U.S. patent application number 09/846589 was filed with the patent office on 2003-09-04 for plant amino acyl-trna synthetase.
Invention is credited to Famodu, Omolayo O., Simmons, Carl R..
Application Number | 20030166241 09/846589 |
Document ID | / |
Family ID | 26786145 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166241 |
Kind Code |
A1 |
Famodu, Omolayo O. ; et
al. |
September 4, 2003 |
Plant amino acyl-tRNA synthetase
Abstract
This invention relates to an isolated nucleic acid fragment
encoding an aminoacyl-tRNA synthetase. The invention also relates
to the construction of a chimeric gene encoding all or a portion of
the aminoacyl-tRNA synthetase, in sense or antisense orientation,
wherein expression of the chimeric gene results in production of
altered levels of the aminoacyl-tRNA synthetase in a transformed
host cell.
Inventors: |
Famodu, Omolayo O.; (Newark,
DE) ; Simmons, Carl R.; (Des Moines, IA) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
1220 N MARKET STREET
P O BOX 2207
WILMINGTON
DE
19899
|
Family ID: |
26786145 |
Appl. No.: |
09/846589 |
Filed: |
May 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09846589 |
May 1, 2001 |
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09352990 |
Jul 14, 1999 |
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6255090 |
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60092866 |
Jul 15, 1998 |
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Current U.S.
Class: |
435/226 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/93 20130101; C12N
15/8274 20130101 |
Class at
Publication: |
435/226 ;
435/69.1; 435/325; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/64; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid fragment encoding an aspartyl-tRNA
synthetase comprising a member selected from the group consisting
of: (a) an isolated nucleic acid fragment encoding an amino acid
sequence that is at least 80% identical to the amino acid sequence
set forth in a member selected from the group consisting of SEQ ID
NO: 2, 4, 6 and 8; (b) an isolated nucleic acid fragment that is
complementary to (a).
2. The isolated nucleic acid fragment of claim 1 wherein nucleic
acid fragment is a functional RNA.
3. The isolated nucleic acid fragment of claim 1 wherein the
nucleotide sequence of the fragment comprises the sequence set
forth in a member selected from the group consisting of SEQ ID NO:
1, 3, 5 and 7.
4. A chimeric gene comprising the nucleic acid fragment of claim 1
operably linked to suitable regulatory sequences.
5. A transformed host cell comprising the chimeric gene of claim
4.
6. An aspartyl-tRNA synthetase polypeptide comprising all or a
substantial portion of the amino acid sequence set forth in a
member selected from the group consisting of SEQ ID NO: 2., 4, 6
and 8
7. An isolated nucleic acid fragment encoding a cysteinyl-tRNA
synthetase comprising a member selected from the group consisting
of: (a) an isolated nucleic acid fragment encoding an amino acid
sequence that is at least 80% identical to the amino acid sequence
set forth in a member selected from the group consisting of SEQ ID
NO: 10, 12 and 14; (b) an isolated nucleic acid fragment that is
complementary to (a).
8. The isolated nucleic acid fragment of claim 7 wherein nucleic
acid fragment is a functional RNA.
9. The isolated nucleic acid fragment of claim 7 wherein the
nucleotide sequence of the fragment comprises the sequence set
forth in a member selected from the group consisting of SEQ ID NO:
9, 11 and 13.
10. A chimeric gene comprising the nucleic acid fragment of claim 7
operably linked to suitable regulatory sequences.
11. A transformed host cell comprising the chimeric gene of claim
10.
12. A cysteinyl-tRNA synthetase polypeptide comprising all or a
substantial portion of the amino acid sequence set forth in a
member selected from the group consisting of SEQ ID NO: 10, 12 and
14.
13. An isolated nucleic acid fragment encoding a tryptophanyl-tRNA
synthetase comprising a member selected from the group consisting
of: (a) an isolated nucleic acid fragment encoding an amino acid
sequence that is at least 80% identical to the amino acid sequence
set forth in a member selected from the group consisting of SEQ ID
NO: 16, 18 and 20; (b) an isolated nucleic acid fragment that is
complementary to (a).
