaaagc tagagtcttg 2160gccattcctg atgatctagc
aaatgtgtca tgcggtgtgg aacagattga agaactgaaa 2220ggattgaacc ttgttgagaa
agatggtggt tcatcttctt ctgacggggc taggaacact 2280aatcctgaaa ctagaaggta
cagtggttcc ttgggtgtag aggatggagc ctatactaat 2340gagatgctcc agtccataga
gatggttact gatgtgctgg actctcttgt gaggagggtt 2400acagtagcag aatctgagtc
tgctgttcaa aaggagaggg cacttttggg agaggaagaa 2460atcagtagga agactatcca
aatcgaaaat ttgtccgtga agttagaaga gatggaacga 2520tttgcttatg ggactaatag
tgttctaaac gaaatgcggg aaaggattga ggaattagtt 2580gaagagacga tgaggcagag
ggaaaaagct gtggaaaacg aagaggagtt gtgtcgtgtg 2640aagagagagt tcgagtcgct
taaaagctac gtcagtactt ttaccaatgt tcgagaaaca 2700cttctttcgt ccgagagaca
attcaaaacc attgaggagc tctttgaacg gttggtcact 2760aagacgacac aattagaagg
ggagaaggca caaaaggagg ttgaagtaca gaaactgatg 2820gaggagaatg tgaaattgac
agcacttctc gacaagaaag aggctcagct tctagctttg 2880aatgaacaat gcaaagttat
ggctttgagt gcatcaaaca tatgactctc taatccaacc 2940gaatctcaag cttcc
295558865DNAArabidopsis
thaliana 58ggctgataaa tatagggaga actatttggg tcacagtatc aaagcccctg
ttggaagatg 60gcaaaaaggt aaagatcttc attggtatgc tagagataaa aagcaaaagg
gttccgagat 120ggatgctatg aaagaagaga ttcaaagagt taaggaacaa gaggagcagg
ccatgaggga 180ggctcttggc ttggcaccaa agtcctctac aaggccacaa ggaaatcgcc
ttgataagca 240agagtttact gaacttgtga agaggggttc gacagcagag gacttaggtg
cagggaatgc 300tgatgctgtg tgggttcacg gtcttggata tgctaaagca ccacgacctt
gggaagatcc 360gagcaccctt gcatcctctc agaaagaaga tgcagattca gcacgcttac
cagcagatac 420atcaggggtc aaaactgttg aagatggacc ggatgatgtt gagagggacc
aaagaaggat 480aggcgtgagg aaaggaaacc tgcaaagaga gagaaggaag aaagacatga
taggcgtgaa 540aaacgcgaaa ggcatgagaa gcgaagcgct cgtgattcag atgatagaaa
gaagcacaag 600aaagagaaga aggagaaaaa aagaaggcat gactctgatt ctgattgaag
cgaattgtcc 660caggatggaa cattttgctc ttcagaggaa gagtggtcgg ctaggtacca
aaatccagct 720accacttctg caagatttaa atctgttgct tatttcattt acgaatcgtg
gagtaaagtg 780ttgttgaaca ttgttgaaaa tgtttgttaa aacacatgaa aaatgtggtt
tgatattata 840acaaaccgag acgctcgttt tagct
86559723DNAArabidopsis thalianamisc_feature(559)..(559)n= a,
c, g, t 59gcaaaagaga gaaacatctg acccggaatc tgacctgaaa acccggaaga
atcgaaaaat 60ggggaaagat ggtctgagcg acgatcaggt ctcgtcgatg aaggaagcct
tcatgctctt 120cgacaccgat ggcgacggca aaatcgcacc gtcagagctc gggatcctca
tgcgatctct 180cggcggaaac ccgacccaag cccagctgaa atccataatc gcatccgaga
atctctcttc 240accgtttgat ttcaacagat tcctcgatct catggcgaaa catctgaaga
cggaaccttt 300cgatcgccag ctccgtgacg cattcaaagt gctcgataag gaaggtaccg
ggttcgttgc 360tgtggcggat ctgaggcata ttctgaccag tatcggagag aagctggagc
ctaatgagtt 420cgatgagtgg atcaaggagg tggatgttgg atccgatgga aagatccggt
atgaagattt 480catagcaagg atggttgcta agtgagatct aatcttttat gttttgaaag
ttgaaatttt 540taagaagaga ttcttttgng gttttttcac ttggttggtt tgatttcgag
cgaatcctaa 600ctaggggttg gtttatcatt gnggaatttg cttactaact ttggcttctt
catggttggg 660tttcaatttt taatggnaaa tggtggctgg gggaattcct aaaaaaaaaa
aaaaaaaaaa 720aaa
72360426DNAArabidopsis thaliana 60caaaaaaaga gatcgcttca
atggagaaac agagtactca accaatttgc ggccaagagg 60ctctccaact tctcaattgc
gtcgcggagt ctcctttcga tcaagagaaa tgcgtccgat 120ttttgcaatc tctcagagaa
tgcgttctat caaagaaagt aaagaagttc tcgataccga 180gtcaagatca cgactctgag
ggagcagctt cagctacaaa gagaccttca taacgttctt 240tgttccgatt ttcttttatc
gtttgagttg taatcatgta attgatttta atgtcatgcc 300ttggattcat aagctgggtc
atgccttgtt tcccctttgt tgtcttgtat gttgaatatt 360gcaaactcta aagagcatat
ttataagaag aaataaaagt ttctacaaaa aaaaaaaaaa 420aaaaaa
426611442DNAArabidopsis
thaliana 61tcaaaatcag aaactttcct tgacaaattt taacaaatct ctttctcgtt
ttctattgaa 60ttctccagta gcgcggtagt tagttttagg tggaagaaga atgacaacta
ctgggtctaa 120ttctaatcac aaccaccatg aaagcaataa taacaacaat aaccctagta
ctaggtcttg 180gggcacggcg gtttcaggtc aatctgtgtc tactagcggc agtatgggct
ctccgtcgag 240ccggagtgag caaaccatca ccgttgttac atctactagc gacactactt
ttcaacgcct 300gaataatttg gacattcaag gtgatgatgc tggttctcaa ggagcttctg
gtgttaagaa 360gaagaagagg ggacagcgtg cggctggtcc agataagact ggaagaggac
tacgtcaatt 420tagtatgaaa ggtcttatct ctttctctgc ccctattatg ctttcatcta
aatgcctttc 480aatttgtgaa aaggtggaaa gcaaaggaag gacaacttac aatgaggttg
cagacgagct 540tgttgctgaa tttgcacttc caaataacga tggaacatcc cctgatcagc
aacagtatga 600tgagaaaaac ataagacgaa gagtatatga tgctttaaac gtcctcatgg
ctatggatat 660aatatccaag gataaaaaag aaattcaatg gagaggtctt cctcggacaa
gcttaagcga 720cattgaagaa ttaaagaacg aacgactctc acttaggaac agaattgaga
agaaaactgc 780atattcccaa gaactggaag aacaagtaat gaacatcatc gatactctcg
gcttatctgc 840ttcctgcctt cagaatctga tacagagaaa tgagcactta tatagctcag
gaaatgctcc 900cagtggcggt gttgctcttc cttttatcct tgtccagact cgtcctcacg
caacagtaga 960agtggagata tcagaagata tgcagctcgt gcattttgat ttcaacagca
ctccatttga 1020gctccacgac gacaattttg tcctcaagac tatgaagttt tgtgatcaac
cgccgcaaca 1080accaaacggt cggaacaaca gccagctggt ttgtcacaat ttcacgccag
aaaaccctaa 1140caaaggcccc agcacaggtc caacaccgca gctggatatg tacgagactc
atcttcaatc 1200gcaacaacat cagcagcatt ctcagctaca aatcattcct atgcctgaga
ctaacaacgt 1260tacttccagc gctgatactg ctccagtgaa atccccgtct cttccaggga
taatgaactc 1320cagcatgaag ccggagaatt gaaacacgta tgaaggcccc ttgtacaatt
tctgtaaaac 1380tgtaaagtag ctcttgaaaa actttacctg gttttttgac gaatagtctg
tttagcggta 1440aa
1442621506DNAArabidopsis thaliana 62atggcgctgc agaacattgg
tgcttccaac cgtaacgatg ccttctacag gtacaagatg 60cctaagatgg ttaccaaaac
cgaaggcaaa ggtaatggca ttaagaccaa cattatcaac 120aatgttgaga ttgccaaagc
cttggctaga ccgccttctt atacgaccaa gtactttggt 180tgtgagcttg gagcgcagtc
taagtttgat gagaagactg ggacgtcgct tgtgaatgga 240gctcacaaca cgtctaagct
tgctgggctt ttggagaatt ttattaagaa gtttgttcag 300tgttatggat gtggtaaccc
ggagactgag attattatta cgaagacgca gatggtgaat 360ctcaagtgtg ctgcttgtgg
gtttatctct gaggtcgaca tgagggataa gttgactaat 420ttcattctca agaacccacc
tgagcagaag aaggtgtcaa aggataagaa agcaatgagg 480aaagctgaga aggagaggct
taaagaaggc gagctagctg atgaggagca gagaaagctg 540aaagctaaga agaaagcatt
gtctaacggc aaggattcta agacgtctaa gaaccattct 600tctgatgagg atataagccc
gaagcatgat gagaatgctc tagaggtgga tgaggatgaa 660gatgatgatg atggtgtcga
gtggcaaact gatacttccc gagaagctgc tgagaaaaga 720atgatggaac agttgagtgc
taaaactgcc gaaatggtga tgctctctgc aatggaagta 780gaagagaaaa aggcgcccaa
aagcaaatct aacgggaacg ttgtgaaaac tgagaatcct 840cctccgcaag agaagaatct
cgtgcaggat atgaaagagt atctgaagaa agggtcacca 900ataagcgcgc tcaaaagttt
catctcgtct ctctctgaac ctcctcaaga catcatggac 960gcactcttca atgctctctt
tgatggtgtg ggaaagggat tcgccaaaga agtgactaag 1020aagaagaatt acttagcggc
tgctgcaaca atgcaagagg atggatcaca gatgcatctg 1080ctcaattcga ttgggacatt
ctgtggaaag aatggaaacg aagaagcttt gaaagaggtg 1140gctctggttc ttaaagcatt
gtacgaccaa gacatcattg aggaagaggt agtgttggat 1200tggtacgaaa agggtctcac
cggagctgac aaaagctcgc cggtttggaa gaatgttaag 1260ccttttgtgg agtggcttca
gagcgctgag tctgagtccg aagaggagga ttgagtcact 1320tttttcttcc ctcctaactt
ttctttgcgg catttcttat aatacttcgt cagttttcag 1380aattcttaaa tctttttgct
gtgttcttat aaagaaacat catctattaa agttgtcttc 1440gtttggattt ggttttgacg
actttgggaa atatttatgt ttaagaaaaa aaaaaaaaaa 1500aaaaaa
1506632631DNAArabidopsis
thaliana 63atggcggcta acaaattcgc gactctgatt catcggaaaa caaaccgaat
cactttaatc 60ctcgtatacg cttttctcga atggtcactc attttcttca ttttgctcaa
ctctctcttt 120tcttatttca tactcagatt cgctgattat ttcggtctta aacgtccttg
tctcttctgc 180tctagactcg atcgtttctt cgatgcttct ggtaaatctc cttctcatcg
agatcttctc 240tgcgatgatc atgctctcca attacattca aaacctgttg aagaatctaa
ttgtggtttc 300ggagaatttc acaatgattt ggttcatcgt ggttgttgcg tagagaagat
aagttcgtca 360ctatgtgctc cgattgagtc tgactttggg aatttagatt atccaattgg
agatgaaggt 420cagatttaca atggtcttaa gtttcctcga tcgatcttcg tctttgaaga
agagaaagta 480ggatctgtaa atttgaatga ttctcaggaa gaaacagagg agaagaaagt
tccccaatct 540catgagaaac ttgaagatga tgatgttgat gaggagtttt catgctatgt
atcaagcttc 600gattgtaaga acaaagaaat tgcaacagag aaggaagaag aaaacagagt
ggatctacct 660atagaggtgg aaactgcaga atcagctccg aaaaacctcg agttctatat
tgatgaagaa 720gactgtcatt tgattccagt tgaattctat aaaccgagtg aagaagttcg
agagatttcc 780gacattaacg gagattttat cctcgatttc ggcgttgagc atgatttcac
ggcggcggcg 840gagacggagg aaatctccga ctttgcttcg ccgggtgaat cgaaaccgga
ggatgcagag 900acgaatctag ttgcttcgga aatggaaaac gacgacgaag aaacagacgc
agaggtttct 960ataggtacag agattcctga tcatgagcaa atcggagata ttccttctca
ccagctcatt 1020cctcaccacg atgacgatga tcatgaggag gaaacgttgg agttcaaaac
agtaacgatt 1080gaaaccaaga tgccagtctt aaacatcaac gaagagcgga ttttagaagc
tcaaggctcg 1140atggaaagct cgcatagtag tctacataac gctatgtttc acttagagca
aagagtatct 1200gttgatggta ttgaatgtcc tgaaggagta ctcactgttg ataagttgaa
gtttgagtta 1260caagaagaga gaaaagcact tcacgcgtta tacgaggagc tggaggtaga
gaggaatgcg 1320tctgctgttg ctgccagtga aacaatggcg atgatcaata ggttgcatga
ggagaaagct 1380gcgatgcaga tggaagcgtt gcagtatcag agaatgatgg aggagcaagc
tgagtttgat 1440caagaagctt tgcagttgtt gaatgagctt atggtgaata gagagaagga
gaatgctgag 1500cttgagaagg agctagaggt gtatagaaag agaatggagg agtatgaagc
taaagagaaa 1560atggggatgt tgaggaggag attgagagat tcctctgttg attcgtatag
aaataatggc 1620gattctgatg agaatagcaa tggagagtta cagtttaaga acgttgaagg
ggttacggat 1680tggaaatata gagagaatga gatggagaat acgccggtgg atgttgtact
tcgtcttgat 1740gagtgtttag atgattatga tggagagagg ctttcgattc ttgggagatt
gaagtttctt 1800gaagagaaac tcacagatct taataacgaa gaggacgacg aggaggaggc
taaaacgttt 1860gagagtaatg gtagcatcaa tggaaatgag catattcatg gcaaagaaac
aaacgggaag 1920cacagagtta tcaagtcaaa gagattactt cccctgtttg atgcggtcga
tggagagatg 1980gaaaacgggt taagtaacgg aaaccatcac gaaaacgggt ttgatgattc
ggagaagggt 2040gagaatgtga cgatagaaga agaagtggat gagctttacg agaggttaga
agctctagag 2100gcagatagag agttcttaag acattgtgtt ggttcattga aaaaaggaga
caaaggtgta 2160catctcctcc atgagattct gcaacatctt cgtgatctaa ggaatatcga
tcttactcgc 2220gtcagagaaa acggagacat gagtttatga gtttgatttt gagttttggg
tttgagtcca 2280ctctttgcat agtgacccaa agaacaagaa aaatcataca ggtatggaag
tgacatgttg 2340cttgtgaggc aaggaacaac gacaaggttt cagatgaaga agaaaacgtt
ctcagaataa 2400aagtatttta agtatatact ctgaggaaaa gtgtcagatc agaatgttcg
tctttcttcg 2460ttcattttca ttattataag ttttgttttt tatattgaag atttatttag
agagagggaa 2520gtgtcagtat aatttcactt ttatatttta tatttgggag ttgtctttat
gagtggtggt 2580aatagaaaaa ggtagaatga tgagtgaaga aaaaaaaaaa aaaaaaaaaa a
2631642743DNAArabidopsis thaliana 64atgtcagacg ctctttctgc
gattccggcc gcagttcatc gcaatctctc cgataaactc 60tatgagaagc gcaaaaatgc
tgcgcttgag cttgagaata ttgtgaagaa tctaacttct 120tcgggtgatc atgacaagat
ctcgaaagtc attgagatgt tgattaagga atttgccaaa 180tctcctcaag ctaatcatcg
gaagggtggt ctaattggct tagctgctgt aactgttggt 240ttgtctacag aagctgctca
atatcttgag caaatagtgc cacctgtgat taattccttt 300tctgatcaag atagccgagt
tcggtactat gcatgtgaag ctctctataa cattgcaaag 360gttgtgcgag gcgatttcat
tattttcttc aataagatat ttgatgcctt atgcaaactc 420tcagcagatt ctgatgccaa
tgtccaaagt gctgctcatc ttttggatcg ccttgttaag 480gatattgtga cggaaagtga
tcagttcagt attgaggaat tcatacctct tttaaaagag 540cgaatgaacg ttctcaaccc
ttacgtccgg caatttctgg ttggatggat cactgttctt 600gatagtgttc cagacattga
catgcttggg tttctgccag actttctcga tgggttattc 660aatatgttga gcgactctag
tcatgaaata cgacagcaag ctgattcagc tctttcagag 720tttcttcaag agataaaaaa
ttcaccatct gtagattatg gtcgcatggc tgaaatactg 780gtgcagaggg ctgcttctcc
tgatgaattc actcgattaa cagccatcac gtggataaac 840gagttcgtaa aacttggggg
agaccagctc gtgcgttatt atgctgacat tcttggggct 900atcttgcctt gcatatctga
caaagaagag aaaatcaggg tggttgctcg tgaaaccaat 960gaagaacttc gttcaatcca
tgttgaaccc tcagatggtt ttgatgttgg cgcaattctc 1020tctgttgcaa ggaggcagct
atcaagtgag tttgaggcta ctcggattga agcattgaat 1080tggatatcaa cacttttaaa
caagcatcgt actgaggtct tgtgcttcct gaatgacata 1140tttgacaccc ttctaaaagc
actatctgat tcttctgatg acgtggtgct cttggttctg 1200gaggttcatg ctggtgtagc
aaaagatcca caacactttc gccagctcat cgtatttctt 1260gtccacaatt tccgagctga
taattctctt ttggaaaggt atctggaaag aacatattat 1320ttagttggtc aaaacatatc
tcgttatagg cgcggtgccc ttattgtccg aagaatgtgt 1380gtacttttgg atgccgaaag
agtctaccga gagctctcta caattcttga gggagaagat 1440aatcttgact ttgcttctac
catggttcag gcattgaatt tgattttgct tacttccccg 1500gagttatcga aactgagaga
actattaaaa ggttcactcg tcaatcgcga agggaaagaa 1560cttttcgttg ccttgtatac
ttcatggtgc cattcaccca tggcaattat aagcctctgc 1620ttattagctc aggcttacca
gcatgcgagt gtcgtgattc aatcattggt agaagaagac 1680attaacgtca aatttctagt
acagcttgat aaattgatcc ggcttctgga aactccaatc 1740tttacttacc ttagattgca
gcttctggaa ccaggaaggt acacatggtt gctgaaaaca 1800ctttatggtc ttcttatgtt
acttcctcag caaagtgcgg cgttcaagat acttaggaca 1860agactcaaaa ctgtgccaac
gtactcattc agtactggaa accaaatagg cagagcaact 1920tcaggagttc ctttctctca
gtataagcat caaaacgagg acggtgactt agaagacgat 1980aacatcaaca gttctcacca
aggaatcaat tttgctgtgc ggctacaaca gttcgaaaac 2040gtacagaatc tacatcgtgg
ccaggcaagg actagagtga actactcata tcactcttcc 2100tcttcttcta catcaaagga
ggtgaggaga tctgaagaac aacaacagca gcagcagcaa 2160caacaacagc aacaacaaca
acaacaacga ccaccacctt cttcgacatc atcatcagtt 2220gcagataaca atagacctcc
atcaagaact tcaagaaaag gccctggtca attacagctt 2280taacctacct ggtaatcata
aataataaat aatattccat ccccgacaat catcatcttc 2340atcttctttg tgtggacacc
accgatccct tttgtctcct gtaaaattgt atatctctct 2400tttttagtaa ctcttcaagt
ttcgacggaa cttgtggaaa agctacggtc gtgtccatca 2460tctctttctc tctgtcgggt
tttttttatt tacgagagat tcttcttcag tccctcagtc 2520tacctttata ttgttttttt
gggggtttct cgtttctttg aatttgtttc attgtttgga 2580gctttttata tttttacctt
atgtggagat gtaagaaaaa gaagtgatca tgtggttttg 2640tgttgttttt ttataactgg
aaaaccacat gagtttgtag aggtcactta ttggatattt 2700tatgtcaaat gatgctcctt
tttacaaaaa aaaaaaaaaa aaa 2743652959DNAArabidopsis
thaliana 65atgtcactct tgttcctcaa tcctccgttt ccctccaatt caatccaccc
aattcctcgt 60cgtgccgccg gaatatcctc cattcgatgc tcaatttctg caccggagaa
gaaaccgagg 120aggaggagga agcagaagcg cggcgacgga gctgagaatg acgactcttt
gtctttcgga 180agtggtgaag ctgtctccgc tctggagagg agtctccgcc tcacttttat
ggacgagctt 240atggaacgag ctagaaatcg agatacttca ggtgtttctg aggttatcta
tgacatgatt 300gctgctgggc ttagccctgg acctcgttct ttccatggtt tggttgtagc
tcacgcgctt 360aacggcgacg aacaaggcgc gatgcactcg ctgagaaagg agctaggtgc
aggccaacgt 420ccgcttcctg agactatgat tgctttggtt cgtctctctg gttcgaaagg
gaatgctacg 480agaggcctag aaatcctcgc cgctatggaa aagcttaagt atgacattcg
tcaagcttgg 540ctcattcttg ttgaggagct catgaggatc aatcacttgg aagatgcgaa
taaagttttc 600ttgaagggtg caagaggtgg gatgagagca acagatcagc tttatgattt
gatgattgaa 660gaagattgca aagctggaga tcattctaat gccttagaca tctcttacga
aatggaggca 720gctggtagaa tggccacaac atttcatttc aactgtcttc ttagtgtgca
ggctacatgt 780gggattcccg aggtagctta tgctacattc gaaaatatgg agtacggtga
aggtttattt 840atgaagcctg acactgagac atataactgg gtgattcaag cctacactag
agccgagtca 900tatgataggg ttcaggatgt tgctgaatta cttggaatga tggttgagga
ccacaaacgt 960gtgcagccaa atgtgaagac ttatgcgctc ttagttgagt gcttcaccaa
atattgtgtc 1020gtgaaggaag cgattagaca ttttcgtgct cttaaaaact ttgaaggagg
aacagtaatt 1080ttacacaatg cagggaattt tgaggatcct ctctctttgt atctcagggc
tttgtgtcga 1140gaaggaagaa ttgttgagct tattgatgct ttagatgcaa tgcgcaaaga
taaccaacct 1200atacctccaa gagccatgat tatgagcaga aagtatcgaa cactagtcag
ctcatggatt 1260gaaccattgc aagaagaagc tgaacttggc tatgagattg attatttagc
gaggtacata 1320gaggaagggg gacttactgg tgaacgcaag cgttgggtac ctcgaagagg
gaaaactcct 1380ttagatcccg atgcttctgg ttttatatac tcaaacccta ttgaaacatc
ctttaaacag 1440agatgccttg aagattggaa agttcaccat aggaagctct tgagaacctt
acagagtgaa 1500ggtcttccag ttctaggaga tgcatcagaa tctgattaca tgagagtggt
ggagagatta 1560cggaacataa taaaaggtcc tgcactgaat cttttgaagc cgaaagcagc
aagcaagatg 1620gttgtatcag agttaaagga agaactcgaa gctcagggtt tgccaattga
tggaacaaga 1680aatgtgcttt accagcgtgt ccaaaaagca aggagaataa acaaatctcg
aggtcgacct 1740ctttgggttc ctccaattga agaagaagag gaggaggtcg atgaagaagt
agacgattta 1800atatgtcgaa tcaagctaca tgaaggagac acagagttct ggaaacgtcg
gtttcttgga 1860gaaggcttga ttgaaacttc agttgaatcc aaggaaacga ctgaatcagt
ggttacaggt 1920gaatcggaga aagcgattga agatatttca aaagaagctg acaatgagga
ggatgatgat 1980gaggaggaac aagaaggaga tgaggatgat gatgaaaatg aagaggaaga
agtggttgtt 2040ccagaaactg agaatcgagc agaaggagaa gatttagtga agaataaggc
agctgacgcg 2100aagaagcatc ttcaaatgat tggagtccaa ctcttgaaag aatccgatga
agcaaacaga 2160acaaagaaac gtgggaagag ggcatctcgt atgacacttg aggatgatgc
agatgaggat 2220tggttccctg aggaaccatt tgaagcattc aaagaaatga gggaaagaaa
agtgttcgat 2280gtggctgaca tgtatacaat agcagacgtt tggggttgga catgggagaa
ggattttaag 2340aacaaaactc caaggaaatg gtcacaagag tgggaagtcg agttggcaat
tgtgctcatg 2400acaaaggtga ttgaattggg tggaattcca acgattggtg attgtgcagt
gatattacga 2460gctgctttaa gagctcccat gccttcagcc ttcttgaaga tcttgcagac
gacacacagt 2520cttggctact catttggcag cccgttgtac gatgagatca tcacattgtg
tttggacctt 2580ggagaacttg atgcagccat cgccatagtt gcagatatgg aaaccacagg
gatcactgtc 2640cctgatcaaa cccttgacaa ggtcatatct gctagacaat ctaatgagag
tccgcggtct 2700gagcctgaag agccagcatc aacagtaagc tcttagttat catatcctct
tctgcttgtt 2760gtgaagtctc tataagaaac agaaatcggt agaaggagct gaatctgtct
tagttatgaa 2820agttttgttc attataagta caagtcatgt agttccgagt gtagaacagt
ttttactagt 2880gttgcaccag gtccctccag tctgatactt aattctttag tgttggatct
ttctatataa 2940gaaaaaaaaa aaaaaaaaa
2959661295DNAArabidopsis thaliana 66aagcttcgaa gtcgatttca
atggaaggtt cctcgtcagc catcgcgagg aagacatggg 60agctagagaa caacattctc
ccagtggaac caaccgattc agcctccgac agtatattcc 120actacgacga cgcttcacaa
gccaaaatcc agcaggagaa gccatgggcc tccgatccta 180actacttcaa gcgcgttcac
atctcagccc ttgctcttct caagatggtg gttcacgctc 240gctccggtgg cacaatcgag
atcatgggtc ttatgcaggg taaaaccgag ggtgatacaa 300tcatcgttat ggatgctttt
gctttgcctg ttgaaggtac tgagactagg gttaatgctc 360agtctgatgc ctatgagtat
atggttgaat actctcagac cagcaagctg gctgggaggt 420tggagaacgt tgttggatgg
tatcactctc accctgggta tggatgttgg ctctcgggta 480ttgatgtttc gacacagatg
cttaaccaac agtatcagga gccattctta gctgttgtta 540ttgatccaac aaggactgtt
tcggctggta aggttgagat tggggcattc agaacatatc 600cagagggaca taagatctcg
gatgatcatg tttctgagta tcagactatc cctcttaaca 660agattgagga ctttggtgta
cattgcaaac agtactactc attggacatc acttatttca 720agtcatctct cgatagtcac
cttctggatc tcctttggaa caagtactgg gtgaacactc 780tttcttcttc cccactgttg
ggcaatggag actatgttgc cgggcaaata tcagacttgg 840ctgagaagct cgagcaagcg
gagagtcagc tcgctaactc ccggtatgga ggaattgcgc 900cagccggtca ccaaaggagg
aaagaggatg agcctcaact cgcgaagata actcgggata 960gtgcaaagat aactgtcgag
caggtccatg gactaatgtc acaggttatc aaagacatct 1020tgttcaattc cgctcgtcag
tccaagaagt ctgctgacga ctcatcagat ccagagccca 1080tgattacatc gtgaagttgg
tctattcttt tgttttttgg ctgcggaaat tgactatcgg 1140tttgacccgg tttatgaggc
aatgcccatt gttccctata tctctagtgt agtatctgct 1200tcagacaaag atctttgggt
tattaaatga cattaacata aatcgatcat tatgtttttg 1260cgttaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaa 129567340PRTArabidopsis
thaliana 67Pro His Ile Arg Asp Glu Glu Thr Lys Lys Pro Asp Ser Val Ser
Ser1 5 10 15Glu Glu Pro
Glu Thr Ile Ile Ile Asp Val Asp Glu Ser Asp Lys Glu 20
25 30Gly Gly Asp Ser Asn Glu Pro Met Phe Val
Gln His Thr Glu Ala Met 35 40
45Leu Glu Glu Ile Glu Gln Met Glu Lys Glu Ile Glu Met Glu Asp Ala 50
55 60Asp Lys Glu Glu Glu Pro Val Ile Asp
Ile Asp Ala Cys Asp Lys Asn65 70 75
80Asn Pro Leu Ala Ala Val Glu Tyr Ile His Asp Met His Thr
Phe Tyr 85 90 95Lys Asn
Phe Glu Lys Leu Ser Cys Val Pro Pro Asn Tyr Met Asp Asn 100
105 110Gln Gln Asp Leu Asn Glu Arg Met Arg
Gly Ile Leu Ile Asp Trp Leu 115 120
125Ile Glu Val His Tyr Lys Phe Glu Leu Met Glu Glu Thr Leu Tyr Leu
130 135 140Thr Ile Asn Val Ile Asp Arg
Phe Leu Ala Val His Gln Ile Val Arg145 150
155 160Lys Lys Leu Gln Leu Val Gly Val Thr Ala Leu Leu
Leu Ala Cys Lys 165 170
175Tyr Glu Glu Val Ser Val Pro Val Val Asp Asp Leu Ile Leu Ile Ser
180 185 190Asp Lys Ala Tyr Ser Arg
Arg Glu Val Leu Asp Met Glu Lys Leu Met 195 200
205Ala Asn Thr Leu Gln Phe Asn Phe Ser Leu Pro Thr Pro Tyr
Val Phe 210 215 220Met Lys Arg Phe Leu
Lys Ala Ala Gln Ser Asp Lys Lys Leu Glu Ile225 230
235 240Leu Ser Phe Phe Met Ile Glu Leu Cys Leu
Val Glu Tyr Glu Met Leu 245 250
255Glu Tyr Leu Pro Ser Lys Leu Ala Ala Ser Ala Ile Tyr Thr Ala Gln
260 265 270Cys Thr Leu Lys Gly
Phe Glu Glu Trp Ser Lys Thr Cys Glu Phe His 275
280 285Thr Gly Tyr Asn Glu Lys Gln Leu Leu Ala Cys Ala
Arg Lys Met Val 290 295 300Ala Phe His
His Lys Ala Gly Thr Gly Lys Leu Thr Gly Val His Arg305
310 315 320Lys Tyr Asn Thr Ser Lys Phe
Cys His Ala Ala Arg Thr Glu Pro Ala 325
330 335Gly Phe Leu Ile 34068145PRTArabidopsis
thaliana 68Pro Asp Ser Gly Thr Ala Ala Gly Gly Ser Asn Ser Asp Pro Phe
Pro1 5 10 15Ala Asn Leu
Arg Val Leu Val Val Asp Asp Asp Pro Thr Cys Leu Met 20
