U.S. patent application number 10/371558 was filed with the patent office on 2003-08-28 for replication protein a and use.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Mahajan, Pramod B..
Application Number | 20030163840 10/371558 |
Document ID | / |
Family ID | 26797450 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030163840 |
Kind Code |
A1 |
Mahajan, Pramod B. |
August 28, 2003 |
Replication protein A and use
Abstract
Methods and compositions for modulating DNA metabolism are
provided. Nucleotide and amino acid sequences encoding a maize
replication protein A subunits are provided. The sequences can be
used in expression cassettes for modulating DNA replication, DNA
repair, and recombination.
Inventors: |
Mahajan, Pramod B.;
(Urbandale, IA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL INC.
7100 N.W. 62ND AVENUE
P.O. BOX 1000
JOHNSTON
IA
50131
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
|
Family ID: |
26797450 |
Appl. No.: |
10/371558 |
Filed: |
February 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10371558 |
Feb 21, 2003 |
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09396149 |
Sep 15, 1999 |
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6538176 |
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60123896 |
Mar 11, 1999 |
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60100690 |
Sep 17, 1998 |
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Current U.S.
Class: |
800/278 ;
435/199; 435/320.1; 435/419; 435/5; 435/6.11; 530/388.1;
536/23.2 |
Current CPC
Class: |
C12N 15/8261 20130101;
C12N 15/8241 20130101; C12N 15/8279 20130101; C12N 15/8263
20130101; Y02A 40/146 20180101; C12N 15/8213 20130101 |
Class at
Publication: |
800/278 ; 435/6;
435/320.1; 435/419; 530/388.1; 536/23.2; 435/199 |
International
Class: |
A01H 001/00; C12N
015/82; C12Q 001/68; C07H 021/04; C12N 009/22; C12N 005/04 |
Claims
What is claimed is:
1. An isolated protein comprising a polypeptide having at least 80%
sequence identity over the full-length of SEQ ID NOS: 12, 14, 16,
18, 20, or 22, wherein the percent sequence identity is determined
by the GAP algorithm under default parameters and wherein the
polypeptide enhances homologous recombination as part of an RPA
complex.
2. The polypeptide of claim 1, wherein the polypeptide has at least
85% sequence identity over the full-length of SEQ ID NOS: 12, 14,
16, 18, 20, or 22.
3. The polypeptide of claim 1, wherein the polypeptide has at least
90% sequence identity over the full-length of SEQ ID NOS: 12, 14,
16, 18, 20, or 22.
4. The polypeptide of claim 1, wherein the polypeptide has at least
95% sequence identity over the full-length of SEQ ID NOS: 12, 14,
16, 18, 20, or 22.
5. The polypeptide of claim 1, wherein the polypeptide is SEQ ID
NOS: 12, 14, 16, 18, 20, or 22.
6. An isolated protein comprising a polypeptide encoded by SEQ ID
NOS: 11, 13, 15, 17, 19, or 21, wherein the polypeptide enhances
homologous recombination as part of an RPA complex.
7. An expression cassette comprising a polynucleotide operably
linked to a promoter, wherein the polynucleotide encodes the
polypeptide of claim 1.
8 A host cell comprising the expression cassette of claim 7.
9. A host cell comprising the polypeptide of claim 1.
10. The host cell of claim 9, wherein the host cell is a plant
cell.
11. The plant cell of claim 10, wherein the plant cell is from a
monocot.
12. The plant cell of claim 11, wherein the monocot is maize,
sorghum, rice, barley, millet, rye, or wheat.
13. The plant cell of claim 10, wherein the plant cell is from a
dicot.
14. The plant cell of claim 13, wherein the dicot is soybean,
canola, alfalfa, sunflower, or safflower.
15. A plant comprising the host cell of claim 10.
16. The plant of claim 15, wherein the plant is a monocot.
17. The plant of claim 16, wherein the monocot is maize, sorghum,
rice, barley, millet, rye, or wheat.
18. The plant of claim 15, wherein the plant is a dicot.
19. The plant of claim 18, wherein the dicot is soybean, canola,
alfalfa, sunflower, or safflower.
20. A seed comprising the host cell of claim 10.
21. The seed of claim 20, wherein the seed is from a monocot.
22. The seed of claim 21, wherein the monocot is maize, sorghum,
rice, barley, millet, rye, or wheat.
23. The seed of claim 20, wherein the seed is from a dicot.
24. The seed of claim 23, wherein the dicot is soybean, canola,
alfalfa, sunflower, or safflower.
25. An antibody that selectively binds to an isolated polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 12, 14, 16, 18, or 20.
26. An antibody that selectively binds to an isolated polypeptide
comprising at least 20 contiguous amino acids of the amino acid
sequence selected from the group consisting of SEQ ID NO: 12, 14,
16, 18, or 20.
27. An isolated polypeptide wherein: (a) the polypeptide comprises
at least 20 contiguous amino acids of a polypeptide selected from
the group consisting of SEQ ID NO: 12, 14, 16, 18, or 20; (b) the
polypeptide, when presented as an immunogen, elicits the production
of an antibody which specifically binds the polypeptide selected
from the group consisting of SEQ ID NO: 12, 14, 16, 18, or 20; (c)
the polypeptide does not bind to antisera raised against the
polypeptide of SEQ ID NO: 12, 14, 16, 18 or 20 after the antisera
has been fully immunosorbed with the polypeptide of SEQ ID NO: 12,
14, 16, 18 or 20; and (d) the polypeptide enhances homologous
recombination as part of an RPA complex.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S.
application Ser. No. 09/396,149 filed Dec. 15, 1999, and claims the
benefit of U.S. Provisional Application Serial No. 60/100,690 filed
Sep. 17, 1998, now abandoned and U.S. Provisional Application
Serial No. 60/123,896 filed Mar. 11, 1999, now abandoned, which are
all herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the genetic manipulation of plants,
particularly to modulating DNA metabolism in transformed plants and
plant cells.
BACKGROUND OF THE INVENTION
[0003] Replication protein A (RPA) is a single-stranded DNA-binding
protein that is required for multiple processes in eukaryotic
cells. RPA from human cells is a stable complex of 70-, 32-, and
14-kDa subunits. Homologues of RPA have been identified in all
eukaryotes examined. However, only human RPA and closely related
homologues can support SV40 DNA replication.
[0004] The RPA complex appears to be highly conserved in all
eukaryotes. The three RPA genes in budding yeast cells are
essential for cell viability. Nevertheless, yeast RPA only
partially substitutes for human RPA in the in vitro replication of
simian virus 40 indicating that species-specific interactions
between RPA and other replication proteins may be important for its
biological activity.
[0005] RPA binds tightly to single stranded DNA as a heterotrimeric
complex. The binding activity has been localized to the 70 kDa
subunit. The affinity of RPA for both double-stranded DNA and RNA
is at least three orders of magnitude lower than it is for
single-stranded DNA. It has been reported that RPA binds
preferentially to the pyrimidine-rich strand of both S. cerevisiae
sequences and the SV40 origin of replication. However, studies
examining the determinants of replication origins in S. cerevisiae
indicate that this preferential binding is not critical for the
initiation of DNA replication.
[0006] Subunits of RPA in the 70-, 32- and 14 kDa ranges have been
identified from various sources. The 32 kDa subunit has also been
referred to as "RPA2", "B", "small", "32 kDa", "P32", "P34", and
"middle" subunit. For the purposes of this invention, the "middle"
subunit is intended as the subunit having a molecular weight of
about 32 kDa.
[0007] The middle subunit of RPA has a role in cell cycle
regulation; single stranded DNA binding; affinity of DNA binding;
species-specificity of DNA binding; DNA recombination, repair,
replication and metabolism; and response to DNA damages. (Anderson
(1966) Calif. Inst. Technol.; Seroussi et al. (1993) J. Biol. Chem.
268:7147-54; Kenny et al. (1989) Proc. Natl. Acad. Sci. USA
86:9757-61; Brush et al. (1995) Methods Enzymol. 262:522-48;
Stigger et al. (1994) Proc. Natl. Acad. Sci. USA 91:579-83;
Philipova et al. (1996) Genes Dev. 10:2222-33).
[0008] Much research has centered on the exploration of the
biochemical and genetic mechanisms by which cell cycle regulation
of DNA synthesis is achieved. While there have been advances in
delineating the existence of cell cycle proteins, more information
is needed on the mechanism of action of DNA replication,
recombination, and repair. Furthermore, methods for regulating or
altering the cell cycle is needed.
RELATED LITERATURE
[0009] Braun et al. (1997) Biochemistry 36:8443-8454; report on the
role of protein-protein interactions and the function of
replication protein A. It is reported that RPA modulates the
activity of DNA polymerase a by multiple mechanisms.
[0010] Loor et al. (1997) Nucleic Acids Research 25:5041-5046
report on the identification of DNA replication in cell cycle
proteins that interact with proliferating cell nuclear antigen.
[0011] Longhese et al. (1994) Molecular and Cellular Biology
14:7884-7890 report that replication factor A is required for in
vivo DNA replication, repair, and recombination.
[0012] Stigger et al. (1998) J. Biol. Chem. 273:9337-9343 provide a
functional analysis of human replication protein A in nucleotide
excision repair.
[0013] Abremova et al. (1997) Proc. Natl. Acad. Sci. USA
94:7186-7191 report that the interaction between replication
protein A and p53 is disrupted after ultraviolet damage in a DNA
repair-dependent manner.
[0014] New et al. (1998) Nature 391:407-410 reports that RAD52
protein stimulates DNA strand exchange by RAD51 and replication
protein A. Stimulation was dependent on the concerted action of
both RAD51 protein and RPA implying that specific protein-protein
interactions between RAD52 protein, RAD51 protein and RPA are
required.
[0015] Dutta et al. (1992) EMBO J 11(6):2189-2199 and Niu et al.
(1997) J. Biol. Chem. 272(19):12634-41 report cell cycle-dependent
phosphorylation of the middle subunit of RPA, implying a role for
the subunit in cell cycle regulation.
[0016] Bochkareva et al. (1998) J. Biol. Chem. 273(7):3932-3936
report the formation of a single stranded DNA binding site on the
human RPA middle subunit.
[0017] Mass et al. (1998) Mol. Cell. Biol. 18(11):6399-6407 report
that the RPA middle subunit contacts nascent simian virus 40 DNA,
particularly the early DNA chain intermediates synthesized by DNA
polymerase alpha-primase (RNA-DNA primers), but not more advanced
products.
[0018] Lavrik et al. (1998) Nucleic Acids Res 26(2):602-607 report
on location of binding of individual subunits of human RPA to DNA
primer-template complexes in various elongation reactions.
[0019] Sibenaller et al. (1998) 37(36):12496-12506 report that
differences in the activity of the middle (32 kDa) and the small
(14 Kda) subunits of RPA are responsible for variations in the
single stranded DNA-binding properties of Saccharomyces cerevisiae
and human RPA, thus implying a role for the subunits in
species-specificity of DNA binding of RPA.
SUMMARY OF THE INVENTION
[0020] Compositions and methods for modulating DNA metabolism in a
host cell is provided. Particularly, the complete cDNA and amino
acid sequence for homologues of maize replication protein A (RPA)
large- and middle subunits are provided. The sequences of the
invention find use in modulating DNA replication, DNA repair, and
recombination.
[0021] Transformed plants can be obtained having altered metabolic
states. The invention has implications in genetic transformation
and gene targeting in plants. Additionally, the methods can be used
to promote cell death particularly in an inducible or
tissue-preferred manner.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 provides a comparison of eukaryotic RPA large subunit
amino acid sequences. Amino acid sequences for the RPA large
subunits from Saccharomyces cerevisiae (Rfal Yeast, SEQ ID NO:10),
Schizosaccharomyces pombe (Rfal_Schpo, SEQ ID NO: 9), Drosophila
melanogaster(Rfal_Drome, SEQ ID NO:8), Homo sapiens (Rfal_Human,
SEQ ID NO: 7), Xenopus laevis (Rfal_Xenla, SEQ ID NO: 6), and Oryza
sativa (024183, SEQ ID NO:5) were compared with the maize RPA LS
homologue 1 (ZMRPALSH1, SEQ ID NO:2) and homologue 2 (ZMRPALSH2,
SEQ ID NO:4) using the GCG PileUp program utilizing default
parameters. The putative zinc finger region is shown in
italics.
[0023] FIG. 2 provides an expression construct for inducible
expression of the maize RPA large or middle subunit antisense
construct.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Nucleotide sequences and proteins useful for modulating DNA
metabolism are provided. The nucleotide and amino acid sequences
correspond to the maize replication protein A (RPA) subunits. RPA
is a single-stranded DNA-binding protein that is required for
multiple processes in DNA metabolism, including DNA replication,
DNA repair, and recombination. The RPA complex generally comprises
subunits of approximately 70, 32, and 14 kDa. By "large subunit",
"middle subunit", and "small subunit" is herein intended a RPA
subunit having the approximate molecular weight of 70-, 32-, and 14
kDa respectively. The sequences of the invention comprise the
large- and middle subunits of the RPA complex. The sequences of the
invention additionally find use in modulating gene expression.
[0025] Compositions of the invention include RPA nucleotide and
amino acid sequences that are involved in modulating DNA
metabolism. In particular, the present invention provides for
isolated nucleic acid molecules comprising nucleotide sequences
encoding the amino acid sequences shown in SEQ ID NOs: 2 and 4 for
the large subunit, and SEQ ID NOs: 12, 14, 16, 18, 20, and 22 for
the middle subunit. SEQ ID NO:2 and SEQ ID NO:4 correspond to the
amino acid sequences for the maize RPA large subunit homologue 1
(ZmRPALSH1) and homologue 2 (ZmRPALSH2). SEQ ID NOs: 12, 14, 16,
18, 20, and 22 correspond to the amino acid sequences for the maize
middle subunit homologue 1 (ZmRPAMSH1); homologues 2 and 3
(ZmRPAMSH2 and ZmRPAMSH3); homologue 4 (ZmRPAMSH4); homologue 5
(ZmRPAMSH5); homologue 6 (ZmRPAMSH6); and homologue 7 (ZmRPAMSH7)
respectively.
[0026] For the large subunit, the present invention alternatively
provides the nucleotide sequences encoding the DNA sequences
deposited in a bacterial host as Patent Deposit Nos. 98754 and
98843. For the large subunits, further are polypeptides having an
amino acid sequence encoded by a nucleic acid molecule described
herein, for example those set forth in SEQ ID NOs: 1 and 3, those
deposited in a bacterial host as Patent Deposit Nos. 98754 and
98843, and fragments and variants thereof.
[0027] Plasmids containing the RPA large subunit nucleotide
sequences of the invention were deposited with the Patent
Depository of the American Type Culture Collection (ATCC),
Manassas, Va., and assigned Patent Deposit Nos. 98754 and 98843.
These deposits will be maintained under the terms of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. These deposits
were made merely as a convenience for those of skill in the art and
are not an admission that a deposit is required under 35 U.S.C.
.sctn.112.
[0028] Nucleotide sequences encoding the amino acid sequences for
the maize RPA large subunit homologue 1 (ZmRPALSH1) and homologue 2
(ZmRPALSH2) are set forth in SEQ ID NOs 1 and 3. Nucleotide
sequences encoding the amino acid sequences for the maize RPA
middle subunit homologue 1 (ZmRPAMSH1); homologues 2 and 3
(ZmRPAMSH2 and ZmRPAMSH3); homologue 4 (ZmRPAMSH4); homologue 5
(ZmRPAMSH5); homologue 6 (ZmRPAMSH6); and homologue 7 (ZmRPAMSH7)
are set forth in SEQ ID NOs: 11, 13, 15, 17, 19, and 21
respectively.
[0029] The invention encompasses isolated or substantially purified
nucleic acid or protein compositions. An "isolated" or "purified"
nucleic acid molecule or protein, or biologically active portion
thereof, is 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. Preferably, an "isolated" nucleic acid is
free of sequences (preferably protein encoding sequences) that
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 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 that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
A protein that is substantially free of cellular material includes
preparations of protein having less than about 30%, 20%, 10%, 5%,
(by dry weight) of contaminating protein. When the protein of the
invention or biologically active portion thereof is recombinantly
produced, preferably culture medium represents less than about 30%,
20%, 10%, or 5% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals.
[0030] RPA binds tightly to single-stranded DNA (ssDNA). The
affinity of binding to double-stranded DNA (dsDNA) is three to four
orders of magnitude lower than the binding affinity for ssDNA.
Because RPA has been found to bind specifically to certain dsDNA
sequences that seem to be involved in the regulation of
transcription, modulation of gene expression may be affected by an
increase or decrease in RPA expression in the host cell.
[0031] RPA has a wide range of activity and therefore uses relating
to DNA metabolism and cell cycle. RPA interacts specifically with
several proteins required for nucleotide excision repair.
Interactions with repair proteins indicate that RPA may be
important for efficient damage recognition and cleavage. RPA
additionally interacts with RAD52 protein, a protein that is
essential for dsDNA-break repair. This interaction appears to be
essential for homologous recombination. In this manner, expression
of the nucleotides of the invention may promote homologous
recombination by recruiting factors which are essential for
recombination to occur. Thus, the methods and compositions of the
invention find use in promoting homologous recombination.
[0032] In one embodiment, genetic manipulation by homologous
recombination can be improved by either expression of the RPA
coding sequences of the invention during transformation, or by
providing RPA protein. RPA protein, for example, may be provided as
a coating to particles during particle bombardment. Alternatively,
DNA constructs providing for the expression of RPA may be included
with the DNA to be transformed. The increase in RPA during
transformation, particularly integration of polynucleotides by
homologous recombination, promotes integration and insertion of the
DNA sequences of interest into the plant genome.
[0033] In the same manner, it may be beneficial to inhibit the
expression or presence of the RPA protein to encourage non-specific
recombination events. In this manner, antibodies, peptides,
antisense oligonucleotides and the like may be utilized to inhibit
the activity of RPA. Alternatively, antisense constructs may be
provided to inhibit the expression of RPA and encourage
non-specific recombination.
[0034] Catalytic RNA molecules or ribozymes can also be used to
inhibit expression of plant genes. It is possible to design
ribozymes that specifically pair with virtually any target RNA and
cleave the phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. In carrying out this
cleavage, the ribozyme is not itself altered, and is thus capable
of recycling and cleaving other molecules, making it a true enzyme.
The inclusion of ribozyme sequences within antisense RNAs confers
RNA-cleaving activity upon them, thereby increasing the activity of
the constructs. The design and use of target RNA-specific ribozymes
is described in Haseloff et al. (1988) Nature 334:585-591.
[0035] A variety of cross-linking agents, alkylating agents and
radical generating species as pendant groups on polynucleotides of
the present invention can be used to bind, label, detect, and/or
cleave nucleic acids. For example, Vlassov, V. V. et al. (1986)
Nucleic Acids Res. 14:4065-4076, describe covalent bonding of a
single-stranded DNA fragment with alkylating derivatives of
nucleotides complementary to target sequences. A report of similar
work by the same group is that by Knorre et al. (1985) Biochimie
67:785-789. Iverson and Dervan also showed sequence-specific
cleavage of single-stranded DNA mediated by incorporation of a
modified nucleotide which was capable of activating cleavage (1987)
J. Am. Chem. Soc. 109:1241-1243). Meyer et al. (1989) J. Am. Chem.
Soc. 111:8517-8519, effect covalent crosslinking to a target
nucleotide using an alkylating agent complementary to the
single-stranded target nucleotide sequence. A photoactivated
crosslinking to single-stranded oligonucleotides mediated by
psoralen was disclosed by Lee et al. (1988) Biochem. 27:3197-3203.
Use of crosslinking in triple-helix forming probes was also
disclosed by Home et al. (1990) J. Am. Chem. Soc. 112:2435-2437.
Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to
single-stranded oligonucleotides has also been described by Webb et
al. (1986) J. Am. Chem. Soc. 108:2764-2765; Webb et al. (1986)
Nucleic Acids Res. 14:7661-7674; Feteritz et al. (1991) J. Am.
Chem. Soc. 113:4000. Various compounds to bind, detect, label,
and/or cleave nucleic acids are known in the art. See, for example,
U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and
5,681,941.
[0036] RPA is required for the replication of chromosomal DNA.
Inhibition of endogenous RPA expression is deleterious to the cell,
organism, or plant. Thus, the constructs of the invention can be
used to selectively kill target cells or tissues. This can be
accomplished through the use of inducible or tissue-preferred
promoters. In this manner, the sequences of the invention may find
use in enhancing pathogen resistance. An antisense construct for
the RPA coding sequence is operably linked to a pathogen-inducible
promoter. Upon contact with the pathogen, the RPA antisense
construct is expressed resulting in cell death and effectively
preventing the invasion of the pathogen.
[0037] The invention is drawn to compositions and methods for
inducing resistance in a plant to plant pests. Accordingly, the
compositions and methods are also useful in protecting plants
against fungal pathogens, viruses, nematodes, insects and the
like.
[0038] By "disease resistance" is intended that the plants avoid
the disease symptoms that are the outcome of plant-pathogen
interactions. That is, pathogens are prevented from causing plant
diseases and the associated disease symptoms, or alternatively, the
disease symptoms caused by the pathogen is minimized or lessened.
The methods of the invention can be utilized to protect plants from
disease, particularly those diseases that are caused by plant
pathogens.
[0039] Pathogens of the invention include, but are not limited to,
viruses or viroids, bacteria, insects, nematodes, fungi, and the
like. Viruses include any plant virus, for example, tobacco or
cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf
mosaic virus, etc. Specific fungal and viral pathogens for the
major crops include: Soybeans: Phytophthora megasperma fsp.
glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia
sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae
(Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium
rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora
manshurica, Colletotrichum dematium (Colletotichum truncatum),
Corynespora cassiicola, Septoria glycines, Phyllosticta soficola,
Alternaria alternata, Pseudomonas syringae p.v. glycinea,
Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,
Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,
Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,
Phakopsora pachyrhizi, Pythium aphamidermatum, Pythium ultimum,
Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines
Fusarium solani; Canola: Albugo candida, Alternaria brassicae,
Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia
sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum,
Peronospora parasitica, Fusarium roseum, Alternaria alternata;
Alfalfa: Clavibater michiganese subsp. insidiosum, Pythium ultimum,
Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium
aphamidermatum, Phytophthora megasperma, Peronospora trifoliorum,
Phoma medicaginis var. medicaginis, Cercospora medicaginis,
Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium,
Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches,
Stemphylium herbarum, Stemphylium alfalfae; Wheat: Pseudomonas
syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas
campestris p.v. translucens, Pseudomonas syringae p.v. syringae,
Alternaria alternata, Cladosporium herbarum, Fusarium graminearum,
Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta
tritici, Cephalosporium gramineum, Collotetrichum graminicola,
Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici,
Puccinia recondita f.sp. tritici, Puccinia striiformis, Pyrenophora
tritici-repentis, Septoria nodorum, Septoria tritici, Septoria
avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani,
Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium
aphamidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris
sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil
Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle
Streak Virus, American Wheat Striate Virus, Claviceps purpurea,
Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia
indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium
gramicola, Pythium aphamidermatum, High Plains Virus, European
wheat striate virus; Sunflower: Plasmophora halstedii, Sclerotinia
sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis
helianthi, Altemaria helianthi, Alternaria zinniae, Botrytis
cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe
cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus
stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia
carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora
cryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var.
subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella
zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis),
Pythium irregulare, Pythium debaryanum, Pythium graminicola,
Pythium splendens, Pythium ultimum, Pythium aphamidermatum,
Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus
heterostrophus), Helminthosporium carbonum I, II & III
(Cochliobolus carbonum), Exserohilum turcicum I, II & III,
Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta
maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis,
Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina,
Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum,
Curvularia lunata, Curvularia inaequalis, Curvularia pallescens,
Clavibacter michiganense subsp. nebraskense, Trichoderma viride,
Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus,
Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae,
Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt
spiroplasma, Diplodia macrospora, Sclerophthora macrospora,
Peronosclerospora sorghi, Peronosclerospora philippinensis,
Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca
reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium
acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize
Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize
Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum
turcicum, Colletotrichum graminicola (Glomerella graminicola),
Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina,
Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v.
holcicola, Pseudomonas andropogonis, Puccinia purpurea,
Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme,
Alternaria alternata, Bipolaris sorghicola, Helminthosporium
sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae
(Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora
sorghicola, Phyllachara sacchari, Sporisorium reilianum
(Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium
sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B,
Claviceps sorghi, Rhizoctonia solani, Acremonium strictum,
Sclerophthona macrospora, Peronosclerospora sorghi,
Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium
graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium
graminicola, etc.