14. The isolated nucleic acid fragment of claim 13 wherein nucleic
acid fragment is a functional RNA.
15. The isolated nucleic acid fragment of claim 13 wherein the
nucleotide sequence of the fragment comprises the sequence set
forth in a member selected from the group consisting of SEQ ID NO:
15, 17 and 19.
16. A chimeric gene comprising the nucleic acid fragment of claim
13 operably linked to suitable regulatory sequences.
17. A transformed host cell comprising the chimeric gene of claim
16.
18. A tryptophanyl-tRNA synthetase polypeptide comprising all or a
substantial portion of the amino acid sequence set forth in a
member selected from the group consisting of SEQ ID NO: 16, 18 and
20.
19. An isolated nucleic acid fragment encoding a tyrosyl-tRNA
synthetase comprising a member selected from the group consisting
of: (a) an isolated nucleic acid fragment encoding an amino acid
sequence that is at least 80% identical to the amino acid sequence
set forth in SEQ ID NO: 22; (b) an isolated nucleic acid fragment
that is complementary to (a).
20. The isolated nucleic acid fragment of claim 19 wherein nucleic
acid fragment is a functional RNA.
21. The isolated nucleic acid fragment of claim 19 wherein the
nucleotide sequence of the fragment comprises the sequence set
forth in SEQ ID NO: 2 1.
22. A chimeric gene comprising the nucleic acid fragment of claim
19 operably linked to suitable regulatory sequences.
23. A transformed host cell comprising the chimeric gene of claim
22.
24. A tyrosyl-tRNA synthetase polypeptide comprising all or a
substantial portion of the amino acid sequence set forth in SEQ ID
NO: 22.
25. A method of altering the level of expression of an
aminoacyl-tRNA synthetase in a host cell comprising: (a)
transforming a host cell with the chimeric gene of any of claims 4,
10, 16 and 22; and (b) growing the transformed host cell produced
in step (a) under conditions that are suitable for expression of
the chimeric gene wherein expression of the chimeric gene results
in production of altered levels of an aminoacyl-tRNA synthetase in
the transformed host cell.
26. A method of obtaining a nucleic acid fragment encoding all or a
substantial portion of the amino acid sequence encoding an
aminoacyl-tRNA synthetase comprising: (a) probing a cDNA or genomic
library with the nucleic acid fragment of any of claims 1, 7,13 and
19; (b) identifying a DNA clone that hybridizes with the nucleic
acid fragment of any of claims 1, 7, 13 and 19; (c) isolating the
DNA clone identified in step (b); and (d) sequencing the cDNA or
genomic fragment that comprises the clone isolated in step (c)
wherein the sequenced nucleic acid fragment encodes all or a
substantial portion of the amino acid sequence encoding an
aminoacyl-tRNA synthetase.
27. A method of obtaining a nucleic acid fragment encoding a
substantial portion of an amino acid sequence encoding an
aminoacyl-tRNA synthetase comprising: (a) synthesizing an
oligonucleotide primer corresponding to a portion of the sequence
set forth in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19
and 21; and (b) amplifying a cDNA insert present in a cloning
vector using the oligonucleotide primer of step (a) and a primer
representing sequences of the cloning vector wherein the amplified
nucleic acid fragment encodes a substantial portion of an amino
acid sequence encoding an aminoacyl-tRNA synthetase.
28. The product of the method of claim 26.
29. The product of the method of claim 27.
30. A method for evaluating at least one compound for its ability
to inhibit the activity of an aminoacyl-tRNA synthetase, the method
comprising the steps of: (a) transforming a host cell with a
chimeric gene comprising a nucleic acid fragment encoding an
aminoacyl-tRNA synthetase, operably linked to suitable regulatory
sequences; (b) growing the transformed host cell under conditions
that are suitable for expression of the chimeric gene wherein
expression of the chimeric gene results in production of the
aminoacyl-tRNA synthetase encoded by the operably linked nucleic
acid fragment in the transformed host cell; (c) optionally
purifying the aminoacyl-tRNA synthetase expressed by the
transformed host cell; (d) treating the aminoacyl-tRNA synthetase
with a compound to be tested; and (e) comparing the activity of the
aminoacyl-tRNA synthetase that has been treated with a test
compound to the activity of an untreated aminoacyl-tRNA synthetase,
thereby selecting compounds with potential for inhibitory activity.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/092,866, filed Jul. 15, 1998.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
fragments encoding aminoacyl-tRNA synthetase in plants and
seeds.