25 30Ile Leu Glu Arg Met Leu Met Thr Cys Leu
Tyr Arg Val Thr Lys Cys 35 40
45Asn Arg Ala Glu Ser Ala Leu Ser Leu Leu Arg Lys Asn Lys Asn Gly 50
55 60Phe Asp Ile Val Ile Ser Asp Val His
Met Pro Asp Met Asp Gly Phe65 70 75
80Lys Leu Leu Glu His Val Gly Leu Glu Met Asp Leu Pro Val
Ile Met 85 90 95Met Ser
Ala Asp Asp Ser Lys Ser Val Val Leu Lys Gly Val Thr His 100
105 110Gly Ala Val Asp Tyr Leu Ile Lys Pro
Val Arg Ile Glu Ala Leu Lys 115 120
125Asn Ile Trp Gln His Val Val Arg Lys Lys Arg Asn Arg Val Glu Trp
130 135 140Phe14569450PRTArabidopsis
thaliana 69Met Gly Lys Glu Asn Ala Val Ser Arg Pro Phe Thr Arg Ser Leu
Ala1 5 10 15Ser Ala Leu
Arg Ala Ser Glu Val Thr Ser Thr Thr Gln Asn Gln Gln 20
25 30Arg Val Asn Thr Lys Arg Pro Ala Leu Glu
Asp Thr Arg Ala Thr Gly 35 40
45Pro Asn Lys Arg Lys Lys Arg Ala Val Leu Gly Glu Ile Thr Asn Val 50
55 60Asn Ser Asn Thr Ala Ile Leu Glu Ala
Lys Asn Ser Lys Gln Ile Lys65 70 75
80Lys Gly Arg Gly His Gly Leu Ala Ser Thr Ser Gln Leu Ala
Thr Ser 85 90 95Val Thr
Ser Glu Val Thr Asp Leu Gln Ser Arg Thr Asp Ala Lys Val 100
105 110Glu Val Ala Ser Asn Thr Ala Gly Asn
Leu Ser Val Ser Lys Gly Thr 115 120
125Asp Asn Thr Ala Asp Asn Cys Ile Glu Ile Trp Asn Ser Arg Leu Pro
130 135 140Pro Arg Pro Leu Gly Arg Ser
Ala Ser Thr Ala Glu Lys Ser Ala Val145 150
155 160Ile Gly Ser Ser Thr Val Pro Asp Ile Pro Lys Phe
Val Asp Ile Asp 165 170
175Ser Asp Asp Lys Asp Pro Leu Leu Cys Cys Leu Tyr Ala Pro Glu Ile
180 185 190His Tyr Asn Leu Arg Val
Ser Glu Leu Lys Arg Arg Pro Leu Pro Asp 195 200
205Phe Met Glu Arg Ile Gln Lys Asp Val Thr Gln Ser Met Arg
Gly Ile 210 215 220Leu Val Asp Trp Leu
Val Glu Val Ser Glu Glu Tyr Thr Leu Ala Ser225 230
235 240Asp Thr Leu Tyr Leu Thr Val Tyr Leu Ile
Asp Trp Phe Leu His Gly 245 250
255Asn Tyr Val Gln Arg Gln Gln Leu Gln Leu Leu Gly Ile Thr Cys Met
260 265 270Leu Ile Ala Ser Lys
Tyr Glu Glu Ile Ser Ala Pro Arg Ile Glu Glu 275
280 285Phe Cys Phe Ile Thr Asp Asn Thr Tyr Thr Arg Asp
Gln Val Leu Glu 290 295 300Met Glu Asn
Gln Val Leu Lys His Phe Ser Phe Gln Ile Tyr Thr Pro305
310 315 320Thr Pro Lys Thr Phe Leu Arg
Arg Phe Leu Arg Ala Ala Gln Ala Ser 325
330 335Arg Leu Ser Pro Ser Leu Glu Val Glu Phe Leu Ala
Ser Tyr Leu Thr 340 345 350Glu
Leu Thr Leu Ile Asp Tyr His Phe Leu Lys Phe Leu Pro Ser Val 355
360 365Val Ala Ala Ser Ala Gly Phe Leu Ala
Lys Trp Thr Met Asp Gln Ser 370 375
380Asn His Pro Trp Asn Pro Thr Leu Glu His Tyr Thr Thr Tyr Lys Ala385
390 395 400Ser Asp Leu Lys
Ala Ser Val His Ala Leu Gln Asp Leu Gln Leu Asn 405
410 415Thr Lys Gly Cys Pro Leu Ser Ala Ile Arg
Met Lys Tyr Arg Gln Glu 420 425
430Lys Tyr Lys Ser Val Ala Val Leu Thr Ser Pro Lys Leu Leu Asp Thr
435 440 445Leu Phe
45070223PRTArabidopsis thaliana 70Met Gly Lys Lys Cys Asp Leu Cys Asn Gly
Val Ala Arg Met Tyr Cys1 5 10
15Glu Ser Asp Gln Ala Ser Leu Cys Trp Asp Cys Asp Gly Lys Val His
20 25 30Gly Ala Asn Phe Leu Val
Ala Lys His Thr Arg Cys Leu Leu Cys Ser 35 40
45Ala Cys Gln Ser Leu Thr Pro Trp Lys Ala Thr Gly Leu Arg
Leu Gly 50 55 60Pro Thr Phe Ser Val
Cys Glu Ser Cys Val Ala Leu Lys Asn Ala Gly65 70
75 80Gly Gly Arg Gly Asn Arg Val Leu Ser Glu
Asn Arg Gly Gln Glu Glu 85 90
95Val Asn Ser Phe Glu Ser Glu Glu Asp Arg Ile Arg Glu Asp His Gly
100 105 110Asp Gly Asp Asp Ala
Glu Ser Tyr Asp Asp Asp Glu Glu Glu Asp Glu 115
120 125Asp Glu Glu Tyr Ser Asp Asp Glu Asp Glu Asp Asp
Asp Glu Asp Gly 130 135 140Asp Asp Glu
Glu Ala Glu Asn Gln Val Val Pro Trp Ser Ala Ala Ala145
150 155 160Gln Val Pro Pro Val Met Ser
Ser Ser Ser Ser Asp Gly Gly Ser Gly 165
170 175Gly Ser Val Thr Lys Arg Thr Arg Ala Arg Glu Asn
Ser Asp Leu Leu 180 185 190Cys
Ser Asp Asp Glu Ile Gly Ser Ser Ser Ala Gln Gly Ser Asn Tyr 195
200 205Ser Arg Pro Leu Lys Arg Ser Ala Phe
Lys Ser Thr Val Val Val 210 215
22071429PRTArabidopsis thaliana 71Met Val Asn Ser Cys Glu Asn Lys Ile Phe
Val Lys Pro Thr Ser Thr1 5 10
15Thr Ile Leu Gln Asp Glu Thr Arg Ser Arg Lys Phe Gly Gln Glu Met
20 25 30Lys Arg Glu Lys Arg Arg
Val Leu Arg Val Ile Asn Gln Asn Leu Ala 35 40
45Gly Ala Arg Val Tyr Pro Cys Val Val Asn Lys Lys Gly Ser
Leu Leu 50 55 60Ser Asn Lys Gln Glu
Glu Glu Glu Gly Cys Gln Lys Lys Lys Phe Asp65 70
75 80Ser Leu Arg Pro Ser Val Thr Arg Ser Gly
Val Glu Glu Glu Thr Asn 85 90
95Lys Lys Leu Lys Pro Ser Val Pro Ser Ala Asn Asp Phe Gly Asp Cys
100 105 110Ile Phe Ile Asp Glu
Glu Glu Ala Thr Leu Asp Leu Pro Met Pro Met 115
120 125Ser Leu Glu Lys Pro Tyr Ile Glu Ala Asp Pro Met
Glu Glu Val Glu 130 135 140Met Glu Asp
Val Thr Val Glu Glu Pro Ile Val Asp Ile Asp Val Leu145
150 155 160Asp Ser Lys Asn Ser Leu Ala
Ala Val Glu Tyr Val Gln Asp Leu Tyr 165
170 175Ala Phe Tyr Arg Thr Met Glu Arg Phe Ser Cys Val
Pro Val Asp Tyr 180 185 190Met
Met Gln Gln Ile Asp Leu Asn Glu Lys Met Arg Ala Ile Leu Ile 195
200 205Asp Trp Leu Ile Glu Val His Asp Lys
Phe Asp Leu Met Asn Glu Thr 210 215
220Leu Phe Leu Thr Val Asn Leu Ile Asp Arg Phe Leu Ser Lys Gln Asn225
230 235 240Val Met Arg Lys
Lys Leu Gln Leu Val Gly Leu Val Ala Leu Leu Leu 245
250 255Ala Cys Lys Tyr Glu Glu Val Ser Val Pro
Val Val Glu Asp Leu Val 260 265
270Leu Ile Ser Asp Lys Ala Tyr Thr Arg Asn Asp Val Leu Glu Met Glu
275 280 285Lys Thr Met Leu Ser Thr Leu
Gln Phe Asn Ile Ser Leu Pro Thr Gln 290 295
300Tyr Pro Phe Leu Lys Arg Phe Leu Lys Ala Ala Gln Ala Asp Lys
Lys305 310 315 320Cys Glu
Val Leu Ala Ser Phe Leu Ile Glu Leu Ala Leu Val Glu Tyr
325 330 335Glu Met Leu Arg Phe Pro Pro
Ser Leu Leu Ala Ala Thr Ser Val Tyr 340 345
350Thr Ala Gln Cys Thr Leu Asp Gly Ser Arg Lys Trp Asn Ser
Thr Cys 355 360 365Glu Phe His Cys
His Tyr Ser Glu Asp Gln Leu Met Glu Cys Ser Arg 370
375 380Lys Leu Val Ser Leu His Gln Arg Ala Ala Thr Gly
Asn Leu Thr Gly385 390 395
400Val Tyr Arg Lys Tyr Ser Thr Ser Lys Phe Gly Tyr Ile Ala Lys Cys
405 410 415Glu Ala Ala His Phe
Leu Val Ser Glu Ser His His Ser 420
42572359PRTArabidopsis thaliana 72Thr Lys Gln Glu Ala Lys Ala Ala Phe Lys
Ser Leu Leu Glu Ser Val1 5 10
15Asn Val His Ser Asp Trp Thr Trp Glu Gln Thr Leu Lys Glu Ile Val
20 25 30His Asp Lys Arg Tyr Gly
Ala Leu Arg Thr Leu Gly Glu Arg Lys Gln 35 40
45Ala Phe Asn Glu Tyr Leu Gly Gln Arg Lys Lys Val Glu Ala
Glu Glu 50 55 60Arg Arg Arg Arg Gln
Lys Lys Ala Arg Glu Glu Phe Val Lys Met Leu65 70
75 80Glu Glu Cys Glu Glu Leu Ser Ser Ser Leu
Lys Trp Ser Lys Ala Met 85 90
95Ser Leu Phe Glu Asn Asp Gln Arg Phe Lys Ala Val Asp Arg Pro Arg
100 105 110Asp Arg Glu Asp Leu
Phe Asp Asn Tyr Ile Val Glu Leu Glu Arg Lys 115
120 125Glu Arg Glu Lys Ala Ala Glu Glu His Arg Gln Tyr
Met Ala Asp Tyr 130 135 140Arg Lys Phe
Leu Glu Thr Cys Asp Tyr Ile Lys Ala Gly Thr Gln Trp145
150 155 160Arg Lys Ile Gln Asp Arg Leu
Glu Asp Asp Asp Arg Cys Ser Cys Leu 165
170 175Glu Lys Ile Asp Arg Leu Ile Gly Phe Glu Glu Tyr
Ile Leu Asp Leu 180 185 190Glu
Lys Glu Glu Glu Glu Leu Lys Arg Val Glu Lys Glu His Val Arg 195
200 205Arg Ala Glu Arg Lys Asn Arg Asp Ala
Phe Arg Thr Leu Leu Glu Glu 210 215
220His Val Ala Ala Gly Ile Leu Thr Ala Lys Thr Tyr Trp Leu Asp Tyr225
230 235 240Cys Ile Glu Leu
Lys Asp Leu Pro Gln Tyr Gln Ala Val Ala Ser Asn 245
250 255Thr Ser Gly Ser Thr Pro Lys Asp Leu Phe
Glu Asp Val Thr Glu Glu 260 265
270Leu Glu Lys Gln Tyr His Glu Asp Lys Ser Tyr Val Lys Asp Ala Met
275 280 285Lys Ser Arg Lys Ile Ser Met
Val Ser Ser Trp Leu Phe Glu Asp Phe 290 295
300Lys Ser Ala Ile Ser Glu Asp Leu Ser Thr Gln Gln Ile Ser Asp
Ile305 310 315 320Asn Leu
Lys Leu Ile Tyr Asp Asp Leu Val Gly Arg Val Lys Glu Lys
325 330 335Glu Glu Lys Glu Ala Arg Lys
Leu Gln Arg Leu Ala Glu Glu Phe Thr 340 345
350Asn Leu Leu His Thr Phe Lys 35573110PRTArabidopsis
thaliana 73Gln Glu Lys Pro Trp Glu Asn Asp Pro His Tyr Phe Lys Arg Val
Lys1 5 10 15Ile Ser Ala
Leu Ala Leu Leu Lys Met Val Val His Ala Arg Ser Gly 20
25 30Gly Thr Ile Glu Ile Met Gly Leu Met Gln
Gly Lys Thr Asp Gly Asp 35 40
45Thr Ile Ile Val Met Asp Ala Phe Ala Leu Pro Val Glu Gly Thr Glu 50
55 60Thr Arg Val Asn Ala Gln Asp Asp Ala
Tyr Glu Tyr Met Val Glu Tyr65 70 75
80Ser Gln Thr Asn Lys Leu Ala Gly Pro Ala Gly Glu Cys Cys
Trp Met 85 90 95Val Ser
Leu Ser Pro Trp Ile Trp Met Leu Ala Leu Arg Tyr 100
105 11074337PRTArabidopsis thaliana 74Val Asp Ser
Pro Asp Ser Thr Ser Asp Asn Ile Phe Tyr Tyr Asp Asp1 5
10 15Thr Ser Gln Thr Arg Phe Gln Gln Glu
Lys Pro Trp Glu Asn Asp Pro 20 25
30His Tyr Phe Lys Arg Val Lys Ile Ser Ala Leu Ala Leu Leu Lys Met
35 40 45Val Val His Ala Arg Ser Gly
Gly Thr Ile Glu Ile Met Gly Leu Met 50 55
60Gln Gly Lys Thr Asp Gly Asp Thr Ile Ile Val Met Asp Ala Phe Ala65
70 75 80Leu Pro Val Glu
Gly Thr Glu Thr Arg Val Asn Ala Gln Asp Asp Ala 85
90 95Tyr Glu Tyr Met Val Glu Tyr Ser Gln Thr
Asn Lys Leu Ala Gly Arg 100 105
110Leu Glu Asn Val Val Gly Trp Tyr His Ser His Pro Gly Tyr Gly Cys
115 120 125Trp Leu Ser Gly Ile Asp Val
Ser Thr Gln Arg Leu Asn Gln Gln His 130 135
140Gln Glu Pro Phe Leu Ala Val Val Ile Asp Pro Thr Arg Thr Val
Ser145 150 155 160Ala Gly
Lys Val Glu Ile Gly Ala Phe Arg Thr Tyr Ser Lys Gly Tyr
165 170 175Lys Pro Pro Asp Glu Pro Val
Ser Glu Tyr Gln Thr Ile Pro Leu Asn 180 185
190Lys Ile Glu Asp Phe Gly Val His Cys Lys Gln Tyr Tyr Ser
Leu Asp 195 200 205Val Thr Tyr Phe
Lys Ser Ser Leu Asp Ser His Leu Leu Asp Leu Leu 210
215 220Trp Asn Lys Tyr Trp Val Asn Thr Leu Ser Ser Ser
Pro Leu Leu Gly225 230 235
240Asn Gly Asp Tyr Val Ala Gly Gln Ile Ser Asp Leu Ala Glu Lys Leu
245 250 255Glu Gln Ala Glu Ser
His Leu Val Gln Ser Arg Phe Gly Gly Val Val 260
265 270Pro Ser Ser Leu His Lys Lys Lys Glu Asp Glu Ser
Gln Leu Thr Lys 275 280 285Ile Thr
Arg Asp Ser Ala Lys Ile Thr Val Glu Gln Val His Gly Leu 290
295 300Met Ser Gln Val Ile Lys Asp Glu Leu Phe Asn
Ser Met Arg Gln Ser305 310 315
320Asn Asn Lys Ser Pro Thr Asp Ser Ser Asp Pro Asp Pro Met Ile Thr
325 330
335Tyr75436PRTArabidopsis thaliana 75Met Tyr Cys Ser Ser Ser Met His Pro
Asn Ala Asn Lys Glu Asn Ile1 5 10
15Ser Thr Ser Asp Val Gln Glu Ser Phe Val Arg Ile Thr Arg Ser
Arg 20 25 30Ala Lys Lys Ala
Met Gly Arg Gly Val Ser Ile Pro Pro Thr Lys Pro 35
40 45Ser Phe Lys Gln Gln Lys Arg Arg Ala Val Leu Lys
Asp Val Ser Asn 50 55 60Thr Ser Ala
Asp Ile Ile Tyr Ser Glu Leu Arg Lys Gly Gly Asn Ile65 70
75 80Lys Ala Asn Arg Lys Cys Leu Lys
Glu Pro Lys Lys Ala Ala Lys Glu 85 90
95Gly Ala Asn Ser Ala Met Asp Ile Leu Val Asp Met His Thr
Glu Lys 100 105 110Ser Lys Leu
Ala Glu Asp Leu Ser Lys Ile Arg Met Ala Glu Ala Gln 115
120 125Asp Val Ser Leu Ser Asn Phe Lys Asp Glu Glu
Ile Thr Glu Gln Gln 130 135 140Glu Asp
Gly Ser Gly Val Met Glu Leu Leu Gln Val Val Asp Ile Asp145
150 155 160Ser Asn Val Glu Asp Pro Gln
Cys Cys Ser Leu Tyr Ala Ala Asp Ile 165
170 175Tyr Asp Asn Ile His Val Ala Glu Leu Gln Gln Arg
Pro Leu Ala Asn 180 185 190Tyr
Met Glu Leu Val Gln Arg Asp Ile Asp Pro Asp Met Arg Lys Ile 195
200 205Leu Ile Asp Trp Leu Val Glu Val Ser
Asp Asp Tyr Lys Leu Val Pro 210 215
220Asp Thr Leu Tyr Leu Thr Val Asn Leu Ile Asp Arg Phe Leu Ser Asn225
230 235 240Ser Tyr Ile Glu
Arg Gln Arg Leu Gln Leu Leu Gly Val Ser Cys Met 245
250 255Leu Ile Ala Ser Lys Tyr Glu Glu Leu Ser
Ala Pro Gly Val Glu Glu 260 265
270Phe Cys Phe Ile Thr Ala Asn Thr Tyr Thr Arg Arg Glu Val Leu Ser
275 280 285Met Glu Ile Gln Ile Leu Asn
Phe Val His Phe Arg Leu Ser Val Pro 290 295
300Thr Thr Lys Thr Phe Leu Arg Arg Phe Ile Lys Ala Ala Gln Ala
Ser305 310 315 320Tyr Lys
Val Pro Phe Ile Glu Leu Glu Tyr Leu Ala Asn Tyr Leu Ala
325 330 335Glu Leu Thr Leu Val Glu Tyr
Ser Phe Leu Arg Phe Leu Pro Ser Leu 340 345
350Ile Ala Ala Ser Ala Val Phe Leu Ala Arg Trp Thr Leu Asp
Gln Thr 355 360 365Asp His Pro Trp
Asn Pro Thr Leu Gln His Tyr Thr Arg Tyr Glu Val 370
375 380Ala Glu Leu Lys Asn Thr Val Leu Ala Met Glu Asp
Leu Gln Leu Asn385 390 395
400Thr Ser Gly Cys Thr Leu Ala Ala Thr Arg Glu Lys Tyr Asn Gln Pro
405 410 415Lys Phe Lys Ser Val
Ala Lys Leu Thr Ser Pro Lys Arg Val Thr Leu 420
425 430Leu Phe Ser Arg 43576254PRTArabidopsis
thaliana 76Met Ala Lys Met Gln Leu Ser Ile Phe Ile Ala Val Val Ala Leu
Ile1 5 10 15Val Cys Ser
Ala Ser Ala Lys Thr Ala Ser Pro Pro Ala Pro Val Leu 20
25 30Pro Pro Thr Pro Ala Pro Ala Pro Ala Pro
Glu Asn Val Asn Leu Thr 35 40
45Glu Leu Leu Ser Val Ala Gly Pro Phe His Thr Phe Leu Asp Tyr Leu 50
55 60Leu Ser Thr Gly Val Ile Glu Thr Phe
Gln Asn Gln Ala Asn Asn Thr65 70 75
80Glu Glu Gly Ile Thr Ile Phe Val Pro Lys Asp Asp Ala Phe
Lys Ala 85 90 95Gln Lys
Asn Pro Pro Leu Ser Asn Leu Thr Lys Asp Gln Leu Lys Gln 100
105 110Leu Val Leu Phe His Ala Leu Pro His
Tyr Tyr Ser Leu Ser Glu Phe 115 120
125Lys Asn Leu Ser Gln Ser Gly Pro Val Ser Thr Phe Ala Gly Gly Gln
130 135 140Tyr Ser Leu Lys Phe Thr Asp
Val Ser Gly Thr Val Arg Ile Asp Ser145 150
155 160Leu Trp Thr Arg Thr Lys Val Ser Ser Ser Val Phe
Ser Thr Asp Pro 165 170
175Val Ala Val Tyr Gln Val Asn Arg Val Leu Leu Pro Glu Ala Ile Phe
180 185 190Gly Thr Asp Val Pro Pro
Met Pro Ala Pro Ala Pro Ala Pro Ile Val 195 200
205Ser Ala Pro Ser Asp Ser Pro Ser Val Ala Asp Ser Glu Gly
Ala Ser 210 215 220Ser Pro Lys Ser Ser
His Lys Asn Ser Gly Gln Lys Leu Leu Leu Ala225 230
235 240Pro Ile Ser Met Val Ile Ser Gly Leu Val
Ala Leu Phe Leu 245 2507786PRTArabidopsis
thaliana 77Met Ala Ile Ser Lys Ala Leu Ile Ala Ser Phe Leu Ile Ser Leu
Leu1 5 10 15Val Leu Gln
Leu Val Gln Ala Asp Val Glu Asn Ser Gln Lys Lys Asn 20
25 30Gly Tyr Ala Lys Lys Ile Asp Cys Gly Ser
Ala Cys Val Ala Arg Leu 35 40
45Gln Ala Phe Glu Glu Ala Glu Ala Val Ser Gln Ser Val Arg Asp Leu 50
55 60Leu Leu Gln Val Gln Leu Cys Ala Ser
Gly Tyr Val Arg Lys Leu Arg65 70 75
80Gln Val Pro Val Leu Arg
8578125PRTArabidopsis thaliana 78Lys Glu Glu Ala Gly Met Tyr Trp Gly Tyr
Lys Val Arg Tyr Ala Ser1 5 10
15Gln Leu Ser Ser Val Phe Lys Glu Cys Pro Phe Glu Gly Gly Tyr Asp
20 25 30Tyr Leu Ile Gly Thr Ser
Glu His Gly Leu Val Ile Ser Ser Ser Glu 35 40
45Leu Lys Ile Pro Thr Phe Arg His Leu Leu Ile Ala Phe Gly
Gly Leu 50 55 60Ala Gly Leu Glu Glu
Ser Ile Glu Asp Asp Asn Gln Tyr Lys Gly Lys65 70
75 80Asn Val Arg Asp Val Phe Asn Val Tyr Leu
Asn Thr Cys Pro His Gln 85 90
95Gly Ser Arg Thr Ile Arg Ala Glu Glu Ala Met Phe Ile Ser Leu Gln
100 105 110Tyr Phe Gln Glu Pro
Ile Ser Arg Ala Val Arg Arg Leu 115 120
12579231PRTArabidopsis thaliana 79Ala Arg Glu Met Gly Lys Lys Asn
Lys Arg Ser Gln Asp Glu Ser Glu1 5 10
15Leu Glu Leu Glu Pro Glu Leu Thr Lys Ile Ile Asp Gly Asp
Ser Lys 20 25 30Lys Lys Lys
Asn Lys Asn Lys Lys Lys Arg Ser His Glu Asp Thr Glu 35
40 45Ile Glu Pro Glu Gln Lys Met Ser Leu Asp Gly
Asp Ser Arg Glu Glu 50 55 60Lys Ile
Lys Lys Lys Arg Lys Asn Lys Asn Gln Glu Glu Glu Pro Glu65
70 75 80Leu Val Thr Glu Lys Thr Lys
Val Gln Glu Glu Glu Lys Gly Asn Val 85 90
95Glu Glu Gly Arg Ala Thr Val Ser Ile Ala Ile Ala Gly
Ser Ile Ile 100 105 110His Asn
Thr Gln Ser Leu Glu Leu Ala Thr Arg Val Ile Ser Leu Ser 115
120 125Leu Tyr Leu Ser Leu Arg Phe Ser Val Phe
Pro Phe Pro Asp Asn Leu 130 135 140Lys
Ser Pro Ser Ser Ile Ser Asn Ile Ser Gln Leu Ala Gly Gln Ile145
150 155 160Ala Arg Ala Ala Thr Ile
Phe Arg Ile Asp Glu Ile Val Val Phe Asp 165
170 175Asn Lys Ser Ser Ser Glu Ile Glu Ser Ala Ala Thr
Asn Ala Ser Asp 180 185 190Ser
Asn Glu Ser Gly Ala Ser Phe Leu Val Arg Ile Leu Lys Tyr Leu 195
200 205Glu Thr Pro Gln Tyr Leu Arg Lys Ser
Leu Phe Pro Lys Gln Asn Asp 210 215
220Leu Arg Tyr Val Gly Met Leu225 23080112PRTArabidopsis
thaliana 80Val Ser Ala Val Trp His Gly Leu Tyr Pro Gly Tyr Ile Ile Phe
Phe1 5 10 15Val Gln Ser
Ala Leu Met Ile Asp Gly Ser Lys Ala Ile Tyr Arg Trp 20
25 30Gln Gln Ala Ile Pro Pro Lys Met Ala Met
Leu Arg Asn Val Leu Val 35 40
45Leu Ile Asn Phe Leu Tyr Thr Val Val Val Leu Asn Tyr Ser Ser Val 50
55 60Gly Phe Met Val Leu Ser Leu His Glu
Thr Leu Val Ala Phe Lys Ser65 70 75
80Val Tyr Tyr Ile Gly Thr Val Ile Pro Ile Ala Val Leu Leu
Leu Ser 85 90 95Tyr Leu
Val Pro Val Lys Pro Val Arg Pro Lys Thr Arg Lys Glu Glu 100
105 11081119PRTArabidopsis
thalianaMISC_FEATURE(97)..(98)Xaa = any amino acid 81Val Phe Glu Tyr Met
Asp Thr Asp Val Lys Lys Phe Ile Arg Ser Phe1 5
10 15Arg Ser Thr Gly Lys Asn Ile Pro Thr Gln Thr
Ile Lys Ser Leu Met 20 25
30Tyr Gln Leu Cys Lys Gly Met Ala Phe Cys His Gly His Gly Ile Leu
35 40 45His Arg Asp Leu Lys Pro His Asn
Leu Leu Met Asp Pro Lys Thr Met 50 55
60Arg Leu Lys Ile Ala Asp Leu Gly Leu Ala Arg Ala Phe Thr Leu Pro65
70 75 80Met Lys Lys Tyr Thr
His Glu Ile Leu Thr Leu Trp Tyr Arg Ala Pro 85
90 95Xaa Xaa Ser Ser Trp Cys His Pro Leu Leu Tyr
Ser Cys Gly Tyr Val 100 105
110Xaa Cys Trp Leu His Ile Cys 11582296PRTArabidopsis thaliana
82Pro Lys Arg Arg Met Ser Met Glu Met Glu Leu Phe Val Thr Pro Glu1
5 10 15Lys Gln Arg Gln His Pro
Ser Val Ser Val Glu Lys Thr Pro Val Arg 20 25
30Arg Lys Leu Ile Val Asp Asp Asp Ser Glu Ile Gly Ser
Glu Lys Lys 35 40 45Gly Gln Ser
Arg Thr Ser Gly Gly Gly Leu Arg Gln Phe Ser Val Met 50
55 60Val Cys Gln Lys Leu Glu Ala Lys Lys Ile Thr Thr
Tyr Lys Glu Val65 70 75
80Ala Asp Glu Ile Ile Ser Asp Phe Ala Thr Ile Lys Gln Asn Ala Glu
85 90 95Lys Pro Leu Asn Glu Asn
Glu Tyr Asn Glu Lys Asn Ile Arg Arg Arg 100
105 110Val Tyr Asp Ala Leu Asn Val Phe Met Ala Leu Asp
Ile Ile Ala Arg 115 120 125Asp Lys
Lys Glu Ile Arg Trp Lys Gly Leu Pro Ile Thr Cys Lys Lys 130
135 140Asp Val Glu Glu Val Lys Met Asp Arg Asn Lys
Val Met Ser Ser Val145 150 155
160Gln Lys Lys Ala Ala Phe Leu Lys Glu Leu Arg Glu Lys Val Ser Ser
165 170 175Leu Glu Ser Leu
Met Ser Arg Asn Gln Glu Met Val Val Lys Thr Gln 180
185 190Gly Pro Ala Glu Gly Phe Thr Leu Pro Phe Ile
Leu Leu Glu Thr Asn 195 200 205Pro
His Ala Val Val Glu Ile Glu Ile Ser Glu Asp Met Gln Leu Val 210
215 220His Leu Asp Phe Asn Ser Thr Pro Phe Ser
Val His Asp Asp Ala Tyr225 230 235
240Ile Leu Lys Leu Met Gln Glu Gln Lys Gln Glu Gln Asn Arg Val
Ser 245 250 255Ser Ser Ser
Ser Thr His His Gln Ser Gln His Ser Ser Ala His Ser 260
265 270Ser Ser Ser Ser Cys Ile Ala Ser Gly Thr
Ser Gly Pro Val Cys Trp 275 280
285Asn Ser Gly Ser Ile Asp Thr Arg 290
29583173PRTArabidopsis thaliana 83Met Gln Pro Thr Glu Thr Ser Gln Pro Ala
Pro Ser Asp Gln Gly Arg1 5 10
15Arg Leu Lys Asp Gln Leu Ser Glu Ser Met Ser Phe Ser Ser Gln Met
20 25 30Lys Lys Glu Asp Asp Glu
Leu Ser Met Lys Ala Leu Ser Ala Phe Lys 35 40
45Ala Lys Glu Glu Glu Ile Glu Lys Lys Lys Met Glu Ile Arg
Glu Arg 50 55 60Val Gln Ala Gln Leu
Gly Arg Val Glu Asp Glu Ser Lys Arg Leu Ala65 70
75 80Met Ile Arg Glu Glu Leu Glu Gly Phe Ala
Asp Pro Met Arg Lys Glu 85 90
95Val Thr Met Val Arg Lys Lys Ile Asp Ser Leu Asp Lys Glu Leu Lys
100 105 110Pro Leu Gly Asn Thr
Val Gln Lys Lys Glu Thr Glu Tyr Lys Asp Ala 115
120 125Leu Glu Ala Phe Asn Glu Lys Asn Lys Glu Lys Val
Glu Leu Ile Thr 130 135 140Lys Leu Gln
Glu Leu Glu Gly Glu Ser Glu Lys Phe Arg Phe Lys Lys145
150 155 160Leu Glu Glu Leu Ser Lys Asn
Ile Asp Leu Thr Lys Pro 165
1708446PRTArabidopsis thaliana 84Gln Lys Gln Ala Pro Gly Ala Gly Asp Val
Pro Ala Thr Ile Gln Glu1 5 10
15Glu Asp Asp Asp Asp Asp Val Pro Asp Leu Val Val Gly Glu Thr Phe
20 25 30Glu Thr Pro Ala Thr Glu
Glu Ala Pro Lys Ala Ala Ala Ser 35 40
4585383PRTArabidopsis thaliana 85Met Glu Asp Asp Asp Glu Ile Gln Ser
Ile Pro Ser Pro Gly Asp Ser1 5 10
15Ser Leu Ser Pro Gln Ala Pro Pro Ser Pro Pro Ile Leu Pro Thr
Asn 20 25 30Asp Val Thr Val
Ala Val Val Lys Lys Pro Gln Pro Gly Leu Ser Ser 35
40 45Gln Ser Pro Ser Met Asn Ala Leu Ala Leu Val Val
His Thr Pro Ser 50 55 60Val Thr Gly
Gly Gly Gly Ser Gly Asn Arg Asn Gly Arg Gly Gly Gly65 70
75 80Gly Gly Ser Gly Gly Gly Gly Gly
Gly Arg Asp Asp Cys Trp Ser Glu 85 90
95Glu Ala Thr Lys Val Leu Ile Glu Ala Trp Gly Asp Arg Phe
Ser Glu 100 105 110Pro Gly Lys
Gly Thr Leu Lys Gln Gln His Trp Lys Glu Val Ala Glu 115
120 125Ile Val Asn Lys Ser Arg Gln Cys Lys Tyr Pro
Lys Thr Asp Ile Gln 130 135 140Cys Lys
Asn Arg Ile Asp Thr Val Lys Lys Lys Tyr Lys Gln Glu Lys145
150 155 160Ala Lys Ile Ala Ser Gly Asp
Gly Pro Ser Lys Trp Val Phe Phe Lys 165
170 175Lys Leu Glu Ser Leu Ile Gly Gly Thr Thr Thr Phe
Ile Ala Ser Ser 180 185 190Lys
Ala Ser Glu Lys Ala Pro Met Gly Gly Ala Leu Gly Asn Ser Arg 195
200 205Ser Ser Met Phe Lys Arg Gln Thr Lys
Gly Asn Gln Ile Val Gln Gln 210 215
220Gln Gln Glu Lys Arg Gly Ser Asp Ser Met Arg Trp His Phe Arg Lys225
230 235 240Arg Ser Ala Ser
Glu Thr Glu Ser Glu Ser Asp Pro Glu Pro Glu Ala 245
250 255Ser Pro Glu Glu Ser Ala Glu Ser Leu Pro
Pro Leu Gln Pro Ile Gln 260 265