[0040] Nematodes include parasitic nematodes such as root-knot,
cyst, and lesion nematodes, including Heterodera and Globodera spp;
particularly Globodera rostochiensis and globodera pailida (potato
cyst nematodes); Heterodera glycines (soybean cyst nematode);
Heterodera schachtii (beet cyst nematode); and Heterodera avenae
(cereal cyst nematode).
[0041] Insect pests include insects selected from the orders
Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,
Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,
Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly
Coleoptera and Lepidoptera. Insect pests of the invention for the
major crops include: Maize: Ostrinia nubilalis, European corn
borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn
earworm; Spodoptera frugiperda, fall armyworm; Diatraea
grandiosella, southwestern corn borer; Elasmopalpus lignosellus,
lesser cornstalk borer; Diatraea saccharalis, surgarcane borer;
Diabrotica virgifera, western corn rootworm; Diabrotica longicomis
barberi, northern corn rootworm; Diabrotica undecimpunctata
howardi, southern corn rootworm; Melanotus spp., wireworms;
Cyclocephala borealis, northern masked chafer (white grub);
Cyclocephala immaculata, southern masked chafer (white grub);
Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn
flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum
maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid;
Blissus leucopterus leucopterus, chinch bug; Melanoplus
femurrubrum, redlegged grasshopper; Melanoplus sanguinipes,
migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza
parvicornis, corn blot leafminer; Anaphothrips obscrurus, grass
thrips; Solenopsis milesta, thief ant; Tetranychus urticae,
twospotted spider mite; Sorghum: Chilo partellus, sorghum borer;
Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn
earworm; Elasmopalpus lignosellus, lesser cornstalk borer; Feltia
subterranea, granulate cutworm; Phyllophaga crinita, white grub;
Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus,
cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle;
Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf
aphid; Sipha flava, yellow sugarcane aphid; Blissus leucopterus
leucopterus, chinch bug; Contarinia sorghicola, sorghum midge;
Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,
twospotted spider mite; Wheat: Pseudaletia unipunctata, army worm;
Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,
lesser cornstalk borer; Agrotis orthogonia, western cutworm;
Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,
cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica
undecimpunctata howardi, southern corn rootworm; Russian wheat
aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English
grain aphid; Melanoplus femurrubrum, red legged grasshopper;
Melanoplus differentialis, differential grasshopper; Melanoplus
sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian
fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat
stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella
fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria
tulipae, wheat curl mite; Sunflower: Suleima helianthana, sunflower
bud moth; Homoeosoma electellum, sunflower moth; zygogramma
exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle;
Neolasioptera murtfeldtiana, sunflower seed midge; Cotton:
Heliothis virescens, cotton budworm; Helicoverpa zea, cotton
bollworm; Spodoptera exigua, beet armyworm; Pectinophora
gossypiella, pink bollworm; Anthonomus grandis grandis, boll
weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus,
cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly;
Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum,
redlegged grasshopper; Melanoplus differentialis, differential
grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca,
tobacco thrips; Tetranychus cinnabarinus, carmine spider mite;
Tetranychus urticae, twospotted spider mite; Rice: Diatraea
saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm;
Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis;
Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae,
rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus
leucopterus leucopterus, chinch bug; Acrosternum hilare, green
stink bug; Soybean: Pseudoplusia includens, soybean looper;
Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra,
green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis
ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis
virescens, cotton budworm; Helicoverpa zea, cotton bollworm;
Epilachna varivestis, Mexican bean beetle; Myzus persicae, green
peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare,
green stink bug; Melanoplus femurrubrum, redlegged grasshopper;
Melanoplus differentialis, differential grasshopper; Hylemya
platura, seedcorn maggot; Sericothrips variabilis, soybean thrips;
Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry
spider mite; Tetranychus urticae, twospotted spider mite; Barley:
Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black
cutworm; Schizaphis graminum, greenbug; Blissus leucopterus
leucopterus, chinch bug; Acrosternum hilare, green stink bug;
Euschistus servus, brown stink bug; Delia platura, seedcorn maggot;
Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat
mite; Oil Seed Rape: Brevicoryne brassicae, cabbage aphid;
Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha
armyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Root
maggots.
[0042] A number of promoters can be used in the practice of the
invention. The promoters can be selected based on the desired
outcome. The nucleic acids can be combined with constitutive,
tissue-preferred, or other promoters for expression in plants.
[0043] A plant promoter can be employed which will direct
expression of a polynucleotide of the present invention in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Such constitutive promoters include, for example,
the core promoter of the Rsyn7 (WO 99/43838); the core CaMV
.sup.35S promoter (Odell et al. (1985) Nature 313:810-812); rice
actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin
(Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and
Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last
et al. (1991) Theor. Appl. Genet 81:581-588); MAS (Velten et al.
(1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026), and the like. Other constitutive promoters include, for
example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; and 5,608,142.
[0044] Alternatively, the plant promoter can direct expression of a
polynucleotide of present invention in a specific tissue or may be
otherwise under more precise environmental or developmental
control. Such promoters are referred to here as "inducible"
promoters. Environmental conditions that may effect transcription
by inducible promoters include pathogen attack, anaerobic
conditions, or the presence of light. Examples of inducible
promoters are the Adhl promoter which is inducible by hypoxia or
cold stress, the Hsp70 promoter which is inducible by heat stress,
and the PPDK promoter which is inducible by light.
[0045] Examples of promoters under developmental control include
promoters that initiate transcription only, or preferentially, in
certain tissues, such as leaves, roots, fruit, seeds, or flowers.
An exemplary promoter is the anther specific promoter 5126 (U.S.
Pat. Nos. 5,689,049 and 5,689,051). The operation of a promoter may
also vary depending on its location in the genome. Thus, an
inducible promoter may become fully or partially constitutive in
certain locations.
[0046] The promoters can be selected based on the desired outcome.
When the genes are expressed at levels to cause cell death, an
inducible promoter or tissue specific promoters can be used to
drive the expression of the genes of the invention. The inducible
promoter must be tightly regulated to prevent unnecessary cell
death, yet be expressed in the presence of a pathogen to prevent
infection and disease symptoms.
[0047] Generally, it will be beneficial to express the gene from an
inducible promoter, particularly from a pathogen-inducible
promoter. Such promoters include those from pathogenesis-related
proteins (PR proteins), which are induced following infection by a
pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,
chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J.
Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656;
and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also U.S. Pat.
No. 6,429,362, herein incorporated by reference.
[0048] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989)
Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al.
(1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al.
(1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad.
Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J.
10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA
91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et
al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386
(nematode-inducible); and the references cited therein. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant Path. 41:189-200).
[0049] Additionally, as pathogens find entry into plants through
wounds or insect damage, a wound-inducible promoter may be used in
the constructions of the invention. Such wound-inducible promoters
include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann.
Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology
14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2
(Stanford et al. (1989) Mol. Gen. Genet 215:200-208); systemin
(McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al.
(1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS
Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J.
6(2):141-150); and the like, herein incorporated by reference.
[0050] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-la
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0051] Where low level expression is desired, weak promoters will
be used. Generally, by "weak promoter" is intended a promoter that
drives expression of a coding sequence at a low level. By low level
is intended at levels of about {fraction (1/1000)} transcripts to
about {fraction (1/100,000)} transcripts to about {fraction
(1/5000,000)} transcripts. Alternatively, it is recognized that
weak promoters also encompasses promoters that are expressed in
only a few cells and not in others to give a total low level of
expression. Where a promoter is expressed at unacceptably high
levels, portions of the promoter sequence can be deleted or
modified to decrease expression levels.
[0052] Such weak constitutive promoters include, for example, the
core promoter of the Rsyn7 (WO 99/43838), the core 35S CaMV
promoter, and the like. Other constitutive promoters include, for
example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, U.S. Pat.
No. 6,177,611, herein incorporated by reference.
[0053] Tissue-preferred promoters can be utilized to target
enhanced RPA expression within a particular plant tissue. In this
aspect of the invention, the antisense constructs are useful for
tissue-preferred expression. Male or female sterility may be
affected by use of the antisense constructs with tissue-preferred
promoters. Although not a limitation, of particular interest are
promoters for male sterility. For example, the anther-preferred
promoter 5126 can be used. See, for example, U.S. Pat. Nos.
5,689,049 and 5,689,051, herein incorporated by reference.
[0054] Tissue-preferred promoters include Yamamoto et al. (1997)
Plant J. 12(2)255-265; Kawamata et al., (1997) Plant Cell Physiol.
38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;
Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.
(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996)
Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant
Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters
can be modified, if necessary, for weak expression.
[0055] Leaf-specific promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0056] Root-specific promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et a. (1992)
Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specific control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-specific promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also Bogusz et al. (1990) Plant Cell
2(7):633-641, where two root-specific promoters isolated from
hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema
tomentosa are described. The promoters of these genes were linked
to a .beta.-glucuronidase reporter gene and introduced into both
the nonlegume Nicotiana tabacum and the legume Lotus corniculatus,
and in both instances root-specific promoter activity was
preserved. Leach and Aoyagi (1991) describe their analysis of the
promoters of the highly expressed rolC and rolD root-inducing genes
of Agrobacterium rhizogenes (see Plant Science (Limerick)
79(1):69-76). They concluded that enhancer and tissue-preferred DNA
determinants are dissociated in those promoters. Teeri et al.
(1989) used gene fusion to lacZ to show that the Agrobacterium
T-DNA gene encoding octopine synthase is especially active in the
epidermis of the root tip and that the TR2' gene is root specific
in the intact plant and stimulated by wounding in leaf tissue, an
especially desirable combination of characteristics for use with an
insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The
TR1' gene, fused to nptII (neomycin phosphotransferase II) showed
similar characteristics. Additional root-preferred promoters
include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant
Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994)
Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876;
5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and
5,023,179.
[0057] "Seed-preferred" promoters include both "seed-specific"
promoters (those promoters active during seed development such as
promoters of seed storage proteins) as well as "seed-germinating"
promoters (those promoters active during seed germination). See
Thompson et al. (1989) BioEssays 10:108, herein incorporated by
reference. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa
zein); milps (myo-inositol-1-phosphate synthase); and celA
(cellulose synthase) (see U.S. Pat. No. 6,225,529, herein
incorporated by reference). Gamma-zein is a preferred
endosperm-specific promoter. Glob-1 is a preferred embryo-specific
promoter. For dicots, seed-specific promoters include, but are not
limited to, bean .beta.-phaseolin, napin, .beta.-conglycinin,
soybean lectin, cruciferin, and the like. For monocots,
seed-specific promoters include, but are not limited to, maize 15
kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1,
shrunken 2, globulin 1, etc.
[0058] Both heterologous and non-heterologous (i.e., endogenous)
promoters can be employed to direct expression of the nucleic acids
of the present invention. These promoters can also be used, for
example, in recombinant expression cassettes to drive expression of
antisense nucleic acids to reduce, increase, or alter RPA content
and/or composition in a desired tissue, or to generate sterile
plants. Optionally, RPA nucleic acids from a variety of sources, as
discussed above can be employed to create male sterile plants. In
optional embodiments, the RPA gene or cDNA is operably linked to an
anther-specific promoter such as 5126, as discussed above.
Preferably, the male sterile plant is maize.
[0059] Thus, in some embodiments, the nucleic acid construct will
comprise a promoter functional in a plant cell, such as in Zea
mays, operably linked to a polynucleotide of the present invention.
Promoters useful in these embodiments include the endogenous
promoters driving expression of a polypeptide of the present
invention.
[0060] In some embodiments, isolated nucleic acids which serve as
promoter or enhancer elements can be introduced in the appropriate
position (generally upstream) of a non-heterologous form of a
polynucleotide of the present invention so as to up or down
regulate expression of a polynucleotide of the present invention.
For example, endogenous promoters can be altered in vivo by
mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.
5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters
can be introduced into a plant cell in the proper orientation and
distance from a RPA gene so as to control the expression of the
gene. Gene expression can be modulated under conditions suitable
for plant growth so as to alter RPA content and/or composition.
Thus, the present invention provides compositions, and methods for
making, heterologous promoters and/or enhancers operably linked to
a native, endogenous (i.e., non-heterologous) form of a
polynucleotide of the present invention.
[0061] Methods for identifying promoters with a particular
expression pattern, in terms of e.g., tissue type, cell type, stage
of development, and/or environmental conditions, are well known in
the art. See, e.g., The Maize Handbook, Chapters 114-115, Freeling
and Walbot, eds., Springer, New York (1994); Corn and Corn
Improvement, 3.sup.rd edition, Chapter 6, Sprague and Dudley, eds.,
American Society of Agronomy, Madison, Wis. (1988). A typical step
in promoter isolation methods is identification of gene products
that are expressed with some degree of specificity in the target
tissue. Amongst the range of methodologies are: differential
hybridization to cDNA libraries; subtractive hybridization;
differential display; differential 2-D protein gel electrophoresis;
DNA probe arrays; and isolation of proteins known to be expressed
with some specificity in the target tissue. Such methods are well
known to those of skill in the art. Commercially available products
for identifying promoters are known in the art such as Clontech's
(Palo Alto, Calif.) Universal GenomeWalker Kit.
[0062] For the protein-based methods, it is helpful to obtain the
amino acid sequence for at least a portion of the identified
protein, and then to use the protein sequence as the basis for
preparing a nucleic acid that can be used as a probe to identify
either genomic DNA directly, or preferably, to identify a cDNA
clone from a library prepared from the target tissue. Once such a
cDNA clone has been identified, that sequence can be used to
identify the sequence at the 5' end of the transcript of the
indicated gene. For differential hybridization, subtractive
hybridization and differential display, the nucleic acid sequence
identified as enriched in the target tissue is used to identify the
sequence at the 5' end of the transcript of the indicated gene.
Once such sequences are identified, starting either from protein
sequences or nucleic acid sequences, any of these sequences
identified as being from the gene transcript can be used to screen
a genomic library prepared from the target organism. Methods for
identifying and confirming the transcriptional start site are well
known in the art.
[0063] In the process of isolating promoters expressed under
particular environmental conditions or stresses, or in specific
tissues, or at particular developmental stages, a number of genes
are identified that are expressed under the desired circumstances,
in the desired tissue, or at the desired stage. Further analysis
will reveal expression of each particular gene in one or more other
tissues of the plant. One can identify a promoter with activity in
the desired tissue or condition but that do not have activity in
any other common tissue.
[0064] To identify the promoter sequence, the 5' portions of the
clones described here are analyzed for sequences characteristic of
promoter sequences. For instance, promoter sequence elements
include the TATA box consensus sequence (TATAAT), which is usually
an AT-rich stretch of 5-10 bp located approximately 20 to 40 base
pairs upstream of the transcription start site. Identification of
the TATA box is well known in the art. For example, one way to
predict the location of this element is to identify the
transcription start site using standard RNA-mapping techniques such
as primer extension, S1 analysis, and/or RNase protection. To
confirm the presence of the AT-rich sequence, a structure-function
analysis can be performed involving mutagenesis of the putative
region and quantification of the mutation's effect on expression of
a linked downstream reporter gene. See, e.g., The Maize Handbook,
Chapter 114, Freeling and Walbot, eds., Springer, New York
(1994).
[0065] In plants, further upstream from the TATA box, at positions
-80 to -100, there is typically a promoter element (i.e., the CAAT
box) with a series of adenines surrounding the trinucleotide G (or
T) N G. J. Messing et al., in Genetic Engineering in Plants,
Kosage, Meredith and Hollaender, eds., pp. 221-227 (1983). In
maize, there no well-conserved CMT box but there are several short,
conserved protein-binding motifs upstream of the TATA box. These
include motifs for the transacting transcription factors involved
in light regulation, anaerobic induction, hormonal regulation, or
anthocyanin biosynthesis, as appropriate for each gene.
[0066] Once promoter and/or gene sequences are known, a region of
suitable size is selected from the genomic DNA that is 5' to the
transcriptional start, or the translational start site, and such
sequences are then linked to a coding sequence. If the
transcriptional start site is used as the point of fusion, any of a
number of possible 5' untranslated regions can be used in between
the transcriptional start site and the partial coding sequence. If
the translational start site at the 3' end of the specific promoter
is used, then it is linked directly to the methionine start codon
of a coding sequence.
[0067] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from the natural gene, from a variety of other plant genes,
or from T-DNA. The 3' end sequence to be added can be derived from,
example, the nopaline synthase or octopine synthase genes, or
alternatively from another plant gene, or less preferably from any
other eukaryotic gene.
[0068] An intron sequence can be added to the 5' untranslated
region or the coding sequence of the partial coding sequence to
increase the amount of the mature message that accumulates in the
cytosol. Inclusion of a spliceable intron in the transcription unit
in both plant and animal expression constructs has been shown to
increase gene expression at both the mRNA and protein levels up to
1000-fold. Buchman et al. (1988) Mol. Cell Biol. 8:4395-4405;
Callis et al. (1987) Genes Dev. 1:1183-1200. Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of maize introns
Adhl-S intron 1, 2, and 6, the Bronze-I intron are known in the
art. See generally, The Maize Handbook, Chapter 116, Freeling and
Walbot, eds., Springer, New York (1994).
[0069] The vector comprising the sequences from a polynucleotide of
the present invention could comprise a selectable marker gene for
the selection of transformed cells or tissues. Selectable marker
genes include genes encoding antibiotic resistance, such as those
encoding neomycin phosphotransferase II (NEO) and hygromycin
phosphotransferase (HPT), as well as genes conferring resistance to
herbicidal compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See
generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;
Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)
Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,
pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987)
Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA
90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956;
Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother 35:1591-1595;
Kleinschmidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993)
Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc.
Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.
Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of
Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill
et al. (1988) Nature 334:721-724. Such disclosures are herein
incorporated by reference.
[0070] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the present
invention.
[0071] Typical vectors useful for expression of genes in higher
plants are well known in the art and include vectors derived from
the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described by Rogers et al. (1987) Meth. in Enzymol. 153:253-277.
These vectors are plant integrating vectors in that on
transformation, the vectors integrate a portion of vector DNA into
the genome of the host plant. Exemplary A. tumefaciens vectors
useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al.
(1987) Gene 61:1-11 and Berger et al. (1989) Proc. Natl. Acad. Sci.
(USA) 86:8402-8406. Another useful vector herein is plasmid
pBI101.2 that is available from Clontech Laboratories, Inc. (Palo
Alto, Calif.).
[0072] As discussed above, a polynucleotide of the present
invention can be expressed in either sense or antisense orientation
as desired. It will be appreciated that control of gene expression
in either sense or antisense orientation can have a direct impact
on the observable plant characteristics. Antisense technology can
be conveniently used for gene expression in plants. To accomplish
this, a nucleic acid segment from the desired gene is cloned and
operably linked to a promoter such that the antisense strand of RNA
will be transcribed. The construct is then transformed into plants
and the antisense strand of RNA is produced. In plant cells, it has
been shown that antisense RNA inhibits gene expression by
preventing the accumulation of mRNA which encodes the enzyme of
interest, see, e.g., Sheehy et al. (1988) Proc. Natl. Acad. Sci.
(USA) 85:8805-8809; and Hiatt et al., U.S. Pat. No. 4,801,340.
[0073] In the methods of the invention, it is recognized that the
entire coding sequence for the RPA construct may be utilized.
Alternatively, portions or fragments of the sequence may be used in
DNA constructs.
[0074] Fragments and variants of the disclosed nucleotide sequences
and proteins encoded thereby are encompassed by the present
invention. By "fragment" is intended a portion of the nucleotide
sequence or a portion of the amino acid sequence and hence protein
encoded thereby. Fragments of a nucleotide sequence may encode
protein fragments that retain the biological activity of the native
protein and hence modulate DNA metabolism. Alternatively, fragments
of a nucleotide sequence that are useful as hybridization probes
generally do not encode fragment proteins retaining biological
activity. Thus, fragments of a nucleotide sequence may range from
at least about 20 nucleotides, about 50 nucleotides, about 100
nucleotides, and up to the full-length nucleotide sequence encoding
the proteins of the invention.
[0075] A fragment of a RPA nucleotide sequence that encodes a
biologically active portion of a RPA protein of the invention will
encode at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous
amino acids, or up to the total number of amino acids present in a
full-length RPA protein of the invention (for example, 623, 617,
273, 273, 273, 318, 273, 273 amino acids for SEQ ID NOs: 2, 4, 12,
14, 16, 18, 20, and 22 respectively. Fragments of a RPA nucleotide
sequence that are useful as hybridization probes for PCR primers
generally need not encode a biologically active portion of a RPA
protein.
[0076] Thus, a fragment of a RPA nucleotide sequence may encode a
biologically active portion of a RPA protein, or it may be a
fragment that can be used as a hybridization probe or PCR primer
using methods disclosed below. A biologically active portion of a
RPA protein can be prepared by isolating a portion of one of the
RPA nucleotide sequences of the invention, expressing the encoded
portion of the RPA protein (e.g., by recombinant expression in
vitro), and assessing the activity of the encoded portion of the
RPA protein. Nucleic acid molecules that are fragments of a RPA
nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900,
1,000 nucleotides, or up to the number of nucleotides present in a
full-length RPA nucleotide sequence disclosed herein (for example,
2497, 2202, 1124, 979, 1051, 1087, 1074, and 1231 nucleotides for
SEQ ID NOs: 1, 3, 11, 13, 15, 17, 19, and 21 respectively.
[0077] By "variants" is intended substantially similar sequences.