BACKGROUND OF THE INVENTION
[0003] All tRNAs have two functions: to chemically link to a
specific amino acid and to recognize a codon in mRNA so that the
linked amino acid can be added to a growing peptide chain during
protein synthesis. In general there is at least one aminoacyl-tRNA
synthetase for each of the twenty amino acids. A specific
aminoacyl-tRNA synthetase links an amino acid to the 2' or 3'
hydroxyl of the adenosine residue at the 3'-terminus of a tRNA
molecule. Once its correct amino acid is attached, a tRNA then
recognizes a codon in mRNA, thus deliverng its amino acid to the
growing polypeptide chain. These enzymatic functions are critical
to gene expression (Neidhart et al. (1975) Annu. Rev. Microbiol.
29:215-250). Mutations in tRNA synthetases often result in
alterations in protein synthesis and in some cases cell death.
[0004] Plants like other cellular organisms have aminoacyl-tRNA
synthetases. However a complete description of the plant
`complement` of aminoacyl-tRNA synthetases has not been published.
It is anticipated that plants will likely have at least forty
aminoacyl-tRNA synthetases. Plants have three sites of protein
synthesis: the cytoplasm, the mitochondria and the chloroplast.
Accordingly, there could be as many as sixty aminoacyl-tRNA
synthetases. Based on knowledge of other eukaryotes the cytoplasmic
and mitochondrial aminoacyl-tRNA synthetases are expected to be
encoded by the same gene. This gene should be nuclearly encoded and
produce two alternate products, one with a mitochondrial specific
transit peptide, and the other without this targeting signal. The
chloroplast is the other site of protein synthesis in plants. Based
on a few examples of known plant chloroplast specific
aminoacyl-tRNA synthetase genes it appears that these genes are
also nuclear-encoded. Chloroplast aminoacyl-tRNA synthetases are
directed to the chloroplast by a transit peptide.
[0005] Because of the central role aminoacyl-tRNA synthetases play
in protein synthesis any agent that inhibits or disrupts
aminoacyl-tRNA synthetase activity is likely to be toxic. Indeed a
number of aminoacyl-tRNA synthetase inhibitors (antibiotics and
herbicides) are known (Zon et al. (1988) Phytochemistry
27(3):711-714 and Heacock et al. (1996) Bioorganic Chemistry
24(3):273-289). Thus it may be possible to develop new herbicides
that target aminoacyl-tRNA synthetases and engineer aminoacyl-tRNA
synthetases that are resistant to such herbicides. Accordingly, the
availability of nucleic acid sequences encoding all or a portion of
these enzymes would facilitate studies to better understand protein
synthesis in plants, provide genetic tools for the manipulation of
gene expression, and provide a possible target for herbicides.
SUMMARY OF THE INVENTION
[0006] The instant invention relates to isolated nucleic acid
fragments encoding aminoacyl-tRNA synthetase. Specifically, this
invention concerns an isolated nucleic acid fragment encoding an
aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase and an
isolated nucleic acid fragment that is substantially similar to an
isolated nucleic acid fragment encoding an aspartyl-tRNA
synthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase
or tyrosyl-tRNA synthetase. In addition, this invention relates to
a nucleic acid fragment that is complementary to the nucleic acid
fragment encoding aspartyl-tRNA synthetase, cysteinyl-tRNA
synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA
synthetase.
[0007] An additional embodiment of the instant invention pertains
to a polypeptide encoding all or a substantial portion of an
aminoacyl-tRNA synthetaseselected from the group consisting of
aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase and tyrosyl-tRNA synthetase.