270Pro Leu Ser Phe His Met Pro Lys Arg Leu Lys Val Asp Lys Ser Gly
275 280 285Gly Gly Gly Ser Gly Val Gly
Asp Val Ala Arg Ala Ile Leu Gly Phe 290 295
300Thr Glu Ala Tyr Glu Lys Ala Glu Thr Ala Lys Leu Lys Leu Met
Ala305 310 315 320Glu Leu
Glu Lys Glu Arg Met Lys Phe Ala Lys Glu Met Glu Leu Gln
325 330 335Arg Met Gln Phe Leu Lys Thr
Gln Leu Glu Ile Thr Gln Asn Asn Gln 340 345
350Glu Glu Glu Glu Arg Ser Arg Gln Arg Gly Glu Arg Arg Ile
Val Asp 355 360 365Asp Asp Asp Asp
Arg Asn Gly Lys Asn Asn Gly Asn Val Ser Ser 370 375
38086131PRTArabidopsis thalianaMISC_FEATURE(70)..(70)Xaa =
any amino acid 86Gly Thr Ser Leu Leu Leu His Ala Ser Ser Ser Ser Ser Ser
Ile Ser1 5 10 15Leu Thr
Ile Pro Ser Asn His Ser Ser Met Ala Thr Val Ser Ser Ser 20
25 30Ser Trp Pro Asn Pro Asn Pro Asn Pro
Asp Ser Thr Ser Ala Ser Asp 35 40
45Ser Asp Ser Thr Phe Pro Ser His Arg Asp Arg Val Asp Glu Pro Asp 50
55 60Ser Leu Asp Ser Phe Xaa Ser Met Ser
Leu Asn Ser Asp Glu Pro Asn65 70 75
80Gln Thr Ser Asn Gln Ser Pro Leu Ser Pro Pro Thr Pro Asn
Leu Pro 85 90 95Val Met
Pro Pro Pro Phe Val Leu Tyr Leu Ser Phe Asn Gln Asp His 100
105 110Ala Cys Phe Ala Cys Xaa His Phe Val
Pro Ser Leu Ser Leu Tyr Leu 115 120
125Ser Ala Thr 13087181PRTArabidopsis thaliana 87Gln Ala His Asp Ser
Arg Ile Ala Cys Phe Ala Leu Thr Gln Asp Gly1 5
10 15His Leu Leu Ala Thr Ala Ser Ser Lys Gly Thr
Leu Val Arg Ile Phe 20 25
30Asn Thr Val Asp Gly Thr Leu Arg Gln Glu Val Arg Arg Gly Ala Asp
35 40 45Arg Ala Glu Ile Tyr Ser Leu Ala
Phe Ser Ser Asn Ala Gln Trp Leu 50 55
60Ala Val Ser Ser Asp Lys Gly Thr Val His Val Phe Gly Leu Lys Val65
70 75 80Asn Ser Gly Ser Gln
Val Lys Asp Ser Ser Arg Ile Ala Pro Asp Ala 85
90 95Thr Pro Ser Ser Pro Ser Ser Ser Leu Ser Leu
Phe Lys Gly Val Leu 100 105
110Pro Arg Tyr Phe Ser Ser Glu Trp Ser Val Ala Gln Phe Arg Leu Val
115 120 125Glu Gly Thr Gln Tyr Ile Ala
Ala Phe Gly His Gln Lys Asn Thr Val 130 135
140Val Ile Leu Gly Met Asp Gly Ser Phe Tyr Arg Cys Gln Phe Asp
Pro145 150 155 160Val Asn
Gly Gly Glu Met Ser Gln Leu Glu Tyr His Asn Cys Leu Lys
165 170 175Pro Pro Ser Val Phe
18088175PRTArabidopsis thaliana 88Met Asp Asp Ser Glu Glu Asp Gln Arg Leu
Pro His His Lys Asp Pro1 5 10
15Lys Glu Phe Val Ser Leu Asp Lys Leu Ala Glu Leu Gly Val Leu Ser
20 25 30Trp Arg Leu Asp Ala Asp
Asn Tyr Glu Thr Asp Glu Asp Leu Lys Lys 35 40
45Ile Arg Glu Ser Arg Gly Tyr Ser Tyr Met Asp Phe Cys Glu
Val Cys 50 55 60Pro Glu Lys Leu Pro
Asn Tyr Glu Val Lys Val Lys Ser Phe Phe Glu65 70
75 80Glu His Leu His Thr Asp Glu Glu Ile Arg
Tyr Cys Val Ala Gly Thr 85 90
95Gly Tyr Phe Asp Val Arg Asp Arg Asn Glu Ala Trp Ile Arg Val Leu
100 105 110Val Lys Lys Gly Gly
Met Ile Val Leu Pro Ala Gly Ile Tyr His Arg 115
120 125Phe Thr Val Asp Ser Asp Asn Tyr Ile Lys Ala Met
Arg Leu Phe Val 130 135 140Gly Glu Pro
Val Trp Thr Pro Tyr Asn Arg Pro His Asp His Leu Pro145
150 155 160Ala Arg Lys Glu Tyr Val Asp
Asn Phe Met Ile Asn Ala Ser Ala 165 170
1758998PRTArabidopsis thaliana 89Thr Ser Phe Pro Ile Thr Arg
Lys Lys Thr Leu Lys Met Asp Gly His1 5 10
15Asp Ser Glu Asp Thr Lys Gln Ser Thr Ala Asp Met Thr
Ala Phe Val 20 25 30Gln Asn
Leu Leu Gln Gln Met Gln Thr Arg Phe Gln Thr Met Ser Asp 35
40 45Ser Ile Ile Thr Lys Ile Asp Asp Met Gly
Gly Arg Ile Asn Glu Leu 50 55 60Glu
Gln Ser Ile Asn Asp Leu Arg Ala Glu Met Gly Val Glu Gly Thr65
70 75 80Pro Pro Pro Ala Ser Lys
Ser Gly Asp Glu Pro Lys Thr Pro Ala Ser 85
90 95Ser Ser90117PRTArabidopsis thaliana 90Ala Gln Val
Arg Ala Lys Met Leu Lys Glu Val Ala Thr Glu Lys Gln1 5
10 15Thr Ala Val Asp Thr His Phe Ala Thr
Ala Lys Lys Leu Ala Gln Glu 20 25
30Gly Asp Ala Leu Phe Val Lys Ile Phe Ala Ile Lys Lys Leu Leu Ala
35 40 45Lys Leu Glu Ala Glu Lys Glu
Ser Val Asp Gly Lys Phe Lys Glu Thr 50 55
60Val Lys Glu Leu Ser His Leu Leu Ala Asp Ala Ser Glu Ala Tyr Glu65
70 75 80Glu Tyr His Gly
Ala Val Arg Lys Ala Lys Asp Glu Gln Ala Ala Glu 85
90 95Glu Phe Ala Lys Glu Ala Thr Gln Ser Ala
Glu Ile Ile Trp Val Lys 100 105
110Phe Leu Ser Ser Leu 11591216PRTArabidopsis thaliana 91Met Glu
Phe Gly Ser Phe Leu Val Ser Leu Gly Thr Ser Phe Val Ile1 5
10 15Phe Val Ile Leu Met Leu Leu Phe
Thr Trp Leu Ser Arg Lys Ser Gly 20 25
30Asn Ala Pro Ile Tyr Tyr Pro Asn Arg Ile Leu Lys Gly Leu Glu
Pro 35 40 45Trp Glu Gly Thr Ser
Leu Thr Arg Asn Pro Phe Ala Trp Met Arg Glu 50 55
60Ala Leu Thr Ser Ser Glu Gln Asp Val Val Asn Leu Ser Gly
Val Asp65 70 75 80Thr
Ala Val His Phe Val Phe Leu Ser Thr Val Leu Gly Ile Phe Ala
85 90 95Cys Ser Ser Leu Leu Leu Leu
Pro Thr Leu Leu Pro Leu Ala Ala Thr 100 105
110Asp Asn Asn Ile Lys Asn Thr Lys Asn Ala Thr Asp Thr Thr
Ser Lys 115 120 125Gly Thr Phe Ser
Gln Leu Asp Asn Leu Ser Met Ala Asn Ile Thr Lys 130
135 140Lys Ser Ser Arg Leu Trp Ala Phe Leu Gly Ala Val
Tyr Trp Ile Ser145 150 155
160Leu Val Thr Tyr Phe Phe Leu Trp Lys Ala Tyr Lys His Val Ser Ser
165 170 175Leu Arg Ala Gln Ala
Leu Met Ser Ala Asp Val Lys Pro Glu Gln Phe 180
185 190Ala Ile Leu Val Arg Asp Met Pro Ala Pro Pro Asp
Gly Arg Arg Gly 195 200 205Arg Glu
Phe Gln Ile Tyr Glu Ser 210 21592328PRTArabidopsis
thaliana 92Val His Thr Pro Ala Gly Glu Leu Gln Arg Gln Ile Arg Ser Trp
Leu1 5 10 15Ala Glu Ser
Phe Glu Phe Leu Ser Val Thr Ala Asp Asp Val Ser Gly 20
25 30Val Thr Thr Gly Gln Leu Glu Leu Leu Ser
Thr Ala Ile Met Asp Gly 35 40
45Trp Met Ala Gly Val Gly Ala Pro Val Pro Pro His Thr Asp Ala Leu 50
55 60Gly Gln Leu Val Ser Glu Tyr Ala Lys
Arg Val Tyr Thr Ser Gln Met65 70 75
80Gln His Leu Lys Asp Ile Ala Gly Thr Leu Ala Ser Glu Glu
Ala Glu 85 90 95Asp Ala
Gly Gln Val Ala Lys Leu Arg Ser Ala Leu Glu Ser Val Asp 100
105 110His Lys Arg Arg Lys Ile Leu Gln Gln
Met Arg Ser Asp Ala Ala Leu 115 120
125Phe Thr Leu Glu Glu Gly Ser Ser Pro Val Gln Asn Pro Ser Thr Ala
130 135 140Ala Glu Asp Ser Arg Leu Ala
Ser Leu Ile Ser Leu Asp Ala Ile Leu145 150
155 160Lys Gln Val Lys Glu Ile Thr Arg Gln Ala Ser Val
His Val Leu Ser 165 170
175Lys Ser Lys Lys Lys Ala Leu Leu Glu Ser Leu Asp Glu Leu Asn Glu
180 185 190Arg Met Pro Ser Leu Leu
Asp Val Asp His Pro Cys Ala Gln Arg Glu 195 200
205Ile Asp Thr Ala His Gln Leu Val Glu Thr Ile Pro Glu Gln
Glu Asp 210 215 220Asn Leu Gln Asp Glu
Lys Arg Pro Ser Ile Asp Ser Ile Ser Ser Thr225 230
235 240Glu Thr Asp Val Ser Gln Trp Asn Val Leu
Gln Phe Asn Thr Gly Gly 245 250
255Ser Ser Ala Pro Phe Ile Ile Lys Cys Gly Ala Asn Ser Asn Ser Glu
260 265 270Leu Val Ile Lys Ala
Asp Ala Arg Ile Gln Glu Pro Lys Gly Gly Glu 275
280 285Ile Val Arg Val Val Pro Arg Pro Ser Val Leu Glu
Asn Met Ser Leu 290 295 300Glu Glu Met
Lys Gln Val Phe Gly Gln Leu Pro Glu Ala Leu Ser Ser305
310 315 320Leu Ala Leu Ala Arg Thr Ala
Asp 3259379PRTArabidopsis thaliana 93Thr Tyr Glu Arg Leu
Pro Ile Glu Glu Glu Gln Gln Gln Glu Gln Pro1 5
10 15Leu Gln Leu Glu Asp Gly Lys Lys Gln Lys Glu
Glu Asn Asp Asp Asn 20 25
30Glu Ser Gly Asn Asn Gly Asn Glu Gly Ser Met Gln Pro Pro Met Tyr
35 40 45Asn Met Pro Pro Asn Phe Ile Pro
Asn Gly His Gln Met Ala Gln His 50 55
60Asp Val Tyr Trp Gly Gly Pro Pro Pro Arg Ala Pro Pro Ser Tyr65
70 7594150PRTArabidopsis thaliana 94Ser Lys Ala
Arg Val Leu Ala Ile Pro Asp Asp Leu Ala Asn Val Ser1 5
10 15Cys Gly Val Glu Gln Ile Glu Glu Leu
Lys Gly Leu Asn Leu Val Glu 20 25
30Lys Asp Gly Gly Ser Ser Ser Ser Asp Gly Ala Arg Asn Thr Asn Pro
35 40 45Glu Thr Arg Arg Tyr Ser Gly
Ser Leu Gly Val Glu Asp Gly Ala Tyr 50 55
60Thr Asn Glu Met Leu Gln Ser Ile Glu Met Val Thr Asp Val Leu Asp65
70 75 80Ser Leu Val Arg
Arg Val Thr Val Ala Glu Ser Glu Ser Ala Val Gln 85
90 95Lys Glu Arg Ala Leu Leu Gly Glu Glu Glu
Ile Ser Arg Lys Thr Ile 100 105
110Gln Ile Glu Asn Leu Ser Val Lys Leu Glu Glu Met Glu Arg Phe Ala
115 120 125Tyr Gly Thr Asn Ser Val Leu
Asn Glu Met Arg Glu Arg Ile Glu Glu 130 135
140Leu Val Glu Glu Thr Met145 15095181PRTArabidopsis
thaliana 95Met Thr Asn Ile Ala Met Ala Asp Ala Leu Lys Ser Leu Glu Ile
Val1 5 10 15Asp Gly Leu
Asp Glu Tyr Met Asn Gln Ser Glu Ser Ser Ala Pro His 20
25 30Ser Pro Thr Ser Val Ala Lys Leu Pro Pro
Ser Thr Ala Thr Arg Thr 35 40
45Thr Arg Arg Lys Thr Thr Thr Lys Ala Glu Pro Gln Pro Ser Ser Gln 50
55 60Leu Val Ser Arg Ser Cys Arg Ser Thr
Ser Lys Ser Leu Ala Gly Asp65 70 75
80Met Asp Gln Glu Asn Ile Asn Lys Asn Val Ala Gln Glu Met
Lys Thr 85 90 95Ser Asn
Val Lys Phe Glu Ala Asn Val Leu Lys Thr Pro Ala Ala Gly 100
105 110Ser Thr Arg Lys Thr Ser Ala Ala Thr
Ser Cys Thr Lys Lys Asp Glu 115 120
125Leu Val Gln Ser Val Tyr Ser Thr Arg Arg Ser Thr Arg Leu Leu Glu
130 135 140Lys Cys Met Ala Asp Leu Ser
Leu Lys Thr Lys Glu Thr Val Asp Asn145 150
155 160Lys Pro Ala Lys Asn Glu Asp Thr Glu Gln Lys Val
Ser Ala Gln Glu 165 170
175Lys Asn Leu Thr Gly 18096163PRTArabidopsis thaliana 96Met
Leu Met Leu Cys Gly Phe Thr Val Leu Asp Met Leu Lys His His1
5 10 15Asp Leu Gly Lys Ile Arg Ala
Pro Leu His Pro Leu Arg Lys Lys Met 20 25
30Gln Ile Gln His Ala Tyr Gln Gln Ile His Gln Gly Ser Lys
Leu Leu 35 40 45Lys Met Asp Arg
Met Met Leu Arg Gly Thr Lys Arg Arg Ile Gly Val 50 55
60Arg Lys Gly Asn Leu Gln Arg Glu Arg Arg Lys Lys Asp
Met Ile Gly65 70 75
80Val Lys Asn Ala Lys Gly Met Arg Ser Glu Ala Leu Val Ile Gln Met
85 90 95Ile Glu Arg Ser Thr Arg
Lys Arg Arg Arg Arg Lys Lys Glu Gly Met 100
105 110Thr Leu Ile Leu Ile Glu Ala Asn Cys Pro Arg Met
Glu His Phe Ala 115 120 125Leu Gln
Arg Lys Ser Gly Arg Leu Gly Thr Lys Ile Gln Leu Pro Leu 130
135 140Leu Gln Asp Leu Asn Leu Leu Leu Ile Ser Phe
Thr Asn Arg Gly Val145 150 155
160Lys Cys Cys97170PRTArabidopsis thaliana 97Gly Thr Arg Gln Lys Arg
Glu Thr Ser Asp Pro Glu Ser Asp Leu Lys1 5
10 15Thr Arg Lys Asn Arg Lys Met Gly Lys Asp Gly Leu
Ser Asp Asp Gln 20 25 30Val
Ser Ser Met Lys Glu Ala Phe Met Leu Phe Asp Thr Asp Gly Asp 35
40 45Gly Lys Ile Ala Pro Ser Glu Leu Gly
Ile Leu Met Arg Ser Leu Gly 50 55
60Gly Asn Pro Thr Gln Ala Gln Leu Lys Ser Ile Ile Ala Ser Glu Asn65
70 75 80Leu Ser Ser Pro Phe
Asp Phe Asn Arg Phe Leu Asp Leu Met Ala Lys 85
90 95His Leu Lys Thr Glu Pro Phe Asp Arg Gln Leu
Arg Asp Ala Phe Lys 100 105
110Val Leu Asp Lys Glu Gly Thr Gly Phe Val Ala Val Ala Asp Leu Arg
115 120 125His Ile Leu Thr Ser Ile Gly
Glu Lys Leu Glu Pro Asn Glu Phe Asp 130 135
140Glu Trp Ile Lys Glu Val Asp Val Gly Ser Asp Gly Lys Ile Arg
Tyr145 150 155 160Glu Asp
Phe Ile Ala Arg Met Val Ala Lys 165
1709838PRTArabidopsis thaliana 98Arg Gly Val Ser Phe Arg Ser Arg Glu Met
Arg Pro Ile Phe Ala Ile1 5 10
15Ser Gln Arg Met Arg Ser Ile Lys Glu Ser Lys Glu Val Leu Asp Thr
20 25 30Glu Ser Arg Ser Arg Leu
3599376PRTArabidopsis thaliana 99Met Thr Thr Thr Gly Ser Asn Ser
Asn His Asn His His Glu Ser Asn1 5 10
15Asn Asn Asn Asn Asn Pro Ser Thr Arg Ser Trp Gly Thr Ala
Val Ser 20 25 30Gly Gln Ser
Val Ser Thr Ser Gly Ser Met Gly Ser Pro Ser Ser Arg 35
40 45Ser Glu Gln Thr Ile Thr Val Val Thr Ser Thr
Ser Asp Thr Thr Phe 50 55 60Gln Arg
Leu Asn Asn Leu Asp Ile Gln Gly Asp Asp Ala Gly Ser Gln65
70 75 80Gly Ala Ser Gly Val Lys Lys
Lys Lys Arg Gly Gln Arg Ala Ala Gly 85 90
95Pro Asp Lys Thr Gly Arg Gly Leu Arg Gln Phe Ser Met
Lys Val Cys 100 105 110Glu Lys
Val Glu Ser Lys Gly Arg Thr Thr Tyr Asn Glu Val Ala Asp 115
120 125Glu Leu Val Ala Glu Phe Ala Leu Pro Asn
Asn Asp Gly Thr Ser Pro 130 135 140Asp
Gln Gln Gln Tyr Asp Glu Lys Asn Ile Arg Arg Arg Val Tyr Asp145
150 155 160Ala Leu Asn Val Leu Met
Ala Met Asp Ile Ile Ser Lys Asp Lys Lys 165
170 175Glu Ile Gln Trp Arg Gly Leu Pro Arg Thr Ser Leu
Ser Asp Ile Glu 180 185 190Glu
Leu Lys Asn Glu Arg Leu Ser Leu Arg Asn Arg Ile Glu Lys Lys 195
200 205Thr Ala Tyr Ser Gln Glu Leu Glu Glu
Gln Arg Asn Glu His Leu Tyr 210 215
220Ser Ser Gly Asn Ala Pro Ser Gly Gly Val Ala Leu Pro Phe Ile Leu225
230 235 240Val Gln Thr Arg
Pro His Ala Thr Val Glu Val Glu Ile Ser Glu Asp 245
250 255Met Gln Leu Val His Phe Asp Phe Asn Ser
Thr Pro Phe Glu Leu His 260 265
270Asp Asp Asn Phe Val Leu Lys Thr Met Lys Phe Cys Asp Gln Pro Pro
275 280 285Gln Gln Pro Asn Gly Arg Asn
Asn Ser Gln Leu Val Cys His Asn Phe 290 295
300Thr Pro Glu Asn Pro Asn Lys Gly Pro Ser Thr Gly Pro Thr Pro
Gln305 310 315 320Leu Asp
Met Tyr Glu Thr His Leu Gln Ser Gln Gln His Gln Gln His
325 330 335Ser Gln Leu Gln Ile Ile Pro
Met Pro Glu Thr Asn Asn Val Thr Ser 340 345
350Ser Ala Asp Thr Ala Pro Val Lys Ser Pro Ser Leu Pro Gly
Ile Met 355 360 365Asn Ser Ser Met
Lys Pro Glu Asn 370 375100145PRTArabidopsis thaliana
100Glu Tyr Leu Lys Lys Gly Ser Pro Ile Ser Ala Leu Lys Ser Phe Ile1
5 10 15Ser Ser Leu Ser Glu Pro
Pro Gln Asp Ile Met Asp Ala Leu Phe Asn 20 25
30Ala Leu Phe Asp Gly Val Gly Lys Gly Phe Ala Lys Glu
Val Thr Lys 35 40 45Lys Lys Asn
Tyr Leu Ala Ala Ala Ala Thr Met Gln Glu Asp Gly Ser 50
55 60Gln Met His Leu Leu Asn Ser Ile Gly Thr Phe Cys
Gly Lys Asn Gly65 70 75
80Asn Glu Glu Ala Leu Lys Glu Val Ala Leu Val Leu Lys Ala Leu Tyr
85 90 95Asp Gln Asp Ile Ile Glu
Glu Glu Val Val Leu Asp Trp Tyr Glu Lys 100
105 110Gly Leu Thr Gly Ala Asp Lys Ser Ser Pro Val Trp
Lys Asn Val Lys 115 120 125Pro Phe
Val Glu Trp Leu Gln Ser Ala Glu Ser Glu Ser Glu Glu Glu 130
135 140Asp145101316PRTArabidopsis thaliana 101Leu
Glu Val Glu Arg Asn Ala Ser Ala Val Ala Ala Ser Glu Thr Met1
5 10 15Ala Met Ile Asn Arg Leu His
Glu Glu Lys Ala Ala Met Gln Met Glu 20 25
30Ala Leu Gln Tyr Gln Arg Met Met Glu Glu Gln Ala Glu Phe
Asp Gln 35 40 45Glu Ala Leu Gln
Leu Leu Asn Glu Leu Met Val Asn Arg Glu Lys Glu 50 55
60Asn Ala Glu Leu Glu Lys Glu Leu Glu Val Tyr Arg Lys
Arg Met Glu65 70 75
80Glu Tyr Glu Ala Lys Glu Lys Met Gly Met Leu Arg Arg Arg Leu Arg
85 90 95Asp Ser Ser Val Asp Ser
Tyr Arg Asn Asn Gly Asp Ser Asp Glu Asn 100
105 110Ser Asn Gly Glu Leu Gln Phe Lys Asn Val Glu Gly
Val Thr Asp Trp 115 120 125Lys Tyr
Arg Glu Asn Glu Met Glu Asn Thr Pro Val Asp Val Val Leu 130
135 140Arg Leu Asp Glu Cys Leu Asp Asp Tyr Asp Gly
Glu Arg Leu Ser Ile145 150 155
160Leu Gly Arg Leu Lys Phe Leu Glu Glu Lys Leu Thr Asp Leu Asn Asn
165 170 175Glu Glu Asp Asp
Glu Glu Glu Ala Lys Thr Phe Glu Ser Asn Gly Ser 180
185 190Ile Asn Gly Asn Glu His Ile His Gly Lys Glu
Thr Asn Gly Lys His 195 200 205Arg
Val Ile Gln Ser Lys Arg Leu Leu Pro Leu Phe Asp Ala Val Asp 210
215 220Gly Glu Met Glu Asn Gly Leu Ser Asn Gly
Asn His His Glu Asn Gly225 230 235
240Phe Asp Asp Ser Glu Lys Gly Glu Asn Val Thr Ile Glu Glu Glu
Val 245 250 255Asp Glu Leu
Tyr Glu Arg Leu Glu Ala Leu Glu Ala Asp Arg Glu Phe 260
265 270Leu Arg His Cys Val Gly Ser Leu Lys Lys
Gly Asp Lys Gly Val His 275 280
285Leu Leu His Glu Ile Leu Gln His Leu Arg Asp Leu Arg Asn Ile Asp 290
295 300Leu Thr Arg Val Arg Glu Asn Gly
Asp Met Ser Leu305 310
315102194PRTArabidopsis thaliana 102Ala Ser Leu Ile Lys Leu Ile Arg Leu
Leu Glu Thr Pro Ile Phe Thr1 5 10
15Tyr Leu Arg Leu Gln Leu Leu Glu Pro Gly Arg Tyr Thr Trp Leu
Leu 20 25 30Lys Thr Leu Tyr
Gly Leu Leu Met Leu Leu Pro Gln Gln Ser Ala Ala 35
40 45Phe Lys Ile Leu Arg Thr Arg Leu Lys Thr Val Pro
Thr Tyr Ser Phe 50 55 60Ser Thr Gly
Asn Gln Ile Gly Arg Ala Thr Ser Gly Val Pro Phe Ser65 70
75 80Gln Tyr Lys His Gln Asn Glu Asp
Gly Asp Leu Glu Asp Asp Asn Ile 85 90
95Asn Ser Ser His Gln Gly Ile Asn Phe Ala Val Arg Leu Gln
Gln Phe 100 105 110Glu Asn Val
Gln Asn Leu His Arg Gly Gln Ala Arg Thr Arg Val Asn 115
120 125Tyr Ser Tyr His Ser Ser Ser Ser Ser Thr Ser
Lys Glu Val Arg Arg 130 135 140Ser Glu
Glu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln145
150 155 160Gln Gln Gln Arg Pro Pro Pro
Ser Ser Thr Ser Ser Ser Val Ala Asp 165
170 175Asn Asn Arg Pro Pro Ser Arg Thr Ser Arg Lys Gly
Pro Gly Gln Leu 180 185 190Gln
Leu103289PRTArabidopsis thaliana 103Leu Ile Glu Thr Ser Val Glu Ser Lys
Glu Thr Thr Glu Ser Val Val1 5 10
15Thr Gly Glu Ser Glu Lys Ala Ile Glu Asp Ile Ser Lys Glu Ala
Asp 20 25 30Asn Glu Glu Asp
Asp Asp Glu Glu Glu Gln Glu Gly Asp Glu Asp Asp 35
40 45Asp Glu Asn Glu Glu Glu Glu Val Val Val Pro Glu
Thr Glu Asn Arg 50 55 60Ala Glu Gly
Glu Asp Leu Val Lys Asn Lys Ala Ala Asp Ala Lys Lys65 70
75 80His Leu Gln Met Ile Gly Val Gln
Leu Leu Lys Glu Ser Asp Glu Ala 85 90
95Asn Arg Thr Lys Lys Arg Gly Lys Arg Ala Ser Arg Met Thr
Leu Glu 100 105 110Asp Asp Ala
Asp Glu Asp Trp Phe Pro Glu Glu Pro Phe Glu Ala Phe 115
120 125Lys Glu Met Arg Glu Arg Lys Val Phe Asp Val
Ala Asp Met Tyr Thr 130 135 140Ile Ala
Asp Val Trp Gly Trp Thr Trp Glu Lys Asp Phe Lys Asn Lys145
150 155 160Thr Pro Arg Lys Trp Ser Gln
Glu Trp Glu Val Glu Leu Ala Ile Val 165
170 175Leu Met Thr Lys Val Ile Glu Leu Gly Gly Ile Pro
Thr Ile Gly Asp 180 185 190Cys
Ala Val Ile Leu Arg Ala Ala Leu Arg Ala Pro Met Pro Ser Ala 195
200 205Phe Leu Lys Ile Leu Gln Thr Thr His
Ser Leu Gly Tyr Ser Phe Gly 210 215
220Ser Pro Leu Tyr Asp Glu Ile Ile Thr Leu Cys Leu Asp Leu Gly Glu225
230 235 240Leu Asp Ala Ala
Ile Ala Ile Val Ala Asp Met Glu Thr Thr Gly Ile 245
250 255Thr Val Pro Asp Gln Thr Leu Asp Lys Val
Ile Ser Ala Arg Gln Ser 260 265
270Asn Glu Ser Pro Arg Ser Glu Pro Glu Glu Pro Ala Ser Thr Val Ser
275 280 285Ser104333PRTArabidopsis
thaliana 104Thr Asp Ser Ala Ser Asp Ser Ile Phe His Tyr Asp Asp Ala Ser
Gln1 5 10 15Ala Lys Ile
Gln Gln Glu Lys Pro Trp Ala Ser Asp Pro Asn Tyr Phe 20
25 30Lys Arg Val His Ile Ser Ala Leu Ala Leu
Leu Lys Met Val Val His 35 40
45Ala Arg Ser Gly Gly Thr Ile Glu Ile Met Gly Leu Met Gln Gly Lys 50
55 60Thr Glu Gly Asp Thr Ile Ile Val Met
Asp Ala Phe Ala Leu Pro Val65 70 75
80Glu Gly Thr Glu Thr Arg Val Asn Ala Gln Ser Asp Ala Tyr
Glu Tyr 85 90 95Met Val
Glu Tyr Ser Gln Thr Ser Lys Leu Ala Gly Arg Leu Glu Asn 100
105 110Val Val Gly Trp Tyr His Ser His Pro
Gly Tyr Gly Cys Trp Leu Ser 115 120
125Gly Ile Asp Val Ser Thr Gln Met Leu Asn Gln Gln Tyr Gln Glu Pro
130 135 140Phe Leu Ala Val Val Ile Asp
Pro Thr Arg Thr Val Ser Ala Gly Lys145 150
155 160Val Glu Ile Gly Ala Phe Arg Thr Tyr Pro Glu Gly
His Lys Ile Ser 165 170
175Asp Asp His Val Ser Glu Tyr Gln Thr Ile Pro Leu Asn Lys Ile Glu
180 185 190Asp Phe Gly Val His Cys
Lys Gln Tyr Tyr Ser Leu Asp Ile Thr Tyr 195 200
205Phe Lys Ser Ser Leu Asp Ser His Leu Leu Asp Leu Leu Trp
Asn Lys 210 215 220Tyr Trp Val Asn Thr
Leu Ser Ser Ser Pro Leu Leu Gly Asn Gly Asp225 230
235 240Tyr Val Ala Gly Gln Ile Ser Asp Leu Ala
Glu Lys Leu Glu Gln Ala 245 250
255Glu Ser Gln Leu Ala Asn Ser Arg Tyr Gly Gly Ile Ala Pro Ala Gly
260 265 270His Gln Arg Arg Lys
Glu Asp Glu Pro Gln Leu Ala Lys Ile Thr Arg 275
280 285Asp Ser Ala Lys Ile Thr Val Glu Gln Val His Gly
Leu Met Ser Gln 290 295 300Val Ile Lys
Asp Ile Leu Phe Asn Ser Ala Arg Gln Ser Lys Lys Ser305
310 315 320Ala Asp Asp Ser Ser Asp Pro
Glu Pro Met Ile Thr Ser 325
330105460PRTArabidopsis thaliana 105Met Val Arg Ser Asp Glu Asn Ser Leu
Gly Leu Ile Gly Ser Met Ser1 5 10
15Leu Gln Gly Thr Leu Asn Arg Ser Ile Leu Leu Leu Lys Ile Lys
Thr 20 25 30Phe Val Leu Phe
Asp Phe Ser Pro Lys Leu Ile Leu Asn Leu Leu Asp 35
40 45Val Gly Gly Gly Val Val Gly Lys Ile Lys Thr Thr
Ala Thr Thr Gly 50 55 60Pro Thr Arg
Arg Ala Leu Ser Thr Ile Asn Lys Asn Ile Thr Glu Ala65 70
75 80Pro Ser Tyr Pro Tyr Ala Val Asn
Lys Arg Ser Val Ser Glu Arg Asp 85 90
95Gly Ile Cys Asn Lys Pro Pro Val His Arg Pro Val Thr Arg
Lys Phe 100 105 110Ala Ala Gln
Leu Ala Asp His Lys Pro His Ile Arg Asp Glu Glu Thr 115
120 125Lys Lys Pro Asp Ser Val Ser Ser Glu Glu Pro
Glu Thr Ile Ile Ile 130 135 140Asp Val
Asp Glu Ser Asp Lys Glu Gly Gly Asp Ser Asn Glu Pro Met145
150 155 160Phe Val Gln His Thr Glu Ala
Met Leu Glu Glu Ile Glu Gln Met Glu 165
170 175Lys Glu Ile Glu Met Glu Asp Ala Asp Lys Glu Glu
Glu Pro Val Ile 180 185 190Asp
Ile Asp Ala Cys Asp Lys Asn Asn Pro Leu Ala Ala Val Glu Tyr 195
200 205Ile His Asp Met His Thr Phe Tyr Lys
Asn Phe Glu Lys Leu Ser Cys 210 215
220Val Pro Pro Asn Tyr Met Asp Asn Gln Gln Asp Leu Asn Glu Arg Met225
230 235 240Arg Gly Ile Leu
Ile Asp Trp Leu Ile Glu Val His Tyr Lys Phe Glu 245
250 255Leu Met Glu Glu Thr Leu Tyr Leu Thr Ile
Asn Val Ile Asp Arg Phe 260 265
270Leu Ala Val His Gln Ile Val Arg Lys Lys Leu Gln Leu Val Gly Val
275 280 285Thr Ala Leu Leu Leu Ala Cys
Lys Tyr Glu Glu Val Ser Val Pro Val 290 295
300Val Asp Asp Leu Ile Leu Ile Ser Asp Lys Ala Tyr Ser Arg Arg
Glu305 310 315 320Val Leu
Asp Met Glu Lys Leu Met Ala Asn Thr Leu Gln Phe Asn Phe
325 330 335Ser Leu Pro Thr Pro Tyr Val
Phe Met Lys Arg Phe Leu Lys Ala Ala 340 345
350Gln Ser Asp Lys Lys Leu Glu Ile Leu Ser Phe Phe Met Ile
Glu Leu 355 360 365Cys Leu Val Glu
Tyr Glu Met Leu Glu Tyr Leu Pro Ser Lys Leu Ala 370