For nucleotide sequences, conservative variants include those
sequences that, because of the degeneracy of the genetic code,
encode the amino acid sequence of one of the RPA polypeptides of
the invention. Such naturally occurring variants including
naturally occurring allelic variants, can be identified with the
use of well-known molecular biology techniques, as, for example,
with polymerase chain reaction (PCR) and hybridization techniques
as outlined below. Variant nucleotide sequences also include
synthetically derived nucleotide sequences, such as those
generated, for example, by using site-directed mutagenesis but
which still encode a RPA protein of the invention. Generally,
variants of a particular nucleotide sequence of the invention will
have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%,
preferably about 90% to 95% or more, and more preferably about 98%
or more sequence identity to that particular nucleotide sequence as
determined by sequence alignment programs described elsewhere
herein using default parameters.
[0078] By "variant" protein is intended a protein derived from the
native protein by deletion (so-called truncation) or addition of
one or more amino acids to the N-terminal and/or C-terminal end of
the native protein; deletion or addition of one or more amino acids
at one or more sites in the native protein; or substitution of one
or more amino acids at one or more sites in the native protein.
Variant proteins encompassed by the present invention are
biologically active, that is they continue to possess the desired
biological activity of the native protein, that is, modulating DNA
metabolism as described herein. Such variants may result from, for
example, genetic polymorphism or from human manipulation.
Biologically active variants of a native RPA protein of the
invention will have at least 40%, 50%, 60%, 70%, generally at least
75%, 80%, 85%, preferably about 90% to 95% or more, and more
preferably about 98% or more sequence identity to the amino acid
sequence for the native protein as determined by sequence alignment
programs described elsewhere herein using default parameters. A
biologically active variant of a protein of the invention may
differ from that protein by as few as 1-15 amino acid residues, as
few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even
1 amino acid residue.
[0079] The proteins of the invention may be altered in various ways
including amino acid substitutions, deletions, truncations, and
insertions. Methods for such manipulations are generally known in
the art. For example, amino acid sequence variants of the RPA
proteins can be prepared by mutations in the DNA. Methods for
mutagenesis and nucleotide sequence alterations are well known in
the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA
82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382;
U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques
in Molecular Biology (MacMillan Publishing Company, New York) and
the references cited therein. Guidance as to appropriate amino acid
substitutions that do not affect biological activity of the protein
of interest may be found in the model of Dayhoff et al. (1978)
Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,
Washington, D.C.), herein incorporated by reference. Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be preferred.
[0080] Thus, the genes and nucleotide sequences of the invention
include both the naturally occurring sequences as well as mutant
forms. Likewise, the proteins of the invention encompass both
naturally occurring proteins as well as variations and modified
forms thereof. Such variants will continue to possess the desired
activity in influencing DNA metabolism. Obviously, the mutations
that will be made in the DNA encoding the variant must not place
the sequence out of reading frame and preferably will not create
complementary regions that could produce secondary mRNA structure.
See, EP Patent Application Publication No. 75,444.
[0081] The deletions, insertions, and substitutions of the protein
sequence encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by routine
screening assays. That is, the activity can be evaluated by
assessing DNA binding, recombination, repair and replication. See,
for example, Braun et al. (1997) Biochemistry 36:8443-8454;
Longhese et al. (1994) Molecular and Cellular Biology 14:7884-7890;
Stigger et al. (1998) J. Biol. Chem. 273:9337-9343; Abremova et al.
(1997) Proc. Natl. Acad. Sci. USA 94:7186-7191; New et al. (1998)
Nature 391:407-410; Bochkareva et al. (1998) J. Biol. Chem.
273(7):3932-6; Mass et al. (1998) Mol. Cell. Biol. 18(11):6399-407;
Lavrik et al. (1998) Nucleic Acids Res 26(2):602-7; Sibenaller et
al. (1998) 37(36):12496-506; Matsunaga et al. (1996) J. Biol. Chem.
271 (19):11047-50; and Sung (1997) Genes & Development
11:1111-21, herein incorporated by reference.
[0082] Variant nucleotide sequences and proteins also encompass
nucleotide sequences and proteins derived from a mutagenic and
recombinogenic procedure such as DNA shuffling. With such a
procedure, one or more different RPA coding sequences can be
manipulated to create a new RPA possessing the desired properties.
In this manner, libraries of recombinant polynucleotides are
generated from a population of related sequence polynucleotides
comprising sequence regions that have substantial sequence identity
and can be homologously recombined in vitro or in vivo. For
example, using this approach, sequence motifs encoding a domain of
interest may be shuffled between the RPA gene of the invention and
other known RPA genes to obtain a new gene coding for a protein
with an improved property of interest, such as an increased K.sub.m
in the case of an enzyme. Strategies for such DNA shuffling are
known in the art. See, for example, Stemmer (1994) Proc. Natl.
Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391;
Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al.
(1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.
Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature
391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
[0083] It is recognized that with these nucleotide sequences,
antisense constructions, complementary to at least a portion of the
messenger RNA (mRNA) for the RPA sequences can be constructed.
Antisense nucleotides are constructed to hybridize with the
corresponding mRNA. Modifications of the antisense sequences may be
made as long as the sequences hybridize to and interfere with
expression of the corresponding mRNA. In this manner, antisense
constructions having 70%, preferably 80%, more preferably 85%
sequence similarity to the corresponding antisense sequences may be
used. Furthermore, portions of the antisense nucleotides may be
used to disrupt the expression of the target gene. Generally,
sequences of at least 50 nucleotides, 100 nucleotides, 200
nucleotides, or greater may be used.
[0084] The nucleotide sequences of the present invention may also
be used in the sense orientation to suppress the expression of
endogenous genes in plants. Methods for suppressing gene expression
in plants using nucleotide sequences in the sense orientation are
known in the art. The methods generally involve transforming plants
with a DNA construct comprising a promoter that drives expression
in a plant operably linked to at least a portion of a nucleotide
sequence that corresponds to the transcript of the endogenous gene.
Typically, such a nucleotide sequence has substantial sequence
identity to the sequence of the transcript of the endogenous gene,
preferably greater than about 65% sequence identity, more
preferably greater than about 85% sequence identity, most
preferably greater than about 95% sequence identity. See, U.S. Pat.
Nos. 5,283,184 and 5,034,323; herein incorporated by reference.
[0085] Use of the polypeptides and proteins, and fragments and
variants thereof, for producing antibodies are also encompassed by
the invention. The invention also encompasses using such antibodies
to determine RPA protein levels, and to modulate one or more
biological activities or interactions of RPA. Methods for the
production of antibodies are known in the art. See, for example,
Harlow and Lane, antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York (1988); and the reference is cited
therein.
[0086] The RPA sequences of the invention may be optimized for
enhanced expression in plants of interest. See, for example,
EPA0359472; WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci.
USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Res.
17:477-498. In this manner, the genes can be synthesized utilizing
plant-preferred codons. See, for example, Murray et al. (1989)
Nucleic Acids Res. 17:477-498, the disclosure of which is
incorporated herein by reference. In this manner, synthetic genes
can also be made based on the distribution of codons a particular
host uses for a particular amino acid. Thus, the nucleotide
sequences can be optimized for expression in any plant. It is
recognized that all or any part of the gene sequence may be
optimized or synthetic. That is, synthetic or partially optimized
sequences may also be used.
[0087] Thus nucleotide sequences of the invention and the proteins
encoded thereby include the native forms as well as variants
thereof. The variant proteins will be substantially homologous and
functionally equivalent to the native proteins. A variant of a
native protein is "substantially homologous" to the native protein
when at least about 80%, more preferably at least about 90%, and
most preferably at least about 95% of its amino acid sequence is
identical to the amino acid sequence of the native protein. By
"functionally equivalent" is intended that the sequence of the
variant defines a chain that produces a protein having
substantially the same biological effect as the native protein of
interest. Such functionally equivalent variants that comprise
substantial sequence variations are also encompassed by the
invention.
[0088] The nucleotide sequences of the invention can be used to
isolate corresponding sequences from other organisms, particularly
other plants, more particularly other monocots. In this manner,
methods such as PCR, hybridization, and the like can be used to
identify such sequences based on their sequence homology to the
sequence set forth herein. Sequences isolated based on their
sequence identity to the entire RPA sequences set forth herein or
to fragments thereof are encompassed by the present invention.
[0089] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any plant of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies
(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR
Methods Manual (Academic Press, New York). Known methods of PCR
include, but are not limited to, methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers,
partially-mismatched primers, and the like.
[0090] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P, or any other detectable marker. Thus, for
example, probes for hybridization can be made by labeling synthetic
oligonucleotides based on the RPA sequences of the invention.
Methods for preparation of probes for hybridization and for
construction of cDNA and genomic libraries are generally known in
the art and are disclosed in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.).
[0091] For example, the entire RPA sequence disclosed herein, or
one or more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding RPA sequences and
messenger RNAs. To achieve specific hybridization under a variety
of conditions, such probes include sequences that are unique among
RPA sequences and are preferably at least about 10 nucleotides in
length, and most preferably at least about 20 nucleotides in
length. Such probes may be used to amplify corresponding RPA
sequences from a chosen plant by PCR. This technique may be used to
isolate additional coding sequences from a desired plant or as a
diagnostic assay to determine the presence of coding sequences in a
plant Hybridization techniques include hybridization screening of
plated DNA libraries (either plaques or colonies; see, for example,
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
[0092] Hybridization of such sequences may be carried out under
stringent conditions. By "stringent conditions" or "stringent
hybridization conditions" is intended conditions under which a
probe will hybridize to its target sequence to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences that are 100% complementary to the probe can be
identified (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length, preferably less than 500 nucleotides in length.
[0093] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times.to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C.
[0094] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6
(log M)+0.41 (%GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, %GC is the percentage of guanosine and cytosine
nucleotides in the DNA, % form is the percentage of formamide in
the hybridization solution, and L is the length of the hybrid in
base pairs. The T.sub.m is the temperature (under defined ionic
strength and pH) at which 50% of a complementary target sequence
hybridizes to a perfectly matched probe. T.sub.m is reduced by
about 1.degree. C. for each 1% of mismatching; thus, T.sub.m,
hybridization, and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with .gtoreq.90% identity are sought, the T.sub.m can be decreased
10.degree. C. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence and its complement at a defined ionic
strength and pH. However, severely stringent conditions can utilize
a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than
the thermal melting point (T.sub.m); moderately stringent
conditions can utilize a hybridization and/or wash at 6, 7, 8, 9,
or 10.degree. C. lower than the thermal melting point (T.sub.m);
low stringency conditions can utilize a hybridization and/or wash
at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal
melting point (T.sub.m). Using the equation, hybridization and wash
compositions, and desired T.sub.m, those of ordinary skill will
understand that variations in the stringency of hybridization
and/or wash solutions are inherently described. If the desired
degree of mismatching results in a T.sub.m of less than 45.degree.
C. (aqueous solution) or 32.degree. C. (formamide solution), it is
preferred to increase the SSC concentration so that a higher
temperature can be used. An extensive guide to the hybridization of
nucleic acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology-Hybridization with Nucleic Acid
Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al.,
eds. (1995) Current Protocols in Molecular Biology, Chapter 2
(Greene Publishing and Wiley-Interscience, New York). See Sambrook
et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold
Spring Harbor Laboratory Press, Plainview, N.Y.).
[0095] Thus, isolated sequences that have promoter activity or
encode for a RPA protein and which hybridize under stringent
conditions to the RPA sequences disclosed herein, or to fragments
thereof, are encompassed by the present invention. Such sequences
will be at least 40% to 50% homologous, about 60% to 70%
homologous, and even about 75%, 80%, 85%, 90%, 95% to 98%
homologous or more with the disclosed sequences. That is, the
sequence identity of sequences may range, sharing at least 40% to
50%, about 60% to 70%, and even about 75%, 80%, 85%, 90%, 95% to
98% or more sequence identity.
[0096] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0097] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0098] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0099] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent identity
between any two sequences can be accomplished using a mathematical
algorithm. Preferred, non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller (1988) CABIOS
4:11-17; the local homology algorithm of Smith et al. (1981) Adv.
Appl. Math. 2:482; the homology alignment algorithm of Needleman
and Wunsch (1970) J. Mol. Biol. 48:443-453; the
search-for-similarity-method of Pearson and Lipman (1988) Proc.
Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0100] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Version 8 (available from Genetics Computer Group
(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using
these programs can be performed using the default parameters. The
CLUSTAL program is well described by Higgins et al. (1988) Gene
73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet
et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)
CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol.
24:307-331. The ALIGN program is based on the algorithm of Myers
and Miller (1988) supra. A PAM120 weight residue table, a gap
length penalty of 12, and a gap penalty of 4 can be used with the
ALIGN program when comparing amino acid sequences. The BLAST
programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based
on the algorithm of Karlin and Altschul (1990) supra. BLAST
nucleotide searches can be performed with the BLASTN program,
score=100, wordlength=12, to obtain nucleotide sequences homologous
to a nucleotide sequence encoding a protein of the invention. BLAST
protein searches can be performed with the BLASTX program,
score=50, wordlength=3, to obtain amino acid sequences homologous
to a protein or polypeptide of the invention. To obtain gapped
alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can
be utilized as described in Altschul et al. (1997) Nucleic Acids
Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used
to perform an iterated search that detects distant relationships
between molecules. See Altschul et al. (1997) supra. When utilizing
BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the
respective programs (e.g., BLASTN for nucleotide sequences, BLASTX
for proteins) can be used. See www.ncbi.nlm.nih.ov. Alignment may
also be performed manually by inspection. Alignment may also be
performed manually by inspection.
[0101] For purposes of the present invention, comparison of
nucleotide or protein sequences for determination of percent
sequence identity to the RPA sequences disclosed herein is
preferably made using the GCG PileUp program, version 10.00, with
its default parameters or any equivalent program. By "equivalent
program" is intended any sequence comparison program that, for any
two sequences in question, generates an alignment having identical
nucleotide or amino acid residue matches and an identical percent
sequence identity when compared to the corresponding alignment
generated by the preferred program.
[0102] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0103] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0104] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70% sequence identity, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95%, compared
to a reference sequence using one of the alignment programs
described using standard parameters. One of skill in the art will
recognize that these values can be appropriately adjusted to
determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
normally means sequence identity of at least 60%, more preferably
at least 70%, 80%, 90%, and most preferably at least 95%.
[0105] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. However, stringent conditions encompass
temperatures in the range of about 1.degree. C. to about 20.degree.
C., depending upon the desired degree of stringency as otherwise
qualified herein. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
polypeptides they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is when the polypeptide encoded by the first nucleic acid
is immunologically cross reactive with the polypeptide encoded by
the second nucleic acid.
[0106] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70% sequence identity to a reference sequence, preferably 80%, more
preferably 85%, most preferably at least 90% or 95% sequence
identity to the reference sequence over a specified comparison
window. Preferably, optimal alignment is conducted using the
homology alignment algorithm of Needleman et al. (1970) J. Mol.
Biol. 48:443. An indication that two peptide sequences are
substantially identical is that one peptide is immunologically
reactive with antibodies raised against the second peptide. Thus, a
peptide is substantially identical to a second peptide, for
example, where the two peptides differ only by a conservative
substitution. Peptides that are "substantially similar" share
sequences as noted above except that residue positions that are not
identical may differ by conservative amino acid changes.
[0107] Using the nucleic acids of the present invention, one may
express a protein of the present invention in a recombinantly
engineered cell such as bacteria, yeast, insect, mammalian, or
preferably plant cells. The cells produce the protein in a
non-natural condition (e.g., in quantity, composition, location,
and/or time), because they have been genetically altered through
human intervention to do so.
[0108] It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of a nucleic acid encoding a protein of the present
invention. No attempt to describe in detail the various methods
known for the expression of proteins in prokaryotes or eukaryotes
will be made.
[0109] In brief summary, the expression of isolated nucleic acids
encoding a protein of the present invention will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter (which is either constitutive or inducible), followed by
incorporation into an expression vector. The vectors can be
suitable for replication and integration in either prokaryotes or
eukaryotes. Typical expression vectors contain transcription and
translation terminators, initiation sequences, and promoters useful
for regulation of the expression of the DNA encoding a protein of
the present invention. To obtain high level expression of a cloned
gene, it is desirable to construct expression vectors which
contain, at the minimum, a strong promoter to direct transcription,
a ribosome binding site for translational initiation, and a
transcription/translation terminator. One of skill would recognize
that modifications can be made to a protein of the present
invention without diminishing its biological activity. Some
modifications may be made to facilitate the cloning, expression, or
incorporation of the targeting molecule into a fusion protein. Such
modifications are well known to those of skill in the art and
include, for example, a methionine added at the amino terminus to
provide an initiation site, or additional amino acids (e.g., poly
His) placed on either terminus to create conveniently located
restriction sites or termination codons or purification
sequences.
[0110] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, include such commonly used promoters as the beta
lactamase (penicillinase) and lactose (lac) promoter systems (Chang
et al. (1977) Nature 198:1056), the tryptophan (trp) promoter
system (Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the
lambda-derived P L promoter and N-gene ribosome binding site
(Shimatake et al. (1981) Nature 292:128). The inclusion of
selection markers in DNA vectors transfected in E. coli is also
useful. Examples of such markers include genes specifying
resistance to ampicillin, tetracycline, or chloramphenicol.
[0111] The vector is selected to allow introduction into the
appropriate host cell. Bacterial vectors are typically of plasmid
or phage origin. Appropriate bacterial cells are infected with
phage vector particles or transfected with naked phage vector DNA.
If a plasmid vector is used, the bacterial cells are transfected
with the plasmid vector DNA. Expression systems for expressing a
protein of the present invention are available using Bacillus sp.
and Salmonella (Palva et al. (1983) Gene 22:229-235; Mosbach et
al., (1983) Nature 302:543-545).
[0112] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. The sequences of the present invention can be
expressed in these eukaryotic systems. In some embodiments,
transformed/transfected plant cells are employed as expression
systems for production of the proteins of the instant
invention.
[0113] Synthesis of heterologous proteins in yeast is well known.
Sherman, F. et al. (1982) Methods in Yeast Genetics, Cold Spring
Harbor Laboratory is a well recognized work describing the various
methods available to produce the protein in yeast. Two widely
utilized yeast for production of eukaryotic proteins are
Saccharomyces cerevisia and Pichia pastoris. Vectors, strains, and
protocols for expression in Saccharomyces and Pichia are known in
the art and available from commercial suppliers (e.g., Invitrogen).
Suitable vectors usually have expression control sequences, such as
promoters, including 3-phosphoglycerate kinase or alcohol oxidase,
and an origin of replication, termination sequences and the like as
desired.
[0114] A protein of the present invention, once expressed, can be
isolated from yeast by lysing the cells and applying standard
protein isolation techniques to the lysates. The monitoring of the
purification process can be accomplished by using Western blot
techniques or radioimmunoassay of other standard immunoassay
techniques.
[0115] The sequences encoding proteins of the present invention can
also be ligated to various expression vectors for use in
transfecting cell cultures of, for instance, mammalian, insect, or
plant origin. Illustrative of cell cultures useful for the
production of the peptides are mammalian cells. Mammalian cell
systems often will be in the form of monolayers of cells although
mammalian cell suspensions may also be used. A number of suitable
host cell lines capable of expressing intact proteins have been
developed in the art, and include the HEK293, BHK21, and CHO cell
lines. Expression vectors for these cells can include expression
control sequences, such as an origin of replication, a promoter
(e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate
kinase promoter)), an enhancer (Queen et al. (1986) Immunol. Rev.
89:49), and necessary processing information sites, such as
ribosome binding sites, RNA splice sites, polyadenylation sites
(e.g., an SV40 large T Ag poly A addition site), and
transcriptional terminator sequences. Other animal cells useful for
production of proteins of the present invention are available, for
instance, from the American Type Culture Collection Catalogue of
Cell Lines and Hybridomas (7th edition, 1992).
[0116] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, armyworm, moth and Drosophila cell lines such as a
Schneider cell line (See Schneider et al. (1987) J. Embryol. Exp.
Morphol. 27:353-365).
[0117] As with yeast, when higher animal or plant host cells are
employed, polyadenylation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenylation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague et al. (1983) J. Virol. 45:773-781).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus-type vectors. Saveria-Campo, M., Bovine
Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning Vol.
II a Practical Approach, D. M. Glover, ed., IRL Press, Arlington,
Va. pp. 213-238 (1985).
[0118] The sequences of the invention can be introduced into any
plant of interest, and used for transformation of any plant
species. The sequences to be introduced may be used in expression
cassettes for expression in the particular plant of interest.
[0119] Plants of interest include, but are not limited to corn (Zea
mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),
particularly those Brassica species useful as sources of seed oil,
alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale
cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,
pearl millet (Pennisetum glaucum), proso millet (Panicum
miliaceum), foxtail millet (Setaria italica), finger millet
(Eleusine coracana)), sunflower (Helianthus annuus), safflower
(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine
max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum),
peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot
esculenta), coffee (Cofea spp.), coconut (Cocos nucifera),
pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa
(Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.),
avocado (Persea americana), fig (Ficus casica), guava (Psidium
guajava), mango (Mangifera indica), olive (Olea europaea), papaya
(Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers.
[0120] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum. Conifers that may be employed in
practicing the present invention include, for example, pines such
as loblolly pine (Pinus taeda), slash pine (Pinus elliotil),
ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta),
and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga
menziesil); Western hemlock (Tsuga canadensis); Sitka spruce (Picea
glauca); redwood (Sequoia sempervirens); true firs such as silver
fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars
such as Western red cedar (Thuja plicata) and Alaska yellow-cedar
(Chamaecyparis nootkatensis). Preferably, plants of the present
invention are crop plants (for example, corn, alfalfa, sunflower,
Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,
millet, tobacco, etc.), more preferably corn and soybean plants,
yet more preferably corn plants.
[0121] Plants of particular interest include grain plants that
provide seeds of interest, oil-seed plants, and leguminous plants.
Seeds of interest include grain seeds, such as corn, wheat, barley,
rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, soybean, garden beans, cowpea, mungbean,
lima bean, fava bean, lentils, chickpea, etc.
[0122] The RPA coding and antisense sequences of the invention are
provided in expression cassettes for expression in the plant of
interest. The cassette will include 5' and 3' regulatory sequences
operably linked to a RPA sequence of the invention. The cassette
may additionally contain at least one additional gene to be
cotransformed into the organism. Alternatively, the additional
gene(s) can be provided on another expression cassette. By
"operably linked" is intended a functional linkage between a
promoter and a second sequence, wherein the promoter sequence
initiates and mediates transcription of the DNA sequence
corresponding to the second sequence. Generally, operably linked
means that the nucleic acid sequences being linked are contiguous
and, where necessary to join two protein coding regions, contiguous
and in the same reading frame.
[0123] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the RPA sequence to be under the
transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
[0124] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region, a RPA DNA sequence of the invention, and a transcriptional
and translational termination region functional in plants. The
transcriptional initiation region, the promoter, may be native or
analogous or foreign or heterologous to the plant host.