[0008] In another embodiment, the instant invention relates to a
chimeric gene encoding an aspartyl-tRNA synthetase, cysteinyl-tRNA
synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA
synthetase, or to a chimeric gene that comprises a nucleic acid
fragment that is complementary to a nucleic acid fragment encoding
an aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, operably
linked to suitable regulatory sequences, wherein expression of the
chimeric gene results in production of levels of the encoded
protein in a transformed host cell that is altered (i.e., increased
or decreased) from the level produced in an untransformed host
cell.
[0009] In a further embodiment, the instant invention concerns a
transformed host cell comprising in its genome a chimeric gene
encoding an aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, operably
linked to suitable regulatory sequences. Expression of the chimeric
gene results in production of altered levels of the encoded protein
in the transformed host cell. The transformed host cell can be of
eukaryotic or prokaryotic origin, and include cells derived from
higher plants and microorganisms. The invention also includes
transformed plants that arise from transformed host cells of higher
plants, and seeds derived from such transformed plants.
[0010] An additional embodiment of the instant invention concerns a
method of altering the level of expression of an aspartyl-tRNA
synthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase
or tyrosyl-tRNA synthetase in a transformed host cell comprising:
a) transforming a host cell with a chimeric gene comprising a
nucleic acid fragment encoding an aspartyl-tRNA synthetase,
cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase or
tyrosyl-tRNA synthetase; and b) growing the transformed host cell
under conditions that are suitable for expression of the chimeric
gene wherein expression of the chimeric gene results in production
of altered levels of aspartyl-tRNA synthetase, cysteinyl-tRNA
synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase
in the transformed host cell.
[0011] An addition embodiment of the instant invention concerns a
method for obtaining a nucleic acid fragment encoding all or a
substantial portion of an amino acid sequence encoding an
aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase.
[0012] A further embodiment of the instant invention is a method
for evaluating at least one compound for its ability to inhibit the
activity of an aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, the method
comprising the steps of: (a) transforming a host cell with a
chimeric gene comprising a nucleic acid fragment encoding an
aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, operably
linked to suitable regulatory sequences; (b) growing the
transformed host cell under conditions that are suitable for
expression of the chimeric gene wherein expression of the chimeric
gene results in production of aspartyl-tRNA synthetase,
cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase or
tyrosyl-tRNA synthetase in the transformed host cell; (c)
optionally purifying the aspartyl-tRNA synthetase, cysteinyl-tRNA
synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase
expressed by the transformed host cell; (d) treating the
aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase with a
compound to be tested; and (e) comparing the activity of the
aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase that has
been treated with a test compound to the activity of an untreated
aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,
tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, thereby
selecting compounds with potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
[0013] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0014] Table 1 lists the polypeptides that are described herein,
the designation of the cDNA clones that comprise the nucleic acid
fragments encoding polypeptides representing all or a substantial
portion of these polypeptides, and the corresponding identifier
(SEQ ID NO:) as used in the attached Sequence Listing. The sequence
descriptions and Sequence Listing attached hereto comply with the
rules governing nucleotide and/or amino acid sequence disclosures
in patent applications as set forth in 37 C.F.R.
.sctn.1.821-1.825.
1TABLE 1 Aninoacyl-tRNA Synthetase SEQ ID NO: (Amino Protein Clone
Designation (Nucleotide) Acid) Aspartyl-tRNA Synthetase
p0094.cssth73r 1 2 Aspartyl-tRNA Synthetase rl0n.pk0015.g11 3 4
Aspartyl-tRNA Synthetase sfl1.pk0046.e8 5 6 Aspartyl-tRNA
Synthetase wle1n.pk0021.e6 7 8 Cysteinyl-tRNA Synthetase
p0119.cmtmt52r 9 10 Cysteinyl-tRNA Synthetase rsl1n.pk016.p18 11 12
Cysteinyl-tRNA Synthetase sfl1.pk0013.f9 13 14 Tryptophanyl-tRNA
p0118.chsbl87r 15 16 Synthetase Tryptophanyl-tRNA sdp4c.pk033.n11
17 18 Synthetase Tryptophanyl-tRNA wlm4.pk0013.c12 19 20 Synthetase
Tyrosyl-tRNA Synthetase cs1.pk0035.d2 21 22
[0015] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Research 13:3021-3030 (1985) and in the
Biochemical Journal 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn.1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0016] In the context of this disclosure, a number of terms shall
be utilized. As used herein, a "nucleic acid fragment" is a polymer
of RNA or DNA that is single- or double-stranded, optionally
containing synthetic, non-natural or altered nucleotide bases. A
nucleic acid fragment in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA or synthetic
DNA.