375 380Ala Ser Ala Ile Tyr Thr Ala Gln Cys Thr Leu Lys
Gly Phe Glu Glu385 390 395
400Trp Ser Lys Thr Cys Glu Phe His Thr Gly Tyr Asn Glu Lys Gln Leu
405 410 415Leu Ala Cys Ala Arg
Lys Met Val Ala Phe His His Lys Ala Gly Thr 420
425 430Gly Lys Leu Thr Gly Val His Arg Lys Tyr Asn Thr
Ser Lys Phe Cys 435 440 445His Ala
Ala Arg Thr Glu Pro Ala Gly Phe Leu Ile 450 455
460106664PRTArabidopsis thaliana 106Met Val Asn Pro Gly His Gly
Arg Gly Pro Asp Ser Gly Thr Ala Ala1 5 10
15Gly Gly Ser Asn Ser Asp Pro Phe Pro Ala Asn Leu Arg
Val Leu Val 20 25 30Val Asp
Asp Asp Pro Thr Cys Leu Met Ile Leu Glu Arg Met Leu Met 35
40 45Thr Cys Leu Tyr Arg Val Thr Lys Cys Asn
Arg Ala Glu Ser Ala Leu 50 55 60Ser
Leu Leu Arg Lys Asn Lys Asn Gly Phe Asp Ile Val Ile Ser Asp65
70 75 80Val His Met Pro Asp Met
Asp Gly Phe Lys Leu Leu Glu His Val Gly 85
90 95Leu Glu Met Asp Leu Pro Val Ile Met Met Ser Ala
Asp Asp Ser Lys 100 105 110Ser
Val Val Leu Lys Gly Val Thr His Gly Ala Val Asp Tyr Leu Ile 115
120 125Lys Pro Val Arg Ile Glu Ala Leu Lys
Asn Ile Trp Gln His Val Val 130 135
140Arg Lys Lys Arg Asn Glu Trp Asn Val Ser Glu His Ser Gly Gly Ser145
150 155 160Ile Glu Asp Thr
Gly Gly Asp Arg Asp Arg Gln Gln Gln His Arg Glu 165
170 175Asp Ala Asp Asn Asn Ser Ser Ser Val Asn
Glu Gly Asn Gly Arg Ser 180 185
190Ser Arg Lys Arg Lys Glu Glu Glu Val Asp Asp Gln Gly Asp Asp Lys
195 200 205Glu Asp Ser Ser Ser Leu Lys
Lys Pro Arg Val Val Trp Ser Val Glu 210 215
220Leu His Gln Gln Phe Val Ala Ala Val Asn Gln Leu Gly Val Asp
Lys225 230 235 240Ala Val
Pro Lys Lys Ile Leu Glu Met Met Asn Val Pro Gly Leu Thr
245 250 255Arg Glu Asn Val Ala Ser His
Leu Gln Lys Tyr Arg Ile Tyr Leu Arg 260 265
270Arg Leu Gly Gly Val Ser Gln His Gln Gly Asn Met Asn His
Ser Phe 275 280 285Met Thr Gly Gln
Asp Gln Ser Phe Gly Pro Leu Ser Ser Leu Asn Gly 290
295 300Phe Asp Leu Gln Ser Leu Ala Val Thr Gly Gln Leu
Pro Pro Gln Ser305 310 315
320Leu Ala Gln Leu Gln Ala Ala Gly Leu Gly Arg Pro Thr Leu Ala Lys
325 330 335Pro Gly Met Ser Val
Ser Pro Leu Val Asp Gln Arg Ser Ile Phe Asn 340
345 350Phe Glu Asn Pro Lys Ile Arg Phe Gly Asp Gly His
Gly Gln Thr Met 355 360 365Asn Asn
Gly Asn Leu Leu His Gly Val Pro Thr Gly Ser His Met Arg 370
375 380Leu Arg Pro Gly Gln Asn Val Gln Ser Ser Gly
Met Met Leu Pro Val385 390 395
400Ala Asp Gln Leu Pro Arg Gly Gly Pro Ser Met Leu Pro Ser Leu Gly
405 410 415Gln Gln Pro Ile
Leu Ser Ser Ser Val Ser Arg Arg Ser Asp Leu Thr 420
425 430Gly Ala Leu Ala Val Arg Asn Ser Ile Pro Glu
Thr Asn Ser Arg Val 435 440 445Leu
Pro Thr Thr His Ser Val Phe Asn Asn Phe Pro Ala Asp Leu Pro 450
455 460Arg Ser Ser Phe Pro Leu Ala Ser Ala Pro
Gly Ile Ser Val Pro Val465 470 475
480Ser Val Ser Tyr Gln Glu Glu Val Asn Ser Ser Asp Ala Lys Gly
Gly 485 490 495Ser Ser Ala
Ala Thr Ala Gly Phe Gly Asn Pro Ser Tyr Asp Ile Phe 500
505 510Asn Asp Phe Pro Gln His Gln Gln His Asn
Lys Asn Ile Ser Asn Lys 515 520
525Leu Asn Asp Trp Asp Leu Arg Asn Met Gly Leu Val Phe Ser Ser Asn 530
535 540Gln Asp Ala Ala Thr Ala Thr Ala
Thr Ala Ala Phe Ser Thr Ser Glu545 550
555 560Ala Tyr Ser Ser Ser Ser Thr Gln Arg Lys Arg Arg
Glu Thr Asp Ala 565 570
575Thr Val Val Gly Glu His Gly Gln Asn Leu Gln Ser Pro Ser Arg Asn
580 585 590Leu Tyr His Leu Asn His
Val Phe Met Asp Gly Gly Ser Val Arg Val 595 600
605Lys Ser Glu Arg Val Ala Glu Thr Val Thr Cys Pro Pro Ala
Asn Thr 610 615 620Leu Phe His Glu Gln
Tyr Asn Gln Glu Asp Leu Met Ser Ala Phe Leu625 630
635 640Lys Gln Glu Gly Ile Pro Ser Val Asp Asn
Glu Phe Glu Phe Asp Gly 645 650
655Tyr Ser Ile Asp Asn Ile Gln Val
660107450PRTArabidopsis thaliana 107Met Gly Lys Glu Asn Ala Val Ser Arg
Pro Phe Thr Arg Ser Leu Ala1 5 10
15Ser Ala Leu Arg Ala Ser Glu Val Thr Ser Thr Thr Gln Asn Gln
Gln 20 25 30Arg Val Asn Thr
Lys Arg Pro Ala Leu Glu Asp Thr Arg Ala Thr Gly 35
40 45Pro Asn Lys Arg Lys Lys Arg Ala Val Leu Gly Glu
Ile Thr Asn Val 50 55 60Asn Ser Asn
Thr Ala Ile Leu Glu Ala Lys Asn Ser Lys Gln Ile Lys65 70
75 80Lys Gly Arg Gly His Gly Leu Ala
Ser Thr Ser Gln Leu Ala Thr Ser 85 90
95Val Thr Ser Glu Val Thr Asp Leu Gln Ser Arg Thr Asp Ala
Lys Val 100 105 110Glu Val Ala
Ser Asn Thr Ala Gly Asn Leu Ser Val Ser Lys Gly Thr 115
120 125Asp Asn Thr Ala Asp Asn Cys Ile Glu Ile Trp
Asn Ser Arg Leu Pro 130 135 140Pro Arg
Pro Leu Gly Arg Ser Ala Ser Thr Ala Glu Lys Ser Ala Val145
150 155 160Ile Gly Ser Ser Thr Val Pro
Asp Ile Pro Lys Phe Val Asp Ile Asp 165
170 175Ser Asp Asp Lys Asp Pro Leu Leu Cys Cys Leu Tyr
Ala Pro Glu Ile 180 185 190His
Tyr Asn Leu Arg Val Ser Glu Leu Lys Arg Arg Pro Leu Pro Asp 195
200 205Phe Met Glu Arg Ile Gln Lys Asp Val
Thr Gln Ser Met Arg Gly Ile 210 215
220Leu Val Asp Trp Leu Val Glu Val Ser Glu Glu Tyr Thr Leu Ala Ser225
230 235 240Asp Thr Leu Tyr
Leu Thr Val Tyr Leu Ile Asp Trp Phe Leu His Gly 245
250 255Asn Tyr Val Gln Arg Gln Gln Leu Gln Leu
Leu Gly Ile Thr Cys Met 260 265
270Leu Ile Ala Ser Lys Tyr Glu Glu Ile Ser Ala Pro Arg Ile Glu Glu
275 280 285Phe Cys Phe Ile Thr Asp Asn
Thr Tyr Thr Arg Asp Gln Val Leu Glu 290 295
300Met Glu Asn Gln Val Leu Lys His Phe Ser Phe Gln Ile Tyr Thr
Pro305 310 315 320Thr Pro
Lys Thr Phe Leu Arg Arg Phe Leu Arg Ala Ala Gln Ala Ser
325 330 335Arg Leu Ser Pro Ser Leu Glu
Val Glu Phe Leu Ala Ser Tyr Leu Thr 340 345
350Glu Leu Thr Leu Ile Asp Tyr His Phe Leu Lys Phe Leu Pro
Ser Val 355 360 365Val Ala Ala Ser
Ala Val Phe Leu Ala Lys Trp Thr Met Asp Gln Ser 370
375 380Asn His Pro Trp Asn Pro Thr Leu Glu His Tyr Thr
Thr Tyr Lys Ala385 390 395
400Ser Asp Leu Lys Ala Ser Val His Ala Leu Gln Asp Leu Gln Leu Asn
405 410 415Thr Lys Gly Cys Pro
Leu Ser Ala Ile Arg Met Lys Tyr Arg Gln Glu 420
425 430Lys Tyr Lys Ser Val Ala Val Leu Thr Ser Pro Lys
Leu Leu Asp Thr 435 440 445Leu Phe
450108901PRTArabidopsis thaliana 108Met Ala Asn Asn Pro Pro Gln Ser
Ser Gly Thr Gln Gly Gln His Phe1 5 10
15Val Pro Ala Ala Ser Gln Pro Phe His Pro Tyr Gly His Val
Pro Pro 20 25 30Asn Val Gln
Ser Gln Pro Pro Gln Tyr Ser Gln Pro Ile Gln Gln Gln 35
40 45Gln Leu Phe Pro Val Arg Pro Gly Gln Pro Val
His Ile Thr Ser Ser 50 55 60Ser Gln
Ala Val Ser Val Pro Tyr Ile Gln Thr Asn Lys Ile Leu Thr65
70 75 80Ser Gly Ser Thr Gln Pro Gln
Pro Asn Ala Pro Pro Met Thr Gly Phe 85 90
95Ala Thr Ser Gly Pro Pro Phe Ser Ser Pro Tyr Thr Phe
Val Pro Ser 100 105 110Ser Tyr
Pro Gln Gln Gln Pro Thr Ser Leu Val Gln Pro Asn Ser Gln 115
120 125Met His Val Ala Gly Val Pro Pro Ala Ala
Asn Thr Trp Pro Val Pro 130 135 140Val
Asn Gln Ser Thr Ser Leu Val Ser Pro Val Gln Gln Thr Gly Gln145
150 155 160Gln Thr Pro Val Ala Val
Ser Thr Asp Pro Gly Asn Leu Thr Pro Gln 165
170 175Ser Ala Ser Asp Trp Gln Glu His Thr Ser Ala Asp
Gly Arg Lys Ala 180 185 190Asp
Ala Ser Thr Val Trp Lys Glu Phe Thr Thr Pro Glu Gly Lys Lys 195
200 205Tyr Tyr Tyr Asn Lys Val Thr Lys Glu
Ser Lys Trp Thr Ile Pro Glu 210 215
220Asp Leu Lys Leu Ala Arg Glu Gln Ala Gln Leu Ala Ser Glu Lys Thr225
230 235 240Ser Leu Ser Glu
Ala Gly Ser Thr Pro Leu Ser His His Ala Ala Ser 245
250 255Ser Ser Asp Leu Ala Val Ser Thr Val Thr
Ser Val Val Pro Ser Thr 260 265
270Ser Ser Ala Leu Thr Gly His Ser Ser Ser Pro Ile Gln Ala Gly Leu
275 280 285Ala Val Pro Val Thr Arg Pro
Pro Ser Val Ala Pro Val Thr Pro Thr 290 295
300Ser Gly Ala Ile Ser Asp Thr Glu Ala Thr Thr Met Tyr Tyr Phe
Ser305 310 315 320Leu Gly
Ser Phe Ala Glu Asn Lys Glu Met Ser Val Asn Gly Lys Ala
325 330 335Asn Leu Ser Pro Ala Gly Asp
Lys Ala Asn Val Glu Glu Pro Met Val 340 345
350Tyr Ala Thr Lys Gln Glu Ala Lys Ala Ala Phe Lys Ser Leu
Leu Glu 355 360 365Ser Val Asn Val
His Ser Asp Trp Thr Trp Glu Gln Thr Leu Lys Glu 370
375 380Ile Val His Asp Lys Arg Tyr Gly Ala Leu Arg Thr
Leu Gly Glu Arg385 390 395
400Lys Gln Ala Phe Asn Glu Tyr Leu Gly Gln Arg Lys Lys Val Glu Ala
405 410 415Glu Glu Arg Arg Arg
Arg Gln Lys Lys Ala Arg Glu Glu Phe Val Lys 420
425 430Met Leu Glu Glu Cys Glu Glu Leu Ser Ser Ser Leu
Lys Trp Ser Lys 435 440 445Ala Met
Ser Leu Phe Glu Asn Asp Gln Arg Phe Lys Ala Val Asp Arg 450
455 460Pro Arg Asp Arg Glu Asp Leu Phe Asp Asn Tyr
Ile Val Glu Leu Glu465 470 475
480Arg Lys Glu Arg Glu Lys Ala Ala Glu Glu His Arg Gln Tyr Met Ala
485 490 495Asp Tyr Arg Lys
Phe Leu Glu Thr Cys Asp Tyr Ile Lys Ala Gly Thr 500
505 510Gln Trp Arg Lys Ile Gln Asp Arg Leu Glu Asp
Asp Asp Arg Cys Ser 515 520 525Cys
Leu Glu Lys Ile Asp Arg Leu Ile Gly Phe Glu Glu Tyr Ile Leu 530
535 540Asp Leu Glu Lys Glu Glu Glu Glu Leu Lys
Arg Val Glu Lys Glu His545 550 555
560Val Arg Arg Ala Glu Arg Lys Asn Arg Asp Ala Phe Arg Thr Leu
Leu 565 570 575Glu Glu His
Val Ala Ala Gly Ile Leu Thr Ala Lys Thr Tyr Trp Leu 580
585 590Asp Tyr Cys Ile Glu Leu Lys Asp Leu Pro
Gln Tyr Gln Ala Val Ala 595 600
605Ser Asn Thr Ser Gly Ser Thr Pro Lys Asp Leu Phe Glu Asp Val Thr 610
615 620Glu Glu Leu Glu Lys Gln Tyr His
Glu Asp Lys Ser Tyr Val Lys Asp625 630
635 640Ala Met Lys Ser Arg Lys Ala Asn Phe Lys Ser Ala
Ile Ser Glu Asp 645 650
655Leu Ser Thr Gln Gln Ile Ser Asp Ile Asn Leu Lys Leu Ile Tyr Asp
660 665 670Asp Leu Val Gly Arg Val
Lys Glu Lys Glu Glu Lys Glu Ala Arg Lys 675 680
685Leu Gln Arg Leu Ala Glu Glu Phe Thr Asn Leu Leu His Thr
Phe Lys 690 695 700Glu Ile Thr Val Ala
Ser Asn Trp Glu Asp Ser Lys Gln Leu Val Glu705 710
715 720Glu Ser Gln Glu Tyr Arg Ser Ile Gly Asp
Glu Ser Val Ser Gln Gly 725 730
735Leu Phe Glu Glu Tyr Ile Thr Ser Leu Gln Glu Lys Ala Lys Glu Lys
740 745 750Glu Arg Lys Arg Asp
Glu Glu Lys Val Arg Lys Glu Lys Glu Arg Asp 755
760 765Glu Lys Glu Lys Arg Lys Asp Lys Asp Lys Glu Arg
Arg Glu Lys Glu 770 775 780Arg Glu Arg
Glu Lys Glu Lys Gly Lys Glu Arg Ser Lys Arg Glu Glu785
790 795 800Ser Asp Gly Glu Thr Ala Met
Asp Val Ser Glu Gly His Lys Asp Glu 805
810 815Lys Arg Lys Gly Lys Asp Arg Asp Arg Lys His Arg
Arg Arg His His 820 825 830Asn
Asn Ser Asp Glu Asp Val Ser Ser Asp Arg Asp Asp Arg Asp Glu 835
840 845Ser Lys Lys Ser Ser Arg Lys His Gly
Asn Asp Arg Lys Lys Ser Arg 850 855
860Lys His Ala Asn Ser Pro Glu Ser Glu Ser Glu Asn Arg His Lys Arg865
870 875 880Gln Lys Lys Glu
Ser Ser Arg Arg Ser Gly Asn Asp Glu Leu Glu Asp 885
890 895Gly Glu Val Gly Glu
900109358PRTArabidopsis thaliana 109Met Glu Gly Ser Ser Ser Thr Ile Ala
Arg Lys Thr Trp Glu Leu Glu1 5 10
15Asn Ser Ile Leu Thr Val Asp Ser Pro Asp Ser Thr Ser Asp Asn
Ile 20 25 30Phe Tyr Tyr Asp
Asp Thr Ser Gln Thr Arg Phe Gln Gln Glu Lys Pro 35
40 45Trp Glu Asn Asp Pro His Tyr Phe Lys Arg Val Lys
Ile Ser Ala Leu 50 55 60Ala Leu Leu
Lys Met Val Val His Ala Arg Ser Gly Gly Thr Ile Glu65 70
75 80Ile Met Gly Leu Met Gln Gly Lys
Thr Asp Gly Asp Thr Ile Ile Val 85 90
95Met Asp Ala Phe Ala Leu Pro Val Glu Gly Thr Glu Thr Arg
Val Asn 100 105 110Ala Gln Asp
Asp Ala Tyr Glu Tyr Met Val Glu Tyr Ser Gln Thr Asn 115
120 125Lys Leu Ala Gly Arg Leu Glu Asn Val Val Gly
Trp Tyr His Ser His 130 135 140Pro Gly
Tyr Gly Cys Trp Leu Ser Gly Ile Asp Val Ser Thr Gln Arg145
150 155 160Leu Asn Gln Gln His Gln Glu
Pro Phe Leu Ala Val Val Ile Asp Pro 165
170 175Thr Arg Thr Val Ser Ala Gly Lys Val Glu Ile Gly
Ala Phe Arg Thr 180 185 190Tyr
Ser Lys Gly Tyr Lys Pro Pro Asp Glu Pro Val Ser Glu Tyr Gln 195
200 205Thr Ile Pro Leu Asn Lys Ile Glu Asp
Phe Gly Val His Cys Lys Gln 210 215
220Tyr Tyr Ser Leu Asp Val Thr Tyr Phe Lys Ser Ser Leu Asp Ser His225
230 235 240Leu Leu Asp Leu
Leu Trp Asn Lys Tyr Trp Val Asn Thr Leu Ser Ser 245
250 255Ser Pro Leu Leu Gly Asn Gly Asp Tyr Val
Ala Gly Gln Ile Ser Asp 260 265
270Leu Ala Glu Lys Leu Glu Gln Ala Glu Ser His Leu Val Gln Ser Arg
275 280 285Phe Gly Gly Val Val Pro Ser
Ser Leu His Lys Lys Lys Glu Asp Glu 290 295
300Ser Gln Leu Thr Lys Ile Thr Arg Asp Ser Ala Lys Ile Thr Val
Glu305 310 315 320Gln Val
His Gly Leu Met Ser Gln Val Ile Lys Asp Glu Leu Phe Asn
325 330 335Ser Met Arg Gln Ser Asn Asn
Lys Ser Pro Thr Asp Ser Ser Asp Pro 340 345
350Asp Pro Met Ile Thr Tyr 35511098PRTArabidopsis
thaliana 110Met Ala Ile Ser Lys Ala Leu Ile Ala Ser Leu Leu Ile Ser Leu
Leu1 5 10 15Val Leu Gln
Leu Val Gln Ala Asp Val Glu Asn Ser Gln Lys Lys Asn 20
25 30Gly Tyr Ala Lys Lys Ile Asp Cys Gly Ser
Ala Cys Val Ala Arg Cys 35 40
45Arg Leu Ser Arg Arg Pro Arg Leu Cys His Arg Ala Cys Gly Thr Cys 50
55 60Cys Tyr Arg Cys Asn Cys Val Pro Pro
Gly Thr Tyr Gly Asn Tyr Asp65 70 75
80Lys Cys Gln Cys Tyr Ala Ser Leu Thr Thr His Gly Gly Arg
Arg Lys 85 90 95Cys
Pro111385PRTArabidopsis thalianaMISC_FEATURE(252)..(253)Xaa = any amino
acid 111Met Gly Lys Lys Asn Lys Arg Ser Gln Asp Glu Ser Glu Leu Glu Leu1
5 10 15Glu Pro Glu Leu
Thr Lys Ile Ile Asp Gly Asp Ser Lys Lys Lys Lys 20
25 30Asn Lys Asn Lys Lys Lys Arg Ser His Glu Asp
Thr Glu Ile Glu Pro 35 40 45Glu
Gln Lys Met Ser Leu Asp Gly Asp Ser Arg Glu Glu Lys Ile Lys 50
55 60Lys Lys Arg Lys Asn Lys Asn Gln Glu Glu
Glu Pro Glu Leu Val Thr65 70 75
80Glu Lys Thr Lys Val Gln Glu Glu Glu Lys Gly Asn Val Glu Glu
Gly 85 90 95Arg Ala Thr
Val Ser Ile Ala Ile Ala Gly Ser Ile Ile His Asn Thr 100
105 110Gln Ser Leu Glu Leu Ala Thr Arg Val Ile
Ser Leu Ser Leu Tyr Leu 115 120
125Ser Leu Arg Phe Ser Val Phe Pro Phe Pro Asp Asn Leu Lys Ser Pro 130
135 140Ser Ser Ile Ser Asn Ile Ser Gln
Leu Ala Gly Gln Ile Ala Arg Ala145 150
155 160Ala Thr Ile Phe Arg Ile Asp Glu Ile Val Val Phe
Asp Asn Lys Ser 165 170
175Ser Ser Glu Ile Glu Ser Ala Ala Thr Asn Ala Ser Asp Ser Asn Glu
180 185 190Ser Gly Ala Ser Phe Leu
Val Arg Ile Leu Lys Tyr Leu Glu Thr Pro 195 200
205Gln Tyr Leu Arg Lys Ser Leu Phe Pro Lys Gln Asn Asp Leu
Arg Tyr 210 215 220Val Gly Met Leu Pro
Gly Met Leu Pro Pro Leu Asp Ala Pro His His225 230
235 240Leu Arg Lys His Glu Trp Glu Gln Tyr Arg
Glu Xaa Xaa Ile Val Pro 245 250
255Pro Ser Lys Pro Arg Glu Glu Ala Gly Met Tyr Trp Gly Tyr Lys Val
260 265 270Arg Tyr Ala Ser Gln
Leu Ser Ser Val Phe Lys Glu Cys Pro Phe Glu 275
280 285Gly Gly Tyr Asp Tyr Leu Ile Gly Thr Ser Glu His
Gly Leu Val Ile 290 295 300Ser Ser Ser
Glu Leu Lys Ile Pro Thr Phe Arg His Leu Leu Ile Ala305
310 315 320Phe Gly Gly Leu Ala Gly Leu
Glu Glu Ser Ile Glu Asp Asp Asn Gln 325
330 335Tyr Lys Gly Lys Asn Val Arg Asp Val Phe Asn Val
Tyr Leu Asn Thr 340 345 350Cys
Pro His Gln Gly Ser Arg Thr Ile Arg Ala Glu Glu Ala Met Phe 355
360 365Ile Ser Leu Gln Tyr Phe Gln Glu Pro
Ile Ser Arg Ala Val Arg Arg 370 375
380Leu385112465PRTArabidopsis thaliana 112Met Glu Leu Leu Asp Met Asn Ser
Met Ala Ala Ser Ile Gly Val Ser1 5 10
15Val Ala Val Leu Arg Phe Leu Leu Cys Phe Val Ala Thr Ile
Pro Ile 20 25 30Ser Phe Leu
Trp Arg Phe Ile Pro Ser Arg Leu Gly Lys His Ile Tyr 35
40 45Ser Ala Ala Ser Gly Ala Phe Leu Ser Tyr Leu
Ser Phe Gly Phe Ser 50 55 60Ser Asn
Leu His Phe Leu Val Pro Met Thr Ile Gly Tyr Ala Ser Met65
70 75 80Ala Ile Tyr Arg Pro Leu Ser
Gly Phe Ile Thr Phe Phe Leu Gly Phe 85 90
95Ala Tyr Leu Ile Gly Cys His Val Phe Tyr Met Ser Gly
Asp Ala Trp 100 105 110Lys Glu
Gly Gly Ile Asp Ser Thr Gly Ala Leu Met Val Leu Thr Leu 115
120 125Lys Val Ile Ser Cys Ser Ile Asn Tyr Asn
Asp Gly Met Leu Lys Glu 130 135 140Glu
Gly Leu Arg Glu Ala Gln Lys Lys Asn Arg Leu Ile Gln Met Pro145
150 155 160Ser Leu Ile Glu Tyr Phe
Gly Tyr Cys Leu Cys Cys Gly Ser His Phe 165
170 175Ala Gly Pro Val Phe Glu Met Lys Asp Tyr Leu Glu
Trp Thr Glu Glu 180 185 190Lys
Gly Ile Trp Ala Val Ser Glu Lys Gly Lys Arg Pro Ser Pro Tyr 195
200 205Gly Ala Met Ile Arg Ala Val Phe Gln
Ala Ala Ile Cys Met Ala Leu 210 215
220Tyr Leu Tyr Leu Val Pro Gln Phe Pro Leu Thr Arg Phe Thr Glu Pro225
230 235 240Val Tyr Gln Glu
Trp Gly Phe Leu Lys Arg Phe Gly Tyr Gln Tyr Met 245
250 255Ala Gly Phe Thr Ala Arg Trp Lys Tyr Tyr
Phe Ile Trp Ser Ile Ser 260 265
270Glu Ala Ser Ile Ile Ile Ser Gly Leu Gly Phe Ser Gly Trp Thr Asp
275 280 285Glu Thr Gln Thr Lys Ala Lys
Trp Asp Arg Ala Lys Asn Val Asp Ile 290 295
300Leu Gly Val Glu Leu Ala Lys Ser Ala Val Gln Ile Pro Leu Phe
Trp305 310 315 320Asn Ile
Gln Val Ser Thr Trp Leu Arg His Tyr Val Tyr Glu Arg Ile
325 330 335Val Lys Pro Gly Lys Lys Ala
Gly Phe Phe Gln Leu Leu Ala Thr Gln 340 345
350Thr Val Ser Ala Val Trp His Gly Leu Tyr Pro Gly Tyr Ile
Ile Phe 355 360 365Phe Val Gln Ser
Ala Leu Met Ile Asp Gly Ser Lys Ala Ile Tyr Arg 370
375 380Trp Gln Gln Ala Ile Pro Pro Lys Met Ala Met Leu
Arg Asn Val Leu385 390 395
400Val Leu Ile Asn Phe Leu Tyr Thr Val Val Val Leu Asn Tyr Ser Ser
405 410 415Val Gly Phe Met Val
Leu Ser Leu His Glu Thr Leu Val Ala Phe Lys 420
425 430Ser Val Tyr Tyr Ile Gly Thr Val Ile Pro Ile Ala
Val Leu Leu Leu 435 440 445Ser Tyr
Leu Val Pro Val Lys Pro Val Arg Pro Lys Thr Arg Lys Glu 450
455 460Glu465113313PRTArabidopsis thaliana 113Met
Asp Glu Gly Val Ile Ala Val Ser Ala Met Asp Ala Phe Glu Lys1
5 10 15Leu Glu Lys Val Gly Glu Gly
Thr Tyr Gly Lys Val Tyr Arg Ala Arg 20 25
30Glu Lys Ala Thr Gly Lys Ile Val Ala Leu Lys Lys Thr Arg
Leu His 35 40 45Glu Asp Glu Glu
Gly Val Pro Ser Thr Thr Leu Arg Glu Ile Ser Ile 50 55
60Leu Arg Met Leu Ala Arg Asp Pro His Val Val Arg Leu
Met Asp Val65 70 75
80Lys Gln Gly Leu Ser Lys Glu Gly Lys Thr Val Leu Tyr Leu Val Phe
85 90 95Glu Tyr Met Asp Thr Asp
Val Lys Lys Phe Ile Arg Ser Phe Arg Ser 100
105 110Thr Gly Lys Asn Ile Pro Thr Gln Thr Ile Lys Ser
Leu Met Tyr Gln 115 120 125Leu Cys
Lys Gly Met Ala Phe Cys His Gly His Gly Ile Leu His Arg 130
135 140Asp Leu Lys Pro His Asn Leu Leu Met Asp Pro
Lys Thr Met Arg Leu145 150 155
160Lys Ile Ala Asp Leu Gly Leu Ala Arg Ala Phe Thr Leu Pro Met Lys
165 170 175Lys Tyr Thr His
Glu Ile Leu Thr Leu Trp Tyr Arg Ala Pro Glu Val 180
185 190Leu Leu Gly Ala Thr His Tyr Ser Thr Ala Val
Asp Met Trp Ser Val 195 200 205Gly
Cys Ile Phe Ala Glu Leu Val Thr Asn Gln Ala Ile Phe Gln Gly 210
215 220Asp Ser Glu Leu Gln Gln Leu Leu His Ile
Phe Lys Leu Phe Gly Thr225 230 235
240Pro Asn Glu Glu Met Trp Pro Gly Val Ser Thr Leu Lys Asn Trp
His 245 250 255Glu Tyr Pro
Gln Trp Lys Pro Ser Thr Leu Ser Ser Ala Val Pro Asn 260
265 270Leu Asp Glu Ala Gly Val Asp Leu Leu Ser
Lys Met Leu Gln Tyr Glu 275 280
285Pro Ala Lys Arg Ile Ser Ala Lys Met Ala Met Glu His Pro Tyr Phe 290
295 300Asp Asp Leu Pro Glu Lys Ser Ser
Leu305 310114292PRTArabidopsis thaliana 114Met Ser Met
Glu Met Glu Leu Phe Val Thr Pro Glu Lys Gln Arg Gln1 5
10 15His Pro Ser Val Ser Val Glu Lys Thr
Pro Val Arg Arg Lys Leu Ile 20 25
30Val Asp Asp Asp Ser Glu Ile Gly Ser Glu Lys Lys Gly Gln Ser Arg
35 40 45Thr Ser Gly Gly Gly Leu Arg
Gln Phe Ser Val Met Val Cys Gln Lys 50 55
60Leu Glu Ala Lys Lys Ile Thr Thr Tyr Lys Glu Val Ala Asp Glu Ile65
70 75 80Ile Ser Asp Phe
Ala Thr Ile Lys Gln Asn Ala Glu Lys Pro Leu Asn 85
90 95Glu Asn Glu Tyr Asn Glu Lys Asn Ile Arg
Arg Arg Val Tyr Asp Ala 100 105
110Leu Asn Val Phe Met Ala Leu Asp Ile Ile Ala Arg Asp Lys Lys Glu
115 120 125Ile Arg Trp Lys Gly Leu Pro
Ile Thr Cys Lys Lys Asp Val Glu Glu 130 135
140Val Lys Met Asp Arg Asn Lys Val Met Ser Ser Val Gln Lys Lys
Ala145 150 155 160Ala Phe
Leu Lys Glu Leu Arg Glu Lys Val Ser Ser Leu Glu Ser Leu
165 170 175Met Ser Arg Asn Gln Glu Met
Val Val Lys Thr Gln Gly Pro Ala Glu 180 185
190Gly Phe Thr Leu Pro Phe Ile Leu Leu Glu Thr Asn Pro His
Ala Val 195 200 205Val Glu Ile Glu
Ile Ser Glu Asp Met Gln Leu Val His Leu Asp Phe 210
215 220Asn Ser Thr Pro Phe Ser Val His Asp Asp Ala Tyr
Ile Leu Lys Leu225 230 235
240Met Gln Glu Gln Lys Gln Glu Gln Asn Arg Val Ser Ser Ser Ser Ser
245 250 255Thr His His Gln Ser
Gln His Ser Ser Ala His Ser Ser Ser Ser Ser 260
265 270Cys Ile Ala Ser Gly Thr Ser Gly Pro Val Cys Trp
Asn Ser Gly Ser 275 280 285Ile Asp
Thr Arg 290115165PRTArabidopsis thaliana 115Met Asn Arg Glu Lys Leu
Met Lys Met Ala Asn Thr Val Arg Thr Gly1 5
10 15Gly Lys Gly Thr Val Arg Arg Lys Lys Lys Ala Val
His Lys Thr Thr 20 25 30Thr
Thr Asp Asp Lys Arg Leu Gln Ser Thr Leu Lys Arg Val Gly Val 35
40 45Asn Ser Ile Pro Ala Ile Glu Glu Val
Asn Ile Phe Lys Asp Asp Val 50 55
60Val Ile Gln Phe Ile Asn Pro Lys Val Gln Ala Ser Ile Ala Ala Asn65
70 75 80Thr Trp Val Val Ser
Gly Thr Pro Gln Thr Lys Lys Leu Gln Asp Ile 85
90 95Leu Pro Gln Ile Ile Ser Gln Leu Gly Pro Asp
Asn Leu Asp Asn Leu 100 105
110Lys Lys Leu Ala Glu Gln Phe Gln Lys Gln Ala Pro Gly Ala Gly Asp
115 120 125Val Pro Ala Thr Ile Gln Glu
Glu Asp Asp Asp Asp Asp Val Pro Asp 130 135
140Leu Val Val Gly Glu Thr Phe Glu Thr Pro Ala Thr Glu Glu Ala
Pro145 150 155 160Lys Ala
Ala Ala Ser 165116432PRTArabidopsis thaliana 116Met Ala
Thr Val Ser Ser Ser Ser Trp Pro Asn Pro Asn Pro Asn Pro1 5