Additionally, the promoter may be the natural sequence or
alternatively a synthetic sequence. By "foreign" is intended that
the transcriptional initiation region is not found in the native
plant into which the transcriptional initiation region is
introduced. As used herein, a chimeric gene comprises a coding
sequence operably linked to a transcription initiation region that
is heterologous to the coding sequence.
[0125] While it may be preferable to express the sequences using
heterologous promoters, the native promoter sequences may be used.
Such constructs would change expression levels of RPA in the plant
or plant cell. Thus, the phenotype of the plant or plant cell is
altered.
[0126] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, or may be derived from another
source. Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also Guerineau et al.,
(1991) Mol. Gen. Genet 262:141-144; Proudfoot (1991) Cell
64:671-674; Sanfacon et al., (1991) Genes Dev. 5:141-149; Mogen et
al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene
91:151-158 Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903;
and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0127] Where appropriate, the gene(s) may be optimized for
increased expression in the transformed plant. That is, the genes
can be synthesized using plant-preferred codons for improved
expression. See, for example, Campbell and Gowri (1990) Plant
Physiol. 92:1-11 for a discussion of host-preferred codon usage.
Methods are available in the art for synthesizing plant-preferred
genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391,
and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein
incorporated by reference.
[0128] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0129] The expression cassettes may additionally contain 5' leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include: picornavirus leaders, for example,
EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et al. (1989) PNAS USA 86:6126-6130); polyvirus leaders, for
example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986);
MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and
human immunoglobulin heavy-chain binding protein (BiP), (Macejak et
al. (1991) Nature 353:90-94); untranslated leader from the coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.
(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV)
(Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,
New York), pp. 237-256); and maize chlorotic mottle virus leader
(MCMV) (Lommel et al. (1991) Virology 81:382-385). See also,
Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods
known to enhance translation can also be utilized, for example,
introns, and the like.
[0130] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0131] The sequences of the present invention can be used to
transform or transfect any plant. In this manner, genetically
modified plants, plant cells, plant tissue, seed, and the like can
be obtained. Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606, Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic
particle acceleration (see, for example, Sanford et al., U.S. Pat.
No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al.,
U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782;
Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells
via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture: Fundamental Methods, ed. Gamborg and Phillips
(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology
6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet.
22:421-477; Sanford et al. (1987) Particulate Science and
Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.
87:671-674 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.
Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl.
Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology
8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA
85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563
(maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat.
Nos. 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol.
91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839
(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)
311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals);
Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349
(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation
of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp.
197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports
9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566
(whisker-mediated transformation); D'Halluin et al. (1992) Plant
Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell
Reports 12:250-255 and Christou and Ford (1995) Annals of Botany
75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology
14:745-750 (maize via Agrobacterium tumefaciens); all of which are
herein incorporated by reference.
[0132] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
expression of the desired phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved.
[0133] 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 be
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.
[0134] A preferred embodiment 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
RPA expression relative to a control plant (i.e., native,
non-transgenic). Backcrossing to a parental plant and out-crossing
with a non-transgenic plant are also contemplated.
[0135] The present invention further provides a method for
modulating (i.e., increasing or decreasing) RPA levels in a plant
or part thereof. Modulation can be effected by increasing or
decreasing the total amount of RPA (i.e., its content) and/or the
ratio of various RPA subunit proteins (i.e., its composition) in
the plant. The method comprises transforming a plant cell with a
recombinant expression cassette comprising a polynucleotide of the
present invention as described above to obtain a transformed plant
cell, growing the transformed plant cell under plant forming
conditions, and inducing expression of a polynucleotide of the
present invention in the plant for a time sufficient to modulate
RPA content and/or composition in the plant or plant part.
[0136] In some embodiments, RPA in a plant may be modulated by
altering, in vivo or in vitro, the promoter of a non-isolated RPA
gene to up- or down-regulate gene expression. In some embodiments,
the coding regions of native RPA genes an be altered via
substitution, addition, insertion, or deletion to decrease activity
of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350;
Zarling et al., PCT/US93/03868. And in some embodiments, an
isolated nucleic acid (e.g., a vector) comprising a promoter
sequence is transfected into a plant cell. Subsequently, a plant
cell comprising the promoter operably linked to a polynucleotide of
the present invention is selected by means known to those of skill
in the art such as, but not limited to, Southern blot, DNA
sequencing, or PCR analysis using primers specific to the promoter
and to the gene and detecting amplicons produced therefrom. A plant
or plant part altered or modified by the foregoing embodiments is
grown under plant forming conditions for a time sufficient to
modulate RPA content and/or composition in the plant. Plant forming
conditions are well known in the art and discussed briefly,
supra.
[0137] In general, content or composition is increased or decreased
by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
relative to a native control plant, plant part, or cell lacking the
aforementioned recombinant expression cassette. Modulation in the
present invention may occur during and/or subsequent to growth of
the plant to the desired stage of development. Modulating nucleic
acid expression temporally and/or in particular tissues can be
controlled by employing the appropriate promoter operably linked to
a polynucleotide of the present invention in, for example, sense or
antisense orientation as discussed in greater detail, supra.
Induction of expression of a polynucleotide of the present
invention can also be controlled by exogenous administration of an
effective amount of inducing compound. Inducible promoters and
inducing compounds that activate expression from these promoters
are well known in the art. In preferred embodiments, RPA is
modulated in monocots, particularly maize.
[0138] The ability of RPA to interact with multiple proteins or
protein complexes allows it to participate and regulate these
multiple pathways of DNA metabolism. For example, it has been shown
in mammalian systems that are RPA interacts with DNA polymerase
alpha (Barun et al. (1997) Biochemistry 36:8443-8454), p53 (Dutta
et al. (1993) Nature 365:79-82), RAD 62 (Park et al. (1996) J.
Biol. Chem. 271:18996-19000).
[0139] Participation of the middle subunit of RPA in
protein-protein interactions has also been shown. Examples of such
interactions include, but are not limited to interactions with XPA
protein and RAD 52 (He et al. (1995) Nature 374:566-69; Matsuda et
al. (1995) J. Biol. Chem. 270:4152-57; Li et al. (1995) Mol. Cell.
Biol. 15:5396402, Park et al. (1996) J. Biol. Chem.
271:18996-19000); and PCNA (Shivji et al. (1995) Biochemistry
34:5011-5017).
[0140] Similarly, yeast RPA has been shown to be involved in
multiple functions in DNA metabolism (Umezu et al. (1998) Genetics
148:989-1005). Therefore, the proteins of the invention may be
useful as a ligand to purify and clone other proteins involved in
DNA recombination, repair, and replication. Particularly, the maize
proteins may be useful to purify other maize proteins involved in
DNA metabolism. For example, the RPA proteins of the invention may
be insolubilized on a solid matrix (e.g. agrose or nylon beads) for
affinity purification, or the RPA cDNA may be used as a bait in a
yeast to-hybrid system. In this manner, other proteins may be used
identified and isolated.
[0141] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
cDNA Cloning
[0142] Total RNA was isolated from corn tissues with TRIzol Reagent
(Life Technology, Inc. Gaithersburg, Md.) using a modification of
the guanidine isothiocyanate/acid-phenol procedure described by
Chomczynski and Sacchi (Chomczynski et al. (1987) Anal. Biochem.
162:156). In brief, plant tissue samples were pulverized in liquid
nitrogen before the addition of the TRIzol Reagent, and then were
further homogenized with a mortar and pestle. Addition of
chloroform by centrifugation was conducted for separation of an
aqueous phase and an organic phase. The total RNA was recovered by
precipitation with isopropyl alcohol from the aqueous phase.
[0143] The selection of poly(A)+ RNA from total RNA was performed
using PolyATract system (Promega Corporation, Madison, Wis.). In
brief, biotinylated oligo (dT) primers were used to hybridize to
the 3' poly(A) tails on mRNA. The hybrids were captured using
streptavidin coupled to paramagnetic particles and a magnetic
separation stand. The mRNA was washed at high stringent condition
and cluted by Rnase-free deionized water.
[0144] Synthesis of the cDNA was performed and unidirectional cDNA
libraries were constructed using the SuperScript Plasmid System
(Life Technology, Inc., Gaithersburg, Md.). First strand of cDNA
was synthesized by priming an oligo(dT) primer containing a Not I
site. The reaction was catalyzed by SuperScript Reverse
Transcriptase II at 45.degree. C. The second strand of cDNA was
labeled with .alpha.-.sup.32P-dCTP and portions of the molecules
smaller than 500 base pairs and unligated adapters were removed by
Sephacryl-S400 chromatography. The selected cDNA molecules were
ligated into pSPORT1 reference vector between the Not I and Sal I
sites.
[0145] Individual colonies were picked and DNA was prepared either
by PCR with M13 forward primers and M13 reverse primers, or by
plasmid miniprep isolation. All the cDNA clones were sequenced
using M13 reverse primers.
[0146] Two maize homologues for RPA large subunit (ZmRPALSH) have
been isolated. The genes map to two different chromosomes as shown
below in Table 1. The amino acid and nucleotide sequences for the
two homologues are set forth in SEQ ID NOs: 1-4.
1TABLE 1 Maize RPA Large Subunit Genes Map to Two Different
Chromosomes Clone ID Chromosome No. Homologue CBPBS68 c9 ZmRPALSH1
CCRBJ83 c9 ZmRPALSH1 CDPGS47 c9 ZmRPALSH1 CHCLE65 c9 ZmRPALSH1
CJLPL35 c9 ZmRPALSH1 COMGE67 c9 ZmRPALSH1 CBAAK06 c9 ZmRPALSH2
CDPGS46 c9 ZmRPALSH2 CERAG93 c9 ZmRPALSH2 COMFY67 c9 ZmRPALSH2
[0147] Ten ESTs, which form two different contigs for maize RPA
large subunit, were used as probes for mapping experiments. Each
contig represents one maize homologue for RPALS.
[0148] Seven maize homologues for RPA middle subunit (ZmRPAMSH)
have been isolated. The genes map to chromosomes 5 as shown below
in Table 2. The nucleotide and amino acid sequences of the seven
homologues are set forth in SEQ ID NOs: 11-22.
2TABLE 2 Maize Homologues of Eukaryotic Replication Protein A
Middle Subunit Map Clone ID Homologue Library Position CCRBK63
ZmRPAMSH-1 P0026 C5 CGEUZ26 ZmRPAMSH-2 P0002 TBD CGEVJ74 ZmRPAMSH-3
P0002 TBD CHSBX01 ZmRPABMS-4 P0118 C5 CIMME04 ZmRPAMSH-5 P0114 C5
CRTBB78 ZmRPAMSH-6 P0041 C5 CVRAP89 ZmRPAMSH-7 P0057 C5 TBD = To be
determined.
Example 2
Transformation and Regeneration of Transgenic Plants
[0149] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing the RPA antisense sequence of
the invention operably linked to a pathogen-inducible promoter
(FIG. 2) plus a plasmid containing the selectable marker gene PAT
(Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to
the herbicide Bialaphos. Transformation is performed as follows.
All media recipes are in the Appendix.
[0150] Preparation of Target Tissue
[0151] The ears are surface sterilized in 30% Chlorox bleach plus
0.5% Micro detergent for 20 minutes, and rinsed two times with
sterile water. The immature embryos are excised and placed embryo
axis side down (scutellum side up), 25 embryos per plate, on 560Y
medium for 4 hours and then aligned within the 2.5-cm target zone
in preparation for bombardment.
[0152] Preparation of DNA
[0153] A plasmid vector comprising the RPA sequence of the
invention operably linked to a ubiquitin promoter is made. This
plasmid DNA plus plasmid DNA containing a PAT selectable marker is
precipitated onto 1.1 .mu.m (average diameter) tungsten pellets
using a CaCl.sub.2 precipitation procedure as follows:
[0154] 100 .mu.l prepared tungsten particles in water
[0155] 10 .mu.l (1 .mu.g) DNA in TrisEDTA buffer (1 .mu.g
total)
[0156] 100 .mu.l 2.5 M CaC1.sub.2
[0157] 10 .mu.l 0.1 M spermidine
[0158] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
[0159] Particle Gun Treatment
[0160] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
[0161] Subsequent Treatment
[0162] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5" pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for expression of the RPA gene of interest.
3APPENDIX Ingredient Amount Unit 272 V D-I H.sub.2O 950.000 Ml MS
Salts (GIBCO 11117-074) 4.300 G Myo-Inositol 0.100 G MS Vitamins
Stock Solution ## 5.000 Ml Sucrose 40.000 G Bacto-Agar @ 6.000 G
Directions: @ = Add after bringing up to volume Dissolve
ingredients in polished D-I H.sub.2O in sequence Adjust to pH 5.6
Bring up to volume with polished D-I H.sub.2O after adjusting pH
Sterilize and cool to 60.degree. C. ## = Dissolve 0.100 g of
Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL;
and 0.400 g of Glycine in 875.00 ml of polished D-I H.sub.2O in
sequence. Bring up to volume with polished D-I H.sub.2O. Make in
400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark
Desiccator. Store for one month, unless contamination or
precipitation occurs, then make fresh stock. Total Volume (L) =
1.00 288 J D-I H.sub.2O 950.000 Ml MS Salts 4.300 G Myo-Inositol
0.100 G MS Vitamins Stock Solution ## 5.000 Ml Zeatin .5 mg/ml
1.000 Ml Sucrose 60.000 G Gelrite @ 3.000 G Indoleacetic Acid 0.5
mg/ml # 2.000 Ml 0.1 mM Abscisic Acid 1.000 Ml Bialaphos 1 mg/ml #
3.000 Ml Directions: @ = Add after bringing up to volume Dissolve
ingredients in polished D-I H.sub.2O in sequence Adjust to pH 5.6