10 15Asp Ser Thr Ser Ala Ser Asp Ser
Asp Ser Thr Phe Pro Ser His Arg 20 25
30Asp Arg Val Asp Glu Pro Asp Ser Leu Asp Ser Phe Ser Ser Met
Ser 35 40 45Leu Asn Ser Asp Glu
Pro Asn Gln Thr Ser Asn Gln Ser Pro Leu Ser 50 55
60Pro Pro Thr Pro Asn Leu Pro Val Met Pro Pro Pro Ser Val
Leu His65 70 75 80Leu
Ser Phe Asn Gln Asp His Ala Cys Phe Ala Val Gly Thr Asp Arg
85 90 95Gly Phe Arg Ile Leu Asn Cys
Asp Pro Phe Arg Glu Ile Phe Arg Arg 100 105
110Asp Phe Asp Arg Gly Gly Gly Val Ala Val Val Glu Met Leu
Phe Arg 115 120 125Cys Asn Ile Leu
Ala Leu Val Gly Gly Gly Pro Asp Pro Gln Tyr Pro 130
135 140Pro Asn Lys Val Met Ile Trp Asp Asp His Gln Gly
Arg Cys Ile Gly145 150 155
160Glu Leu Ser Phe Arg Ser Asp Val Arg Ser Val Arg Leu Arg Arg Asp
165 170 175Arg Ile Ile Val Val
Leu Glu Gln Lys Ile Phe Val Tyr Asn Phe Ser 180
185 190Asp Leu Lys Leu Met His Gln Ile Glu Thr Ile Ala
Asn Pro Lys Gly 195 200 205Leu Cys
Ala Val Ser Gln Gly Val Gly Ser Met Val Leu Val Cys Pro 210
215 220Gly Leu Gln Lys Gly Gln Val Arg Ile Glu His
Tyr Ala Ser Lys Arg225 230 235
240Thr Lys Phe Val Met Ala His Asp Ser Arg Ile Ala Cys Phe Ala Leu
245 250 255Thr Gln Asp Gly
His Leu Leu Ala Thr Ala Ser Ser Lys Gly Thr Leu 260
265 270Val Arg Ile Phe Asn Thr Val Asp Gly Thr Leu
Arg Gln Glu Ser Gly 275 280 285Thr
Ser Glu Asp Glu Ile Gly Lys Glu Gly Ala Asp Arg Ala Glu Ile 290
295 300Tyr Ser Leu Ala Phe Ser Ser Asn Ala Gln
Trp Leu Ala Val Ser Ser305 310 315
320Asp Lys Gly Thr Val His Val Phe Gly Leu Lys Val Asn Ser Gly
Ser 325 330 335Gln Val Lys
Asp Ser Ser Arg Ile Ala Pro Asp Ala Thr Pro Ser Ser 340
345 350Pro Ser Ser Ser Leu Ser Leu Phe Lys Val
Leu Pro Arg Tyr Phe Ser 355 360
365Ser Glu Trp Ser Val Ala Gln Phe Arg Leu Val Glu Gly Thr Gln Tyr 370
375 380Ile Ala Ala Phe Gly His Gln Lys
Asn Thr Val Val Ile Leu Gly Met385 390
395 400Asp Gly Ser Phe Tyr Arg Cys Gln Phe Asp Pro Val
Asn Gly Gly Glu 405 410
415Met Ser Gln Leu Glu Tyr His Asn Cys Leu Lys Pro Pro Ser Val Phe
420 425 430117559PRTArabidopsis
thaliana 117Met Asp Val Gly Val Thr Thr Ala Lys Ser Ile Leu Glu Lys Pro
Leu1 5 10 15Lys Leu Leu
Thr Glu Glu Asp Ile Ser Gln Leu Thr Arg Glu Asp Cys 20
25 30Arg Lys Phe Leu Lys Glu Lys Gly Phe Phe
Phe Phe Leu Ser Pro Phe 35 40
45Phe Ser Gly Leu Ile Val Phe Asp Glu Trp Arg Leu Thr Arg Val Glu 50
55 60Thr Gly Met Arg Arg Pro Ser Trp Asn
Lys Ser Gln Ala Ile Gln Gln65 70 75
80Val Leu Ser Leu Lys Ala Leu Tyr Glu Pro Gly Asp Asp Ser
Gly Ala 85 90 95Gly Ile
Leu Arg Lys Ile Leu Val Ser Gln Pro Pro Asn Pro Pro Arg 100
105 110Val Thr Thr Thr Leu Ile Glu Pro Arg
Asn Glu Leu Glu Ala Cys Gly 115 120
125Arg Ile Pro Leu Gln Glu Asp Asp Gly Ala Cys His Arg Arg Asp Ser
130 135 140Pro Arg Ser Ala Glu Phe Ser
Gly Ser Ser Gly Gln Phe Val Ala Asp145 150
155 160Lys Asp Ser His Lys Thr Val Ser Val Ser Pro Arg
Ser Pro Ala Glu 165 170
175Thr Asn Ala Val Val Gly Gln Met Thr Ile Phe Tyr Ser Gly Lys Val
180 185 190Asn Val Tyr Asp Gly Val
Pro Pro Glu Lys Ala Arg Ser Ile Met His 195 200
205Phe Ala Ala Asn Pro Ile Asp Leu Pro Glu Asn Gly Ile Phe
Ala Ser 210 215 220Ser Arg Met Ile Ser
Lys Pro Met Ser Lys Glu Lys Met Val Glu Leu225 230
235 240Pro Gln Tyr Gly Leu Glu Lys Ala Pro Ala
Ser Arg Asp Ser Asp Val 245 250
255Glu Gly Gln Ala Asn Arg Lys Val Ser Leu Gln Arg Tyr Leu Glu Lys
260 265 270Arg Lys Asp Arg Phe
Ser Lys Thr Lys Lys Ala Pro Gly Val Ala Ser 275
280 285Ser Ser Leu Glu Met Phe Leu Asn Arg Gln Pro Arg
Met Asn Ala Ala 290 295 300Tyr Ser Gln
Asn Leu Ser Gly Thr Gly His Cys Glu Ser Pro Glu Asn305
310 315 320Gln Thr Lys Ser Pro Asn Ile
Ser Val Asp Leu Asn Ser Asp Leu Asn 325
330 335Ser Glu Gly Ala Lys Arg Thr Gly Asp Gly Thr Thr
Gly Gln Lys Ala 340 345 350Gly
Arg Thr Ile Ser Cys Ser Tyr Asn Met Thr Lys Thr Ser Arg Gly 355
360 365Thr Arg Trp Val Lys Arg Ser Arg Glu
Glu Val Ile Gln Ala Trp Tyr 370 375
380Met Asp Asp Ser Glu Glu Asp Gln Arg Leu Pro His His Lys Asp Pro385
390 395 400Lys Glu Phe Val
Ser Leu Asp Lys Leu Ala Glu Leu Gly Val Leu Ser 405
410 415Trp Arg Leu Asp Ala Asp Asn Tyr Glu Thr
Asp Glu Asp Leu Lys Lys 420 425
430Ile Arg Glu Ser Arg Gly Tyr Ser Tyr Met Asp Phe Cys Glu Val Cys
435 440 445Pro Glu Lys Leu Pro Asn Tyr
Glu Val Lys Val Lys Ser Phe Phe Glu 450 455
460Glu His Leu His Thr Asp Glu Glu Ile Arg Tyr Cys Val Ala Gly
Thr465 470 475 480Gly Tyr
Phe Asp Val Arg Asp Arg Asn Glu Ala Trp Ile Arg Val Leu
485 490 495Val Lys Lys Gly Gly Met Ile
Val Leu Pro Ala Gly Ile Tyr His Arg 500 505
510Phe Thr Val Asp Ser Asp Asn Tyr Ile Lys Ala Met Arg Leu
Phe Val 515 520 525Gly Glu Pro Val
Trp Thr Pro Tyr Asn Arg Pro His Asp His Leu Pro 530
535 540Ala Arg Lys Glu Tyr Val Asp Asn Phe Met Ile Asn
Ala Ser Ala545 550 55511886PRTArabidopsis
thaliana 118Met Asp Gly His Asp Ser Lys Asp Thr Lys Gln Ser Thr Ala Asp
Met1 5 10 15Thr Ala Phe
Val Gln Asn Leu Leu Gln Gln Met Gln Thr Arg Phe Gln 20
25 30Thr Met Ser Asp Ser Ile Ile Thr Lys Ile
Asp Asp Met Gly Gly Arg 35 40
45Ile Asn Glu Leu Glu Gln Ser Ile Asn Asp Leu Arg Ala Glu Met Gly 50
55 60Val Glu Gly Thr Pro Pro Pro Ala Ser
Lys Ser Gly Asp Glu Pro Lys65 70 75
80Thr Pro Ala Ser Ser Ser
85119784PRTArabidopsis thaliana 119Met Glu Ile Tyr Thr Met Lys Thr Asn
Phe Leu Val Leu Ala Leu Ser1 5 10
15Leu Cys Ile Leu Leu Ser Ser Phe His Glu Val Ser Cys Gln Asp
Asp 20 25 30Gly Ser Gly Leu
Ser Asn Leu Asp Leu Ile Glu Arg Asp Tyr Gln Asp 35
40 45Ser Val Asn Ala Leu Gln Gly Lys Asp Asp Glu Asp
Gln Ser Ala Lys 50 55 60Ile Gln Ser
Glu Asn Gln Asn Asn Thr Thr Val Thr Asp Lys Asn Thr65 70
75 80Ile Ser Leu Ser Leu Ser Asp Glu
Ser Glu Val Gly Ser Val Ser Asp 85 90
95Glu Ser Val Gly Arg Ser Ser Leu Leu Asp Gln Ile Lys Leu
Glu Phe 100 105 110Glu Ala His
His Asn Ser Ile Asn Gln Ala Gly Ser Asp Gly Val Lys 115
120 125Ala Glu Ser Lys Asp Asp Asp Glu Glu Leu Ser
Ala His Arg Gln Lys 130 135 140Met Leu
Glu Glu Ile Glu His Glu Phe Glu Ala Ala Ser Asp Ser Leu145
150 155 160Lys Gln Leu Lys Thr Asp Asp
Val Asn Glu Gly Asn Asp Glu Glu His 165
170 175Ser Ala Lys Arg Gln Ser Leu Leu Glu Glu Ile Glu
Arg Glu Phe Glu 180 185 190Ala
Ala Thr Lys Glu Leu Glu Gln Leu Lys Val Asn Asp Phe Thr Gly 195
200 205Asp Lys Asp Asp Glu Glu His Ser Ala
Lys Arg Lys Ser Met Leu Glu 210 215
220Ala Ile Glu Arg Glu Phe Glu Ala Ala Met Glu Gly Ile Glu Ala Leu225
230 235 240Lys Val Ser Asp
Ser Thr Gly Ser Gly Asp Asp Glu Glu Gln Ser Ala 245
250 255Lys Arg Leu Ser Met Leu Glu Glu Ile Glu
Arg Glu Phe Glu Ala Ala 260 265
270Ser Lys Gly Leu Glu Gln Leu Arg Ala Ser Asp Ser Thr Ala Asp Asn
275 280 285Asn Glu Glu Glu His Ala Ala
Lys Gly Gln Ser Leu Leu Glu Glu Ile 290 295
300Glu Arg Glu Phe Glu Ala Ala Thr Glu Ser Leu Lys Gln Leu Gln
Val305 310 315 320Asp Asp
Ser Thr Glu Asp Lys Glu His Cys Lys Ala Leu Phe Phe Leu
325 330 335Leu Ser Ala Ile Leu Ser Leu
Trp Leu Ser Glu Ser Gly Phe Glu Cys 340 345
350Ile Val Val Thr Ala Ala Lys Arg Gln Ser Leu Leu Glu Glu
Ile Glu 355 360 365Arg Glu Phe Glu
Ala Ala Thr Lys Asp Leu Lys Gln Leu Asn Asp Phe 370
375 380Thr Glu Gly Ser Ala Asp Asp Glu Gln Ser Ala Lys
Arg Asn Lys Met385 390 395
400Leu Glu Asp Ile Glu Arg Glu Phe Glu Ala Ala Thr Ile Gly Leu Glu
405 410 415Gln Leu Lys Ala Asn
Asp Phe Ser Glu Gly Asn Asn Asn Glu Glu Gln 420
425 430Ser Ala Lys Arg Lys Ser Met Leu Glu Glu Ile Glu
Arg Glu Phe Glu 435 440 445Ala Ala
Ile Gly Gly Leu Lys Gln Ile Lys Val Asp Asp Ser Arg Asn 450
455 460Leu Glu Glu Glu Ser Ala Lys Arg Lys Ile Ile
Leu Glu Glu Met Glu465 470 475
480Arg Glu Phe Glu Glu Ala His Ser Gly Ile Asn Ala Lys Ala Asp Lys
485 490 495Glu Glu Ser Ala
Lys Lys Gln Ser Gly Ser Ala Ile Pro Glu Val Leu 500
505 510Gly Leu Gly Gln Ser Gly Gly Cys Ser Cys Ser
Lys Gln Asp Glu Asp 515 520 525Ser
Ser Ile Val Ile Pro Thr Lys Tyr Ser Ile Glu Asp Ile Leu Ser 530
535 540Glu Glu Ser Ala Val Gln Gly Thr Glu Thr
Ser Ser Leu Thr Ala Ser545 550 555
560Leu Thr Gln Leu Val Glu Asn His Arg Lys Glu Lys Glu Ser Leu
Leu 565 570 575Gly His Arg
Val Leu Thr Ser Pro Ser Ile Ala Ser Ser Thr Ser Glu 580
585 590Ser Ser Ala Thr Ser Glu Thr Val Glu Thr
Leu Arg Ala Lys Leu Asn 595 600
605Glu Leu Arg Gly Leu Thr Ala Arg Glu Leu Val Thr Arg Lys Asp Phe 610
615 620Gly Gln Ile Leu Ile Thr Ala Ala
Ser Phe Glu Glu Leu Ser Ser Ala625 630
635 640Pro Ile Ser Tyr Ile Ser Arg Leu Ala Lys Tyr Arg
Asn Val Ile Lys 645 650
655Glu Gly Leu Glu Ala Ser Glu Arg Val His Ile Ala Gln Val Arg Ala
660 665 670Lys Met Leu Lys Glu Val
Ala Thr Glu Lys Gln Thr Ala Val Asp Thr 675 680
685His Phe Ala Thr Ala Lys Lys Leu Ala Gln Glu Gly Asp Ala
Leu Phe 690 695 700Val Lys Ile Phe Ala
Ile Lys Lys Leu Leu Ala Lys Leu Glu Ala Glu705 710
715 720Lys Glu Ser Val Asp Gly Lys Phe Lys Glu
Thr Val Lys Glu Leu Ser 725 730
735His Leu Leu Ala Asp Ala Ser Glu Ala Tyr Glu Glu Tyr His Gly Ala
740 745 750Val Arg Lys Ala Lys
Asp Glu Gln Ala Ala Glu Glu Phe Ala Lys Glu 755
760 765Ala Thr Gln Ser Ala Glu Ile Ile Trp Val Lys Phe
Leu Ser Ser Leu 770 775
780120724PRTArabidopsis thaliana 120Met Glu Phe Gly Ser Phe Leu Val Ser
Leu Gly Thr Ser Phe Val Ile1 5 10
15Phe Val Ile Leu Met Leu Leu Phe Thr Trp Leu Ser Arg Lys Ser
Gly 20 25 30Asn Ala Pro Ile
Tyr Tyr Pro Asn Arg Ile Leu Lys Gly Leu Glu Pro 35
40 45Trp Glu Gly Thr Ser Leu Thr Arg Asn Pro Phe Ala
Trp Met Arg Glu 50 55 60Ala Leu Thr
Ser Ser Glu Gln Asp Val Val Asn Leu Ser Gly Val Asp65 70
75 80Thr Ala Val His Phe Val Phe Leu
Ser Thr Val Leu Gly Ile Phe Ala 85 90
95Cys Ser Ser Leu Leu Leu Leu Pro Thr Leu Leu Pro Leu Ala
Ala Thr 100 105 110Asp Asn Asn
Ile Lys Asn Thr Lys Asn Ala Thr Asp Thr Thr Ser Lys 115
120 125Gly Thr Phe Ser Gln Leu Asp Asn Leu Ser Met
Ala Asn Ile Thr Lys 130 135 140Lys Ser
Ser Arg Leu Trp Ala Phe Leu Gly Ala Val Tyr Trp Ile Ser145
150 155 160Leu Val Thr Tyr Phe Phe Leu
Trp Lys Ala Tyr Lys His Val Ser Ser 165
170 175Leu Arg Ala Gln Ala Leu Met Ser Ala Asp Val Lys
Pro Glu Gln Phe 180 185 190Ala
Ile Leu Val Arg Asp Met Pro Ala Pro Pro Asp Gly Gln Thr Gln 195
200 205Lys Glu Phe Ile Asp Ser Tyr Phe Arg
Glu Ile Tyr Pro Glu Thr Phe 210 215
220Tyr Arg Ser Leu Val Ala Thr Glu Asn Ser Lys Val Asn Lys Ile Trp225
230 235 240Glu Lys Leu Glu
Gly Tyr Lys Lys Lys Leu Ala Arg Ala Glu Ala Ile 245
250 255Leu Ala Ala Thr Asn Asn Arg Pro Thr Asn
Lys Thr Gly Phe Cys Gly 260 265
270Leu Val Gly Lys Gln Val Asp Ser Ile Glu Tyr Tyr Thr Glu Leu Ile
275 280 285Asn Glu Ser Val Ala Lys Leu
Glu Thr Glu Gln Lys Ala Val Leu Ala 290 295
300Glu Lys Gln Gln Thr Ala Ala Val Val Phe Phe Thr Thr Arg Val
Ala305 310 315 320Ala Ala
Ser Ala Ala Gln Ser Leu His Cys Gln Met Val Asp Lys Trp
325 330 335Thr Val Thr Glu Ala Pro Glu
Pro Arg Gln Leu Leu Trp Gln Asn Leu 340 345
350Asn Ile Lys Leu Phe Ser Arg Ile Ile Arg Gln Tyr Phe Ile
Tyr Phe 355 360 365Phe Val Ala Val
Thr Ile Leu Phe Tyr Met Ile Pro Ile Ala Phe Val 370
375 380Ser Ala Ile Thr Thr Leu Lys Asn Leu Gln Arg Ile
Ile Pro Phe Ile385 390 395
400Lys Pro Val Val Glu Ile Thr Ala Ile Arg Thr Val Leu Glu Ser Phe
405 410 415Leu Pro Gln Ile Ala
Leu Ile Val Phe Leu Ala Met Leu Pro Lys Leu 420
425 430Leu Leu Phe Leu Ser Lys Ala Glu Gly Ile Pro Ser
Gln Ser His Ala 435 440 445Ile Arg
Ala Ala Ser Gly Lys Tyr Phe Tyr Phe Ser Val Phe Asn Val 450
455 460Phe Ile Gly Val Thr Leu Ala Gly Thr Leu Phe
Asn Thr Val Lys Asp465 470 475
480Ile Ala Lys Asn Pro Lys Leu Asp Met Ile Ile Asn Leu Leu Ala Thr
485 490 495Ser Leu Pro Lys
Ser Ala Thr Phe Phe Leu Thr Tyr Val Ala Leu Lys 500
505 510Phe Phe Ile Gly Tyr Gly Leu Glu Leu Ser Arg
Ile Ile Pro Leu Ile 515 520 525Ile
Phe His Leu Lys Lys Lys Tyr Leu Cys Lys Thr Glu Ala Glu Val 530
535 540Lys Glu Ala Trp Tyr Pro Gly Asp Leu Ser
Tyr Ala Thr Arg Val Pro545 550 555
560Gly Asp Met Leu Ile Leu Thr Ile Thr Phe Cys Tyr Ser Val Ile
Ala 565 570 575Pro Leu Ile
Leu Ile Phe Gly Ile Thr Tyr Phe Gly Leu Gly Trp Leu 580
585 590Val Leu Arg Asn Gln Ala Leu Lys Val Tyr
Val Pro Ser Tyr Glu Ser 595 600
605Tyr Gly Arg Met Trp Pro His Ile His Gln Arg Ile Leu Ala Ala Leu 610
615 620Phe Leu Phe Gln Val Val Met Phe
Gly Tyr Leu Gly Ala Lys Thr Phe625 630
635 640Phe Tyr Thr Ala Leu Val Ile Pro Leu Ile Ile Thr
Ser Leu Ile Phe 645 650
655Gly Tyr Val Cys Arg Gln Lys Phe Tyr Gly Gly Phe Glu His Thr Ala
660 665 670Leu Glu Val Ala Cys Arg
Glu Leu Lys Gln Ser Pro Asp Leu Glu Glu 675 680
685Ile Phe Arg Ala Tyr Ile Pro His Ser Leu Ser Ser His Lys
Pro Glu 690 695 700Glu His Glu Phe Lys
Gly Ala Met Ser Arg Tyr Gln Asp Phe Asn Ala705 710
715 720Ile Ala Gly Val1211313PRTArabidopsis
thaliana 121Met Ala Glu Gln Lys Ser Thr Asn Met Trp Asn Trp Glu Val Thr
Gly1 5 10 15Phe Glu Ser
Lys Lys Ser Pro Ser Ser Glu Glu Gly Val His Arg Thr 20
25 30Pro Ser Ser Met Leu Arg Arg Tyr Ser Ile
Pro Lys Asn Ser Leu Pro 35 40
45Pro His Ser Ser Glu Leu Ala Ser Lys Val Gln Ser Leu Lys Asp Lys 50
55 60Val Gln Leu Ala Lys Asp Asp Tyr Val
Gly Leu Arg Gln Glu Ala Thr65 70 75
80Asp Leu Gln Glu Tyr Ser Asn Ala Lys Leu Glu Arg Val Thr
Arg Tyr 85 90 95Leu Gly
Val Leu Ala Asp Lys Ser Arg Lys Leu Asp Gln Tyr Ala Leu 100
105 110Glu Thr Glu Ala Arg Ile Ser Pro Leu
Ile Asn Glu Lys Lys Arg Leu 115 120
125Phe Asn Asp Leu Leu Thr Thr Lys Gly Ala His Leu Pro Phe Pro Thr
130 135 140Ser Phe Ser Ile Leu Thr Ser
Ile Asp Ile Asp His Thr Arg Pro Leu145 150
155 160Phe Glu Asp Glu Gly Pro Ser Ile Ile Glu Phe Pro
Asp Asn Cys Thr 165 170
175Ile Arg Val Asn Thr Ser Asp Asp Thr Leu Ser Asn Pro Lys Lys Glu
180 185 190Phe Glu Phe Asp Arg Val
Tyr Gly Pro Gln Val Gly Gln Ala Ser Leu 195 200
205Phe Ser Asp Val Gln Pro Phe Val Gln Ser Ala Leu Asp Gly
Ser Asn 210 215 220Val Ser Ile Phe Ala
Tyr Gly Gln Thr His Ala Gly Lys Thr Tyr Thr225 230
235 240Met Val Ala Pro Pro Phe Pro Phe Leu Ser
Glu Ile Arg Tyr Arg Ser 245 250
255Cys Leu Asp Leu Asn Met Ile Gly Lys Phe Met Asp Val His Ser Lys
260 265 270Phe Met Asp Glu Gly
Ser Asn Gln Asp Arg Gly Leu Tyr Ala Arg Cys 275
280 285Phe Glu Glu Leu Met Asp Leu Ala Asn Ser Asp Ser
Thr Ser Ala Ser 290 295 300Gln Phe Ser
Phe Ser Val Ser Val Phe Glu Leu Tyr Asn Glu Gln Val305
310 315 320Arg Asp Leu Leu Ser Gly Cys
Gln Ser Asn Leu Pro Lys Ile Asn Met 325
330 335Gly Leu Arg Glu Ser Val Ile Glu Leu Ser Gln Glu
Lys Val Asp Asn 340 345 350Pro
Ser Glu Phe Met Arg Val Leu Asn Ser Ala Phe Gln Asn Arg Gly 355
360 365Asn Asp Lys Ser Lys Ser Thr Val Thr
His Leu Ile Val Ser Ile His 370 375
380Ile Cys Tyr Ser Asn Thr Ile Thr Arg Glu Asn Val Ile Ser Lys Leu385
390 395 400Ser Leu Val Asp
Leu Ala Gly Ser Glu Gly Leu Thr Val Glu Asp Asp 405
410 415Asn Gly Asp His Val Thr Asp Leu Leu His
Val Thr Asn Ser Ile Ser 420 425
430Ala Leu Gly Asp Val Leu Ser Ser Leu Thr Ser Lys Arg Asp Thr Ile
435 440 445Pro Tyr Glu Asn Ser Phe Leu
Thr Arg Ile Leu Ala Asp Ser Leu Gly 450 455
460Gly Ser Ser Lys Thr Leu Met Ile Val Asn Ile Cys Pro Ser Ala
Arg465 470 475 480Asn Leu
Ser Glu Ile Met Ser Cys Leu Asn Tyr Ala Ala Arg Ala Arg
485 490 495Asn Thr Val Pro Ser Leu Gly
Asn Arg Asp Thr Ile Lys Lys Trp Arg 500 505
510Asp Val Ala Asn Asp Ala Arg Lys Glu Val Leu Glu Lys Glu
Arg Glu 515 520 525Asn Gln Arg Leu
Lys Gln Glu Val Thr Gly Leu Lys Gln Ala Leu Lys 530
535 540Glu Ala Asn Asp Gln Cys Val Leu Leu Tyr Asn Glu
Val Gln Arg Ala545 550 555
560Trp Arg Val Ser Phe Thr Leu Gln Ser Asp Leu Lys Ser Glu Asn Ala
565 570 575Met Val Val Asp Lys
His Lys Ile Glu Lys Glu Gln Asn Phe Gln Leu 580
585 590Arg Asn Gln Ile Ala Gln Leu Leu Gln Leu Glu Gln
Glu Gln Lys Leu 595 600 605Gln Ala
Gln Gln Gln Asp Ser Thr Ile Gln Asn Leu Gln Ser Lys Val 610
615 620Lys Asp Leu Glu Ser Gln Leu Ser Lys Ala Leu
Lys Ser Asp Met Thr625 630 635
640Arg Ser Arg Asp Pro Leu Glu Pro Gln Pro Arg Ala Ala Glu Asn Thr
645 650 655Leu Asp Ser Ser
Ala Val Thr Lys Lys Leu Glu Glu Glu Leu Lys Lys 660
665 670Arg Asp Ala Leu Ile Glu Arg Leu His Glu Glu
Asn Glu Lys Leu Phe 675 680 685Asp
Arg Leu Thr Glu Lys Ser Val Ala Ser Ser Thr Gln Val Ser Ser 690
695 700Pro Ser Ser Lys Ala Ser Pro Thr Val Gln
Pro Ala Asp Val Asp Arg705 710 715
720Lys Asn Ser Ala Gly Thr Leu Pro Ser Ser Val Asp Lys Asn Glu
Gly 725 730 735Thr Ile Thr
Leu Val Lys Ser Ser Ser Glu Leu Val Lys Thr Thr Pro 740
745 750Ala Gly Glu Tyr Leu Thr Ala Ala Leu Asn
Asp Phe Asp Pro Glu Gln 755 760
765Tyr Glu Gly Leu Ala Ala Ile Ala Asp Gly Ala Asn Lys Leu Leu Met 770
775 780Leu Val Leu Ala Ala Val Ile Lys
Ala Gly Ala Ser Arg Glu His Glu785 790
795 800Ile Leu Ala Glu Ile Arg Asp Ser Val Phe Ser Phe
Ile Arg Lys Met 805 810
815Glu Pro Arg Arg Val Met Asp Thr Met Leu Val Ser Arg Val Arg Ile
820 825 830Leu Tyr Ile Arg Ser Leu
Leu Ala Arg Ser Pro Glu Leu Gln Ser Ile 835 840
845Lys Val Ser Pro Val Glu Arg Phe Leu Glu Lys Pro Tyr Thr
Gly Arg 850 855 860Thr Arg Ser Ser Ser
Gly Ser Ser Ser Pro Gly Arg Ser Pro Val Arg865 870
875 880Tyr Tyr Asp Glu Gln Ile Tyr Gly Phe Lys
Val Asn Leu Lys Pro Glu 885 890
895Lys Lys Ser Lys Leu Val Ser Val Val Ser Arg Ile Arg Gly His Asp
900 905 910Gln Asp Thr Gly Arg
Gln Gln Val Thr Gly Gly Lys Leu Arg Glu Ile 915
920 925Gln Asp Glu Ala Lys Ser Phe Ala Ile Gly Asn Lys
Pro Leu Ala Ala 930 935 940Leu Phe Val
His Thr Pro Ala Gly Glu Leu Gln Arg Gln Ile Arg Ser945
950 955 960Trp Leu Ala Glu Ser Phe Glu
Phe Leu Ser Val Thr Ala Asp Asp Val 965
970 975Ser Gly Val Thr Thr Gly Gln Leu Glu Leu Leu Ser
Thr Ala Ile Met 980 985 990Asp
Gly Trp Met Ala Gly Val Gly Ala Ala Val Pro Pro His Thr Asp 995
1000 1005Ala Leu Gly Gln Leu Leu Ser Glu Tyr
Ala Lys Arg Val Tyr Thr 1010 1015
1020Ser Gln Met Gln His Leu Lys Asp Ile Ala Gly Thr Leu Ala Ser
1025 1030 1035Glu Glu Ala Glu Asp Ala
Gly Gln Val Ala Lys Leu Arg Ser Ala 1040 1045
1050Leu Glu Ser Val Asp His Lys Arg Arg Lys Ile Leu Gln Gln
Met 1055 1060 1065Arg Ser Asp Ala Ala
Leu Phe Thr Leu Glu Glu Gly Ser Ser Pro 1070 1075
1080Val Gln Asn Pro Ser Thr Ala Ala Glu Asp Ser Arg Leu
Ala Ser 1085 1090 1095Leu Ile Ser Leu
Asp Ala Ile Leu Lys Gln Val Lys Glu Ile Thr 1100
1105 1110Arg Gln Ala Ser Val His Val Leu Ser Lys Ser
Lys Lys Lys Ala 1115 1120 1125Leu Leu
Glu Ser Leu Asp Glu Leu Asn Glu Arg Met Pro Ser Leu 1130
1135 1140Leu Asp Val Asp His Pro Cys Ala Gln Arg
Glu Ile Asp Thr Ala 1145 1150 1155His
Gln Leu Val Glu Thr Ile Pro Glu Gln Glu Asp Asn Leu Gln 1160
1165 1170Asp Glu Lys Arg Pro Ser Ile Asp Ser
Ile Ser Ser Thr Glu Thr 1175 1180
1185Asp Val Ser Gln Trp Asn Val Leu Gln Phe Asn Thr Gly Gly Ser
1190 1195 1200Ser Ala Pro Phe Ile Ile
Lys Cys Gly Ala Asn Ser Asn Ser Glu 1205 1210
1215Leu Val Ile Lys Ala Asp Ala Arg Ile Gln Glu Pro Lys Gly
Gly 1220 1225 1230Glu Ile Val Arg Val
Val Pro Arg Pro Ser Val Leu Glu Asn Met 1235 1240
1245Ser Leu Glu Glu Met Lys Gln Val Phe Gly Gln Leu Pro
Glu Ala 1250 1255 1260Leu Ser Ser Leu
Ala Leu Ala Arg Thr Ala Asp Gly Thr Arg Ala 1265
1270 1275Arg Tyr Ser Arg Leu Tyr Arg Thr Leu Ala Met
Lys Val Pro Ser 1280 1285 1290Leu Arg
Asp Leu Val Gly Glu Leu Glu Lys Gly Gly Val Leu Lys 1295
1300 1305Asp Thr Lys Ser Thr
1310122310PRTArabidopsis thaliana 122Met Ala Asn Pro Trp Trp Val Gly Asn
Val Ala Ile Gly Gly Val Glu1 5 10
15Ser Pro Val Thr Ser Ser Ala Pro Ser Leu His His Arg Asn Ser
Asn 20 25 30Asn Asn Asn Pro
Pro Thr Met Thr Arg Ser Asp Pro Arg Leu Asp His 35
40 45Asp Phe Thr Thr Asn Asn Ser Gly Ser Pro Asn Thr
Gln Thr Gln Ser 50 55 60Gln Glu Glu
Gln Asn Ser Arg Asp Glu Gln Pro Ala Val Glu Pro Gly65 70
75 80Ser Gly Ser Gly Ser Thr Gly Arg
Arg Pro Arg Gly Arg Pro Pro Gly 85 90
95Ser Lys Asn Lys Pro Lys Ser Pro Val Val Val Thr Lys Glu
Ser Pro 100 105 110Asn Ser Leu
Gln Ser His Val Leu Glu Ile Ala Thr Gly Ala Asp Val 115
120 125Ala Glu Ser Leu Asn Ala Phe Ala Arg Arg Arg
Gly Arg Gly Val Ser 130 135 140Val Leu
Ser Gly Ser Gly Leu Val Thr Asn Val Thr Leu Arg Gln Pro145
150 155 160Ala Ala Ser Gly Gly Val Val
Ser Leu Arg Gly Gln Phe Glu Ile Leu 165
170 175Ser Met Cys Gly Ala Phe Leu Pro Thr Ser Gly Ser
Pro Ala Ala Ala 180 185 190Ala
Gly Leu Thr Ile Tyr Leu Ala Gly Ala Gln Gly Gln Val Val Gly 195
200 205Gly Gly Val Ala Gly Pro Leu Ile Ala
Ser Gly Pro Val Ile Val Ile 210 215
220Ala Ala Thr Phe Cys Asn Ala Thr Tyr Glu Arg Leu Pro Ile Glu Glu225
230 235 240Glu Gln Gln Gln
Glu Gln Pro Leu Gln Leu Glu Asp Gly Lys Lys Gln 245
250 255Lys Glu Glu Asn Asp Asp Asn Glu Ser Gly
Asn Asn Gly Asn Glu Gly 260 265
270Ser Met Gln Pro Pro Met Tyr Asn Met Pro Pro Asn Phe Ile Pro Asn
275 280 285Gly His Gln Met Ala Gln His
Asp Val Tyr Trp Gly Gly Pro Pro Pro 290 295
300Arg Ala Pro Pro Ser Tyr305
310123964PRTArabidopsis thaliana 123Met Ala Leu Asn Leu Arg Gln Lys Gln
Thr Glu Cys Val Ile Arg Met1 5 10
15Leu Asn Leu Asn Gln Pro Leu Asn Pro Ser Gly Thr Ala Asn Glu
Glu 20 25 30Val Tyr Lys Ile
Leu Ile Tyr Asp Arg Phe Cys Gln Asn Ile Leu Ser 35
40 45Pro Leu Thr His Val Lys Asp Leu Arg Lys His Gly
Val Thr Leu Phe 50 55 60Phe Leu Ile
Asp Lys Asp Arg Gln Pro Val His Asp Val Pro Ala Val65 70
75 80Tyr Phe Val Gln Pro Thr Glu Ser
Asn Leu Gln Arg Ile Ile Ala Asp 85 90
95Ala Ser Arg Ser Leu Tyr Asp Thr Phe His Leu Asn Phe Ser
Ser Ser 100 105 110Ile Pro Arg
Lys Phe Leu Glu Glu Leu Ala Ser Gly Thr Leu Lys Ser 115
120 125Gly Ser Val Glu Lys Val Ser Lys Val His Asp
Gln Tyr Leu Glu Phe 130 135 140Val Thr
Leu Glu Asp Asn Leu Phe Ser Leu Ala Gln Gln Ser Thr Tyr145
150 155 160Val Gln Met Asn Asp Pro Ser
Ala Gly Glu Lys Glu Ile Asn Glu Ile 165
170 175Ile Glu Arg Val Ala Ser Gly Leu Phe Cys Val Leu
Val Thr Leu Gly 180 185 190Val
Val Pro Val Ile Arg Cys Pro Ser Gly Gly Pro Ala Glu Met Val 195
200 205Ala Ser Leu Leu Asp Gln Lys Leu Arg
Asp His Leu Leu Ser Lys Asn 210 215
220Asn Leu Phe Thr Glu Gly Gly Gly Phe Met Ser Ser Phe Gln Arg Pro225
230 235 240Leu Leu Cys Ile
Phe Asp Arg Asn Phe Glu Leu Ser Val Gly Ile Gln 245
250 255His Asp Phe Arg Tyr Arg Pro Leu Val His
Asp Val Leu Gly Leu Lys 260 265
270Leu Asn Gln Leu Lys Val Gln Gly Glu Lys Gly Pro Pro Lys Ser Phe
275 280 285Glu Leu Asp Ser Ser Asp Pro
Phe Trp Ser Ala Asn Ser Thr Leu Glu 290 295
300Phe Pro Asp Val Ala Val Glu Ile Glu Thr Gln Leu Asn Lys Tyr
Lys305 310 315 320Arg Asp
Val Glu Glu Val Asn Lys Lys Thr Gly Gly Gly Ser Gly Ala
325 330 335Glu Phe Asp Gly Thr Asp Leu
Ile Gly Asn Ile His Thr Glu His Leu 340 345
350Met Asn Thr Val Lys Ser Leu Pro Glu Leu Thr Glu Arg Lys
Lys Val 355 360 365Ile Asp Lys His
Thr Asn Ile Ala Thr Ala Leu Leu Gly Gln Ile Lys 370
375 380Glu Arg Ser Ile Asp Ala Phe Thr Lys Lys Glu Ser
Asp Met Met Met385 390 395
400Arg Gly Gly Ile Asp Arg Thr Glu Leu Met Ala Ala Leu Lys Gly Lys
405 410 415Gly Thr Lys Met Asp
Lys Leu Arg Phe Ala Ile Met Tyr Leu Ile Ser 420
425 430Thr Glu Thr Ile Asn Gln Ser Glu Val Glu Ala Val
Glu Ala Ala Leu 435 440 445Asn Glu
Ala Glu Ala Asp Thr Ser Ala Phe Gln Tyr Val Lys Lys Ile 450
455 460Lys Ser Leu Asn Ala Ser Phe Ala Ala Thr Ser
Ala Asn Ser Ala Ser465 470 475
480Arg Ser Asn Ile Val Asp Trp Ala Glu Lys Leu Tyr Gly Gln Ser Ile
485 490 495Ser Ala Val Thr
Ala Gly Val Lys Asn Leu Leu Ser Ser Asp Gln Gln 500
505 510Leu Ala Val Thr Arg Thr Val Glu Ala Leu Thr
Glu Gly Lys Pro Asn 515 520 525Pro
Glu Ile Asp Ser Tyr Arg Phe Leu Asp Pro Arg Ala Pro Lys Ser 530
535 540Ser Ser Ser Gly Gly Ser His Val Lys Gly
Pro Phe Arg Glu Ala Ile545 550 555
560Val Phe Met Ile Gly Gly Gly Asn Tyr Val Glu Tyr Gly Ser Leu
Gln 565 570 575Glu Leu Thr
Gln Arg Gln Leu Thr Val Lys Asn Val Ile Tyr Gly Ala 580
585 590Thr Glu Ile Leu Asn Gly Gly Glu Leu Val
Glu Gln Leu Gly Leu Leu 595 600
605Gly Lys Lys Met Gly Leu Gly Gly Pro Val Ala Ser Thr Leu Lys Arg 610
615 620Leu Gly Met Ala Gly Lys Glu Glu
Thr Asp Val Ser Ala Gln Gly Ser625 630
635 640Leu Thr Arg Glu Ala Thr Glu Ile Trp Arg Ser Glu
Leu Glu Ser Arg 645 650
655Arg Phe Gln Val Asp Ser Leu Glu Ala Glu Leu Val Asp Val Lys Ala
660 665 670Tyr Leu Glu Phe Gly Ser
Glu Glu Asp Ala Arg Lys Glu Leu Gly Val 675 680
685Leu Ser Gly Arg Val Arg Ser Thr Ala Thr Met Leu Arg Tyr
Leu Arg 690 695 700Ser Lys Ala Arg Val
Leu Ala Ile Pro Asp Asp Leu Ala Asn Val Ser705 710
715 720Cys Gly Val Glu Gln Ile Glu Glu Leu Lys
Gly Leu Asn Leu Val Glu 725 730
735Lys Asp Gly Gly Ser Ser Ser Ser Asp Gly Ala Arg Asn Thr Asn Pro
740 745 750Glu Thr Arg Arg Tyr
Ser Gly Ser Leu Gly Val Glu Asp Gly Ala Tyr 755
760 765Thr Asn Glu Met Leu Gln Ser Ile Glu Met Val Thr
Asp Val Leu Asp 770 775 780Ser Leu Val
Arg Arg Val Thr Val Ala Glu Ser Glu Ser Ala Val Gln785
790 795 800Lys Glu Arg Ala Leu Leu Gly
Glu Glu Glu Ile Ser Arg Lys Thr Ile 805
810 815Gln Ile Glu Asn Leu Ser Val Lys Leu Glu Glu Met
Glu Arg Phe Ala 820 825 830Tyr
Gly Thr Asn Ser Val Leu Asn Glu Met Arg Glu Arg Ile Glu Glu 835
840 845Leu Val Glu Glu Thr Met Arg Gln Arg
Glu Lys Ala Val Glu Asn Glu 850 855
860Glu Glu Leu Cys Arg Val Lys Arg Glu Phe Glu Ser Leu Lys Ser Tyr865
870 875 880Val Ser Thr Phe
Thr Asn Val Arg Glu Thr Leu Leu Ser Ser Glu Arg 885
890 895Gln Phe Lys Thr Ile Glu Glu Leu Phe Glu
Arg Leu Val Thr Lys Thr 900 905
910Thr Gln Leu Glu Gly Glu Lys Ala Gln Lys Glu Val Glu Val Gln Lys
915 920 925Leu Met Glu Glu Asn Val Lys
Leu Thr Ala Leu Leu Asp Lys Lys Glu 930 935
940Ala Gln Leu Leu Ala Leu Asn Glu Gln Cys Lys Val Met Ala Leu
Ser945 950 955 960Ala Ser
Asn Ile124222PRTArabidopsis thaliana 124Met Asp Ala Met Lys Glu Glu Ile
Gln Arg Val Lys Glu Gln Glu Glu1 5 10
15Gln Ala Met Arg Glu Ala Leu Gly Leu Ala Pro Lys Ser Ser
Thr Arg 20 25 30Pro Gln Gly
Asn Arg Leu Asp Lys Gln Glu Phe Thr Glu Leu Val Lys 35
40 45Arg Gly Ser Thr Ala Glu Asp Leu Gly Ala Gly
Asn Ala Asp Ala Val 50 55 60Trp Val
His Gly Leu Gly Tyr Ala Lys Ala Pro Arg Pro Trp Glu Asp65
70 75 80Pro Ser Thr Leu Ala Ser Ser
Gln Lys Glu Asp Ala Asp Ser Ala Arg 85 90
95Leu Pro Ala Asp Thr Ser Gly Val Lys Thr Val Glu Asp
Gly Pro Asp 100 105 110Asp Val
Glu Arg Asp Gln Arg Arg Ile Gly Val Arg Lys Gly Asn Leu 115
120 125Gln Arg Glu Arg Arg Lys Lys Asp Met Ile
Gly Val Lys Asn Ala Lys 130 135 140Gly
Met Arg Ser Glu Ala Leu Val Ile Gln Met Ile Glu Arg Ser Thr145
150 155 160Arg Lys Arg Arg Arg Arg
Lys Lys Glu Gly Met Thr Leu Ile Leu Ile 165
170 175Glu Ala Asn Cys Pro Arg Met Glu His Phe Ala Leu
Gln Arg Lys Ser 180 185 190Gly
Arg Leu Gly Thr Lys Ile Gln Leu Pro Leu Leu Gln Asp Leu Asn 195
200 205Leu Leu Leu Ile Ser Phe Thr Asn Arg
Gly Val Lys Cys Cys 210 215
220125148PRTArabidopsis thaliana 125Met Gly Lys Asp Gly Leu Ser Asp Asp
Gln Val Ser Ser Met Lys Glu1 5 10
15Ala Phe Met Leu Phe Asp Thr Asp Gly Asp Gly Lys Ile Ala Pro
Ser 20 25 30Glu Leu Gly Ile
Leu Met Arg Ser Leu Gly Gly Asn Pro Thr Gln Ala 35
40 45Gln Leu Lys Ser Ile Ile Ala Ser Glu Asn Leu Ser
Ser Pro Phe Asp 50 55 60Phe Asn Arg
Phe Leu Asp Leu Met Ala Lys His Leu Lys Thr Glu Pro65 70
75 80Phe Asp Arg Gln Leu Arg Asp Ala
Phe Lys Val Leu Asp Lys Glu Gly 85 90
95Thr Gly Phe Val Ala Val Ala Asp Leu Arg His Ile Leu Thr
Ser Ile 100 105 110Gly Glu Lys
Leu Glu Pro Asn Glu Phe Asp Glu Trp Ile Lys Glu Val 115
120 125Asp Val Gly Ser Asp Gly Lys Ile Arg Tyr Glu
Asp Phe Ile Ala Arg 130 135 140Met Val
Ala Lys14512670PRTArabidopsis thaliana 126Met Glu Lys Gln Ser Thr Gln Pro
Ile Cys Gly Gln Glu Ala Leu Gln1 5 10
15Leu Leu Asn Cys Val Ala Glu Ser Pro Phe Asp Gln Glu Lys
Cys Val 20 25 30Arg Phe Leu
Gln Ser Leu Arg Glu Cys Val Leu Ser Lys Lys Val Lys 35
40 45Lys Phe Ser Ile Pro Ser Gln Asp His Asp Ser
Glu Gly Ala Ala Ser 50 55 60Ala Thr
Lys Arg Pro Ser65 70127385PRTArabidopsis thaliana 127Met
Thr Thr Thr Gly Ser Asn Ser Asn His Asn His His Glu Ser Asn1
5 10 15Asn Asn Asn Asn Asn Pro Ser
Thr Arg Ser Trp Gly Thr Ala Val Ser 20 25
30Gly Gln Ser Val Ser Thr Ser Gly Ser Met Gly Ser Pro Ser
Ser Arg 35 40 45Ser Glu Gln Thr
Ile Thr Val Val Thr Ser Thr Ser Asp Thr Thr Phe 50 55
60Gln Arg Leu Asn Asn Leu Asp Ile Gln Gly Asp Asp Ala
Gly Ser Gln65 70 75
80Gly Ala Ser Gly Val Lys Lys Lys Lys Arg Gly Gln Arg Ala Ala Gly
85 90 95Pro Asp Lys Thr Gly Arg
Gly Leu Arg Gln Phe Ser Met Lys Val Cys 100
105 110Glu Lys Val Glu Ser Lys Gly Arg Thr Thr Tyr Asn
Glu Val Ala Asp 115 120 125Glu Leu
Val Ala Glu Phe Ala Leu Pro Asn Asn Asp Gly Thr Ser Pro 130
135 140Asp Gln Gln Gln Tyr Asp Glu Lys Asn Ile Arg
Arg Arg Val Tyr Asp145 150 155
160Ala Leu Asn Val Leu Met Ala Met Asp Ile Ile Ser Lys Asp Lys Lys
165 170 175Glu Ile Gln Trp
Arg Gly Leu Pro Arg Thr Ser Leu Ser Asp Ile Glu 180
185 190Glu Leu Lys Asn Glu Arg Leu Ser Leu Arg Asn
Arg Ile Glu Lys Lys 195 200 205Thr
Ala Tyr Ser Gln Glu Leu Glu Glu Gln Tyr Val Gly Leu Gln Asn 210
215 220Leu Ile Gln Arg Asn Glu His Leu Tyr Ser
Ser Gly Asn Ala Pro Ser225 230 235
240Gly Gly Val Ala Leu Pro Phe Ile Leu Val Gln Thr Arg Pro His
Ala 245 250 255Thr Val Glu
Val Glu Ile Ser Glu Asp Met Gln Leu Val His Phe Asp 260
265 270Phe Asn Ser Thr Pro Phe Glu Leu His Asp
Asp Asn Phe Val Leu Lys 275 280
285Thr Met Lys Phe Cys Asp Gln Pro Pro Gln Gln Pro Asn Gly Arg Asn 290
295 300Asn Ser Gln Leu Val Cys His Asn
Phe Thr Pro Glu Asn Pro Asn Lys305 310
315 320Gly Pro Ser Thr Gly Pro Thr Pro Gln Leu Asp Met
Tyr Glu Thr His 325 330
335Leu Gln Ser Gln Gln His Gln Gln His Ser Gln Leu Gln Ile Ile Pro
340 345 350Met Pro Glu Thr Asn Asn
Val Thr Ser Ser Ala Asp Thr Ala Pro Val 355 360
365Lys Ser Pro Ser Leu Pro Gly Ile Met Asn Ser Ser Met Lys
Pro Glu 370 375
380Asn385128437PRTArabidopsis thaliana 128Met Ala Leu Gln Asn Ile Gly Ala
Ser Asn Arg Asn Asp Ala Phe Tyr1 5 10
15Arg Tyr Lys Met Pro Lys Met Val Thr Lys Thr Glu Gly Lys
Gly Asn 20 25 30Gly Ile Lys
Thr Asn Ile Ile Asn Asn Val Glu Ile Ala Lys Ala Leu 35
40 45Ala Arg Pro Pro Ser Tyr Thr Thr Lys Tyr Phe
Gly Cys Glu Leu Gly 50 55 60Ala Gln
Ser Lys Phe Asp Glu Lys Thr Gly Thr Ser Leu Val Asn Gly65
70 75 80Ala His Asn Thr Ser Lys Leu
Ala Gly Leu Leu Glu Asn Phe Ile Lys 85 90
95Lys Phe Val Gln Cys Tyr Gly Cys Gly Asn Pro Glu Thr
Glu Ile Ile 100 105 110Ile Thr
Lys Thr Gln Met Val Asn Leu Lys Cys Ala Ala Cys Gly Phe 115
120 125Ile Ser Glu Val Asp Met Arg Asp Lys Leu
Thr Asn Phe Ile Leu Lys 130 135 140Asn
Pro Pro Glu Gln Lys Lys Val Ser Lys Asp Lys Lys Ala Met Arg145
150 155 160Lys Ala Glu Lys Glu Arg
Leu Lys Glu Gly Glu Leu Ala Asp Glu Glu 165
170 175Gln Arg Lys Leu Lys Ala Lys Lys Lys Ala Leu Ser
Asn Gly Lys Asp 180 185 190Ser
Lys Thr Ser Lys Asn His Ser Ser Asp Glu Asp Ile Ser Pro Lys 195
200 205His Asp Glu Asn Ala Leu Glu Val Asp
Glu Asp Glu Asp Asp Asp Asp 210 215
220Gly Val Glu Trp Gln Thr Asp Thr Ser Arg Glu Ala Ala Glu Lys Arg225
230 235 240Met Met Glu Gln
Leu Ser Ala Lys Thr Ala Glu Met Val Met Leu Ser 245
250 255Ala Met Glu Val Glu Glu Lys Lys Ala Pro
Lys Ser Lys Ser Asn Gly 260 265
270Asn Val Val Lys Thr Glu Asn Pro Pro Pro Gln Glu Lys Asn Leu Val
275 280 285Gln Asp Met Lys Glu Tyr Leu
Lys Lys Gly Ser Pro Ile Ser Ala Leu 290 295
300Lys Ser Phe Ile Ser Ser Leu Ser Glu Pro Pro Gln Asp Ile Met
Asp305 310 315 320Ala Leu
Phe Asn Ala Leu Phe Asp Gly Val Gly Lys Gly Phe Ala Lys
325 330 335Glu Val Thr Lys Lys Lys Asn
Tyr Leu Ala Ala Ala Ala Thr Met Gln 340 345
350Glu Asp Gly Ser Gln Met His Leu Leu Asn Ser Ile Gly Thr
Phe Cys 355 360 365Gly Lys Asn Gly
Asn Glu Glu Ala Leu Lys Glu Val Ala Leu Val Leu 370
375 380Lys Ala Leu Tyr Asp Gln Asp Ile Ile Glu Glu Glu
Val Val Leu Asp385 390 395
400Trp Tyr Glu Lys Gly Leu Thr Gly Ala Asp Lys Ser Ser Pro Val Trp
405 410 415Lys Asn Val Lys Pro
Phe Val Glu Trp Leu Gln Ser Ala Glu Ser Glu 420
425 430Ser Glu Glu Glu Asp
435129749PRTArabidopsis thaliana 129Met Ala Ala Asn Lys Phe Ala Thr Leu
Ile His Arg Lys Thr Asn Arg1 5 10
15Ile Thr Leu Ile Leu Val Tyr Ala Phe Leu Glu Trp Ser Leu Ile
Phe 20 25 30Phe Ile Leu Leu
Asn Ser Leu Phe Ser Tyr Phe Ile Leu Arg Phe Ala 35
40 45Asp Tyr Phe Gly Leu Lys Arg Pro Cys Leu Phe Cys
Ser Arg Leu Asp 50 55 60Arg Phe Phe
Asp Ala Ser Gly Lys Ser Pro Ser His Arg Asp Leu Leu65 70
75 80Cys Asp Asp His Ala Leu Gln Leu
His Ser Lys Pro Val Glu Glu Ser 85 90
95Asn Cys Gly Phe Gly Glu Phe His Asn Asp Leu Val His Arg
Gly Cys 100 105 110Cys Val Glu
Lys Ile Ser Ser Ser Leu Cys Ala Pro Ile Glu Ser Asp 115
120 125Phe Gly Asn Leu Asp Tyr Pro Ile Gly Asp Glu
Gly Gln Ile Tyr Asn 130 135 140Gly Leu
Lys Phe Pro Arg Ser Ile Phe Val Phe Glu Glu Glu Lys Val145
150 155 160Gly Ser Val Asn Leu Asn Asp
Ser Gln Glu Glu Thr Glu Glu Lys Lys 165
170 175Val Pro Gln Ser His Glu Lys Leu Glu Asp Asp Asp
Val Asp Glu Glu 180 185 190Phe
Ser Cys Tyr Val Ser Ser Phe Asp Cys Lys Asn Lys Glu Ile Ala 195
200 205Thr Glu Lys Glu Glu Glu Asn Arg Val
Asp Leu Pro Ile Glu Val Glu 210 215
220Thr Ala Glu Ser Ala Pro Lys Asn Leu Glu Phe Tyr Ile Asp Glu Glu225
230 235 240Asp Cys His Leu
Ile Pro Val Glu Phe Tyr Lys Pro Ser Glu Glu Val 245
250 255Arg Glu Ile Ser Asp Ile Asn Gly Asp Phe
Ile Leu Asp Phe Gly Val 260 265
270Glu His Asp Phe Thr Ala Ala Ala Glu Thr Glu Glu Ile Ser Asp Phe
275 280 285Ala Ser Pro Gly Glu Ser Lys
Pro Glu Asp Ala Glu Thr Asn Leu Val 290 295
300Ala Ser Glu Met Glu Asn Asp Asp Glu Glu Thr Asp Ala Glu Val
Ser305 310 315 320Ile Gly
Thr Glu Ile Pro Asp His Glu Gln Ile Gly Asp Ile Pro Ser
325 330 335His Gln Leu Ile Pro His His
Asp Asp Asp Asp His Glu Glu Glu Thr 340 345
350Leu Glu Phe Lys Thr Val Thr Ile Glu Thr Lys Met Pro Val
Leu Asn 355 360 365Ile Asn Glu Glu
Arg Ile Leu Glu Ala Gln Gly Ser Met Glu Ser Ser 370
375 380His Ser Ser Leu His Asn Ala Met Phe His Leu Glu
Gln Arg Val Ser385 390 395
400Val Asp Gly Ile Glu Cys Pro Glu Gly Val Leu Thr Val Asp Lys Leu
405 410 415Lys Phe Glu Leu Gln
Glu Glu Arg Lys Ala Leu His Ala Leu Tyr Glu 420
425 430Glu Leu Glu Val Glu Arg Asn Ala Ser Ala Val Ala
Ala Ser Glu Thr 435 440 445Met Ala
Met Ile Asn Arg Leu His Glu Glu Lys Ala Ala Met Gln Met 450
455 460Glu Ala Leu Gln Tyr Gln Arg Met Met Glu Glu
Gln Ala Glu Phe Asp465 470 475
480Gln Glu Ala Leu Gln Leu Leu Asn Glu Leu Met Val Asn Arg Glu Lys
485 490 495Glu Asn Ala Glu
Leu Glu Lys Glu Leu Glu Val Tyr Arg Lys Arg Met 500
505 510Glu Glu Tyr Glu Ala Lys Glu Lys Met Gly Met
Leu Arg Arg Arg Leu 515 520 525Arg
Asp Ser Ser Val Asp Ser Tyr Arg Asn Asn Gly Asp Ser Asp Glu 530
535 540Asn Ser Asn Gly Glu Leu Gln Phe Lys Asn
Val Glu Gly Val Thr Asp545 550 555
560Trp Lys Tyr Arg Glu Asn Glu Met Glu Asn Thr Pro Val Asp Val
Val 565 570 575Leu Arg Leu
Asp Glu Cys Leu Asp Asp Tyr Asp Gly Glu Arg Leu Ser 580
585 590Ile Leu Gly Arg Leu Lys Phe Leu Glu Glu
Lys Leu Thr Asp Leu Asn 595 600
605Asn Glu Glu Asp Asp Glu Glu Glu Ala Lys Thr Phe Glu Ser Asn Gly 610
615 620Ser Ile Asn Gly Asn Glu His Ile
His Gly Lys Glu Thr Asn Gly Lys625 630
635 640His Arg Val Ile Lys Ser Lys Arg Leu Leu Pro Leu
Phe Asp Ala Val 645 650
655Asp Gly Glu Met Glu Asn Gly Leu Ser Asn Gly Asn His His Glu Asn
660 665 670Gly Phe Asp Asp Ser Glu
Lys Gly Glu Asn Val Thr Ile Glu Glu Glu 675 680
685Val Asp Glu Leu Tyr Glu Arg Leu Glu Ala Leu Glu Ala Asp
Arg Glu 690 695 700Phe Leu Arg His Cys
Val Gly Ser Leu Lys Lys Gly Asp Lys Gly Val705 710
715 720His Leu Leu His Glu Ile Leu Gln His Leu
Arg Asp Leu Arg Asn Ile 725 730
735Asp Leu Thr Arg Val Arg Glu Asn Gly Asp Met Ser Leu
740 745130742PRTArabidopsis thaliana 130Met Ser Asp Ala
Leu Ser Ala Ile Pro Ala Ala Val His Arg Asn Leu1 5
10 15Ser Asp Lys Leu Tyr Glu Lys Arg Lys Asn
Ala Ala Leu Glu Leu Glu 20 25
30Asn Ile Val Lys Asn Leu Thr Ser Ser Gly Asp His Asp Lys Ile Ser
35 40 45Lys Val Ile Glu Met Leu Ile Lys
Glu Phe Ala Lys Ser Pro Gln Ala 50 55
60Asn His Arg Lys Gly Gly Leu Ile Gly Leu Ala Ala Val Thr Val Gly65
70 75 80Leu Ser Thr Glu Ala
Ala Gln Tyr Leu Glu Gln Ile Val Pro Pro Val 85
90 95Ile Asn Ser Phe Ser Asp Gln Asp Ser Arg Val
Arg Tyr Tyr Ala Cys 100 105
110Glu Ala Leu Tyr Asn Ile Ala Lys Val Val Arg Gly Asp Phe Ile Ile
115 120 125Phe Phe Asn Lys Ile Phe Asp
Ala Leu Cys Lys Leu Ser Ala Asp Ser 130 135
140Asp Ala Asn Val Gln Ser Ala Ala His Leu Leu Asp Arg Leu Val
Lys145 150 155 160Asp Ile
Val Thr Glu Ser Asp Gln Phe Ser Ile Glu Glu Phe Ile Pro
165 170 175Leu Leu Lys Glu Arg Met Asn
Val Leu Asn Pro Tyr Val Arg Gln Phe 180 185
190Leu Val Gly Trp Ile Thr Val Leu Asp Ser Val Pro Asp Ile
Asp Met 195 200 205Leu Gly Phe Leu
Pro Asp Phe Leu Asp Gly Leu Phe Asn Met Leu Ser 210
215 220Asp Ser Ser His Glu Ile Arg Gln Gln Ala Asp Ser
Ala Leu Ser Glu225 230 235
240Phe Leu Gln Glu Ile Lys Asn Ser Pro Ser Val Asp Tyr Gly Arg Met
245 250 255Ala Glu Ile Leu Val
Gln Arg Ala Ala Ser Pro Asp Glu Phe Thr Arg 260
265 270Leu Thr Ala Ile Thr Trp Ile Asn Glu Phe Val Lys
Leu Gly Gly Asp 275 280 285Gln Leu
Val Arg Tyr Tyr Ala Asp Ile Leu Gly Ala Ile Leu Pro Cys 290
295 300Ile Ser Asp Lys Glu Glu Lys Ile Arg Val Val
Ala Arg Glu Thr Asn305 310 315
320Glu Glu Leu Arg Ser Ile His Val Glu Pro Ser Asp Gly Phe Asp Val
325 330 335Gly Ala Ile Leu
Ser Val Ala Arg Arg Gln Leu Ser Ser Glu Phe Glu 340
345 350Ala Thr Arg Ile Glu Ala Leu Asn Trp Ile Ser
Thr Leu Leu Asn Lys 355 360 365His
Arg Thr Glu Val Leu Cys Phe Leu Asn Asp Ile Phe Asp Thr Leu 370
375 380Leu Lys Ala Leu Ser Asp Ser Ser Asp Asp
Val Val Leu Leu Val Leu385 390 395
400Glu Val His Ala Gly Val Ala Lys Asp Pro Gln His Phe Arg Gln
Leu 405 410 415Ile Val Phe
Leu Val His Asn Phe Arg Ala Asp Asn Ser Leu Leu Glu 420
425 430Arg Gly Ala Leu Ile Val Arg Arg Met Cys
Val Leu Leu Asp Ala Glu 435 440
445Arg Val Tyr Arg Glu Leu Ser Thr Ile Leu Glu Gly Glu Asp Asn Leu 450
455 460Asp Phe Ala Ser Thr Met Val Gln
Ala Leu Asn Leu Ile Leu Leu Thr465 470
475 480Ser Pro Glu Leu Ser Lys Leu Arg Glu Leu Leu Lys
Gly Ser Leu Val 485 490
495Asn Arg Glu Gly Lys Glu Leu Phe Val Ala Leu Tyr Thr Ser Trp Cys
500 505 510His Ser Pro Met Ala Ile
Ile Ser Leu Cys Leu Leu Ala Gln Ala Tyr 515 520
525Gln His Ala Ser Val Val Ile Gln Ser Leu Val Glu Glu Asp
Ile Asn 530 535 540Val Lys Phe Leu Val
Gln Leu Asp Lys Leu Ile Arg Leu Leu Glu Thr545 550
555 560Pro Ile Phe Thr Tyr Leu Arg Leu Gln Leu
Leu Glu Pro Gly Arg Tyr 565 570
575Thr Trp Leu Leu Lys Thr Leu Tyr Gly Leu Leu Met Leu Leu Pro Gln
580 585 590Gln Ser Ala Ala Phe
Lys Ile Leu Arg Thr Arg Leu Lys Thr Val Pro 595
600 605Thr Tyr Ser Phe Ser Thr Gly Asn Gln Ile Gly Arg
Ala Thr Ser Gly 610 615 620Val Pro Phe
Ser Gln Tyr Lys His Gln Asn Glu Asp Gly Asp Leu Glu625
630 635 640Asp Asp Asn Ile Asn Ser Ser
His Gln Gly Ile Asn Phe Ala Val Arg 645
650 655Leu Gln Gln Phe Glu Asn Val Gln Asn Leu His Arg
Gly Gln Ala Arg 660 665 670Thr
Arg Val Asn Tyr Ser Tyr His Ser Ser Ser Ser Ser Thr Ser Lys 675
680 685Glu Val Arg Arg Ser Glu Glu Gln Gln
Gln Gln Gln Gln Gln Gln Gln 690 695
700Gln Gln Gln Gln Gln Gln Gln Arg Pro Pro Pro Ser Ser Thr Ser Ser705
710 715 720Ser Val Ala Asp
Asn Asn Arg Pro Pro Ser Arg Thr Ser Arg Lys Gly 725
730 735Pro Gly Gln Leu Gln Leu
740131911PRTArabidopsis thaliana 131Met Ser Leu Leu Phe Leu Asn Pro Pro
Phe Pro Ser Asn Ser Ile His1 5 10
15Pro Ile Pro Arg Arg Ala Ala Gly Ile Ser Ser Ile Arg Cys Ser
Ile 20 25 30Ser Ala Pro Glu
Lys Lys Pro Arg Arg Arg Arg Lys Gln Lys Arg Gly 35
40 45Asp Gly Ala Glu Asn Asp Asp Ser Leu Ser Phe Gly
Ser Gly Glu Ala 50 55 60Val Ser Ala
Leu Glu Arg Ser Leu Arg Leu Thr Phe Met Asp Glu Leu65 70
75 80Met Glu Arg Ala Arg Asn Arg Asp
Thr Ser Gly Val Ser Glu Val Ile 85 90
95Tyr Asp Met Ile Ala Ala Gly Leu Ser Pro Gly Pro Arg Ser
Phe His 100 105 110Gly Leu Val
Val Ala His Ala Leu Asn Gly Asp Glu Gln Gly Ala Met 115
120 125His Ser Leu Arg Lys Glu Leu Gly Ala Gly Gln
Arg Pro Leu Pro Glu 130 135 140Thr Met
Ile Ala Leu Val Arg Leu Ser Gly Ser Lys Gly Asn Ala Thr145
150 155 160Arg Gly Leu Glu Ile Leu Ala
Ala Met Glu Lys Leu Lys Tyr Asp Ile 165
170 175Arg Gln Ala Trp Leu Ile Leu Val Glu Glu Leu Met
Arg Ile Asn His 180 185 190Leu
Glu Asp Ala Asn Lys Val Phe Leu Lys Gly Ala Arg Gly Gly Met 195
200 205Arg Ala Thr Asp Gln Leu Tyr Asp Leu
Met Ile Glu Glu Asp Cys Lys 210 215
220Ala Gly Asp His Ser Asn Ala Leu Asp Ile Ser Tyr Glu Met Glu Ala225
230 235 240Ala Gly Arg Met
Ala Thr Thr Phe His Phe Asn Cys Leu Leu Ser Val 245
250 255Gln Ala Thr Cys Gly Ile Pro Glu Val Ala
Tyr Ala Thr Phe Glu Asn 260 265
270Met Glu Tyr Gly Glu Gly Leu Phe Met Lys Pro Asp Thr Glu Thr Tyr
275 280 285Asn Trp Val Ile Gln Ala Tyr
Thr Arg Ala Glu Ser Tyr Asp Arg Val 290 295
300Gln Asp Val Ala Glu Leu Leu Gly Met Met Val Glu Asp His Lys
Arg305 310 315 320Val Gln
Pro Asn Val Lys Thr Tyr Ala Leu Leu Val Glu Cys Phe Thr
325 330 335Lys Tyr Cys Val Val Lys Glu
Ala Ile Arg His Phe Arg Ala Leu Lys 340 345
350Asn Phe Glu Gly Gly Thr Val Ile Leu His Asn Ala Gly Asn
Phe Glu 355 360 365Asp Pro Leu Ser
Leu Tyr Leu Arg Ala Leu Cys Arg Glu Gly Arg Ile 370
375 380Val Glu Leu Ile Asp Ala Leu Asp Ala Met Arg Lys
Asp Asn Gln Pro385 390 395
400Ile Pro Pro Arg Ala Met Ile Met Ser Arg Lys Tyr Arg Thr Leu Val
405 410 415Ser Ser Trp Ile Glu
Pro Leu Gln Glu Glu Ala Glu Leu Gly Tyr Glu 420
425 430Ile Asp Tyr Leu Ala Arg Tyr Ile Glu Glu Gly Gly
Leu Thr Gly Glu 435 440 445Arg Lys
Arg Trp Val Pro Arg Arg Gly Lys Thr Pro Leu Asp Pro Asp 450
455 460Ala Ser Gly Phe Ile Tyr Ser Asn Pro Ile Glu
Thr Ser Phe Lys Gln465 470 475
480Arg Cys Leu Glu Asp Trp Lys Val His His Arg Lys Leu Leu Arg Thr