Bring up to volume with polished D-I H.sub.2O after adjusting pH
Sterilize and cool to 60.degree. C. Add 3.5 g/L of Gelrite for cell
biology. ## = Dissolve 0.100 g of Nicotinic Acid; 0.020 g of
Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in
875.00 ml of polished D-I H.sub.2O in sequence. Bring up to volume
with polished D-I H.sub.2O. Make in 400 ml portions. Thiamine.HCL
& Pyridoxine.HCL are in Dark Desiccator. Store for one month,
unless contamination or precipitation occurs, then make fresh
stock. Total Volume (L) = 1.00 560 R D-I Water, Filtered 950.000 Ml
CHU (N6) Basal Salts (SIGMA C-1416) 4.000 G Eriksson's Vitamin Mix
(1000X SIGMA-1511 1.000 Ml Thiamine.HCL 0.4 mg/ml 1.250 Ml Sucrose
30.000 G 2,4-D 0.5 mg/ml 4.000 Ml Gelrite @ 3.000 G Silver Nitrate
2 mg/ml # 0.425 Ml Bialaphos 1 mg/ml # 3.000 Ml Directions: @ = Add
after bringing up to volume # = Add after sterilizing and cooling
to temp. Dissolve ingredients in D-I H.sub.2O in sequence Adjust to
pH 5.8 with KOH Bring up to volume with D-I H.sub.2O Sterilize and
cool to room temp. Total Volume (L) = 1.00 560 Y D-I Water,
Filtered 950.000 Ml CHU (N6) Basal Salts (SIGMA C-1416) 4.000 G
Eriksson's Vitamin Mix (1000X SIGMA-1511 1.000 Ml Thiamine.HCL 0.4
mg/ml 1.250 Ml Sucrose 120.000 G 2,4-D 0.5 mg/ml 2.000 Ml L-Proline
2.880 G Gelrite @ 2.000 G Silver Nitrate 2 mg/ml # 4.250 Ml
Directions: @ = Add after bringing up to volume # = Add after
sterilizing and cooling to temp. Dissolve ingredients in D-I
H.sub.2O in sequence Adjust to pH 5.8 with KOH Bring up to volume
with D-I H.sub.2O Sterilize and cool to room temp. **Autoclave less
time because of increased sucrose** Total Volume (L) = 1.00 All
publications and patent applications mentioned in the specification
are indicative of the level of those skilled in the art to which
this invention pertains. All publications and patent applications
are herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference.
[0163] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
22 1 2497 DNA Zea Mays CDS (157)...(2025) Coding sequence for the
Maize RPA Large Subunit Homologue-1 1 ccttatcata ttataagcgc
gcgtagcctt ggcagctcga cgcatcttcg cctccgctca 60 acgctcgccc
acgcccccag cccccaccga tccacgagaa accttctcgc ctccgcggga 120
cgattcgcca gggagagcaa aggtagcaga ggcgcc atg gac gct gcc aag tcg 174
Met Asp Ala Ala Lys Ser 1 5 gtg acg ccg ggc gcc gtg tcc tac atc ctg
gcg cac ccg tct acg ggc 222 Val Thr Pro Gly Ala Val Ser Tyr Ile Leu
Ala His Pro Ser Thr Gly 10 15 20 tcc gat ggc gcc gtg tcg gat ctc
gtc gtt cag gtc ctc gat ctc aag 270 Ser Asp Gly Ala Val Ser Asp Leu
Val Val Gln Val Leu Asp Leu Lys 25 30 35 tcc atc ggc atg ggc agc
cgg ttc agt ttc acg gca tcc gat ggg aac 318 Ser Ile Gly Met Gly Ser
Arg Phe Ser Phe Thr Ala Ser Asp Gly Asn 40 45 50 gac aaa atc aag
gcg atg ctc ccc act tac ttt gcg tcg gag gtc cac 366 Asp Lys Ile Lys
Ala Met Leu Pro Thr Tyr Phe Ala Ser Glu Val His 55 60 65 70 tcc ggc
aat ctg aag aat ttc ggt ctc atc cgc atc ctc gac tac act 414 Ser Gly
Asn Leu Lys Asn Phe Gly Leu Ile Arg Ile Leu Asp Tyr Thr 75 80 85
tgc aac tcc gtc aaa ggc aac gct gac aaa gtc ctg att gtc gtc aaa 462
Cys Asn Ser Val Lys Gly Asn Ala Asp Lys Val Leu Ile Val Val Lys 90
95 100 tgc gag act gtg tgc gaa gcg ctc gac gcc gag atc aac ggc gag
gcc 510 Cys Glu Thr Val Cys Glu Ala Leu Asp Ala Glu Ile Asn Gly Glu
Ala 105 110 115 aag aaa gag gat cct cca att gtg ctg aag cct aaa gac
gaa ggc tca 558 Lys Lys Glu Asp Pro Pro Ile Val Leu Lys Pro Lys Asp
Glu Gly Ser 120 125 130 gtc gtg gct gag gaa aca aat tct ccc cca ctc
gtg atg aag cct aag 606 Val Val Ala Glu Glu Thr Asn Ser Pro Pro Leu
Val Met Lys Pro Lys 135 140 145 150 caa gag gtg aag tcc gcg tcc cag
atc gtg act gag cag cgt gga aat 654 Gln Glu Val Lys Ser Ala Ser Gln
Ile Val Thr Glu Gln Arg Gly Asn 155 160 165 gct gct cct gcc acg cgc
ctt tcc atg aca agg agg gtc cat ccc ttg 702 Ala Ala Pro Ala Thr Arg
Leu Ser Met Thr Arg Arg Val His Pro Leu 170 175 180 atc act ctg aac
ccc tac cag ggt aac tgg gtc att aag gtg cgg gtc 750 Ile Thr Leu Asn
Pro Tyr Gln Gly Asn Trp Val Ile Lys Val Arg Val 185 190 195 acg agc
aaa ggc aat ctg aga acc tac agg aat gct cgt gga gaa ggc 798 Thr Ser
Lys Gly Asn Leu Arg Thr Tyr Arg Asn Ala Arg Gly Glu Gly 200 205 210
tgc gtc ttc aac gta gag ctt act gat gag gat ggc acc cag atc cag 846
Cys Val Phe Asn Val Glu Leu Thr Asp Glu Asp Gly Thr Gln Ile Gln 215
220 225 230 gcc acc atg ttt aac gag gct gca aag aag ttc tat cca att
ttt gag 894 Ala Thr Met Phe Asn Glu Ala Ala Lys Lys Phe Tyr Pro Ile
Phe Glu 235 240 245 ctg gga aag gtc tat tat gtc tca aaa gga tct ctt
aga att gcc aac 942 Leu Gly Lys Val Tyr Tyr Val Ser Lys Gly Ser Leu
Arg Ile Ala Asn 250 255 260 aag cag ttc aag aca gtc aaa aat gac tat
gag ttg tca cta aac gag 990 Lys Gln Phe Lys Thr Val Lys Asn Asp Tyr
Glu Leu Ser Leu Asn Glu 265 270 275 aat gct att gtt gaa gaa gca gag
ggg gag act ttc ctt cca cca gtg 1038 Asn Ala Ile Val Glu Glu Ala
Glu Gly Glu Thr Phe Leu Pro Pro Val 280 285 290 caa tac aac ctt gtc
aag att gat cag cta gga cca tac gtc ggt ggc 1086 Gln Tyr Asn Leu
Val Lys Ile Asp Gln Leu Gly Pro Tyr Val Gly Gly 295 300 305 310 agg
gag ctt gta gat att gtt ggt gtg gtt cag agc gta tct ccc aca 1134
Arg Glu Leu Val Asp Ile Val Gly Val Val Gln Ser Val Ser Pro Thr 315
320 325 ctc agt gtt agg aga aag att gac aac gag aca ata ccg aag cgt
gac 1182 Leu Ser Val Arg Arg Lys Ile Asp Asn Glu Thr Ile Pro Lys
Arg Asp 330 335 340 att gtt gta gca gac gac tct ggc aaa act gtt act
att tct ctc tgg 1230 Ile Val Val Ala Asp Asp Ser Gly Lys Thr Val
Thr Ile Ser Leu Trp 345 350 355 aat gat ctt gct act acg act ggc caa
gag ctt ttg gac atg gtt gac 1278 Asn Asp Leu Ala Thr Thr Thr Gly
Gln Glu Leu Leu Asp Met Val Asp 360 365 370 agt tcg cct gtt gtt gcg
ata aag agc cta aaa gta tct gac ttc caa 1326 Ser Ser Pro Val Val
Ala Ile Lys Ser Leu Lys Val Ser Asp Phe Gln 375 380 385 390 ggc gtg
tct ctt tca act att ggc aga agt act ctc gag att aat cct 1374 Gly
Val Ser Leu Ser Thr Ile Gly Arg Ser Thr Leu Glu Ile Asn Pro 395 400
405 gac ctg cct gag gct aag aat ctt aag tcc tgg tat gac tct gaa ggc
1422 Asp Leu Pro Glu Ala Lys Asn Leu Lys Ser Trp Tyr Asp Ser Glu
Gly 410 415 420 aaa gat act tca ctg gca cca atc agt gca gaa gcg ggt
gcc aca cgc 1470 Lys Asp Thr Ser Leu Ala Pro Ile Ser Ala Glu Ala
Gly Ala Thr Arg 425 430 435 gct ggt ggt ttc aag tcc atg tat tct gat
aga gtt ttt ctg tct cac 1518 Ala Gly Gly Phe Lys Ser Met Tyr Ser
Asp Arg Val Phe Leu Ser His 440 445 450 atc acc agt gat cct gct atg
ggc cag gaa aag cct gtt ttc ttc agt 1566 Ile Thr Ser Asp Pro Ala
Met Gly Gln Glu Lys Pro Val Phe Phe Ser 455 460 465 470 ctg tac gcc
atc ata agc cac atc aag cct gat cag aat atg tgg tac 1614 Leu Tyr
Ala Ile Ile Ser His Ile Lys Pro Asp Gln Asn Met Trp Tyr 475 480 485
cgt gct tgc acg acc tgt aac aag aag gtg act gaa gct ttt ggg tct
1662 Arg Ala Cys Thr Thr Cys Asn Lys Lys Val Thr Glu Ala Phe Gly
Ser 490 495 500 gga tac tgg tgc gag ggg tgc caa aag aat gac tct gag
tgc tcg ctg 1710 Gly Tyr Trp Cys Glu Gly Cys Gln Lys Asn Asp Ser
Glu Cys Ser Leu 505 510 515 agg tac atc atg gtg atc aag ctc tcc gat
ccc act ggt gag gct tgg 1758 Arg Tyr Ile Met Val Ile Lys Leu Ser
Asp Pro Thr Gly Glu Ala Trp 520 525 530 gtg tcc gtg ttc aac gag cat
gcg gag aag atc att ggc tgc agc gcc 1806 Val Ser Val Phe Asn Glu
His Ala Glu Lys Ile Ile Gly Cys Ser Ala 535 540 545 550 gac gag ctt
gat cgg atc agg aaa gag gag ggg gac gac agc tac gtt 1854 Asp Glu
Leu Asp Arg Ile Arg Lys Glu Glu Gly Asp Asp Ser Tyr Val 555 560 565
ctc aag ctc aag gaa gcc acc tgg gtt cct cac ctg ttc cgc gtc agc
1902 Leu Lys Leu Lys Glu Ala Thr Trp Val Pro His Leu Phe Arg Val
Ser 570 575 580 gtc aca cag cat gaa tac atg aac gag aag agg cag aga
atc acc gtg 1950 Val Thr Gln His Glu Tyr Met Asn Glu Lys Arg Gln
Arg Ile Thr Val 585 590 595 agg ggt gaa gca ccg gtc gac ttc gca gct
gag tcc aag tac ttg ctt 1998 Arg Gly Glu Ala Pro Val Asp Phe Ala
Ala Glu Ser Lys Tyr Leu Leu 600 605 610 gaa gag atc gcg aag ctc acc
gct tgc tagaagacgc agtctttctg 2045 Glu Glu Ile Ala Lys Leu Thr Ala
Cys 615 620 gtggttcttg aaggactggc ccccgatatg tctccctctc agtttttctt
ttgagctcca 2105 gtaacttgat tactgttctg tgtgttgctc tcactgggtt
ttagcacttc tgtaaggtat 2165 atgtagatgc tagtttacct tggtgtcaag
gaacagatgc tattataagc cttgcaaaat 2225 tgcagttcca attccgtgta
tctgcaacct tgagcaaata gggaaagatt atgagtacta 2285 attgatgatg
ttaggtcgct gcagctaaca agtgtttggt ttttagtgac tactgtttag 2345
tccctatatt ttattctatt ttagtattta aggttgcgtt tggttgcgtc gactagacat
2405 gttgtgcgtg tccgatgagt ctattattga agcacaaaat tgggaataaa
aaaaaaaaaa 2465 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2497 2 623 PRT
Zea Mays 2 Met Asp Ala Ala Lys Ser Val Thr Pro Gly Ala Val Ser Tyr
Ile Leu 1 5 10 15 Ala His Pro Ser Thr Gly Ser Asp Gly Ala Val Ser
Asp Leu Val Val 20 25 30 Gln Val Leu Asp Leu Lys Ser Ile Gly Met
Gly Ser Arg Phe Ser Phe 35 40 45 Thr Ala Ser Asp Gly Asn Asp Lys
Ile Lys Ala Met Leu Pro Thr Tyr 50 55 60 Phe Ala Ser Glu Val His
Ser Gly Asn Leu Lys Asn Phe Gly Leu Ile 65 70 75 80 Arg Ile Leu Asp
Tyr Thr Cys Asn Ser Val Lys Gly Asn Ala Asp Lys 85 90 95 Val Leu
Ile Val Val Lys Cys Glu Thr Val Cys Glu Ala Leu Asp Ala 100 105 110
Glu Ile Asn Gly Glu Ala Lys Lys Glu Asp Pro Pro Ile Val Leu Lys 115
120 125 Pro Lys Asp Glu Gly Ser Val Val Ala Glu Glu Thr Asn Ser Pro
Pro 130 135 140 Leu Val Met Lys Pro Lys Gln Glu Val Lys Ser Ala Ser
Gln Ile Val 145 150 155 160 Thr Glu Gln Arg Gly Asn Ala Ala Pro Ala
Thr Arg Leu Ser Met Thr 165 170 175 Arg Arg Val His Pro Leu Ile Thr
Leu Asn Pro Tyr Gln Gly Asn Trp 180 185 190 Val Ile Lys Val Arg Val
Thr Ser Lys Gly Asn Leu Arg Thr Tyr Arg 195 200 205 Asn Ala Arg Gly
Glu Gly Cys Val Phe Asn Val Glu Leu Thr Asp Glu 210 215 220 Asp Gly
Thr Gln Ile Gln Ala Thr Met Phe Asn Glu Ala Ala Lys Lys 225 230 235
240 Phe Tyr Pro Ile Phe Glu Leu Gly Lys Val Tyr Tyr Val Ser Lys Gly
245 250 255 Ser Leu Arg Ile Ala Asn Lys Gln Phe Lys Thr Val Lys Asn
Asp Tyr 260 265 270 Glu Leu Ser Leu Asn Glu Asn Ala Ile Val Glu Glu
Ala Glu Gly Glu 275 280 285 Thr Phe Leu Pro Pro Val Gln Tyr Asn Leu
Val Lys Ile Asp Gln Leu 290 295 300 Gly Pro Tyr Val Gly Gly Arg Glu
Leu Val Asp Ile Val Gly Val Val 305 310 315 320 Gln Ser Val Ser Pro
Thr Leu Ser Val Arg Arg Lys Ile Asp Asn Glu 325 330 335 Thr Ile Pro
Lys Arg Asp Ile Val Val Ala Asp Asp Ser Gly Lys Thr 340 345 350 Val
Thr Ile Ser Leu Trp Asn Asp Leu Ala Thr Thr Thr Gly Gln Glu 355 360
365 Leu Leu Asp Met Val Asp Ser Ser Pro Val Val Ala Ile Lys Ser Leu
370 375 380 Lys Val Ser Asp Phe Gln Gly Val Ser Leu Ser Thr Ile Gly
Arg Ser 385 390 395 400 Thr Leu Glu Ile Asn Pro Asp Leu Pro Glu Ala
Lys Asn Leu Lys Ser 405 410 415 Trp Tyr Asp Ser Glu Gly Lys Asp Thr
Ser Leu Ala Pro Ile Ser Ala 420 425 430 Glu Ala Gly Ala Thr Arg Ala
Gly Gly Phe Lys Ser Met Tyr Ser Asp 435 440 445 Arg Val Phe Leu Ser
His Ile Thr Ser Asp Pro Ala Met Gly Gln Glu 450 455 460 Lys Pro Val
Phe Phe Ser Leu Tyr Ala Ile Ile Ser His Ile Lys Pro 465 470 475 480
Asp Gln Asn Met Trp Tyr Arg Ala Cys Thr Thr Cys Asn Lys Lys Val 485
490 495 Thr Glu Ala Phe Gly Ser Gly Tyr Trp Cys Glu Gly Cys Gln Lys
Asn 500 505 510 Asp Ser Glu Cys Ser Leu Arg Tyr Ile Met Val Ile Lys
Leu Ser Asp 515 520 525 Pro Thr Gly Glu Ala Trp Val Ser Val Phe Asn
Glu His Ala Glu Lys 530 535 540 Ile Ile Gly Cys Ser Ala Asp Glu Leu
Asp Arg Ile Arg Lys Glu Glu 545 550 555 560 Gly Asp Asp Ser Tyr Val
Leu Lys Leu Lys Glu Ala Thr Trp Val Pro 565 570 575 His Leu Phe Arg
Val Ser Val Thr Gln His Glu Tyr Met Asn Glu Lys 580 585 590 Arg Gln
Arg Ile Thr Val Arg Gly Glu Ala Pro Val Asp Phe Ala Ala 595 600 605
Glu Ser Lys Tyr Leu Leu Glu Glu Ile Ala Lys Leu Thr Ala Cys 610 615
620 3 2202 DNA Zea Mays CDS (91)...(1941) Coding Region for Maize
RPA Large Subunit Homologue-2 3 acgttccccc cacgccccaa cctatccacg
cgaaaccttc tttcccccgg gagacgattc 60 gtcagggaga ggaaagaggc
aagaggggcc atg gac gct gcc aag ttg gtg acg 114 Met Asp Ala Ala Lys
Leu Val Thr 1 5 ccg gtc gct gtg tct cac att ctg gcg cac ccg tcg gcg
ggc tcc gac 162 Pro Val Ala Val Ser His Ile Leu Ala His Pro Ser Ala
Gly Ser Asp 10 15 20 ggc gca gtg acc gat ctc gtc gtt cag gtc ctc
gac ctg aag tcc gtc 210 Gly Ala Val Thr Asp Leu Val Val Gln Val Leu
Asp Leu Lys Ser Val 25 30 35 40 ggc acg ggc agc cgg ttc agt ttc aca
gca act gac ggg aag gat aag 258 Gly Thr Gly Ser Arg Phe Ser Phe Thr
Ala Thr Asp Gly Lys Asp Lys 45 50 55 atc aag gcg atg ctt ccc acc
aac ttc ggg tcg gag gtc cgc tct ggc 306 Ile Lys Ala Met Leu Pro Thr
Asn Phe Gly Ser Glu Val Arg Ser Gly 60 65 70 aac ctg aag aac ctc
ggc ctc atc cgc atc atc gac tac act tgc aac 354 Asn Leu Lys Asn Leu
Gly Leu Ile Arg Ile Ile Asp Tyr Thr Cys Asn 75 80 85 gtc gtc aaa
ggc aaa gat gac aaa gtc ttg gtt gtc atc aaa tgc gag 402 Val Val Lys
Gly Lys Asp Asp Lys Val Leu Val Val Ile Lys Cys Glu 90 95 100 ctt
gtg tgc caa gcg ctt gac gcc gag atc aac ggc gag gcc aaa aaa 450 Leu
Val Cys Gln Ala Leu Asp Ala Glu Ile Asn Gly Glu Ala Lys Lys 105 110
115 120 gag gag cct cca att gtg ctg aag cct aag gac gaa tgc gtg ggc
gtg 498 Glu Glu Pro Pro Ile Val Leu Lys Pro Lys Asp Glu Cys Val Gly
Val 125 130 135 act tcc cca ctc gct atg aag ccc aag cag gag gtg aag
tct gcg tcc 546 Thr Ser Pro Leu Ala Met Lys Pro Lys Gln Glu Val Lys
Ser Ala Ser 140 145 150 cag atc gtg aat gag cag cgt gga aat act gct
cct gtc aag ccc ctt 594 Gln Ile Val Asn Glu Gln Arg Gly Asn Thr Ala
Pro Val Lys Pro Leu 155 160 165 tcc atg aca aag agg gtc cat cct ttg
atc act ctg aac ccc tac cag 642 Ser Met Thr Lys Arg Val His Pro Leu
Ile Thr Leu Asn Pro Tyr Gln 170 175 180 ggt aac tgg gtc att aag gtg
cgg gtc acg agc aaa ggc aac ctg aga 690 Gly Asn Trp Val Ile Lys Val
Arg Val Thr Ser Lys Gly Asn Leu Arg 185 190 195 200 acc tac agg aat
gct cgc gga gaa ggc tgt gtc ttc aat gta gag ctc 738 Thr Tyr Arg Asn
Ala Arg Gly Glu Gly Cys Val Phe Asn Val Glu Leu 205 210 215 acc gat
gag gat ggc acc cag atc caa gcc acc atg ttt aat gac gct 786 Thr Asp
Glu Asp Gly Thr Gln Ile Gln Ala Thr Met Phe Asn Asp Ala 220 225 230
gca aag aag ttc tat ccg att ttt gag ctg gga aag gtc tat tat gtc 834
Ala Lys Lys Phe Tyr Pro Ile Phe Glu Leu Gly Lys Val Tyr Tyr Val 235
240 245 tca aaa gga tct ctt aga att gct aac aag cag ttc aag act gtc
caa 882 Ser Lys Gly Ser Leu Arg Ile Ala Asn Lys Gln Phe Lys Thr Val
Gln 250 255 260 aat gac tac gag atg tca cta aac gag aat gct att gtt
gaa gaa gca 930 Asn Asp Tyr Glu Met Ser Leu Asn Glu Asn Ala Ile Val
Glu Glu Ala 265 270 275 280 gag ggg gag act tgc att ccg caa gtg caa
tac aac ctt gtc aag att 978 Glu Gly Glu Thr Cys Ile Pro Gln Val Gln
Tyr Asn Leu Val Lys Ile 285 290 295 gat caa cta gga tca tat gtc ggt
ggc agg gaa ctt gta gat att gtt 1026 Asp Gln Leu Gly Ser Tyr Val
Gly Gly Arg Glu Leu Val Asp Ile Val 300 305 310 ggt gtg gtt cag agc
gta tct ccc aca ctc agt gtc agg aga aag att 1074 Gly Val Val Gln
Ser Val Ser Pro Thr Leu Ser Val Arg Arg Lys Ile 315 320 325 gac aac
gag aca ata ccg aag cgt gac att gtt gtg gcg gat gac tct 1122 Asp
Asn Glu Thr Ile Pro Lys Arg Asp Ile Val Val Ala Asp Asp Ser 330 335
340 ggc aaa act gtt agt atc tct ctt tgg aat gat ctt gct act acg act
1170 Gly Lys Thr Val Ser Ile Ser Leu Trp Asn Asp Leu Ala Thr Thr
Thr 345 350 355 360 ggg caa gag ctt ttg gac atg gct gac agt tcg cct
gtt gtt gcg ata 1218 Gly Gln Glu Leu Leu Asp Met Ala Asp Ser Ser
Pro Val Val Ala Ile 365 370 375 aag agc cta aaa gtg tct gac ttt caa
ggc gtg tct ctt tct act gta 1266 Lys Ser Leu Lys Val Ser Asp Phe
Gln Gly Val Ser Leu Ser Thr Val 380 385 390 ggc aaa agt act ctt gcg
att aat cct gat cta cac gag gct cag aat 1314 Gly Lys Ser Thr Leu
Ala Ile Asn Pro Asp Leu His Glu Ala Gln Asn 395
400 405 ctc aag tca tgg tat gac tct gaa ggc aaa gat act tcg ctg gca
cca 1362 Leu Lys Ser Trp Tyr Asp Ser Glu Gly Lys Asp Thr Ser Leu
Ala Pro 410 415 420 att ggt gca gaa atg ggt gcc gca cgg gcc ggt ggc
ttc aag tcc acg 1410 Ile Gly Ala Glu Met Gly Ala Ala Arg Ala Gly
Gly Phe Lys Ser Thr 425 430 435 440 tat tct gat aga gtt ttt ctg tct
cac att act agt gat cct gcc atg 1458 Tyr Ser Asp Arg Val Phe Leu
Ser His Ile Thr Ser Asp Pro Ala Met 445 450 455 ggc cag gaa aag cct
gtt ttc ttc agt ttg tat gcc acc ata agc cac 1506 Gly Gln Glu Lys
Pro Val Phe Phe Ser Leu Tyr Ala Thr Ile Ser His 460 465 470 atc aag
cct gac cag aac atg tgg tac cgt gct tgc aag acc tgc aac 1554 Ile
Lys Pro Asp Gln Asn Met Trp Tyr Arg Ala Cys Lys Thr Cys Asn 475 480