485 490 495Leu Gln Ser Glu
Gly Leu Pro Val Leu Gly Asp Ala Ser Glu Ser Asp 500
505 510Tyr Met Arg Val Val Glu Arg Leu Arg Asn Ile
Ile Lys Gly Pro Ala 515 520 525Leu
Asn Leu Leu Lys Pro Lys Ala Ala Ser Lys Met Val Val Ser Glu 530
535 540Leu Lys Glu Glu Leu Glu Ala Gln Gly Leu
Pro Ile Asp Gly Thr Arg545 550 555
560Asn Val Leu Tyr Gln Arg Val Gln Lys Ala Arg Arg Ile Asn Lys
Ser 565 570 575Arg Gly Arg
Pro Leu Trp Val Pro Pro Ile Glu Glu Glu Glu Glu Glu 580
585 590Val Asp Glu Glu Val Asp Asp Leu Ile Cys
Arg Ile Lys Leu His Glu 595 600
605Gly Asp Thr Glu Phe Trp Lys Arg Arg Phe Leu Gly Glu Gly Leu Ile 610
615 620Glu Thr Ser Val Glu Ser Lys Glu
Thr Thr Glu Ser Val Val Thr Gly625 630
635 640Glu Ser Glu Lys Ala Ile Glu Asp Ile Ser Lys Glu
Ala Asp Asn Glu 645 650
655Glu Asp Asp Asp Glu Glu Glu Gln Glu Gly Asp Glu Asp Asp Asp Glu
660 665 670Asn Glu Glu Glu Glu Val
Val Val Pro Glu Thr Glu Asn Arg Ala Glu 675 680
685Gly Glu Asp Leu Val Lys Asn Lys Ala Ala Asp Ala Lys Lys
His Leu 690 695 700Gln Met Ile Gly Val
Gln Leu Leu Lys Glu Ser Asp Glu Ala Asn Arg705 710
715 720Thr Lys Lys Arg Gly Lys Arg Ala Ser Arg
Met Thr Leu Glu Asp Asp 725 730
735Ala Asp Glu Asp Trp Phe Pro Glu Glu Pro Phe Glu Ala Phe Lys Glu
740 745 750Met Arg Glu Arg Lys
Val Phe Asp Val Ala Asp Met Tyr Thr Ile Ala 755
760 765Asp Val Trp Gly Trp Thr Trp Glu Lys Asp Phe Lys
Asn Lys Thr Pro 770 775 780Arg Lys Trp
Ser Gln Glu Trp Glu Val Glu Leu Ala Ile Val Leu Met785
790 795 800Thr Lys Val Ile Glu Leu Gly
Gly Ile Pro Thr Ile Gly Asp Cys Ala 805
810 815Val Ile Leu Arg Ala Ala Leu Arg Ala Pro Met Pro
Ser Ala Phe Leu 820 825 830Lys
Ile Leu Gln Thr Thr His Ser Leu Gly Tyr Ser Phe Gly Ser Pro 835
840 845Leu Tyr Asp Glu Ile Ile Thr Leu Cys
Leu Asp Leu Gly Glu Leu Asp 850 855
860Ala Ala Ile Ala Ile Val Ala Asp Met Glu Thr Thr Gly Ile Thr Val865
870 875 880Pro Asp Gln Thr
Leu Asp Lys Val Ile Ser Ala Arg Gln Ser Asn Glu 885
890 895Ser Pro Arg Ser Glu Pro Glu Glu Pro Ala
Ser Thr Val Ser Ser 900 905
910132357PRTArabidopsis thaliana 132Met Glu Gly Ser Ser Ser Ala Ile Ala
Arg Lys Thr Trp Glu Leu Glu1 5 10
15Asn Asn Ile Leu Pro Val Glu Pro Thr Asp Ser Ala Ser Asp Ser
Ile 20 25 30Phe His Tyr Asp
Asp Ala Ser Gln Ala Lys Ile Gln Gln Glu Lys Pro 35
40 45Trp Ala Ser Asp Pro Asn Tyr Phe Lys Arg Val His
Ile Ser Ala Leu 50 55 60Ala Leu Leu
Lys Met Val Val His Ala Arg Ser Gly Gly Thr Ile Glu65 70
75 80Ile Met Gly Leu Met Gln Gly Lys
Thr Glu Gly Asp Thr Ile Ile Val 85 90
95Met Asp Ala Phe Ala Leu Pro Val Glu Gly Thr Glu Thr Arg
Val Asn 100 105 110Ala Gln Ser
Asp Ala Tyr Glu Tyr Met Val Glu Tyr Ser Gln Thr Ser 115
120 125Lys Leu Ala Gly Arg Leu Glu Asn Val Val Gly
Trp Tyr His Ser His 130 135 140Pro Gly
Tyr Gly Cys Trp Leu Ser Gly Ile Asp Val Ser Thr Gln Met145
150 155 160Leu Asn Gln Gln Tyr Gln Glu
Pro Phe Leu Ala Val Val Ile Asp Pro 165
170 175Thr Arg Thr Val Ser Ala Gly Lys Val Glu Ile Gly
Ala Phe Arg Thr 180 185 190Tyr
Pro Glu Gly His Lys Ile Ser Asp Asp His Val Ser Glu Tyr Gln 195
200 205Thr Ile Pro Leu Asn Lys Ile Glu Asp
Phe Gly Val His Cys Lys Gln 210 215
220Tyr Tyr Ser Leu Asp Ile Thr Tyr Phe Lys Ser Ser Leu Asp Ser His225
230 235 240Leu Leu Asp Leu
Leu Trp Asn Lys Tyr Trp Val Asn Thr Leu Ser Ser 245
250 255Ser Pro Leu Leu Gly Asn Gly Asp Tyr Val
Ala Gly Gln Ile Ser Asp 260 265
270Leu Ala Glu Lys Leu Glu Gln Ala Glu Ser Gln Leu Ala Asn Ser Arg
275 280 285Tyr Gly Gly Ile Ala Pro Ala
Gly His Gln Arg Arg Lys Glu Asp Glu 290 295
300Pro Gln Leu Ala Lys Ile Thr Arg Asp Ser Ala Lys Ile Thr Val
Glu305 310 315 320Gln Val
His Gly Leu Met Ser Gln Val Ile Lys Asp Ile Leu Phe Asn
325 330 335Ser Ala Arg Gln Ser Lys Lys
Ser Ala Asp Asp Ser Ser Asp Pro Glu 340 345
350Pro Met Ile Thr Ser 35513357DNAartificial
sequenceprimer 133ggggacaagt ttgtacaaaa aagcaggctt cacaatggtt agatcagatg
aaaatag 5713454DNAartificial sequenceprimer 134ggggaccact
ttgtacaaga aagctgggtt cttattaata ttaaatcaga aacc
5413552DNAartificial sequenceprimer 135ggggacaagt ttgtacaaaa aagcaggctt
cacaatggta aatccgggtc ac 5213651DNAartificial sequenceprimer
136ggggaccact ttgtacaaga aagctgggtt ttctgtagtc agacctggat a
5113755DNAartificial sequenceprimer 137ggggacaagt ttgtacaaaa aagcaggctt
cacaatgggg aaggaaaatg ctgtg 5513854DNAartificial sequenceprimer
138ggggaccact ttgtacaaga aagctgggtc cttcagaata gcgtgtcaag tagc
5413954DNAartificial sequenceprimer 139ggggacaagt ttgtacaaaa aagcaggctt
cacaatgggg aagaagtgtg attt 5414050DNAartificial sequenceprimer
140ggggaccact ttgtacaaga aagctgggtt gtgagttaaa caacaaccgt
5014155DNAartificial sequenceprimer 141ggggacaagt ttgtacaaaa aagcaggctt
cacaatggtt aactcatgcg agaac 5514252DNAartificial sequenceprimer
142ggggaccact ttgtacaaga aagctgggtt ggattaagaa tgatgagact ca
5214352DNAartificial sequenceprimer 143ggggacaagt ttgtacaaaa aagcaggctt
cacaatggcg aataatcctc cg 5214451DNAartificial sequenceprimer
144ggggaccact ttgtacaaga aagctgggtc actatcactc cccaacttct c
5114551DNAartificial sequenceprimer 145ggggacaagt ttgtacaaaa aagcaggctt
cacaatggag ggttcgtcgt c 5114649DNAartificial sequenceprimer
146ggggaccact ttgtacaaga aagctgggtc caaaagaaga gcaacttca
4914756DNAartificial sequenceprimer 147ggggacaagt ttgtacaaaa aagcaggctt
cacaatgtat tgctcttctt cgatgc 5614852DNAartificial sequenceprimer
148ggggaccact ttgtacaaga aagctgggtg cttggtgtca tcttgagaat ag
5214954DNAartificial sequenceprimer 149ggggacaagt ttgtacaaaa aagcaggctt
cacaatggca aagatgcaat tatc 5415050DNAartificial sequenceprimer
150ggggaccact ttgtacaaga aagctgggta accatctgat cacaagaaca
5015158DNAartificial sequenceprimer 151ggggacaagt ttgtacaaaa aagcaggctt
cacaatggct atttcaaaag ctcttatc 5815248DNAartificial sequenceprimer
152ggggaccact ttgtacaaga aagctgggtg aggctagcgt agcactgg
4815354DNAartificial sequenceprimer 153ggggacaagt ttgtacaaaa aagcaggctt
cacaatgggg aagaagaaca agag 5415451DNAartificial sequenceprimer
154ggggaccact ttgtacaaga aagctgggtg cttctttgac tctttttatc g
5115555DNAartificial sequenceprimer 155ggggacaagt ttgtacaaaa aagcaggctt
cacaatggaa ttgcttgaca tgaac 5515653DNAartificial sequenceprimer
156ggggaccact ttgtacaaga aagctgggtc aacattattc ttcttttctg gtc
5315754DNAartificial sequenceprimer 157ggggacaagt ttgtacaaaa aagcaggctt
cacaatggac gagggagtta tagc 5415850DNAartificial sequenceprimer
158ggggaccact ttgtacaaga aagctgggtc cttagagaga ggacttttct
5015955DNAartificial sequenceprimer 159ggggacaagt ttgtacaaaa aagcaggctt
cacaatggag ttgtttgtca ctcca 5516049DNAartificial sequenceprimer
160ggggaccact ttgtacaaga aagctgggtt cagcgagtat caatggatc
4916152DNAartificial sequenceprimer 161ggggacaagt ttgtacaaaa aagcaggctt
cacaatgcaa ccgacagaga cg 5216249DNAartificial sequenceprimer
162ggggaccact ttgtacaaga aagctgggtg ctcgtccaac actaaggtt
4916355DNAartificial sequenceprimer 163ggggacaagt ttgtacaaaa aagcaggctt
cacaatgaat agggaaaagt tgatg 5516447DNAartificial sequenceprimer
164ggggaccact ttgtacaaga aagctgggtc ctctaagaag cagcagc
4716552DNAartificial sequenceprimer 165ggggacaagt ttgtacaaaa aagcaggctt
cacaatggag gacgacgacg ag 5216650DNAartificial sequenceprimer
166ggggaccact ttgtacaaga aagctgggtt gtcagctact tacattgccg
5016751DNAartificial sequenceprimer 167ggggacaagt ttgtacaaaa aagcaggctt
cacaatggcc accgtatctt c 5116848DNAartificial sequenceprimer
168ggggaccact ttgtacaaga aagctgggtg attagaaaac tgaaggcg
4816956DNAartificial sequenceprimer 169ggggacaagt ttgtacaaaa aagcaggctt
cacaatggat gtaggagtta ctacgg 5617047DNAartificial sequenceprimer
170ggggaccact ttgtacaaga aagctgggtc taagccgagg cattgat
4717156DNAartificial sequenceprimer 171ggggacaagt ttgtacaaaa aagcaggctt
cacaatggat ggtcatgatt ctaagg 5617249DNAartificial sequenceprimer
172ggggaccact ttgtacaaga aagctgggtt taagaggaac tagccggtg
4917352DNAartificial sequenceprimer 173ggggacaagt ttgtacaaaa aagcaggctt
cacaatggaa atctacacca tg 5217451DNAartificial sequenceprimer
174ggggaccact ttgtacaaga aagctgggta actaaagaga actaagaaac t
5117553DNAartificial sequenceprimer 175ggggacaagt ttgtacaaaa aagcaggctt
cacaatggag tttggatctt ttc 5317647DNAartificial sequenceprimer
176ggggaccact ttgtacaaga aagctgggtc tctcaagctt taaacgc
4717754DNAartificial sequenceprimer 177ggggacaagt ttgtacaaaa aagcaggctt
cacaatggcg gagcagaaga gtac 5417854DNAartificial sequenceprimer
178ggggaccact ttgtacaaga aagctgggtc ctatcatgtc gattttgtat cttt
5417951DNAartificial sequenceprimer 179ggggacaagt ttgtacaaaa aagcaggctt
cacaatggcg aatccttggt g 5118048DNAartificial sequenceprimer
180ggggaccact ttgtacaaga aagctgggtt caatacgaag gaggagca
4818153DNAartificial sequenceprimer 181ggggacaagt ttgtacaaaa aagcaggctt
cacaatggct ctcaatctcc gtc 5318254DNAartificial sequenceprimer
182ggggaccact ttgtacaaga aagctgggtg gattagagag tcatatgttt gatg
5418352DNAartificial sequenceprimer 183ggggacaagt ttgtacaaaa aagcaggctt
cacaatgctg atgctgtgtg gg 5218452DNAartificial sequenceprimer
184ggggaccact ttgtacaaga aagctgggtt ttcaacaatg ttcaacaaca ct
5218554DNAartificial sequenceprimer 185ggggacaagt ttgtacaaaa aagcaggctt
cacaatggtg aagttgatga tacg 5418649DNAartificial sequenceprimer
186ggggaccact ttgtacaaga aagctgggtt ttagtgcaac caaagagtc
4918756DNAartificial sequenceprimer 187ggggacaagt ttgtacaaaa aagcaggctt
cacaatggag aaacagagta ctcaac 5618851DNAartificial sequenceprimer
188ggggaccact ttgtacaaga aagctgggtt tatgaaggtc tctttgtagc t
5118958DNAartificial sequenceprimer 189ggggacaagt ttgtacaaaa aagcaggctt
cacaatgaca actactgggt ctaattct 5819047DNAartificial sequenceprimer
190ggggaccact ttgtacaaga aagctgggtt caattctccg gcttcat
4719152DNAartificial sequenceprimer 191ggggacaagt ttgtacaaaa aagcaggctt
cacaatggcg ctgcagaaca tt 5219251DNAartificial sequenceprimer
192ggggaccact ttgtacaaga aagctgggtg caaagaaaag ttaggaggga a
5119353DNAartificial sequenceprimer 193ggggacaagt ttgtacaaaa aagcaggctt
cacaatggcg gctaacaaat tcg 5319449DNAartificial sequenceprimer
194ggggaccact ttgtacaaga aagctgggtg tcgttgttcc ttgcctcac
4919557DNAartificial sequenceprimer 195ggggacaagt ttgtacaaaa aagcaggctt
cacaatgtca ctcttgttcc tcaatcc 5719654DNAartificial sequenceprimer
196ggggaccact ttgtacaaga aagctgggtc cttctaccga tttctgtttc ttat
5419753DNAartificial sequenceprimer 197ggggacaagt ttgtacaaaa aagcaggctt
cacaatggaa ggttcctcgt cag 5319848DNAartificial sequenceprimer
198ggggaccact ttgtacaaga aagctgggtt cacgatgtaa tcatgggc
4819912PRTartificial sequencemotif 199Glu Glu Thr Ala Arg Phe Gln Pro Gly
Tyr Arg Ser1 5 1020010PRTartificial
sequencemotif 200Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu1 5
102017PRTartificial sequencemotif 201Asp Tyr Lys Asp
Asp Asp Lys1 52029PRTartificial sequencemotif 202Tyr Pro
Tyr Asp Val Pro Asp Tyr Ala1 520312PRTartificial
sequencemotif 203Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys1
5 1020411PRTartificial sequencemotif 204Tyr Thr
Asp Ile Glu Met Asn Arg Leu Gly Lys1 5
10205131PRTArabidopsis thaliana 205Asn Arg Ile Leu Trp Lys Gly Val Asp
Ala Cys Pro Gly Asp Glu Asp1 5 10
15Ala Asp Val Ser Val Leu Gln Leu Gln Ala Glu Ile Glu Asn Leu
Ala 20 25 30Leu Glu Glu Gln
Ala Leu Asp Asn Gln Ile Arg Gln Thr Glu Glu Arg 35
40 45Leu Arg Asp Leu Ser Glu Asn Glu Lys Asn Gln Lys
Trp Leu Phe Val 50 55 60Thr Glu Glu
Asp Ile Lys Ser Leu Pro Gly Phe Gln Asn Gln Thr Leu65 70
75 80Ile Ala Val Lys Ala Pro His Gly
Thr Thr Leu Glu Val Pro Asp Pro 85 90
95Asp Glu Ala Ala Asp His Pro Gln Arg Arg Tyr Arg Ile Ile
Leu Arg 100 105 110Ser Thr Met
Gly Pro Ile Asp Val Tyr Leu Val Ser Glu Phe Glu Gly 115
120 125Lys Phe Glu 130206385PRTArabidopsis
thaliana 206Met Ser Glu Glu Val Pro Gln Gln Phe Pro Ser Ser Lys Arg Gln
Leu1 5 10 15His Pro Ser
Leu Ser Ser Met Lys Pro Pro Leu Val Ala Pro Gly Glu 20
25 30Tyr His Arg Phe Asp Ala Ala Glu Thr Arg
Gly Gly Gly Ala Val Ala 35 40
45Asp Gln Val Val Ser Asp Ala Ile Val Ile Lys Ser Thr Leu Lys Arg 50
55 60Lys Thr Asp Leu Val Asn Gln Ile Val
Glu Val Asn Glu Leu Asn Thr65 70 75
80Gly Val Leu Gln Thr Pro Val Ser Gly Lys Gly Gly Lys Ala
Lys Lys 85 90 95Thr Ser
Arg Ser Ala Lys Ser Asn Lys Ser Gly Thr Leu Ala Ser Gly 100
105 110Ser Asn Ala Gly Ser Pro Gly Asn Asn
Phe Ala Gln Ala Gly Thr Cys 115 120
125Arg Tyr Asp Ser Ser Leu Gly Leu Leu Thr Lys Lys Phe Ile Asn Leu
130 135 140Ile Lys Gln Ala Glu Asp Gly
Ile Leu Asp Leu Asn Lys Ala Ala Asp145 150
155 160Thr Leu Glu Val Gln Lys Arg Arg Ile Tyr Asp Ile
Thr Asn Val Leu 165 170
175Glu Gly Ile Gly Leu Ile Glu Lys Thr Leu Lys Asn Arg Ile Gln Trp
180 185 190Lys Gly Leu Asp Val Ser
Lys Pro Gly Glu Thr Ile Glu Ser Ile Ala 195 200
205Asn Leu Gln Asp Glu Val Gln Asn Leu Ala Ala Glu Glu Ala
Arg Leu 210 215 220Asp Asp Gln Ile Arg
Glu Ser Gln Glu Arg Leu Thr Ser Leu Ser Glu225 230
235 240Asp Glu Asn Asn Lys Arg Leu Leu Phe Val
Thr Glu Asn Asp Ile Lys 245 250
255Asn Leu Pro Cys Phe Gln Asn Lys Thr Leu Ile Ala Val Lys Ala Pro
260 265 270His Gly Thr Thr Leu
Glu Val Pro Asp Pro Asp Glu Ala Gly Gly Tyr 275
280 285Gln Arg Arg Tyr Arg Ile Ile Leu Arg Ser Thr Met
Gly Pro Ile Asp 290 295 300Val Tyr Leu
Val Ser Gln Phe Glu Glu Ser Phe Glu Asp Ile Pro Gln305
310 315 320Ala Asp Glu Pro Ser Asn Val
Pro Asp Glu Pro Ser Asn Val Pro Asp 325
330 335Glu Pro Ser Asn Leu Pro Ser Thr Ser Gly Leu Pro
Glu Asn His Asp 340 345 350Val
Ser Met Pro Met Lys Glu Glu Ser Thr Glu Arg Asn Met Glu Thr 355
360 365Gln Glu Val Asp Asp Thr Gln Arg Val
Tyr Ser Asp Ile Glu Ser His 370 375
380Asp385207127PRTArabidopsis thaliana 207Met Ser Glu Glu Val Pro Gln Gln
Phe Pro Ser Ser Lys Arg Gln Leu1 5 10
15His Pro Ser Leu Ser Ser Met Lys Pro Pro Leu Val Ala Pro
Gly Glu 20 25 30Tyr His Arg
Phe Asp Ala Ala Glu Thr Arg Gly Gly Gly Ala Val Ala 35
40 45Asp Gln Val Val Ser Asp Ala Ile Val Ile Lys
Ser Thr Leu Lys Arg 50 55 60Lys Thr
Asp Leu Val Asn Gln Ile Val Glu Val Asn Glu Leu Asn Thr65
70 75 80Gly Val Leu Gln Thr Pro Val
Ser Gly Lys Gly Gly Lys Ala Lys Lys 85 90
95Thr Ser Arg Ser Ala Lys Ser Asn Lys Ser Gly Thr Leu
Ala Ser Gly 100 105 110Ser Asn
Ala Gly Ser Pro Gly Asn Asn Phe Ala Gln Ala Gly Thr 115
120 125208142PRTArabidopsis thaliana 208Met Ser Met
Glu Met Glu Leu Phe Val Thr Pro Glu Lys Gln Arg Gln1 5
10 15His Pro Ser Val Ser Val Glu Lys Thr
Pro Val Arg Arg Lys Leu Ile 20 25
30Val Asp Asp Asp Ser Glu Ile Gly Ser Glu Lys Lys Gly Gln Ser Arg
35 40 45Thr Ser Gly Gly Gly Leu Arg
Gln Phe Ser Val Met Val Cys Gln Lys 50 55
60Leu Glu Ala Lys Lys Ile Thr Thr Tyr Lys Glu Val Ala Asp Glu Ile65
70 75 80Ile Ser Asp Phe
Ala Thr Ile Lys Gln Asn Ala Glu Lys Pro Leu Asn 85
90 95Glu Asn Glu Tyr Asn Glu Lys Asn Ile Arg
Arg Arg Val Tyr Asp Ala 100 105
110Leu Asn Val Phe Met Ala Leu Asp Ile Ile Ala Arg Asp Lys Lys Glu
115 120 125Ile Arg Trp Lys Gly Leu Pro
Ile Thr Cys Lys Lys Asp Val 130 135
140209101PRTArabidopsis thaliana 209Glu Lys Lys Gly Gln Ser Arg Thr Ser
Gly Gly Gly Leu Arg Gln Phe1 5 10
15Ser Val Met Val Cys Gln Lys Leu Glu Ala Lys Lys Ile Thr Thr
Tyr 20 25 30Lys Glu Val Ala
Asp Glu Ile Ile Ser Asp Phe Ala Thr Ile Lys Gln 35
40 45Asn Ala Glu Lys Pro Leu Asn Glu Asn Glu Tyr Asn
Glu Lys Asn Ile 50 55 60Arg Arg Arg
Val Tyr Asp Ala Leu Asn Val Phe Met Ala Leu Asp Ile65 70
75 80Ile Ala Arg Asp Lys Lys Glu Ile
Arg Trp Lys Gly Leu Pro Ile Thr 85 90
95Cys Lys Lys Asp Val 100210251PRTArabidopsis
thaliana 210Glu Lys Lys Gly Gln Ser Arg Thr Ser Gly Gly Gly Leu Arg Gln
Phe1 5 10 15Ser Val Met
Val Cys Gln Lys Leu Glu Ala Lys Lys Ile Thr Thr Tyr 20
25 30Lys Glu Val Ala Asp Glu Ile Ile Ser Asp
Phe Ala Thr Ile Lys Gln 35 40
45Asn Ala Glu Lys Pro Leu Asn Glu Asn Glu Tyr Asn Glu Lys Asn Ile 50
55 60Arg Arg Arg Val Tyr Asp Ala Leu Asn
Val Phe Met Ala Leu Asp Ile65 70 75
80Ile Ala Arg Asp Lys Lys Glu Ile Arg Trp Lys Gly Leu Pro
Ile Thr 85 90 95Cys Lys
Lys Asp Val Glu Glu Val Lys Met Asp Arg Asn Lys Val Met 100
105 110Ser Ser Val Gln Lys Lys Ala Ala Phe
Leu Lys Glu Leu Arg Glu Lys 115 120
125Val Ser Ser Leu Glu Ser Leu Met Ser Arg Asn Gln Glu Met Val Val
130 135 140Lys Thr Gln Gly Pro Ala Glu
Gly Phe Thr Leu Pro Phe Ile Leu Leu145 150
155 160Glu Thr Asn Pro His Ala Val Val Glu Ile Glu Ile
Ser Glu Asp Met 165 170
175Gln Leu Val His Leu Asp Phe Asn Ser Thr Pro Phe Ser Val His Asp
180 185 190Asp Ala Tyr Ile Leu Lys
Leu Met Gln Glu Gln Lys Gln Glu Gln Asn 195 200
205Arg Val Ser Ser Ser Ser Ser Thr His His Gln Ser Gln His
Ser Ser 210 215 220Ala His Ser Ser Ser
Ser Ser Cys Ile Ala Ser Gly Thr Ser Gly Pro225 230
235 240Val Cys Trp Asn Ser Gly Ser Ile Asp Thr
Arg 245 250211172PRTArabidopsis thaliana
211Ile Ile Ala Arg Asp Lys Lys Glu Ile Arg Trp Lys Gly Leu Pro Ile1
5 10 15Thr Cys Lys Lys Asp Val
Glu Glu Val Lys Met Asp Arg Asn Lys Val 20 25
30Met Ser Ser Val Gln Lys Lys Ala Ala Phe Leu Lys Glu
Leu Arg Glu 35 40 45Lys Val Ser
Ser Leu Glu Ser Leu Met Ser Arg Asn Gln Glu Met Val 50
55 60Val Lys Thr Gln Gly Pro Ala Glu Gly Phe Thr Leu
Pro Phe Ile Leu65 70 75
80Leu Glu Thr Asn Pro His Ala Val Val Glu Ile Glu Ile Ser Glu Asp
85 90 95Met Gln Leu Val His Leu
Asp Phe Asn Ser Thr Pro Phe Ser Val His 100
105 110Asp Asp Ala Tyr Ile Leu Lys Leu Met Gln Glu Gln
Lys Gln Glu Gln 115 120 125Asn Arg
Val Ser Ser Ser Ser Ser Thr His His Gln Ser Gln His Ser 130
135 140Ser Ala His Ser Ser Ser Ser Ser Cys Ile Ala
Ser Gly Thr Ser Gly145 150 155
160Pro Val Cys Trp Asn Ser Gly Ser Ile Asp Thr Arg
165 17021293PRTArabidopsis thaliana 212Ile Ile Ala Arg
Asp Lys Lys Glu Ile Arg Trp Lys Gly Leu Pro Ile1 5
10 15Thr Cys Lys Lys Asp Val Glu Glu Val Lys
Met Asp Arg Asn Lys Val 20 25
30Met Ser Ser Val Gln Lys Lys Ala Ala Phe Leu Lys Glu Leu Arg Glu
35 40 45Lys Val Ser Ser Leu Glu Ser Leu
Met Ser Arg Asn Gln Glu Met Val 50 55
60Val Lys Thr Gln Gly Pro Ala Glu Gly Phe Thr Leu Pro Phe Ile Leu65
70 75 80Leu Glu Thr Asn Pro
His Ala Val Val Glu Ile Glu Ile 85
90213121PRTArabidopsis thaliana 213Ser Leu Glu Ser Leu Met Ser Arg Asn
Gln Glu Met Val Val Lys Thr1 5 10
15Gln Gly Pro Ala Glu Gly Phe Thr Leu Pro Phe Ile Leu Leu Glu
Thr 20 25 30Asn Pro His Ala
Val Val Glu Ile Glu Ile Ser Glu Asp Met Gln Leu 35
40 45Val His Leu Asp Phe Asn Ser Thr Pro Phe Ser Val
His Asp Asp Ala 50 55 60Tyr Ile Leu
Lys Leu Met Gln Glu Gln Lys Gln Glu Gln Asn Arg Val65 70
75 80Ser Ser Ser Ser Ser Thr His His
Gln Ser Gln His Ser Ser Ala His 85 90
95Ser Ser Ser Ser Ser Cys Ile Ala Ser Gly Thr Ser Gly Pro
Val Cys 100 105 110Trp Asn Ser
Gly Ser Ile Asp Thr Arg 115
120214193PRTArabidopsis thaliana 214Met Thr Thr Thr Gly Ser Asn Ser Asn
His Asn His His Glu Ser Asn1 5 10
15Asn Asn Asn Asn Asn Pro Ser Thr Arg Ser Trp Gly Thr Ala Val
Ser 20 25 30Gly Gln Ser Val
Ser Thr Ser Gly Ser Met Gly Ser Pro Ser Ser Arg 35
40 45Ser Glu Gln Thr Ile Thr Val Val Thr Ser Thr Ser
Asp Thr Thr Phe 50 55 60Gln Arg Leu
Asn Asn Leu Asp Ile Gln Gly Asp Asp Ala Gly Ser Gln65 70
75 80Gly Ala Ser Gly Val Lys Lys Lys
Lys Arg Gly Gln Arg Ala Ala Gly 85 90
95Pro Asp Lys Thr Gly Arg Gly Leu Arg Gln Phe Ser Met Lys
Val Cys 100 105 110Glu Lys Val
Glu Ser Lys Gly Arg Thr Thr Tyr Asn Glu Val Ala Asp 115
120 125Glu Leu Val Ala Glu Phe Ala Leu Pro Asn Asn
Asp Gly Thr Ser Pro 130 135 140Asp Gln
Gln Gln Tyr Asp Glu Lys Asn Ile Arg Arg Arg Val Tyr Asp145
150 155 160Ala Leu Asn Val Leu Met Ala
Met Asp Ile Ile Ser Lys Asp Lys Lys 165
170 175Glu Ile Gln Trp Arg Gly Leu Pro Arg Thr Ser Leu
Ser Asp Ile Glu 180 185
190Glu21581PRTArabidopsis thaliana 215Gly Leu Pro Arg Thr Ser Leu Ser Asp
Ile Glu Glu Leu Lys Asn Glu1 5 10
15Arg Leu Ser Leu Arg Asn Arg Ile Glu Lys Lys Thr Ala Tyr Ser
Gln 20 25 30Glu Leu Glu Glu
Gln Tyr Val Gly Leu Gln Asn Leu Ile Gln Arg Asn 35
40 45Glu His Leu Tyr Ser Ser Gly Asn Ala Pro Ser Gly
Gly Val Ala Leu 50 55 60Pro Phe Ile
Leu Val Gln Thr Arg Pro His Ala Thr Val Glu Val Glu65 70
75 80Ile216204PRTArabidopsis thaliana
216Gly Leu Pro Arg Thr Ser Leu Ser Asp Ile Glu Glu Leu Lys Asn Glu1
5 10 15Arg Leu Ser Leu Arg Asn
Arg Ile Glu Lys Lys Thr Ala Tyr Ser Gln 20 25
30Glu Leu Glu Glu Gln Tyr Val Gly Leu Gln Asn Leu Ile
Gln Arg Asn 35 40 45Glu His Leu
Tyr Ser Ser Gly Asn Ala Pro Ser Gly Gly Val Ala Leu 50
55 60Pro Phe Ile Leu Val Gln Thr Arg Pro His Ala Thr
Val Glu Val Glu65 70 75
80Ile Ser Glu Asp Met Gln Leu Val His Phe Asp Phe Asn Ser Thr Pro
85 90 95Phe Glu Leu His Asp Asp
Asn Phe Val Leu Lys Thr Met Lys Phe Cys 100
105 110Asp Gln Pro Pro Gln Gln Pro Asn Gly Arg Asn Asn
Ser Gln Leu Val 115 120 125Cys His
Asn Phe Thr Pro Glu Asn Pro Asn Lys Gly Pro Ser Thr Gly 130
135 140Pro Thr Pro Gln Leu Asp Met Tyr Glu Thr His
Leu Gln Ser Gln Gln145 150 155
160His Gln Gln His Ser Gln Leu Gln Ile Ile Pro Met Pro Glu Thr Asn
165 170 175Asn Val Thr Ser
Ser Ala Asp Thr Ala Pro Val Lys Ser Pro Ser Leu 180
185 190Pro Gly Ile Met Asn Ser Ser Met Lys Pro Glu
Asn 195 200217420PRTArabidopsis thaliana 217Met
Ser Gly Val Val Arg Ser Ser Pro Gly Ser Ser Gln Pro Pro Pro1
5 10 15Pro Pro Pro His His Pro Pro
Ser Ser Pro Val Pro Val Thr Ser Thr 20 25
30Pro Val Ile Pro Pro Ile Arg Arg His Leu Ala Phe Ala Ser
Thr Lys 35 40 45Pro Pro Phe His
Pro Ser Asp Asp Tyr His Arg Phe Asn Pro Ser Ser 50 55
60Leu Ser Asn Asn Asn Asp Arg Ser Phe Val His Gly Cys
Gly Val Val65 70 75
80Asp Arg Glu Glu Asp Ala Val Val Val Arg Ser Pro Ser Arg Lys Arg
85 90 95Lys Ala Thr Met Asp Met
Val Val Ala Pro Ser Asn Asn Gly Phe Thr 100
105 110Ser Ser Gly Phe Thr Asn Ile Pro Ser Ser Pro Cys
Gln Thr Pro Arg 115 120 125Lys Gly
Gly Arg Val Asn Ile Lys Ser Lys Ala Lys Gly Asn Lys Ser 130
135 140Thr Pro Gln Thr Pro Ile Ser Thr Asn Ala Gly
Ser Pro Ile Thr Leu145 150 155
160Thr Pro Ser Gly Ser Cys Arg Tyr Asp Ser Ser Leu Gly Leu Leu Thr
165 170 175Lys Lys Phe Val
Asn Leu Ile Lys Gln Ala Lys Asp Gly Met Leu Asp 180
185 190Leu Asn Lys Ala Ala Glu Thr Leu Glu Val Gln
Lys Arg Arg Ile Tyr 195 200 205Asp
Ile Thr Asn Val Leu Glu Gly Ile Asp Leu Ile Glu Lys Pro Phe 210
215 220Lys Asn Arg Ile Leu Trp Lys Gly Val Asp
Ala Cys Pro Gly Asp Glu225 230 235
240Asp Ala Asp Val Ser Val Leu Gln Leu Gln Ala Glu Ile Glu Asn
Leu 245 250 255Ala Leu Glu
Glu Gln Ala Leu Asp Asn Gln Ile Arg Gln Thr Glu Glu 260
265 270Arg Leu Arg Asp Leu Ser Glu Asn Glu Lys
Asn Gln Lys Trp Leu Phe 275 280
285Val Thr Glu Glu Asp Ile Lys Ser Leu Pro Gly Phe Gln Asn Gln Thr 290
295 300Leu Ile Ala Val Lys Ala Pro His
Gly Thr Thr Leu Glu Val Pro Asp305 310
315 320Pro Asp Glu Ala Ala Asp His Pro Gln Arg Arg Tyr
Arg Ile Ile Leu 325 330
335Arg Ser Thr Met Gly Pro Ile Asp Val Tyr Leu Val Ser Glu Phe Glu
340 345 350Gly Lys Phe Glu Asp Thr
Asn Gly Ser Gly Ala Ala Pro Pro Ala Cys 355 360
365Leu Pro Ile Ala Ser Ser Ser Gly Ser Thr Gly His His Asp
Ile Glu 370 375 380Ala Leu Thr Val Asp
Asn Pro Glu Thr Ala Ile Val Ser His Asp His385 390
395 400Pro His Pro Gln Pro Gly Asp Thr Ser Asp
Leu Asn Tyr Leu Gln Glu 405 410
415Gln Val Gly Gly 420218324PRTArabidopsis thaliana
218Pro Ser Gly Ser Cys Arg Tyr Asp Ser Ser Leu Gly Leu Leu Thr Lys1
5 10 15Lys Phe Val Asn Leu Ile
Lys Gln Ala Lys Asp Gly Met Leu Asp Leu 20 25
30Asn Lys Ala Ala Glu Thr Leu Glu Val Gln Lys Arg Arg
Ile Tyr Asp 35 40 45Ile Thr Asn
Val Leu Glu Gly Ile Asp Leu Ile Glu Lys Pro Phe Lys 50
55 60Asn Arg Ile Leu Trp Lys Gly Val Asp Ala Cys Pro
Gly Asp Glu Asp65 70 75
80Ala Asp Val Ser Val Leu Gln Leu Gln Ala Glu Ile Glu Asn Leu Ala
85 90 95Leu Glu Glu Gln Ala Leu
Asp Asn Gln Ile Arg Gln Thr Glu Glu Arg 100
105 110Leu Arg Asp Leu Ser Glu Asn Glu Lys Asn Gln Lys
Trp Leu Phe Val 115 120 125Thr Glu
Glu Asp Ile Lys Ser Leu Pro Gly Phe Gln Asn Gln Thr Leu 130
135 140Ile Ala Val Lys Ala Pro His Gly Thr Thr Leu
Glu Val Pro Asp Pro145 150 155
160Asp Glu Ala Ala Asp His Pro Gln Arg Arg Tyr Arg Ile Ile Leu Arg
165 170 175Ser Thr Met Gly
Pro Ile Asp Val Tyr Leu Val Ser Glu Phe Glu Gly 180
185 190Lys Phe Glu Asp Thr Asn Gly Ser Gly Ala Ala
Pro Pro Ala Cys Leu 195 200 205Pro
Ile Ala Ser Ser Ser Gly Ser Thr Gly His His Asp Ile Glu Ala 210
215 220Leu Thr Val Asp Asn Pro Glu Thr Ala Ile
Val Ser His Asp His Pro225 230 235
240His Pro Gln Pro Gly Asp Thr Ser Asp Leu Asn Tyr Leu Gln Glu
Gln 245 250 255Val Gly Gly
Met Leu Lys Ile Thr Pro Ser Asp Val Glu Asn Asp Glu 260
265 270Ser Asp Tyr Trp Leu Leu Ser Asn Ala Glu
Ile Ser Met Thr Asp Ile 275 280
285Trp Lys Thr Asp Ser Gly Ile Asp Trp Asp Tyr Gly Ile Ala Asp Val 290
295 300Ser Thr Pro Pro Pro Gly Met Gly
Glu Ile Ala Pro Thr Ala Val Asp305 310
315 320Ser Thr Pro Arg21938PRTArabidopsis thaliana 219Met
Ser Gly Val Val Arg Ser Ser Pro Gly Ser Ser Gln Pro Pro Pro1
5 10 15Pro Pro Pro His His Pro Pro
Ser Ser Pro Val Pro Val Thr Ser Thr 20 25
30Pro Val Ile Pro Pro Ile 35220142PRTArabidopsis
thaliana 220Met Ser Met Glu Met Glu Leu Phe Val Thr Pro Glu Lys Gln Arg
Gln1 5 10 15His Pro Ser
Val Ser Val Glu Lys Thr Pro Val Arg Arg Lys Leu Ile 20
25 30Val Asp Asp Asp Ser Glu Ile Gly Ser Glu
Lys Lys Gly Gln Ser Arg 35 40
45Thr Ser Gly Gly Gly Leu Arg Gln Phe Ser Val Met Val Cys Gln Lys 50
55 60Leu Glu Ala Lys Lys Ile Thr Thr Tyr
Lys Glu Val Ala Asp Glu Ile65 70 75
80Ile Ser Asp Phe Ala Thr Ile Lys Gln Asn Ala Glu Lys Pro
Leu Asn 85 90 95Glu Asn
Glu Tyr Asn Glu Lys Asn Ile Arg Arg Arg Val Tyr Asp Ala 100
105 110Leu Asn Val Phe Met Ala Leu Asp Ile
Ile Ala Arg Asp Lys Lys Glu 115 120
125Ile Arg Trp Lys Gly Leu Pro Ile Thr Cys Lys Lys Asp Val 130
135 140221150PRTArabidopsis thaliana 221Glu Glu
Val Lys Met Asp Arg Asn Lys Val Met Ser Ser Val Gln Lys1 5
10 15Lys Ala Ala Phe Leu Lys Glu Leu
Arg Glu Lys Val Ser Ser Leu Glu 20 25
30Ser Leu Met Ser Arg Asn Gln Glu Met Val Val Lys Thr Gln Gly
Pro 35 40 45Ala Glu Gly Phe Thr
Leu Pro Phe Ile Leu Leu Glu Thr Asn Pro His 50 55
60Ala Val Val Glu Ile Glu Ile Ser Glu Asp Met Gln Leu Val
His Leu65 70 75 80Asp
Phe Asn Ser Thr Pro Phe Ser Val His Asp Asp Ala Tyr Ile Leu
85 90 95Lys Leu Met Gln Glu Gln Lys
Gln Glu Gln Asn Arg Val Ser Ser Ser 100 105
110Ser Ser Thr His His Gln Ser Gln His Ser Ser Ala His Ser
Ser Ser 115 120 125Ser Ser Cys Ile
Ala Ser Gly Thr Ser Gly Pro Val Cys Trp Asn Ser 130
135 140Gly Ser Ile Asp Thr Arg145
15022271PRTArabidopsis thaliana 222Glu Glu Val Lys Met Asp Arg Asn Lys
Val Met Ser Ser Val Gln Lys1 5 10
15Lys Ala Ala Phe Leu Lys Glu Leu Arg Glu Lys Val Ser Ser Leu
Glu 20 25 30Ser Leu Met Ser
Arg Asn Gln Glu Met Val Val Lys Thr Gln Gly Pro 35
40 45Ala Glu Gly Phe Thr Leu Pro Phe Ile Leu Leu Glu
Thr Asn Pro His 50 55 60Ala Val Val
Glu Ile Glu Ile65 70223262PRTArabidopsis thaliana 223Met
Thr Thr Thr Gly Ser Asn Ser Asn His Asn His His Glu Ser Asn1
5 10 15Asn Asn Asn Asn Asn Pro Ser
Thr Arg Ser Trp Gly Thr Ala Val Ser 20 25
30Gly Gln Ser Val Ser Thr Ser Gly Ser Met Gly Ser Pro Ser
Ser Arg 35 40 45Ser Glu Gln Thr
Ile Thr Val Val Thr Ser Thr Ser Asp Thr Thr Phe 50 55
60Gln Arg Leu Asn Asn Leu Asp Ile Gln Gly Asp Asp Ala
Gly Ser Gln65 70 75
80Gly Ala Ser Gly Val Lys Lys Lys Lys Arg Gly Gln Arg Ala Ala Gly
85 90 95Pro Asp Lys Thr Gly Arg
Gly Leu Arg Gln Phe Ser Met Lys Val Cys 100
105 110Glu Lys Val Glu Ser Lys Gly Arg Thr Thr Tyr Asn
Glu Val Ala Asp 115 120 125Glu Leu
Val Ala Glu Phe Ala Leu Pro Asn Asn Asp Gly Thr Ser Pro 130
135 140Asp Gln Gln Gln Tyr Asp Glu Lys Asn Ile Arg
Arg Arg Val Tyr Asp145 150 155
160Ala Leu Asn Val Leu Met Ala Met Asp Ile Ile Ser Lys Asp Lys Lys
165 170 175Glu Ile Gln Trp
Arg Gly Leu Pro Arg Thr Ser Leu Ser Asp Ile Glu 180
185 190Glu Leu Lys Asn Glu Arg Leu Ser Leu Arg Asn
Arg Ile Glu Lys Lys 195 200 205Thr
Ala Tyr Ser Gln Glu Leu Glu Glu Gln Tyr Val Gly Leu Gln Asn 210
215 220Leu Ile Gln Arg Asn Glu His Leu Tyr Ser
Ser Gly Asn Ala Pro Ser225 230 235
240Gly Gly Val Ala Leu Pro Phe Ile Leu Val Gln Thr Arg Pro His
Ala 245 250 255Thr Val Glu
Val Glu Ile 26022451PRTArabidopsis thaliana 224Gly Val Asp Ala
Cys Pro Gly Asp Glu Asp Ala Asp Val Ser Val Leu1 5
10 15Gln Leu Gln Ala Glu Ile Glu Asn Leu Ala
Leu Glu Glu Gln Ala Leu 20 25
30Asp Asn Gln Ile Arg Gln Thr Glu Glu Arg Leu Arg Asp Leu Ser Glu
35 40 45Asn Glu Lys
50225121PRTArabidopsis thaliana 225Gly Val Asp Ala Cys Pro Gly Asp Glu
Asp Ala Asp Val Ser Val Leu1 5 10
15Gln Leu Gln Ala Glu Ile Glu Asn Leu Ala Leu Glu Glu Gln Ala
Leu 20 25 30Asp Asn Gln Ile
Arg Gln Thr Glu Glu Arg Leu Arg Asp Leu Ser Glu 35
40 45Asn Glu Lys Asn Gln Lys Trp Leu Phe Val Thr Glu
Glu Asp Ile Lys 50 55 60Ser Leu Pro
Gly Phe Gln Asn Gln Thr Leu Ile Ala Val Lys Ala Pro65 70
75 80His Gly Thr Thr Leu Glu Val Pro
Asp Pro Asp Glu Ala Ala Asp His 85 90
95Pro Gln Arg Arg Tyr Arg Ile Ile Leu Arg Ser Thr Met Gly
Pro Ile 100 105 110Asp Val Tyr
Leu Val Ser Glu Phe Glu 115 12022650PRTArabidopsis
thaliana 226Gly Leu Asp Val Ser Lys Pro Gly Glu Thr Ile Glu Ser Ile Ala
Asn1 5 10 15Leu Gln Asp
Glu Val Gln Asn Leu Ala Ala Glu Glu Ala Arg Leu Asp 20
25 30Asp Gln Ile Arg Glu Ser Gln Glu Arg Leu
Thr Ser Leu Ser Glu Asp 35 40
45Glu Asn 50227118PRTArabidopsis thaliana 227Gly Leu Asp Val Ser Lys
Pro Gly Glu Thr Ile Glu Ser Ile Ala Asn1 5
10 15Leu Gln Asp Glu Val Gln Asn Leu Ala Ala Glu Glu
Ala Arg Leu Asp 20 25 30Asp
Gln Ile Arg Glu Ser Gln Glu Arg Leu Thr Ser Leu Ser Glu Asp 35
40 45Glu Asn Asn Lys Arg Leu Leu Phe Val
Thr Glu Asn Asp Ile Lys Asn 50 55
60Leu Pro Cys Phe Gln Asn Lys Thr Leu Ile Ala Val Lys Ala Pro His65
70 75 80Gly Thr Thr Leu Glu
Val Pro Asp Pro Asp Glu Ala Gly Gly Tyr Gln 85
90 95Arg Arg Tyr Arg Ile Ile Leu Arg Ser Thr Met
Gly Pro Ile Asp Val 100 105
110Tyr Leu Val Ser Gln Phe 115228393DNAArabidopsis thaliana
228aatcgaatac tttggaaggg agttgatgcg tgtcctggcg atgaggatgc tgacgtatct
60gtattacagc tgcaggcaga aattgaaaac ctcgccctcg aagagcaagc attagacaac
120caaatcagac aaacagagga aagattaaga gacctgagcg aaaatgaaaa gaatcagaaa
180tggctttttg taactgaaga ggatatcaag agtttaccag gtttccagaa ccagactctg
240atagccgtca aagctcctca tggcacaact ttggaagtgc ctgatccaga tgaagcggct
300gaccacccac aaaggagata caggatcatt cttagaagta caatgggacc tattgacgta
360tacctcgtca gcgaatttga agggaaattc gaa
393229516DNAArabidopsis thaliana 229attattgcaa gggataaaaa ggaaatccgg
tggaaaggac ttcctattac ctgcaaaaag 60gatgtggaag aagtcaagat ggatcgtaat
aaagttatga gcagtgtgca aaagaaggct 120gcttttctta aagagttgag agaaaaggtc
tcaagtcttg agagtcttat gtcgagaaat 180caagagatgg ttgtgaagac tcaaggccca
gcagaaggat ttaccttacc attcattcta 240cttgagacaa accctcacgc agtagtcgaa
atcgagattt ctgaagatat gcaacttgta 300cacctcgact tcaatagcac acctttctcg
gtccatgatg atgcttacat tttgaaactg 360atgcaagaac agaagcaaga acagaacaga
gtatcttctt cttcatctac acatcaccaa 420tctcaacata gctccgctca ttcttcatcc
agttcttgca ttgcttctgg aacctcaggc 480ccggtttgct ggaactcggg atccattgat
actcgc 516230276DNAArabidopsis thaliana
230ggtcttcctc ggacaagctt aagcgacatt gaagaattaa agaacgaacg actctcactt
60aggaacagaa ttgagaagaa aactgcatat tcccaagaac tggaagaaca agtaatgaac
120atcatcgata ctctcggctt atctgcttcc tgccttcaga atctgataca gagaaatgag
180cacttatata gctcaggaaa tgctcccagt ggcggtgttg ctcttccttt tatccttgtc
240cagactcgtc ctcacgcaac agtagaagtg gagata
276231645DNAArabidopsis thaliana 231ggtcttcctc ggacaagctt aagcgacatt
gaagaattaa agaacgaacg actctcactt 60aggaacagaa ttgagaagaa aactgcatat
tcccaagaac tggaagaaca agtaatgaac 120atcatcgata ctctcggctt atctgcttcc
tgccttcaga atctgataca gagaaatgag 180cacttatata gctcaggaaa tgctcccagt
ggcggtgttg ctcttccttt tatccttgtc 240cagactcgtc ctcacgcaac agtagaagtg
gagatatcag aagatatgca gctcgtgcat 300tttgatttca acagcactcc atttgagctc
cacgacgaca attttgtcct caagactatg 360aagttttgtg atcaaccgcc gcaacaacca
aacggtcgga acaacagcca gctggtttgt 420cacaatttca cgccagaaaa ccctaacaaa
ggccccagca caggtccaac accgcagctg 480gatatgtacg agactcatct tcaatcgcaa
caacatcagc agcattctca gctacaaatc 540attcctatgc ctgagactaa caacgttact
tccagcgctg atactgctcc agtgaaatcc 600ccgtctcttc cagggataat gaactccagc
atgaagccgg agaat 645232450DNAArabidopsis thaliana
232gaagaagtca agatggatcg taataaagtt atgagcagtg tgcaaaagaa ggctgctttt
60cttaaagagt tgagagaaaa ggtctcaagt cttgagagtc ttatgtcgag aaatcaagag
120atggttgtga agactcaagg cccagcagaa ggatttacct taccattcat tctacttgag
180acaaaccctc acgcagtagt cgaaatcgag atttctgaag atatgcaact tgtacacctc
240gacttcaata gcacaccttt ctcggtccat gatgatgctt acattttgaa actgatgcaa
300gaacagaagc aagaacagaa cagagtatct tcttcttcat ctacacatca ccaatctcaa
360catagctccg ctcattcttc atccagttct tgcattgctt ctggaacctc aggcccggtt
420tgctggaact cgggatccat tgatactcgc
450233213DNAArabidopsis thaliana 233gaagaagtca agatggatcg taataaagtt
atgagcagtg tgcaaaagaa ggctgctttt 60cttaaagagt tgagagaaaa ggtctcaagt
cttgagagtc ttatgtcgag aaatcaagag 120atggttgtga agactcaagg cccagcagaa
ggatttacct taccattcat tctacttgag 180acaaaccctc acgcagtagt cgaaatcgag
att 213234870DNAArabidopsis thaliana
234atgacaacta ctgggtctaa ttctaatcac aaccaccatg aaagcaataa taacaacaat
60aaccctagta ctaggtcttg gggcacggcg gtttcaggtc aatctgtgtc tactagcggc
120agtatgggct ctccgtcgag ccggagtgag caaaccatca ccgttgttac atctactagc
180gacactactt ttcaacgcct gaataatttg gacattcaag gtgatgatgc tggttctcaa
240ggagcttctg gtgttaagaa gaagaagagg ggacagcgtg cggctggtcc agataagact
300ggaagaggac tacgtcaatt tagtatgaaa ggtcttatct ctttctctgc ccctattatg
360ctttcatcta aatgcctttc aatttgtgaa aaggtggaaa gcaaaggaag gacaacttac
420aatgaggttg cagacgagct tgttgctgaa tttgcacttc caaataacga tggaacatcc
480cctgatcagc aacagtatga tgagaaaaac ataagacgaa gagtatatga tgctttaaac
540gtcctcatgg ctatggatat aatatccaag gataaaaaag aaattcaatg gagaggtctt
600cctcggacaa gcttaagcga cattgaagaa ttaaagaacg aacgactctc acttaggaac
660agaattgaga agaaaactgc atattcccaa gaactggaag aacaagtaat gaacatcatc
720gatactctcg gcttatctgc ttcctgcctt cagaatctga tacagagaaa tgagcactta
780tatagctcag gaaatgctcc cagtggcggt gttgctcttc cttttatcct tgtccagact
840cgtcctcacg caacagtaga agtggagata
870235153DNAArabidopsis thaliana 235ggagttgatg cgtgtcctgg cgatgaggat
gctgacgtat ctgtattaca gctgcaggca 60gaaattgaaa acctcgccct cgaagagcaa
gcattagaca accaaatcag acaaacagag 120gaaagattaa gagacctgag cgaaaatgaa
aag 153236363DNAArabidopsis thaliana
236ggagttgatg cgtgtcctgg cgatgaggat gctgacgtat ctgtattaca gctgcaggca
60gaaattgaaa acctcgccct cgaagagcaa gcattagaca accaaatcag acaaacagag
120gaaagattaa gagacctgag cgaaaatgaa aagaatcaga aatggctttt tgtaactgaa
180gaggatatca agagtttacc aggtttccag aaccagactc tgatagccgt caaagctcct
240catggcacaa ctttggaagt gcctgatcca gatgaagcgg ctgaccaccc acaaaggaga
300tacaggatca ttcttagaag tacaatggga cctattgacg tatacctcgt cagcgaattt
360gaa
363237150DNAArabidopsis thaliana 237ggtctcgatg tctcaaaacc aggagaaaca
atcgaaagca tagctaacct acaggatgaa 60gtacaaaacc tcgcagctga ggaggcaaga
ttagatgacc aaatcagaga atcacaagaa 120agattaacaa gcttgagtga ggatgaaaac
150238354DNAArabidopsis thaliana
238ggtctcgatg tctcaaaacc aggagaaaca atcgaaagca tagctaacct acaggatgaa
60gtacaaaacc tcgcagctga ggaggcaaga ttagatgacc aaatcagaga atcacaagaa
120agattaacaa gcttgagtga ggatgaaaac aacaaaaggt tactgttcgt cactgaaaac
180gacattaaga acctaccatg cttccagaat aagacgctga tagctgtaaa ggcaccgcat
240ggaacaactc ttgaggttcc agatcctgat gaggctggtg gttatcagag gaggtacaga
300atcattctga gaagcacaat gggaccaata gacgtgtacc tagtcagtca attc
354239426DNAArabidopsis thaliana 239atgagtatgg agatggagtt gtttgtcact
ccagagaagc agaggcaaca tccttcagtg 60agcgttgaga aaactccagt gagaaggaaa
ttgattgttg atgatgattc tgaaattgga 120tcagagaaga aagggcaatc aagaacttct
ggaggcgggc ttcgtcaatt cagtgttatg 180gtttgtcaga agttggaagc caagaagata
actacttaca aggaggttgc agacgaaatt 240atttcagatt ttgccacaat taagcaaaac
gcagagaagc ctttgaatga aaatgagtac 300aatgagaaga acataaggcg gagagtctac
gatgcgctca atgtgttcat ggcgttggat 360attattgcaa gggataaaaa ggaaatccgg
tggaaaggac ttcctattac ctgcaaaaag 420gatgtg
4262407PRTartificial sequencemotif
240Met Lys Val Cys Glu Lys Val1 52418PRTartificial
sequencemotif 241Leu Asn Val Leu Met Ala Met Asp1
52428PRTartificial sequencemotif 242Phe Asn Ser Thr Pro Phe Glu Leu1
524330DNAartificial sequenceprimer 243atagaattca tgaaagtttg
tgaaaaggtg 3024433DNAartificial
sequenceprimer 244atagaattcc tgaatgttct catggcaatg gat
3324533DNAartificial sequenceprimer 245ataggatccc
agctcaaaag gagtgctatt gaa
3324629DNAartificial sequencemotif 246ggggacaagt ttgtacaaaa aagcaggct
292475DNAartificial sequencemotif
247tcaca
524829DNAartificial sequencemotif 248ggggaccact ttgtacaaga aagctgggt
2924927DNAartificial sequenceprimer
249atagaattca tgtccggtgt cgtacga
2725030DNAartificial sequenceprimer 250ataggatccc acctccaatg tttctgcagc
3025130DNAartificial sequenceprimer
251atagaattcg agaagaaagg gcaatcaaga
3025230DNAartificial sequenceprimer 252atactgcaga gaaatctcga tttcgactac
3025325DNAartificial sequenceprimer
253gccactctca tagggttctc catcg
2525425DNAartificial sequenceprimer 254ggcatgcctc caagatcctt gaagt
2525522DNAartificial sequenceprimer
255gggtcttggt cgttttactg tt
2225625DNAartificial sequenceprimer 256ccaagacgat gacaacagat acagc
2525721DNAartificial sequenceprimer
257ataaactaaa tcttcgctga a
2125821DNAartificial sequenceprimer 258caaacgcgga tctgaaaaac t
2125918DNAartificial sequenceprimer
259tctctcttcc aaatctcc
1826020DNAartificial sequenceprimer 260aagtctctca ctttctcact
2026125DNAartificial sequenceprimer
261ctaagctctc aagatcaaag gctta
2526225DNAartificial sequenceprimer 262ttaacattgc aaagagtttc aaggt
252634PRTartificial sequencemotif
263Thr Pro Trp Lys1264289PRTArabidopsis thaliana 264Met Gly Lys Tyr Ile
Arg Lys Ser Lys Ile Asp Gly Ala Gly Ala Gly1 5
10 15Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Glu Ser Ser Ile Ala 20 25
30Leu Met Asp Val Val Ser Pro Ser Ser Ser Ser Ser Leu Gly Val Leu
35 40 45Thr Arg Ala Lys Ser Leu Ala Leu
Gln Gln Gln Gln Gln Arg Cys Leu 50 55
60Leu Gln Lys Pro Ser Ser Pro Ser Ser Leu Pro Pro Thr Ser Ala Ser65
70 75 80Pro Asn Pro Pro Ser
Lys Gln Lys Met Lys Lys Lys Gln Gln Gln Met 85
90 95Asn Asp Cys Gly Ser Tyr Leu Gln Leu Arg Ser
Arg Arg Leu Gln Lys 100 105
110Lys Pro Pro Ile Val Val Ile Arg Ser Thr Lys Arg Arg Lys Gln Gln
115 120 125Arg Arg Asn Glu Thr Cys Gly
Arg Asn Pro Asn Pro Arg Ser Asn Leu 130 135
140Asp Ser Ile Arg Gly Asp Gly Ser Arg Ser Asp Ser Val Ser Glu
Ser145 150 155 160Val Val
Phe Gly Lys Asp Lys Asp Leu Ile Ser Glu Ile Asn Lys Asp
165 170 175Pro Thr Phe Gly Gln Asn Phe
Phe Asp Leu Glu Glu Glu His Thr Gln 180 185
190Ser Phe Asn Arg Thr Thr Arg Glu Ser Thr Pro Cys Ser Leu
Ile Arg 195 200 205Arg Pro Glu Ile
Met Thr Thr Pro Gly Ser Ser Thr Lys Leu Asn Ile 210
215 220Cys Val Ser Glu Ser Asn Gln Arg Glu Asp Ser Leu
Ser Arg Ser His225 230 235
240Arg Arg Arg Pro Thr Thr Pro Glu Met Asp Glu Phe Phe Ser Gly Ala
245 250 255Glu Glu Glu Gln Gln
Lys Gln Phe Ile Glu Lys Tyr Asn Phe Asp Pro 260
265 270Val Asn Glu Gln Pro Leu Pro Gly Arg Phe Glu Trp
Thr Lys Val Asp 275 280 285Asp
26520DNAartificial sequenceprimer 265cgggccccaa ataatgattt
2026618DNAartificial sequenceprimer
266gacacgggcc agagctgc
1826710PRTartificial sequencemotif 267Arg Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa
Asn1 5 102688PRTartificial sequencemotif
268Met Arg Xaa Ile Leu Xaa Asp Trp1 52698PRTartificial
sequencemotif 269Lys Tyr Glu Glu Xaa Xaa Xaa Pro1
52709PRTartificial sequencemotif 270Gly Xaa Gly Xaa Xaa Gly Xaa Val Tyr1
527110PRTartificial sequencemotif 271His Arg Asp Xaa Lys Xaa
Xaa Asn Xaa Leu1 5 1027212PRTartificial
sequencemotif 272Asp Xaa Xaa Xaa Ser Xaa Gly Xaa Xaa Xaa Xaa Glu1
5 102735PRTartificial sequencemotif 273Thr Pro
Xaa Xaa Xaa1 52744PRTartificial sequencemotif 274Ser Pro
Xaa Xaa12754PRTartificial sequencemotif 275Ser Pro Xaa
Xaa12764PRTartificial sequencemotif 276Ser Pro Xaa Xaa12777PRTartificial
sequencemotif 277Pro Lys Lys Lys Arg Lys Val1
527816PRTartificial sequencemotif 278Lys Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Lys Lys Lys Lys1 5 10
152795PRTartificial sequencemotif 279Lys Arg Pro Arg Pro1
52809PRTartificial sequencemotif 280Pro Ala Ala Lys Arg Val Lys
Leu Asp1 52814PRTartificial sequencemotif 281Arg Xaa Xaa
Phe12825PRTartificial sequencemotif 282Leu Xaa Cys Xaa Glu1
52835PRTartificial sequencemotif 283Leu Xaa Ser Xaa Glu1
528417PRTartificial sequencemotif 284Asp Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Glu Xaa Xaa Xaa Asp Leu Phe1 5 10
15Asp28517PRTartificial sequencemotif 285Asp Tyr Xaa Xaa Xaa Xaa
Xaa Xaa Asp Xaa Xaa Xaa Xaa Asp Met Trp1 5
10 15Glu28635PRTartificial sequencemotif 286Xaa Xaa Lys
Asn Ile Arg Xaa Arg Val Xaa Asp Ala Leu Asn Val Xaa1 5
10 15Met Ala Xaa Xaa Xaa Ile Xaa Xaa Xaa
Lys Lys Glu Ile Xaa Trp Xaa 20 25
30Gly Leu Pro 3528753PRTartificial sequencemotif 287Xaa Xaa
Gly Leu Arg Xaa Phe Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Lys Xaa 20 25
30Xaa Xaa Xaa Lys Xaa Xaa Thr Thr Xaa Tyr Xaa Glu Val Ala Asp
Glu 35 40 45Xaa Xaa Xaa Xaa Phe
502889PRTartificial sequencemotif 288Xaa Xaa Xaa Xaa Lys Xaa Xaa Xaa
Glu1 528927PRTartificial sequencemotif 289Xaa Xaa Xaa Xaa
Lys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Xaa1 5
10 15Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Arg
Asn 20 2529046PRTartificial sequencemotif
290Xaa Pro Phe Ile Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa 20 25
30Xaa Phe Xaa Xaa His Asp Asp Xaa Xaa Xaa Leu Xaa Xaa
Met 35 40 45
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