485 aag aag gtg act gaa act ttt gga tct gga tac tgg tgc gag gga tgc
1602 Lys Lys Val Thr Glu Thr Phe Gly Ser Gly Tyr Trp Cys Glu Gly
Cys 490 495 500 caa aag aat gac tcg gaa tgc tca ctg aga tac atc atg
gtc atc aag 1650 Gln Lys Asn Asp Ser Glu Cys Ser Leu Arg Tyr Ile
Met Val Ile Lys 505 510 515 520 gtc tcc gat cct act ggc gag gca tgg
ttc tct gtg ttc aac gag cat 1698 Val Ser Asp Pro Thr Gly Glu Ala
Trp Phe Ser Val Phe Asn Glu His 525 530 535 gca gag aag atc att ggc
tgc agc gcc gac gag ctt gat cgg atc agg 1746 Ala Glu Lys Ile Ile
Gly Cys Ser Ala Asp Glu Leu Asp Arg Ile Arg 540 545 550 aaa gag gag
ggg gac gac agt tat gtt ctg aag ctc aag gaa gcc acc 1794 Lys Glu
Glu Gly Asp Asp Ser Tyr Val Leu Lys Leu Lys Glu Ala Thr 555 560 565
tgg gtt cct cac ctg ttc cgc gtc agc gtc aca cag cat gaa tac aat
1842 Trp Val Pro His Leu Phe Arg Val Ser Val Thr Gln His Glu Tyr
Asn 570 575 580 aac gag aaa agg cag aga atc act gtg agg agt gaa gcg
ccg gtc gag 1890 Asn Glu Lys Arg Gln Arg Ile Thr Val Arg Ser Glu
Ala Pro Val Glu 585 590 595 600 cac gca gct gaa tcc aag tac ctg ctt
gaa cag ata gcg aag ctt act 1938 His Ala Ala Glu Ser Lys Tyr Leu
Leu Glu Gln Ile Ala Lys Leu Thr 605 610 615 gct tgatagtaga
agatgcaacc ttactgcaaa tagcgaggat tattaggact 1991 Ala aattgatggt
gtcaggtcat tgcggcccta agctttagct ctctatcagc agtcagatgt 2051
attaaccatt ccctgctcta atagtcatct atcagcagtc agatgtattt aaccaaaaaa
2111 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagggcgg
ccgctctaga 2171 ggatccaagc ttacgtacgc gtgcatgcga c 2202 4 617 PRT
Zea Mays 4 Met Asp Ala Ala Lys Leu Val Thr Pro Val Ala Val Ser His
Ile Leu 1 5 10 15 Ala His Pro Ser Ala Gly Ser Asp Gly Ala Val Thr
Asp Leu Val Val 20 25 30 Gln Val Leu Asp Leu Lys Ser Val Gly Thr
Gly Ser Arg Phe Ser Phe 35 40 45 Thr Ala Thr Asp Gly Lys Asp Lys
Ile Lys Ala Met Leu Pro Thr Asn 50 55 60 Phe Gly Ser Glu Val Arg
Ser Gly Asn Leu Lys Asn Leu Gly Leu Ile 65 70 75 80 Arg Ile Ile Asp
Tyr Thr Cys Asn Val Val Lys Gly Lys Asp Asp Lys 85 90 95 Val Leu
Val Val Ile Lys Cys Glu Leu Val Cys Gln Ala Leu Asp Ala 100 105 110
Glu Ile Asn Gly Glu Ala Lys Lys Glu Glu Pro Pro Ile Val Leu Lys 115
120 125 Pro Lys Asp Glu Cys Val Gly Val Thr Ser Pro Leu Ala Met Lys
Pro 130 135 140 Lys Gln Glu Val Lys Ser Ala Ser Gln Ile Val Asn Glu
Gln Arg Gly 145 150 155 160 Asn Thr Ala Pro Val Lys Pro Leu Ser Met
Thr Lys Arg Val His Pro 165 170 175 Leu Ile Thr Leu Asn Pro Tyr Gln
Gly Asn Trp Val Ile Lys Val Arg 180 185 190 Val Thr Ser Lys Gly Asn
Leu Arg Thr Tyr Arg Asn Ala Arg Gly Glu 195 200 205 Gly Cys Val Phe
Asn Val Glu Leu Thr Asp Glu Asp Gly Thr Gln Ile 210 215 220 Gln Ala
Thr Met Phe Asn Asp Ala Ala Lys Lys Phe Tyr Pro Ile Phe 225 230 235
240 Glu Leu Gly Lys Val Tyr Tyr Val Ser Lys Gly Ser Leu Arg Ile Ala
245 250 255 Asn Lys Gln Phe Lys Thr Val Gln Asn Asp Tyr Glu Met Ser
Leu Asn 260 265 270 Glu Asn Ala Ile Val Glu Glu Ala Glu Gly Glu Thr
Cys Ile Pro Gln 275 280 285 Val Gln Tyr Asn Leu Val Lys Ile Asp Gln
Leu Gly Ser Tyr Val Gly 290 295 300 Gly Arg Glu Leu Val Asp Ile Val
Gly Val Val Gln Ser Val Ser Pro 305 310 315 320 Thr Leu Ser Val Arg
Arg Lys Ile Asp Asn Glu Thr Ile Pro Lys Arg 325 330 335 Asp Ile Val
Val Ala Asp Asp Ser Gly Lys Thr Val Ser Ile Ser Leu 340 345 350 Trp
Asn Asp Leu Ala Thr Thr Thr Gly Gln Glu Leu Leu Asp Met Ala 355 360
365 Asp Ser Ser Pro Val Val Ala Ile Lys Ser Leu Lys Val Ser Asp Phe
370 375 380 Gln Gly Val Ser Leu Ser Thr Val Gly Lys Ser Thr Leu Ala
Ile Asn 385 390 395 400 Pro Asp Leu His Glu Ala Gln Asn Leu Lys Ser
Trp Tyr Asp Ser Glu 405 410 415 Gly Lys Asp Thr Ser Leu Ala Pro Ile
Gly Ala Glu Met Gly Ala Ala 420 425 430 Arg Ala Gly Gly Phe Lys Ser
Thr Tyr Ser Asp Arg Val Phe Leu Ser 435 440 445 His Ile Thr Ser Asp
Pro Ala Met Gly Gln Glu Lys Pro Val Phe Phe 450 455 460 Ser Leu Tyr
Ala Thr Ile Ser His Ile Lys Pro Asp Gln Asn Met Trp 465 470 475 480
Tyr Arg Ala Cys Lys Thr Cys Asn Lys Lys Val Thr Glu Thr Phe Gly 485
490 495 Ser Gly Tyr Trp Cys Glu Gly Cys Gln Lys Asn Asp Ser Glu Cys
Ser 500 505 510 Leu Arg Tyr Ile Met Val Ile Lys Val Ser Asp Pro Thr
Gly Glu Ala 515 520 525 Trp Phe Ser Val Phe Asn Glu His Ala Glu Lys
Ile Ile Gly Cys Ser 530 535 540 Ala Asp Glu Leu Asp Arg Ile Arg Lys
Glu Glu Gly Asp Asp Ser Tyr 545 550 555 560 Val Leu Lys Leu Lys Glu
Ala Thr Trp Val Pro His Leu Phe Arg Val 565 570 575 Ser Val Thr Gln
His Glu Tyr Asn Asn Glu Lys Arg Gln Arg Ile Thr 580 585 590 Val Arg
Ser Glu Ala Pro Val Glu His Ala Ala Glu Ser Lys Tyr Leu 595 600 605
Leu Glu Gln Ile Ala Lys Leu Thr Ala 610 615 5 630 PRT Oryza sativa
5 Met Asp Ser Asp Ala Ala Pro Ser Val Thr Pro Gly Ala Val Ala Phe 1
5 10 15 Val Leu Glu Asn Ala Ser Pro Asp Ala Ala Thr Gly Val Pro Val
Pro 20 25 30 Glu Ile Val Leu Gln Val Val Asp Leu Lys Pro Ile Gly
Thr Arg Phe 35 40 45 Thr Phe Leu Ala Ser Asp Gly Lys Asp Lys Ile
Lys Thr Met Leu Leu 50 55 60 Thr Gln Leu Ala Pro Glu Val Arg Ser
Gly Asn Ile Gln Asn Leu Gly 65 70 75 80 Val Ile Arg Val Leu Asp Tyr
Thr Cys Asn Thr Ile Gly Glu Lys Gln 85 90 95 Glu Lys Val Leu Ile
Ile Thr Lys Leu Glu Val Val Phe Lys Ala Leu 100 105 110 Asp Ser Glu
Ile Lys Cys Glu Ala Glu Lys Gln Glu Glu Lys Pro Ala 115 120 125 Ile
Leu Leu Ser Pro Lys Glu Glu Ser Val Val Leu Ser Lys Pro Thr 130 135
140 Asn Ala Pro Pro Leu Pro Pro Val Val Leu Lys Pro Lys Gln Glu Val
145 150 155 160 Lys Ser Ala Ser Gln Ile Val Asn Glu Gln Arg Gly Asn
Ala Ala Pro 165 170 175 Ala Ala Arg Leu Ala Met Thr Arg Arg Val His
Pro Leu Ile Ser Leu 180 185 190 Asn Pro Tyr Gln Gly Asn Trp Ile Ile
Lys Val Arg Val Thr Ser Lys 195 200 205 Gly Asn Leu Arg Thr Tyr Lys
Asn Ala Arg Gly Glu Gly Cys Val Phe 210 215 220 Asn Val Glu Leu Thr
Asp Val Asp Gly Thr Gln Ile Gln Ala Thr Met 225 230 235 240 Phe Asn
Glu Ala Ala Lys Lys Phe Tyr Pro Met Phe Glu Leu Gly Lys 245 250 255
Val Tyr Tyr Ile Ser Lys Gly Ser Leu Arg Val Ala Asn Lys Gln Phe 260
265 270 Lys Thr Val His Asn Asp Tyr Glu Met Thr Leu Asn Glu Asn Ala
Val 275 280 285 Val Glu Glu Ala Glu Gly Glu Thr Phe Ile Pro Gln Ile
Gln Tyr Asn 290 295 300 Phe Val Lys Ile Asp Gln Leu Gly Pro Tyr Val
Gly Gly Arg Glu Leu 305 310 315 320 Val Asp Val Ile Gly Val Val Gln
Ser Val Ser Pro Thr Leu Ser Val 325 330 335 Arg Arg Lys Ile Asp Asn
Glu Thr Ile Pro Lys Arg Asp Ile Val Val 340 345 350 Ala Asp Asp Ser
Ser Lys Thr Val Thr Ile Ser Leu Trp Asn Asp Leu 355 360 365 Ala Thr
Thr Thr Gly Gln Glu Leu Leu Asp Met Val Asp Ser Ala Pro 370 375 380
Ile Ile Ala Ile Lys Ser Leu Lys Val Ser Asp Phe Gln Gly Leu Ser 385
390 395 400 Leu Ser Thr Val Gly Arg Ser Thr Ile Val Val Asn Pro Asp
Leu Pro 405 410 415 Glu Ala Glu Gln Leu Arg Ala Trp Tyr Asp Ser Glu
Gly Lys Gly Thr 420 425 430 Ser Met Ala Ser Ile Gly Ser Asp Met Gly
Ala Ser Arg Val Gly Gly 435 440 445 Ala Arg Ser Met Tyr Ser Asp Arg
Val Phe Leu Ser His Ile Thr Ser 450 455 460 Asp Pro Asn Leu Gly Gln
Asp Lys Pro Val Phe Phe Ser Leu Asn Ala 465 470 475 480 Tyr Ile Ser
Leu Ile Lys Pro Asp Gln Thr Met Trp Tyr Arg Ala Cys 485 490 495 Lys
Thr Cys Asn Lys Lys Val Thr Glu Ala Met Gly Ser Gly Tyr Trp 500 505
510 Cys Glu Gly Cys Gln Lys Asn Asp Ala Glu Cys Ser Leu Arg Tyr Ile
515 520 525 Met Val Ile Lys Val Ser Asp Pro Thr Gly Glu Ala Trp Leu
Ser Leu 530 535 540 Phe Asn Asp Gln Ala Glu Arg Ile Val Gly Cys Ser
Ala Asp Glu Leu 545 550 555 560 Asp Arg Ile Arg Lys Glu Glu Gly Asp
Asp Ser Tyr Leu Leu Lys Leu 565 570 575 Lys Glu Ala Thr Trp Val Pro
His Leu Phe Arg Val Ser Val Thr Gln 580 585 590 Asn Glu Tyr Met Asn
Glu Lys Arg Gln Arg Ile Thr Val Arg Ser Glu 595 600 605 Ala Pro Val
Asp His Ala Ala Glu Ala Lys Tyr Met Leu Glu Glu Ile 610 615 620 Ala
Lys Leu Thr Gly Cys 625 630 6 609 PRT Xenopus laevis 6 Met Ala Leu
Pro Gln Leu Ser Glu Gly Ala Ile Ser Ala Met Leu Gly 1 5 10 15 Gly
Asp Ser Ser Cys Lys Pro Thr Leu Gln Val Ile Asn Ile Arg Pro 20 25
30 Ile Asn Thr Gly Asn Gly Pro Pro Arg Tyr Arg Leu Leu Met Ser Asp
35 40 45 Gly Leu Asn Thr Leu Ser Ser Phe Met Leu Ala Thr Gln Leu
Asn Ser 50 55 60 Leu Val Asp Asn Asn Leu Leu Ala Thr Asn Cys Ile
Cys Gln Val Ser 65 70 75 80 Arg Phe Ile Val Asn Asn Leu Lys Asp Gly
Arg Arg Val Ile Ile Val 85 90 95 Met Glu Leu Asp Val Leu Lys Ser
Ala Asp Leu Val Met Gly Lys Ile 100 105 110 Gly Asn Pro Gln Pro Tyr
Asn Asp Gly Gln Pro Gln Pro Ala Ala Pro 115 120 125 Ala Pro Ala Ser
Ala Pro Ala Pro Ala Pro Ser Lys Leu Gln Asn Asn 130 135 140 Ser Ala
Pro Pro Pro Ser Met Asn Arg Gly Thr Ser Lys Leu Phe Gly 145 150 155
160 Gly Gly Ser Leu Leu Asn Thr Pro Gly Gly Ser Gln Ser Lys Val Val
165 170 175 Pro Ile Ala Ser Leu Asn Pro Tyr Gln Ser Lys Trp Thr Val
Arg Ala 180 185 190 Arg Val Thr Asn Lys Gly Gln Ile Arg Thr Trp Ser
Asn Ser Arg Gly 195 200 205 Glu Gly Lys Leu Phe Ser Ile Glu Met Val
Asp Glu Ser Gly Glu Ile 210 215 220 Arg Ala Thr Ala Phe Asn Glu Gln
Ala Asp Lys Phe Phe Ser Ile Ile 225 230 235 240 Glu Val Asn Lys Val
Tyr Tyr Phe Ser Lys Gly Thr Leu Lys Ile Ala 245 250 255 Asn Lys Gln
Tyr Thr Ser Val Lys Asn Asp Tyr Glu Met Thr Phe Asn 260 265 270 Ser
Glu Thr Ser Val Ile Pro Cys Asp Asp Ser Ala Asp Val Pro Met 275 280
285 Val Gln Phe Glu Phe Val Ser Ile Gly Glu Leu Glu Ser Lys Asn Lys
290 295 300 Asp Thr Val Leu Asp Ile Ile Gly Val Cys Lys Asn Val Glu
Glu Val 305 310 315 320 Thr Lys Val Thr Ile Lys Ser Asn Asn Arg Glu
Val Ser Lys Arg Ser 325 330 335 Ile His Leu Met Asp Ser Ser Gly Lys
Val Val Ser Thr Thr Leu Trp 340 345 350 Gly Glu Asp Ala Asp Lys Phe
Asp Gly Ser Arg Gln Pro Val Val Ala 355 360 365 Ile Lys Gly Ala Arg
Leu Ser Asp Phe Gly Gly Arg Ser Leu Ser Val 370 375 380 Leu Ser Ser
Ser Thr Val Met Ile Asn Pro Asp Ile Pro Glu Ala Phe 385 390 395 400
Lys Leu Arg Ala Trp Phe Asp Ser Glu Gly Gln Val Val Glu Gly Thr 405
410 415 Ser Ile Ser Glu Ser Arg Gly Gly Gly Thr Gly Gly Gly Asn Thr
Asn 420 425 430 Trp Lys Ser Leu Leu Glu Val Lys Asn Glu Asn Leu Gly
His Gly Glu 435 440 445 Lys Ala Asp Tyr Phe Thr Ser Val Ala Thr Ile
Val Tyr Leu Arg Lys 450 455 460 Glu Asn Cys Leu Tyr Gln Ala Cys Pro
Ser Gln Asp Cys Asn Lys Lys 465 470 475 480 Val Ile Asp Gln Gln Asn
Gly Leu Phe Arg Cys Glu Lys Cys Asn Lys 485 490 495 Glu Phe Pro Asn
Phe Lys Tyr Arg Leu Ile Leu Ser Ala Asn Ile Ala 500 505 510 Asp Phe
Gly Glu Asn Gln Trp Ile Thr Cys Phe Gln Glu Ser Ala Glu 515 520 525
Ser Ile Leu Gly Gln Asn Ala Thr Tyr Leu Gly Glu Leu Lys Glu Lys 530
535 540 Asn Glu Gln Ala Tyr Asp Glu Val Phe Gln Asn Ala Asn Phe Arg
Ser 545 550 555 560 Tyr Thr Phe Arg Ala Arg Val Lys Leu Glu Thr Tyr
Asn Asp Glu Ser 565 570 575 Arg Ile Lys Ala Thr Ala Val Asp Val Lys
Pro Val Asp His Lys Glu 580 585 590 Tyr Ser Arg Arg Leu Ile Met Asn
Ile Arg Lys Met Ala Thr Gln Gly 595 600 605 Val 7 616 PRT Homo
sapiens 7 Met Val Gly Gln Leu Ser Glu Gly Ala Ile Ala Ala Ile Met
Gln Lys 1 5 10 15 Gly Asp Thr Asn Ile Lys Pro Ile Leu Gln Val Ile
Asn Ile Arg Pro 20 25 30 Ile Thr Thr Gly Asn Ser Pro Pro Arg Tyr
Arg Leu Leu Met Ser Asp 35 40 45 Gly Leu Asn Thr Leu Ser Ser Phe
Met Leu Ala Thr Gln Leu Asn Pro 50 55 60 Leu Val Glu Glu Glu Gln
Leu Ser Ser Asn Cys Val Cys Gln Ile His 65 70 75 80 Arg Phe Ile Val
Asn Thr Leu Lys Asp Gly Arg Arg Val Val Ile Leu 85 90 95 Met Glu
Leu Glu Val Leu Lys Ser Ala Glu Ala Val Gly Val Lys Ile 100 105 110
Gly Asn Pro Val Pro Tyr Asn Glu Gly Leu Gly Gln Pro Gln Val Ala 115
120 125 Pro Pro Ala Pro Ala Ala Ser Pro Ala Ala Ser Ser Arg Pro Gln
Pro 130 135 140 Gln Asn Gly Ser Ser Gly Met Gly Ser Thr Val Ser Lys
Ala Tyr Gly 145 150 155 160 Ala Ser Lys Thr Phe Gly Lys Ala Ala Gly
Pro Ser Leu Ser His Thr 165 170 175 Ser Gly Gly Thr Gln Ser Lys Val
Val Pro Ile Ala Ser Leu Thr Pro 180 185 190 Tyr Gln Ser Lys Trp
Thr
Ile Cys Ala Arg Val Thr Asn Lys Ser Gln 195 200 205 Ile Arg Thr Trp
Ser Asn Ser Arg Gly Glu Gly Lys Leu Phe Ser Leu 210 215 220 Glu Leu
Val Asp Glu Ser Gly Glu Ile Arg Ala Thr Ala Phe Asn Glu 225 230 235
240 Gln Val Asp Lys Phe Phe Pro Leu Ile Glu Val Asn Lys Val Tyr Tyr
245 250 255 Phe Ser Lys Gly Thr Leu Lys Ile Ala Asn Lys Gln Phe Thr
Ala Val 260 265 270 Lys Asn Asp Tyr Glu Met Thr Phe Asn Asn Glu Thr
Ser Val Met Pro 275 280 285 Cys Glu Asp Asp His His Leu Pro Thr Val
Gln Phe Asp Phe Thr Gly 290 295 300 Ile Asp Asp Leu Glu Asn Lys Ser
Lys Asp Ser Leu Val Asp Ile Ile 305 310 315 320 Gly Ile Cys Lys Ser
Tyr Glu Asp Ala Thr Lys Ile Thr Val Arg Ser 325 330 335 Asn Asn Arg
Glu Val Ala Lys Arg Asn Ile Tyr Leu Met Asp Thr Ser 340 345 350 Gly
Lys Val Val Thr Ala Thr Leu Trp Gly Glu Asp Ala Asp Lys Phe 355 360
365 Asp Gly Ser Arg Gln Pro Val Leu Ala Ile Lys Gly Ala Arg Val Ser
370 375 380 Asp Phe Gly Gly Arg Ser Leu Ser Val Leu Ser Ser Ser Thr
Ile Ile 385 390 395 400 Ala Asn Pro Asp Ile Pro Glu Ala Tyr Lys Leu
Arg Gly Trp Phe Asp 405 410 415 Ala Glu Gly Gln Ala Leu Asp Gly Val
Ser Ile Ser Asp Leu Lys Ser 420 425 430 Gly Gly Val Gly Gly Ser Asn
Thr Asn Trp Lys Thr Leu Tyr Glu Val 435 440 445 Lys Ser Glu Asn Leu
Gly Gln Gly Asp Lys Pro Asp Tyr Phe Ser Ser 450 455 460 Val Ala Thr
Val Val Tyr Leu Arg Lys Glu Asn Cys Met Tyr Gln Ala 465 470 475 480
Cys Pro Thr Gln Asp Cys Asn Lys Lys Val Ile Asp Gln Gln Asn Gly 485
490 495 Leu Tyr Arg Cys Glu Lys Cys Asp Thr Glu Phe Pro Asn Phe Lys
Tyr 500 505 510 Arg Met Ile Leu Ser Val Asn Ile Ala Asp Phe Gln Glu
Asn Gln Trp 515 520 525 Val Thr Cys Phe Gln Glu Ser Ala Glu Ala Ile
Leu Gly Gln Asn Ala 530 535 540 Ala Tyr Leu Gly Glu Leu Lys Asp Lys
Asn Glu Gln Ala Phe Glu Glu 545 550 555 560 Val Phe Gln Asn Ala Asn
Phe Arg Ser Phe Ile Phe Arg Val Arg Val 565 570 575 Lys Val Glu Thr
Tyr Asn Asp Glu Ser Arg Ile Lys Ala Thr Val Met 580 585 590 Asp Val
Lys Pro Val Asp Tyr Arg Glu Tyr Gly Arg Arg Leu Val Met 595 600 605
Ser Ile Arg Arg Ser Ala Leu Met 610 615 8 603 PRT Drosophila
melanogaster 8 Met Val Leu Ala Ser Leu Ser Thr Gly Val Ile Ala Arg
Ile Met His 1 5 10 15 Gly Glu Val Val Asp Ala Pro Val Leu Gln Ile
Leu Ala Ile Lys Lys 20 25 30 Ile Asn Ser Ala Ala Asp Ser Glu Arg
Tyr Arg Ile Leu Ile Ser Asp 35 40 45 Gly Lys Tyr Phe Asn Ser Tyr
Ala Met Leu Ala Ser Gln Leu Asn Val 50 55 60 Met Gln His Asn Gly
Glu Leu Glu Glu Phe Thr Ile Val Gln Leu Asp 65 70 75 80 Lys Tyr Val
Thr Ser Leu Val Gly Lys Asp Gly Ala Gly Lys Arg Val 85 90 95 Leu
Ile Ile Ser Glu Leu Thr Val Val Asn Pro Gly Ala Glu Val Lys 100 105
110 Ser Lys Ile Gly Glu Pro Val Thr Tyr Glu Asn Ala Ala Lys Gln Asp
115 120 125 Leu Ala Pro Lys Pro Ala Val Thr Ser Asn Ser Lys Pro Ile
Ala Lys 130 135 140 Lys Glu Pro Ser His Asn Asn Asn Asn Asn Ile Val
Met Asn Ser Ser 145 150 155 160 Ile Asn Ser Gly Met Thr His Pro Ile
Ser Ser Leu Ser Pro Tyr Gln 165 170 175 Asn Lys Trp Val Ile Lys Ala
Arg Val Thr Ser Lys Ser Gly Ile Arg 180 185 190 Thr Trp Ser Asn Ala
Arg Gly Glu Gly Lys Leu Phe Ser Met Asp Leu 195 200 205 Met Asp Glu
Ser Gly Glu Ile Arg Ala Thr Ala Phe Lys Glu Gln Cys 210 215 220 Asp
Lys Phe Tyr Asp Leu Ile Gln Val Asp Ser Val Tyr Tyr Ile Ser 225 230
235 240 Lys Cys Gln Leu Lys Pro Ala Asn Lys Gln Tyr Ser Ser Leu Asn
Asn 245 250 255 Ala Tyr Glu Met Thr Phe Ser Gly Glu Thr Val Val Gln
Leu Cys Glu 260 265 270 Asp Thr Asp Asp Asp Pro Ile Pro Glu Ile Lys
Tyr Asn Leu Val Pro 275 280 285 Ile Ser Asp Val Ser Gly Met Glu Asn
Lys Ala Ala Val Asp Thr Ile 290 295 300 Gly Ile Cys Lys Glu Val Gly
Glu Leu Gln Ser Phe Val Ala Arg Thr 305 310 315 320 Thr Asn Lys Glu
Phe Lys Lys Arg Asp Ile Thr Leu Val Asp Met Ser 325 330 335 Asn Ser
Ala Ile Ser Leu Thr Leu Trp Gly Asp Asp Ala Val Asn Phe 340 345 350
Asp Gly His Val Gln Pro Val Ile Leu Val Lys Gly Thr Arg Ile Asn 355
360 365 Glu Phe Asn Gly Gly Lys Ser Leu Ser Leu Gly Gly Gly Ser Ile
Met 370 375 380 Lys Ile Asn Pro Asp Ile Pro Glu Ala His Lys Leu Arg
Gly Trp Phe 385 390 395 400 Asp Asn Gly Gly Gly Asp Ser Val Ala Asn
Met Val Ser Ala Arg Thr 405 410 415 Gly Gly Gly Ser Phe Ser Thr Glu
Trp Met Thr Leu Lys Asp Ala Arg 420 425 430 Ala Arg Asn Leu Gly Ser
Gly Asp Lys Pro Asp Tyr Phe Gln Cys Lys 435 440 445 Ala Val Val His
Ile Val Lys Gln Glu Asn Ala Phe Tyr Arg Ala Cys 450 455 460 Pro Gln
Ser Asp Cys Asn Lys Lys Val Val Asp Glu Gly Asn Asp Gln 465 470 475
480 Phe Arg Cys Glu Lys Cys Asn Ala Leu Phe Pro Asn Phe Lys Tyr Arg
485 490 495 Leu Leu Ile Asn Met Ser Ile Gly Asp Trp Thr Ser Asn Arg
Trp Val 500 505 510 Ser Ser Phe Asn Glu Val Gly Glu Gln Leu Leu Gly
His Thr Ser Gln 515 520 525 Glu Val Gly Glu Ala Leu Glu Asn Asp Pro
Ala Lys Ala Glu Gln Ile 530 535 540 Phe Ser Ala Leu Asn Phe Thr Ser
His Ile Phe Lys Leu Arg Cys Lys 545 550 555 560 Asn Glu Val Tyr Gly
Asp Met Thr Arg Asn Lys Leu Thr Val Gln Ser 565 570 575 Val Ala Pro
Ile Asn His Lys Glu Tyr Asn Lys His Leu Leu Lys Glu 580 585 590 Leu
Gln Glu Leu Thr Gly Ile Gly Ser Ser Asn 595 600 9 609 PRT
Schizosaccharomyces pombe 9 Met Ala Glu Arg Leu Ser Val Gly Ala Leu
Arg Ile Ile Asn Thr Ser 1 5 10 15 Asp Ala Ser Ser Phe Pro Pro Asn
Pro Ile Leu Gln Val Leu Thr Val 20 25 30 Lys Glu Leu Asn Ser Asn
Pro Thr Ser Gly Ala Pro Lys Arg Tyr Arg 35 40 45 Val Val Leu Ser
Asp Ser Ile Asn Tyr Ala Gln Ser Met Leu Ser Thr 50 55 60 Gln Leu
Asn His Leu Val Ala Glu Asn Lys Leu Gln Lys Gly Ala Phe 65 70 75 80
Val Gln Leu Thr Gln Phe Thr Val Asn Val Met Lys Glu Arg Lys Ile 85
90 95 Leu Ile Val Leu Gly Leu Asn Val Leu Thr Glu Leu Gly Val Met
Asp 100 105 110 Lys Ile Gly Asn Pro Ala Gly Leu Glu Thr Val Asp Ala
Leu Arg Gln 115 120 125 Gln Gln Asn Glu Gln Asn Asn Ala Ser Ala Pro
Arg Thr Gly Ile Ser 130 135 140 Thr Ser Thr Asn Ser Phe Tyr Gly Asn
Asn Ala Ala Ala Thr Ala Pro 145 150 155 160 Ala Pro Pro Pro Met Met
Lys Lys Pro Ala Ala Pro Asn Ser Leu Ser 165 170 175 Thr Ile Ile Tyr
Pro Ile Glu Gly Leu Ser Pro Tyr Gln Asn Lys Trp 180 185 190 Thr Ile
Arg Ala Arg Val Thr Asn Lys Ser Glu Val Lys His Trp His 195 200 205
Asn Gln Arg Gly Glu Gly Lys Leu Phe Ser Val Asn Leu Leu Asp Glu 210
215 220 Ser Gly Glu Ile Arg Ala Thr Gly Phe Asn Asp Gln Val Asp Ala
Phe 225 230 235 240 Tyr Asp Ile Leu Gln Glu Gly Ser Val Tyr Tyr Ile
Ser Arg Cys Arg 245 250 255 Val Asn Ile Ala Lys Lys Gln Tyr Thr Asn
Val Gln Asn Glu Tyr Glu 260 265 270 Leu Met Phe Glu Arg Asp Thr Glu
Ile Arg Lys Ala Glu Asp Gln Thr 275 280 285 Ala Val Pro Val Ala Lys
Phe Ser Phe Val Ser Leu Gln Glu Val Gly 290 295 300 Asp Val Ala Lys
Asp Ala Val Ile Asp Val Ile Gly Val Leu Gln Asn 305 310 315 320 Val
Gly Pro Val Gln Gln Ile Thr Ser Arg Ala Thr Ser Arg Gly Phe 325 330
335 Asp Lys Arg Asp Ile Thr Ile Val Asp Gln Thr Gly Tyr Glu Met Arg
340 345 350 Val Thr Leu Trp Gly Lys Thr Ala Ile Glu Phe Ser Val Ser
Glu Glu 355 360 365 Ser Ile Leu Ala Phe Lys Gly Val Lys Val Asn Asp
Phe Gln Gly Arg 370 375 380 Ser Leu Ser Met Leu Thr Ser Ser Thr Met
Ser Val Asp Pro Asp Ile 385 390 395 400 Gln Glu Ser His Leu Leu Lys
Gly Trp Tyr Asp Gly Gln Gly Arg Gly 405 410 415 Gln Glu Phe Ala Lys
His Ser Val Ile Ser Ser Thr Leu Ser Thr Thr 420 425 430 Gly Arg Ser
Ala Glu Arg Lys Asn Ile Ala Glu Val Gln Ala Glu His 435 440 445 Leu
Gly Met Ser Glu Thr Pro Asp Tyr Phe Ser Leu Lys Gly Thr Ile 450 455
460 Val Tyr Ile Arg Lys Lys Asn Val Ser Tyr Pro Ala Cys Pro Ala Ala
465 470 475 480 Asp Cys Asn Lys Lys Val Phe Asp Gln Gly Gly Ser Trp
Arg Cys Glu 485 490 495 Lys Cys Asn Lys Glu Tyr Asp Ala Pro Gln Tyr
Arg Tyr Ile Ile Thr 500 505 510 Ile Ala Val Gly Asp His Thr Gly Gln
Leu Trp Leu Asn Val Phe Asp 515 520 525 Asp Val Gly Lys Leu Ile Met
His Lys Thr Ala Asp Glu Leu Asn Asp 530 535 540 Leu Gln Glu Asn Asp
Glu Asn Ala Phe Met Asn Cys Met Ala Glu Ala 545 550 555 560 Cys Tyr
Met Pro Tyr Ile Phe Gln Cys Arg Ala Lys Gln Asp Asn Phe 565 570 575
Lys Gly Glu Met Arg Val Arg Tyr Thr Val Met Ser Ile Asn Gln Met 580
585 590 Asp Trp Lys Glu Glu Ser Lys Arg Leu Ile Asn Phe Ile Glu Ser
Ala 595 600 605 Gln 10 621 PRT Saccharomyces cerevisiae 10 Met Ser
Ser Val Gln Leu Ser Arg Gly Asp Phe His Ser Ile Phe Thr 1 5 10 15
Asn Lys Gln Arg Tyr Asp Asn Pro Thr Gly Gly Val Tyr Gln Val Tyr 20
25 30 Asn Thr Arg Lys Ser Asp Gly Ala Asn Ser Asn Arg Lys Asn Leu
Ile 35 40 45 Met Ile Ser Asp Gly Ile Tyr His Met Lys Ala Leu Leu
Arg Asn Gln 50 55 60 Ala Ala Ser Lys Phe Gln Ser Met Glu Leu Gln
Arg Gly Asp Ile Ile 65 70 75 80 Arg Val Ile Ile Ala Glu Pro Ala Ile
Val Arg Glu Arg Lys Lys Tyr 85 90 95 Val Leu Leu Val Asp Asp Phe
Glu Leu Val Gln Ser Arg Ala Asp Met 100 105 110 Val Asn Gln Thr Ser
Thr Phe Leu Asp Asn Tyr Phe Ser Glu His Pro 115 120 125 Asn Glu Thr
Leu Lys Asp Glu Asp Ile Thr Asp Ser Gly Asn Val Ala 130 135 140 Asn
Gln Thr Asn Ala Ser Asn Ala Gly Val Pro Asp Met Leu His Ser 145 150
155 160 Asn Ser Asn Leu Asn Ala Asn Glu Arg Lys Phe Ala Asn Glu Asn
Pro 165 170 175 Asn Ser Gln Lys Thr Arg Pro Ile Phe Ala Ile Glu Gln
Leu Ser Pro 180 185 190 Tyr Gln Asn Val Trp Thr Ile Lys Ala Arg Val
Ser Tyr Lys Gly Glu 195 200 205 Ile Lys Thr Trp His Asn Gln Arg Gly
Asp Gly Lys Leu Phe Asn Val 210 215 220 Asn Phe Leu Asp Thr Ser Gly
Glu Ile Arg Ala Thr Ala Phe Asn Asp 225 230 235 240 Phe Ala Thr Lys
Phe Asn Glu Ile Leu Gln Glu Gly Lys Val Tyr Tyr 245 250 255 Val Ser
Lys Ala Lys Leu Gln Pro Ala Lys Pro Gln Phe Thr Asn Leu 260 265 270
Thr His Pro Tyr Glu Leu Asn Leu Asp Arg Asp Thr Val Ile Glu Glu 275
280 285 Cys Phe Asp Glu Ser Asn Val Pro Lys Thr His Phe Asn Phe Ile
Lys 290 295 300 Leu Asp Ala Ile Gln Asn Gln Glu Val Asn Ser Asn Val
Asp Val Leu 305 310 315 320 Gly Ile Ile Gln Thr Ile Asn Pro His Phe
Glu Leu Thr Ser Arg Ala 325 330 335 Gly Lys Lys Phe Asp Arg Arg Asp
Ile Thr Ile Val Asp Asp Ser Gly 340 345 350 Phe Ser Ile Ser Val Gly
Leu Trp Asn Gln Gln Ala Leu Asp Phe Asn 355 360 365 Leu Pro Glu Gly
Ser Val Ala Ala Ile Lys Gly Val Arg Val Thr Asp 370 375 380 Phe Gly
Gly Lys Ser Leu Ser Met Gly Phe Ser Ser Thr Leu Ile Pro 385 390 395
400 Asn Pro Glu Ile Pro Glu Ala Tyr Ala Leu Lys Gly Trp Tyr Asp Ser
405 410 415 Lys Gly Arg Asn Ala Asn Phe Ile Thr Leu Lys Gln Glu Pro
Gly Met 420 425 430 Gly Gly Gln Ser Ala Ala Ser Leu Thr Lys Phe Ile
Ala Gln Arg Ile 435 440 445 Thr Ile Ala Arg Ala Gln Ala Glu Asn Leu
Gly Arg Ser Glu Lys Gly 450 455 460 Asp Phe Phe Ser Val Lys Ala Ala
Ile Ser Phe Leu Lys Val Asp Asn 465 470 475 480 Phe Ala Tyr Pro Ala
Cys Ser Asn Glu Asn Cys Asn Lys Lys Val Leu 485 490 495 Glu Gln Pro
Asp Gly Thr Trp Arg Cys Glu Lys Cys Asp Thr Asn Asn 500 505 510 Ala
Arg Pro Asn Trp Arg Tyr Ile Leu Thr Ile Ser Ile Ile Asp Glu 515 520
525 Thr Asn Gln Leu Trp Leu Thr Leu Phe Asp Asp Gln Ala Lys Gln Leu
530 535 540 Leu Gly Val Asp Ala Asn Thr Leu Met Ser Leu Lys Glu Glu
Asp Pro 545 550 555 560 Asn Glu Phe Thr Lys Ile Thr Gln Ser Ile Gln
Met Asn Glu Tyr Asp 565 570 575 Phe Arg Ile Arg Ala Arg Glu Asp Thr
Tyr Asn Asp Gln Ser Arg Ile 580 585 590 Arg Tyr Thr Val Ala Asn Leu
His Ser Leu Asn Tyr Arg Ala Glu Ala 595 600 605 Asp Tyr Leu Ala Asp
Glu Leu Ser Lys Ala Leu Leu Ala 610 615 620 11 1124 DNA Zea mays
misc_feature (0)...(0) Maize RPA Middle Subunit Homologue-1 11
tcgacccacg cgtccgatcc tcccatctgc gcacccgcaa gcctattcgc cgcacctcct
60 caggtgaccg ggaag atg atg ccg ttg agc caa acc gac ttc tcg ccg tcg
111 Met Met Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser 1 5 10 cag ttc
acc tcc tcc cag aat gcc gcc gcc gac tcc acc acg cct tcc 159 Gln Phe
Thr Ser Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser 15 20 25
aag atg cgc ggc gcg tcc agc acc atg ccg ctc acc gtg aag cag gtc 207
Lys Met Arg Gly Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln Val 30
35 40 gtc gac gcg cag cag tct ggc acg ggc gag aag ggc gct ccg ttc
atc 255 Val Asp Ala Gln Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe
Ile 45 50 55 60 gtc aat ggc gtc gag atg gct aac att cga ctt gtg ggg
atg gtc aat 303 Val Asn Gly Val Glu Met Ala Asn Ile Arg Leu Val Gly
Met Val Asn 65 70 75 gcc aag gtg gag cgg acg acc gat gtg acc ttc
acg ctc gac gat ggc 351 Ala Lys Val Glu Arg Thr Thr Asp Val Thr Phe
Thr Leu Asp Asp Gly 80 85 90 acc ggc cgc ctc gat ttc atc aga tgg
gtg aat
gat gct tca gat tct 399 Thr Gly Arg Leu Asp Phe Ile Arg Trp Val Asn
Asp Ala Ser Asp Ser 95 100 105 ttt gaa act gct gct att cag aat ggt
atg tac att gcg gtc att gga 447 Phe Glu Thr Ala Ala Ile Gln Asn Gly
Met Tyr Ile Ala Val Ile Gly 110 115 120 agc ctc aag gga ctg caa gag
agg aag cgt gct act gct ttc tca atc 495 Ser Leu Lys Gly Leu Gln Glu
Arg Lys Arg Ala Thr Ala Phe Ser Ile 125 130 135 140 agg cct ata acc
gat ttc aat gag gtt acg ctg cat ttc att cag tgt 543 Arg Pro Ile Thr
Asp Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys 145 150 155 gtt cgg
atg cat ata gag aac att gaa tta aag gct ggc agt cct gca 591 Val Arg
Met His Ile Glu Asn Ile Glu Leu Lys Ala Gly Ser Pro Ala 160 165 170
cga atc agt tct tct atg gga gtg tca ttc tca aat gga ttc agt gaa 639
Arg Ile Ser Ser Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu 175
180 185 tca agc aca ccg aca tct ttg aaa tcc agt ccc gca ccg gtg acc
agc 687 Ser Ser Thr Pro Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr
Ser 190 195 200 ggg tca tcc gat act gat ctg cac acg cag gtc ctg aat
ttt ttt aat 735 Gly Ser Ser Asp Thr Asp Leu His Thr Gln Val Leu Asn
Phe Phe Asn 205 210 215 220 gaa cca gcg aac ctc gag agt gag cat ggg
gtg cac gtt gat gaa gta 783 Glu Pro Ala Asn Leu Glu Ser Glu His Gly
Val His Val Asp Glu Val 225 230 235 ctc aag cgg ttc aaa ctt ttg ccg
aag aag cag atc acg gat gct att 831 Leu Lys Arg Phe Lys Leu Leu Pro
Lys Lys Gln Ile Thr Asp Ala Ile 240 245 250 gat tac aat atg gac tcg
ggg cgt ctt tac tca aca att gat gaa ttc 879 Asp Tyr Asn Met Asp Ser
Gly Arg Leu Tyr Ser Thr Ile Asp Glu Phe 255 260 265 cac tac aag gca
act taaccgattt gaaggccagc ctgctggaaa tggcagagga 934 His Tyr Lys Ala
Thr 270 ctaagtatca cttgtactaa accaaagtct ggaaatgtca tgttgtgtca
tgaaatgcat 994 ggttggttta tggaaacatt tatatcttgt atcaactagt
tgatttgtat ctcgtgtcaa 1054 cttaatgact gagccaagaa aaggaagatg
tagaggccga cagaaaaaaa aaaaaaaaaa 1114 aaaaaaaaaa 1124 12 273 PRT
Zea mays 12 Met Met Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe
Thr Ser 1 5 10 15 Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser
Lys Met Arg Gly 20 25 30 Ala Ser Ser Thr Met Pro Leu Thr Val Lys
Gln Val Val Asp Ala Gln 35 40 45 Gln Ser Gly Thr Gly Glu Lys Gly
Ala Pro Phe Ile Val Asn Gly Val 50 55 60 Glu Met Ala Asn Ile Arg
Leu Val Gly Met Val Asn Ala Lys Val Glu 65 70 75 80 Arg Thr Thr Asp
Val Thr Phe Thr Leu Asp Asp Gly Thr Gly Arg Leu 85 90 95 Asp Phe
Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe Glu Thr Ala 100 105 110
Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly 115
120 125 Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser Ile Arg Pro Ile
Thr 130 135 140 Asp Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys Val
Arg Met His 145 150 155 160 Ile Glu Asn Ile Glu Leu Lys Ala Gly Ser
Pro Ala Arg Ile Ser Ser 165 170 175 Ser Met Gly Val Ser Phe Ser Asn
Gly Phe Ser Glu Ser Ser Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser
Pro Ala Pro Val Thr Ser Gly Ser Ser Asp 195 200 205 Thr Asp Leu His
Thr Gln Val Leu Asn Phe Phe Asn Glu Pro Ala Asn 210 215 220 Leu Glu
Ser Glu His Gly Val His Val Asp Glu Val Leu Lys Arg Phe 225 230 235
240 Lys Leu Leu Pro Lys Lys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met
245 250 255 Asp Ser Gly Arg Leu Tyr Ser Thr Ile Asp Glu Phe His Tyr
Lys Ala 260 265 270 Thr 13 979 DNA Zea mays misc_feature (0)...(0)
Maize RPA Middle Subunit Homologue-2 and 3 13 ttcggcacga gcgcacctcc
tcaggtgacc gggaag atg atg ccg ttg agc caa 54 Met Met Pro Leu Ser
Gln 1 5 acc gac ttc tcg ccg tcg cag ttc acc tcc tcc cag aat gcc gcc
gcc 102 Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser Ser Gln Asn Ala Ala
Ala 10 15 20 gac tcc acc acg cct tcc aag atg cgc ggc gcg tcc agc
acc atg ccg 150 Asp Ser Thr Thr Pro Ser Lys Met Arg Gly Ala Ser Ser
Thr Met Pro 25 30 35 ctc acc gtg aag cag gtc gtc gac gcg cag cag
tct ggc acg ggc gac 198 Leu Thr Val Lys Gln Val Val Asp Ala Gln Gln
Ser Gly Thr Gly Asp 40 45 50 aag ggc gct ccg ttc atc gtc aat ggc
gtc gag atg gct aac att cga 246 Lys Gly Ala Pro Phe Ile Val Asn Gly
Val Glu Met Ala Asn Ile Arg 55 60 65 70 ctt gtg ggg atg gtc aat gcc
aag gtg gag cgg acg acc gat gtg acc 294 Leu Val Gly Met Val Asn Ala
Lys Val Glu Arg Thr Thr Asp Val Thr 75 80 85 ttc acg ctc gac gat
ggc acc ggc cgc ctc gat ttc atc aga tgg gtg 342 Phe Thr Leu Asp Asp
Gly Thr Gly Arg Leu Asp Phe Ile Arg Trp Val 90 95 100 aat gat gct
tca gat tct ttt gaa act gct gct att cag aat ggt atg 390 Asn Asp Ala
Ser Asp Ser Phe Glu Thr Ala Ala Ile Gln Asn Gly Met 105 110 115 tac
att gcg gtc att gga agc ctc aag gga ctg caa gag agg aag cgt 438 Tyr
Ile Ala Val Ile Gly Ser Leu Lys Gly Leu Gln Glu Arg Lys Arg 120 125
130 gct act gct ttc tca atc agg cct ata acc gat ttc aat gag gtt acg
486 Ala Thr Ala Phe Ser Ile Arg Pro Ile Thr Asp Phe Asn Glu Val Thr
135 140 145 150 ctg cat ttc att cag tgt gtt cgg atg cat ata gag aac
att gaa tta 534 Leu His Phe Ile Gln Cys Val Arg Met His Ile Glu Asn
Ile Glu Leu 155 160 165 aag gct ggc agt cct gca cga atc agt tct tct
atg gga gtg tca ttc 582 Lys Ala Gly Ser Pro Ala Arg Ile Ser Ser Ser
Met Gly Val Ser Phe 170 175 180 tca aat gga ttc agt gaa tca agc aca
ccg aca tct ttg aaa tcc agt 630 Ser Asn Gly Phe Ser Glu Ser Ser Thr
Pro Thr Ser Leu Lys Ser Ser 185 190 195 ccc gca ccg gtg acc agc ggg
tca tcc gat act gat ctg cac acg cag 678 Pro Ala Pro Val Thr Ser Gly
Ser Ser Asp Thr Asp Leu His Thr Gln 200 205 210 gtc ctg aat ttt ttt
aat gaa cca gcg aac ctc gag agt gag cat ggg 726 Val Leu Asn Phe Phe
Asn Glu Pro Ala Asn Leu Glu Ser Glu His Gly 215 220 225 230 gtg cac
gtt gat gaa gta ctc aag cgg ttc aaa ctt ttg ccg aag aag 774 Val His
Val Asp Glu Val Leu Lys Arg Phe Lys Leu Leu Pro Lys Lys 235 240 245
cag atc acg gat gct att gat tac aat atg gac tcg ggg cgt ctt tac 822
Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met Asp Ser Gly Arg Leu Tyr 250
255 260 tca aca att gat gaa ttc cac tac aag gca act taaccgattt
gaaggccagc 875 Ser Thr Ile Asp Glu Phe His Tyr Lys Ala Thr 265 270
ctgctggaaa tggcagagga ctaagtatca cttgtactaa accaaagtct ggaaatgtca
935 tgttgtgtca tgaaatgcat ggttggttta tggaaacaaa aaaa 979 14 273 PRT
Zea mays 14 Met Met Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe
Thr Ser 1 5 10 15 Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser
Lys Met Arg Gly 20 25 30 Ala Ser Ser Thr Met Pro Leu Thr Val Lys
Gln Val Val Asp Ala Gln 35 40 45 Gln Ser Gly Thr Gly Asp Lys Gly
Ala Pro Phe Ile Val Asn Gly Val 50 55 60 Glu Met Ala Asn Ile Arg
Leu Val Gly Met Val Asn Ala Lys Val Glu 65 70 75 80 Arg Thr Thr Asp
Val Thr Phe Thr Leu Asp Asp Gly Thr Gly Arg Leu 85 90 95 Asp Phe
Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe Glu Thr Ala 100 105 110
Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly 115
120 125 Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser Ile Arg Pro Ile
Thr 130 135 140 Asp Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys Val
Arg Met His 145 150 155 160 Ile Glu Asn Ile Glu Leu Lys Ala Gly Ser
Pro Ala Arg Ile Ser Ser 165 170 175 Ser Met Gly Val Ser Phe Ser Asn
Gly Phe Ser Glu Ser Ser Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser
Pro Ala Pro Val Thr Ser Gly Ser Ser Asp 195 200 205 Thr Asp Leu His
Thr Gln Val Leu Asn Phe Phe Asn Glu Pro Ala Asn 210 215 220 Leu Glu
Ser Glu His Gly Val His Val Asp Glu Val Leu Lys Arg Phe 225 230 235
240 Lys Leu Leu Pro Lys Lys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met
245 250 255 Asp Ser Gly Arg Leu Tyr Ser Thr Ile Asp Glu Phe His Tyr
Lys Ala 260 265 270 Thr 15 1051 DNA Zea mays misc_feature (0)...(0)
Maize RPA Middle Subunit Homologue-4 15 tcgacccacg cgtccgatcc
tcccatctgc gcacccgcaa gcctattcgc cgcacctcct 60 caggtgaccg ggaag atg
atg ccg ttg agc caa acc gac ttc tcg ccg tcg 111 Met Met Pro Leu Ser
Gln Thr Asp Phe Ser Pro Ser 1 5 10 cag ttc acc tcc tcc cag aat gcc
gcc gcc gac tcc acc acg cct tcc 159 Gln Phe Thr Ser Ser Gln Asn Ala
Ala Ala Asp Ser Thr Thr Pro Ser 15 20 25 aag atg cgc ggc gcg tcc
agc acc atg ccg ctc acc gtg aag cag gtc 207 Lys Met Arg Gly Ala Ser
Ser Thr Met Pro Leu Thr Val Lys Gln Val 30 35 40 gtc gac gcg cag
cag tct ggc acg ggc gag aag ggc gct ccg ttc atc 255 Val Asp Ala Gln
Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile 45 50 55 60 gtc aat
ggc gtc gag atg gct aac att cga ctt gtg ggg atg gtc aat 303 Val Asn
Gly Val Glu Met Ala Asn Ile Arg Leu Val Gly Met Val Asn 65 70 75
gcc aag gtg gag cgg acg acc gat gtg acc ttc acg ctc gac gat ggc 351
Ala Lys Val Glu Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly 80
85 90 acc ggc cgc ctc gat ttc atc aga tgg gtg aat gat gct tca gat
tct 399 Thr Gly Arg Leu Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp
Ser 95 100 105 ttt gaa act gct gct att cag aat ggt atg tac att gcg
gtc att gga 447 Phe Glu Thr Ala Ala Ile Gln Asn Gly Met Tyr Ile Ala
Val Ile Gly 110 115 120 agc ctc aag gga ctg caa gag agg aag cgt gct
act gct ttc tca atc 495 Ser Leu Lys Gly Leu Gln Glu Arg Lys Arg Ala
Thr Ala Phe Ser Ile 125 130 135 140 agg cct ata acc gat ttc aat gag
gtt acg ctg cat ttc att cag tgt 543 Arg Pro Ile Thr Asp Phe Asn Glu
Val Thr Leu His Phe Ile Gln Cys 145 150 155 gtt cgg atg cat ata gag
aac act gaa tta aag gct ggc agt cct gca 591 Val Arg Met His Ile Glu
Asn Thr Glu Leu Lys Ala Gly Ser Pro Ala 160 165 170 cga atc aat tct
tct atg gga gtg tca ttc tca aat gga ttc agt gaa 639 Arg Ile Asn Ser
Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu 175 180 185 tca agc
aca ccg aca tct ttg aaa tcc agt ccc gca ccg gtg acc agc 687 Ser Ser
Thr Pro Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr Ser 190 195 200
ggg tca tcc gat act gat ctg cac acg cag gtc ctg aat ttt ttt aat 735
Gly Ser Ser Asp Thr Asp Leu His Thr Gln Val Leu Asn Phe Phe Asn 205
210 215 220 gaa cca gcg aac ctc gag agt gag cat ggg gtg cac gtt gat
gaa gta 783 Glu Pro Ala Asn Leu Glu Ser Glu His Gly Val His Val Asp
Glu Val 225 230 235 ctc aag cgg ttc aaa ctt ttg ccg aag aag cag atc
acg gat gct att 831 Leu Lys Arg Phe Lys Leu Leu Pro Lys Lys Gln Ile
Thr Asp Ala Ile 240 245 250 gat tac aat atg gac tcg ggg cgt ctt tac
tca aca att gat gaa ttc 879 Asp Tyr Asn Met Asp Ser Gly Arg Leu Tyr
Ser Thr Ile Asp Glu Phe 255 260 265 cac tac aag gca act taaccgattt
gaaggtcagc ctgctggaaa tggcagagga 934 His Tyr Lys Ala Thr 270
ctaagtatca cttgtactaa accaaagtct ggaaatgtca tgttgtgtca tgaaatgcat
994 ggttggttta tggaaacaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa
1051 16 273 PRT Zea mays 16 Met Met Pro Leu Ser Gln Thr Asp Phe Ser
Pro Ser Gln Phe Thr Ser 1 5 10 15 Ser Gln Asn Ala Ala Ala Asp Ser
Thr Thr Pro Ser Lys Met Arg Gly 20 25 30 Ala Ser Ser Thr Met Pro
Leu Thr Val Lys Gln Val Val Asp Ala Gln 35 40 45 Gln Ser Gly Thr
Gly Glu Lys Gly Ala Pro Phe Ile Val Asn Gly Val 50 55 60 Glu Met
Ala Asn Ile Arg Leu Val Gly Met Val Asn Ala Lys Val Glu 65 70 75 80
Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly Thr Gly Arg Leu 85
90 95 Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe Glu Thr
Ala 100 105 110 Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly Ser
Leu Lys Gly 115 120 125 Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser
Ile Arg Pro Ile Thr 130 135 140 Asp Phe Asn Glu Val Thr Leu His Phe
Ile Gln Cys Val Arg Met His 145 150 155 160 Ile Glu Asn Thr Glu Leu
Lys Ala Gly Ser Pro Ala Arg Ile Asn Ser 165 170 175 Ser Met Gly Val
Ser Phe Ser Asn Gly Phe Ser Glu Ser Ser Thr Pro 180 185 190 Thr Ser
Leu Lys Ser Ser Pro Ala Pro Val Thr Ser Gly Ser Ser Asp 195 200 205
Thr Asp Leu His Thr Gln Val Leu Asn Phe Phe Asn Glu Pro Ala Asn 210
215 220 Leu Glu Ser Glu His Gly Val His Val Asp Glu Val Leu Lys Arg
Phe 225 230 235 240 Lys Leu Leu Pro Lys Lys Gln Ile Thr Asp Ala Ile
Asp Tyr Asn Met 245 250 255 Asp Ser Gly Arg Leu Tyr Ser Thr Ile Asp
Glu Phe His Tyr Lys Ala 260 265 270 Thr 17 1087 DNA Zea mays
misc_feature (0)...(0) Maize RPA Middle Subunit Homologue-5 17
aattccgggg ccgacccacg cgtccgcatc gatcctccca tctgcgcacc cgcaagccta
60 ttcgccgcac ctcctcaggt gaccgggaag atg atg ccg ttg agc caa acc gac
114 Met Met Pro Leu Ser Gln Thr Asp 1 5 ttc tcg ccg tcg cag ttc acc
tcc tcc cag aat gcc gcc gcc gac tcc 162 Phe Ser Pro Ser Gln Phe Thr
Ser Ser Gln Asn Ala Ala Ala Asp Ser 10 15 20 acc acg cct tcc aag
atg cgc ggc gcg tcc agc acc atg ccg ctc acc 210 Thr Thr Pro Ser Lys
Met Arg Gly Ala Ser Ser Thr Met Pro Leu Thr 25 30 35 40 gtg aag car
gtc gtc gac gcg cag cag tct ggc acg ggc gag aag ggc 258 Val Lys Xaa
Val Val Asp Ala Gln Gln Ser Gly Thr Gly Glu Lys Gly 45 50 55 gct
ccg ttc atc gtc aat ggc gtc gag atg gct aac att cga ctt gtg 306 Ala
Pro Phe Ile Val Asn Gly Val Glu Met Ala Asn Ile Arg Leu Val 60 65
70 ggg atg gtc aat gcc aag gtg gag cgg acg acc gat gtg acc ttc acg
354 Gly Met Val Asn Ala Lys Val Glu Arg Thr Thr Asp Val Thr Phe Thr
75 80 85 ctc gac gat ggc acc ggc cgc ctc gat ttc atc aga tgg gtg
aat gat 402 Leu Asp Asp Gly Thr Gly Arg Leu Asp Phe Ile Arg Trp Val
Asn Asp 90 95 100 gct tca gat tct ttt gaa act gct gct att cag aat
ggt atg tac att 450 Ala Ser Asp Ser Phe Glu Thr Ala Ala Ile Gln Asn
Gly Met Tyr Ile 105 110 115 120 gcg gtc att gga agc ctc aag gga ctg
caa gag agg aag cgt gct act 498 Ala Val Ile Gly Ser Leu Lys Gly Leu
Gln Glu Arg Lys Arg Ala Thr 125 130 135 gct ttc tca atc agg cct ata
acc gat ttc aat gag gtt acg ctg cat 546 Ala Phe Ser Ile Arg Pro Ile
Thr Asp Phe Asn Glu Val Thr Leu His 140 145 150 ttc att cag tgt gtt
cgg atg cat ata gag aac act gaa tta aag gct 594 Phe Ile Gln Cys Val
Arg Met His Ile Glu Asn Thr Glu Leu Lys Ala 155 160 165 ggc agt
cct
gca cga atc aat tct tct atg gga gtg tca ttc tca aat 642 Gly Ser Pro
Ala Arg Ile Asn Ser Ser Met Gly Val Ser Phe Ser Asn 170 175 180 gga
ttc agt gaa tca agc aca ccg aca tct ttg aaa tcc agt ccc gca 690 Gly
Phe Ser Glu Ser Ser Thr Pro Thr Ser Leu Lys Ser Ser Pro Ala 185 190
195 200 ccg gtg acc agc ggg tca tcc gat act gat ctg cac acg cag gtc
ctg 738 Pro Val Thr Ser Gly Ser Ser Asp Thr Asp Leu His Thr Gln Val
Leu 205 210 215 aat ttt ttt aat gaa cca gcg aac ctc gag agt gag cat
ggg gtg cac 786 Asn Phe Phe Asn Glu Pro Ala Asn Leu Glu Ser Glu His
Gly Val His 220 225 230 gtt gat gaa gta ctc aag cgg ttc aac ttt tgc
cga aga agc aga tca 834 Val Asp Glu Val Leu Lys Arg Phe Asn Phe Cys
Arg Arg Ser Arg Ser 235 240 245 cgg atg cta ttg att aca ata tgg act
cgg ggc gtc ttt act caa caa 882 Arg Met Leu Leu Ile Thr Ile Trp Thr
Arg Gly Val Phe Thr Gln Gln 250 255 260 ttg atg aat tcc act aca agg
caa ctt aac cga ttt gaa ggt cag cct 930 Leu Met Asn Ser Thr Thr Arg
Gln Leu Asn Arg Phe Glu Gly Gln Pro 265 270 275 280 gct gga aat ggc
aga gga cta agt atc act tgt act aaa cca aag tct 978 Ala Gly Asn Gly
Arg Gly Leu Ser Ile Thr Cys Thr Lys Pro Lys Ser 285 290 295 gga aat
gtc atg ttg tgt cat gaa atg cat ggt tgg ttt atg gaa aca 1026 Gly
Asn Val Met Leu Cys His Glu Met His Gly Trp Phe Met Glu Thr 300 305
310 ttt ata tct tgt atc aac tagttgattt gtatctcttg tgtcaaaaaa 1074
Phe Ile Ser Cys Ile Asn 315 aaaaaaaaaa aaa 1087 18 318 PRT Zea mays
VARIANT (1)...(318) Xaa = Any Amino Acid 18 Met Met Pro Leu Ser Gln
Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser 1 5 10 15 Ser Gln Asn Ala
Ala Ala Asp Ser Thr Thr Pro Ser Lys Met Arg Gly 20 25 30 Ala Ser
Ser Thr Met Pro Leu Thr Val Lys Xaa Val Val Asp Ala Gln 35 40 45
Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile Val Asn Gly Val 50
55 60 Glu Met Ala Asn Ile Arg Leu Val Gly Met Val Asn Ala Lys Val
Glu 65 70 75 80 Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly Thr
Gly Arg Leu 85 90 95 Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp
Ser Phe Glu Thr Ala 100 105 110 Ala Ile Gln Asn Gly Met Tyr Ile Ala
Val Ile Gly Ser Leu Lys Gly 115 120 125 Leu Gln Glu Arg Lys Arg Ala
Thr Ala Phe Ser Ile Arg Pro Ile Thr 130 135 140 Asp Phe Asn Glu Val
Thr Leu His Phe Ile Gln Cys Val Arg Met His 145 150 155 160 Ile Glu
Asn Thr Glu Leu Lys Ala Gly Ser Pro Ala Arg Ile Asn Ser 165 170 175
Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu Ser Ser Thr Pro 180
185 190 Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr Ser Gly Ser Ser
Asp 195 200 205 Thr Asp Leu His Thr Gln Val Leu Asn Phe Phe Asn Glu
Pro Ala Asn 210 215 220 Leu Glu Ser Glu His Gly Val His Val Asp Glu
Val Leu Lys Arg Phe 225 230 235 240 Asn Phe Cys Arg Arg Ser Arg Ser
Arg Met Leu Leu Ile Thr Ile Trp 245 250 255 Thr Arg Gly Val Phe Thr
Gln Gln Leu Met Asn Ser Thr Thr Arg Gln 260 265 270 Leu Asn Arg Phe
Glu Gly Gln Pro Ala Gly Asn Gly Arg Gly Leu Ser 275 280 285 Ile Thr
Cys Thr Lys Pro Lys Ser Gly Asn Val Met Leu Cys His Glu 290 295 300
Met His Gly Trp Phe Met Glu Thr Phe Ile Ser Cys Ile Asn 305 310 315
19 1074 DNA Zea mays misc_feature (0)...(0) Maize RPA Middle
Subunit Homologue-6 19 gacccacgcg tccgcgcaag cctattcgcc gcacctcctc
aggtgaccgg gaag atg 57 Met 1 atg ccg ttg agc caa acc gac ttc tcg
ccg tcg cag ttc acc tcc tcc 105 Met Pro Leu Ser Gln Thr Asp Phe Ser
Pro Ser Gln Phe Thr Ser Ser 5 10 15 cag aat gcc gcc gcc gac tcc acc
acg cct tcc aag atg cgc ggc gcg 153 Gln Asn Ala Ala Ala Asp Ser Thr
Thr Pro Ser Lys Met Arg Gly Ala 20 25 30 tcc agc acc atg ccg ctc
acc gtg aag cag gtc gtc gac gcg cag cag 201 Ser Ser Thr Met Pro Leu
Thr Val Lys Gln Val Val Asp Ala Gln Gln 35 40 45 tct ggc acg ggc
gag aag ggc gct ccg ttc atc gtc aat ggc gtc gag 249 Ser Gly Thr Gly
Glu Lys Gly Ala Pro Phe Ile Val Asn Gly Val Glu 50 55 60 65 atg gct
aac att cga ctt gtg ggg atg gtc aat gcc aag gtg gag cgg 297 Met Ala
Asn Ile Arg Leu Val Gly Met Val Asn Ala Lys Val Glu Arg 70 75 80
acg acc gat gtg acc ttc acg ctc gac gat ggc acc ggc cgc ctc gat 345
Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly Thr Gly Arg Leu Asp 85
90 95 ttc atc aga tgg gtg aat gat gct tca gat tct ttt gaa act gct
gct 393 Phe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe Glu Thr Ala
Ala 100 105 110 att cag aat ggt atg tac att gcg gtc att gga agc ctc
aag gga ctg 441 Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly Ser Leu
Lys Gly Leu 115 120 125 caa gag agg aag cgt gct act gct ttc tca atc
agg cct ata acc gat 489 Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser Ile
Arg Pro Ile Thr Asp 130 135 140 145 ttc aat gag gtt acg ctg cat ttc
att cag tgt gtt cgg atg cat ata 537 Phe Asn Glu Val Thr Leu His Phe
Ile Gln Cys Val Arg Met His Ile 150 155 160 gag aac act gaa tta aag
gct ggc agt cct gca cga atc aat tct tct 585 Glu Asn Thr Glu Leu Lys
Ala Gly Ser Pro Ala Arg Ile Asn Ser Ser 165 170 175 atg gga gtg tca
ttc tca aat gga ttc agt gaa tca agc aca ccg aca 633 Met Gly Val Ser
Phe Ser Asn Gly Phe Ser Glu Ser Ser Thr Pro Thr 180 185 190 tct ttg
aaa tcc agt ccc gca ccg gtg acc agc ggg tca tcc gat act 681 Ser Leu
Lys Ser Ser Pro Ala Pro Val Thr Ser Gly Ser Ser Asp Thr 195 200 205
gat ctg cac acg cag gtc ctg aat ttt ttt aat gaa cca gcg aac ctc 729
Asp Leu His Thr Gln Val Leu Asn Phe Phe Asn Glu Pro Ala Asn Leu 210
215 220 225 gag agt gag cat ggg gtg cac gtt gat gaa gta ctc aag cgg
ttc aaa 777 Glu Ser Glu His Gly Val His Val Asp Glu Val Leu Lys Arg
Phe Lys 230 235 240 ctt ttg ccg aag aag cag atc acg gat gct att gat
tac aat atg gac 825 Leu Leu Pro Lys Lys Gln Ile Thr Asp Ala Ile Asp
Tyr Asn Met Asp 245 250 255 tcg ggg cgt ctt tac tca aca att gat gaa
ttc cac tac aag gca act 873 Ser Gly Arg Leu Tyr Ser Thr Ile Asp Glu
Phe His Tyr Lys Ala Thr 260 265 270 taaccgattt gaaggtcagc
ctgctggaaa tggcagagga ctaagtatca cttgtactaa 933 accaaagtct
ggaaatgtca tgttgtgtca tgaaatgcat ggttggttta tggaaacatt 993
tatatcttgt atcaactagt tgatttgtat ctcttgtgtc aacttaatga ctgagccaac
1053 aaaaggaaaa aaaaaaaaaa a 1074 20 273 PRT Zea mays 20 Met Met
Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser 1 5 10 15
Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser Lys Met Arg Gly 20
25 30 Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln Val Val Asp Ala
Gln 35 40 45 Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile Val
Asn Gly Val 50 55 60 Glu Met Ala Asn Ile Arg Leu Val Gly Met Val
Asn Ala Lys Val Glu 65 70 75 80 Arg Thr Thr Asp Val Thr Phe Thr Leu
Asp Asp Gly Thr Gly Arg Leu 85 90 95 Asp Phe Ile Arg Trp Val Asn
Asp Ala Ser Asp Ser Phe Glu Thr Ala 100 105 110 Ala Ile Gln Asn Gly
Met Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly 115 120 125 Leu Gln Glu
Arg Lys Arg Ala Thr Ala Phe Ser Ile Arg Pro Ile Thr 130 135 140 Asp
Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys Val Arg Met His 145 150
155 160 Ile Glu Asn Thr Glu Leu Lys Ala Gly Ser Pro Ala Arg Ile Asn
Ser 165 170 175 Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu Ser
Ser Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr
Ser Gly Ser Ser Asp 195 200 205 Thr Asp Leu His Thr Gln Val Leu Asn
Phe Phe Asn Glu Pro Ala Asn 210 215 220 Leu Glu Ser Glu His Gly Val
His Val Asp Glu Val Leu Lys Arg Phe 225 230 235 240 Lys Leu Leu Pro
Lys Lys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met 245 250 255 Asp Ser
Gly Arg Leu Tyr Ser Thr Ile Asp Glu Phe His Tyr Lys Ala 260 265 270
Thr 21 1231 DNA Zea mays misc_feature (0)...(0) Maize RPA Middle
Subunit Homologue-7 21 tcccgggtcg acccacgcgt ccgcgatcct cccatctgcg
cacccgcaag cctattcgcc 60 gcacctcctc aggtgaccgg gaag atg atg ccg ttg
agc caa acc gac ttc 111 Met Met Pro Leu Ser Gln Thr Asp Phe 1 5 tcg
ccg tcg cag ttc acc tcc tcc cag aat gcc gcc gcc gac tcc acc 159 Ser
Pro Ser Gln Phe Thr Ser Ser Gln Asn Ala Ala Ala Asp Ser Thr 10 15
20 25 acg cct tcc aag atg cgc ggc gcg tcc agc acc atg ccg ctc acc
gtg 207 Thr Pro Ser Lys Met Arg Gly Ala Ser Ser Thr Met Pro Leu Thr
Val 30 35 40 aag cag gtc gtc gac gcg cag cag tct ggc acg ggc gag
aag ggc gct 255 Lys Gln Val Val Asp Ala Gln Gln Ser Gly Thr Gly Glu
Lys Gly Ala 45 50 55 ccg ttc atc gtc aat ggc gtc gag atg gct aac
att cga ctt gtg ggg 303 Pro Phe Ile Val Asn Gly Val Glu Met Ala Asn
Ile Arg Leu Val Gly 60 65 70 atg gtc aat gcc aag gtg gag cgg acg
acc gat gtg acc ttc acg ctc 351 Met Val Asn Ala Lys Val Glu Arg Thr
Thr Asp Val Thr Phe Thr Leu 75 80 85 gac gat ggc acc ggc cgc ctc
gat ttc atc aga tgg gtg aat gat gct 399 Asp Asp Gly Thr Gly Arg Leu
Asp Phe Ile Arg Trp Val Asn Asp Ala 90 95 100 105 tca gat tct ttt
gaa act gct gct att cag aat ggt atg tac att gcg 447 Ser Asp Ser Phe
Glu Thr Ala Ala Ile Gln Asn Gly Met Tyr Ile Ala 110 115 120 gtc att
gga agc ctc aag gga ctg caa gag agg aag cgt gct act gct 495 Val Ile
Gly Ser Leu Lys Gly Leu Gln Glu Arg Lys Arg Ala Thr Ala 125 130 135
ttc tca atc agg cct ata acc gat ttc aat gag gtt acg ctg cat ttc 543
Phe Ser Ile Arg Pro Ile Thr Asp Phe Asn Glu Val Thr Leu His Phe 140
145 150 att cag tgt gtt cgg atg cat ata gag aac act gaa tta aag gct
ggc 591 Ile Gln Cys Val Arg Met His Ile Glu Asn Thr Glu Leu Lys Ala
Gly 155 160 165 agt cct gca cga atc aat tct tct atg gga gtg tca ttc
tca aat gga 639 Ser Pro Ala Arg Ile Asn Ser Ser Met Gly Val Ser Phe
Ser Asn Gly 170 175 180 185 ttc agt gaa tca agc aca ccg aca tct ttg
aaa tcc agt ccc gca ccg 687 Phe Ser Glu Ser Ser Thr Pro Thr Ser Leu
Lys Ser Ser Pro Ala Pro 190 195 200 gtg acc agc ggg tca tcc gat act
gat ctg cac acg cag gtc ctg aat 735 Val Thr Ser Gly Ser Ser Asp Thr
Asp Leu His Thr Gln Val Leu Asn 205 210 215 ttt ttt aat gaa cca gcg
aac ctc gag agt gag cat ggg gtg cac gtt 783 Phe Phe Asn Glu Pro Ala
Asn Leu Glu Ser Glu His Gly Val His Val 220 225 230 gat gaa gta ctc
aag cgg ttc aaa ctt ttg ccg aag aag cag atc acg 831 Asp Glu Val Leu
Lys Arg Phe Lys Leu Leu Pro Lys Lys Gln Ile Thr 235 240 245 gat gct
att gat tac aat atg gac tcg ggg cgt ctt tac tca aca att 879 Asp Ala
Ile Asp Tyr Asn Met Asp Ser Gly Arg Leu Tyr Ser Thr Ile 250 255 260
265 gat gaa ttc cac tac aag gca act taaccgattt gaaggtcagc
ctgctggaaa 933 Asp Glu Phe His Tyr Lys Ala Thr 270 tggcagagga
ctaagtatca cttgtactaa accaaagtct ggaaatgtca tgttgtgtca 993
tgaaatgcat ggttggttta tggaaacatt tatatcttgt atcaactagt tgatttgtat
1053 ctcttgtgtc aacttaatga ctgagccaac aaaaggaaga tgtagaggca
gacagacatt 1113 tgtagattgg ctgatagctg attcgggtag ctggtccaat
tgcaatctgg ggcccaataa 1173 ttcagatgca aaagcagaaa gatatttcaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1231 22 273 PRT Zea mays 22 Met Met
Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser 1 5 10 15
Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser Lys Met Arg Gly 20
25 30 Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln Val Val Asp Ala
Gln 35 40 45 Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile Val
Asn Gly Val 50 55 60 Glu Met Ala Asn Ile Arg Leu Val Gly Met Val
Asn Ala Lys Val Glu 65 70 75 80 Arg Thr Thr Asp Val Thr Phe Thr Leu
Asp Asp Gly Thr Gly Arg Leu 85 90 95 Asp Phe Ile Arg Trp Val Asn
Asp Ala Ser Asp Ser Phe Glu Thr Ala 100 105 110 Ala Ile Gln Asn Gly
Met Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly 115 120 125 Leu Gln Glu
Arg Lys Arg Ala Thr Ala Phe Ser Ile Arg Pro Ile Thr 130 135 140 Asp
Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys Val Arg Met His 145 150
155 160 Ile Glu Asn Thr Glu Leu Lys Ala Gly Ser Pro Ala Arg Ile Asn
Ser 165 170 175 Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu Ser
Ser Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr
Ser Gly Ser Ser Asp 195 200 205 Thr Asp Leu His Thr Gln Val Leu Asn
Phe Phe Asn Glu Pro Ala Asn 210 215 220 Leu Glu Ser Glu His Gly Val
His Val Asp Glu Val Leu Lys Arg Phe 225 230 235 240 Lys Leu Leu Pro
Lys Lys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met 245 250 255 Asp Ser
Gly Arg Leu Tyr Ser Thr Ile Asp Glu Phe His Tyr Lys Ala 260 265 270
Thr
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References