[0017] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the polypeptide encoded by the
nucleotide sequence. "Substantially similar" also refers to nucleic
acid fragments wherein changes in one or more nucleotide bases does
not affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by gene silencing through for example
antisense or co-suppression technology. "Substantially similar"
also refers to modifications of the nucleic acid fragments of the
instant invention such as deletion or insertion of one or more
nucleotides that do not substantially affect the functional
properties of the resulting transcript vis--vis the ability to
mediate gene silencing or alteration of the functional properties
of the resulting protein molecule. It is therefore understood that
the invention encompasses more than the specific exemplary
nucleotide or amino acid sequences and includes functional
equivalents thereof.
[0018] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by nucleic acid fragments
that do not share 100% sequence identity with the gene to be
suppressed. Moreover, alterations in a nucleic acid fragment which
result in the production of a chemically equivalent amino acid at a
given site, but do not effect the functional properties of the
encoded polypeptide, are well known in the art. Thus, a codon for
the amino acid alanine, a hydrophobic amino acid, may be
substituted by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue, such as valine,
leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine, can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the polypeptide molecule would also not be expected to alter the
activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
[0019] Moreover, substantially similar nucleic acid fragments may
also be characterized by their ability to hybridize. Estimates of
such homology are provided by either DNA-DNA or DNA-RNA
hybridization under conditions of stringency as is well understood
by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic
Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions
can be adjusted to screen for moderately similar fragments, such as
homologous sequences from distantly related organisms, to highly
similar fragments, such as genes that duplicate functional enzymes
from closely related organisms. Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a
series of washes starting with 6.times. SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2.times. SSC, 0.5% SDS
at 45.degree. C. for 30 min, and then repeated twice with
0.2.times. SSC, 0.5% SDS at 50.degree. C. for 30 min. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times. SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times. SSC, 0.1%
SDS at 65.degree. C.
[0020] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Preferred are those nucleic acid
fragments whose nucleotide sequences encode amino acid sequences
that are 80% identical to the amino acid sequences reported herein.
More preferred nucleic acid fragments encode amino acid sequences
that are 90% identical to the amino acid sequences reported herein.
Most preferred are nucleic acid fragments that encode amino acid
sequences that are 95% identical to the amino acid sequences
reported herein. Sequence alignments and percent identity
calculations were performed using the Megalign program of the
LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). Multiple alignment of the sequences was performed using the
Clustal method of alignment (Higgins and Sharp (1989) CABIOS.
5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5.
[0021] A "substantial portion" of an amino acid or nucleotide
sequence comprises an amino acid or a nucleotide sequence that is
sufficient to afford putative identification of the protein or gene
that the amino acid or nucleotide sequence comprises. Amino acid
and nucleotide sequences can be evaluated either manually by one
skilled in the art, or by using computer-based sequence comparison
and identification tools that employ algorithms such as BLAST
(Basic Local Alignment Search Tool; Altschul et al. (1993) J Mol.
Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/- ). In
general, a sequence of ten or more contiguous amino acids or thirty
or more contiguous nucleotides is necessary in order to putatively
identify a polypeptide or nucleic acid sequence as homologous to a
known protein or gene. Moreover, with respect to nucleotide
sequences, gene-specific oligonucleotide probes comprising 30 or
more contiguous nucleotides may be used in sequence-dependent
methods of gene identification (e.g., Southern hybridization) and
isolation (e.g., in situ hybridization of bacterial colonies or
bacteriophage plaques). In addition, short oligonucleotides of 12
or more nucleotides may be used as amplification primers in PCR in
order to obtain a particular nucleic acid fragment comprising the
primers. Accordingly, a "substantial portion" of a nucleotide
sequence comprises a nucleotide sequence that will afford specific
identification and/or isolation of a nucleic acid fragment
comprising the sequence. The instant specification teaches amino
acid and nucleotide sequences encoding polypeptides that comprise
one or more particular plant proteins. The skilled artisan, having
the benefit of the sequences as reported herein, may now use all or
a substantial portion of the disclosed sequences for purposes known
to those skilled in this art. Accordingly, the instant invention
comprises the complete sequences as reported in the accompanying
Sequence Listing, as well as substantial portions of those
sequences as defined above.
[0022] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment comprising a
nucleotide sequence that encodes all or a substantial portion of
the amino acid sequences set forth herein. The skilled artisan is
well aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a nucleic acid fragment for improved
expression in a host cell, it is desirable to design the nucleic
acid fragment such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0023] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized
using procedures known to those skilled in the art. These building
blocks are ligated and annealed to form larger nucleic acid
fragments which may then be enzymatically assembled to construct
the entire desired nucleic acid fragment. "Chemically synthesized",
as related to nucleic acid fragment, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
nucleic acid fragments may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the nucleic acid fragments can be tailored for optimal gene
expression based on optimization of nucleotide sequence to reflect
the codon bias of the host cell. The skilled artisan appreciates
the likelihood of successful gene expression if codon usage is
biased towards those codons favored by the host. Determination of
preferred codons can be based on a survey of genes derived from the
host cell where sequence information is available.
[0024] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers any gene
that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature.
"Endogenous gene" refers to a native gene in its natural location
in the genome of an organism. A "foreign" gene refers to a gene not
normally found in the host organism, but that is introduced into
the host organism by gene transfer. Foreign genes can comprise
native genes inserted into a non-native organism, or chimeric
genes. A "transgene" is a gene that has been introduced into the
genome by a transformation procedure.
[0025] "Coding sequence" refers to a nucleotide sequence that codes
for a specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
[0026] "Promoter" refers to a nucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a nucleotide sequence which can
stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic nucleotide segments. It is understood by those skilled in
the art that different promoters may direct the expression of a
gene in different tissues or cell types, or at different stages of
development, or in response to different environmental conditions.
Promoters which cause a nucleic acid fragment to be expressed in
most cell types at most times are commonly referred to as
"constitutive promoters". New promoters of various types useful in
plant cells are constantly being discovered; numerous examples may
be found in the compilation by Okamuro and Goldberg (1989)
Biochemistry of Plants 15:1-82. It is further recognized that since
in most cases the exact boundaries of regulatory sequences have not
been completely defined, nucleic acid fragments of different
lengths may have identical promoter activity.
[0027] The "translation leader sequence" refers to a nucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Molecular Biotechnology
3:225).
[0028] The "3' non-coding sequences" refer to nucleotide sequences
located downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
[0029] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into polypeptide by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to an RNA transcript that includes the mRNA and
so can be translated into a polypeptide by the cell. "Antisense
RNA" refers to an RNA transcript that is complementary to all or
part of a target primary transcript or mRNA and that blocks the
expression of a target gene (see U.S. Pat. No. 5,107,065,
incorporated herein by reference). The complementarity of an
antisense RNA may be with any part of the specific nucleotide
sequence, i.e., at the 5' non-coding sequence, 3' non-coding
sequence, introns, or the coding sequence. "Functional RNA" refers
to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may
not be translated but yet has an effect on cellular processes.
[0030] The term "operably linked" refers to the association of two
or more nucleic acid fragments on a single nucleic acid fragment so
that the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0031] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein. "Overexpression" refers to the production of a
gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference).
[0032] "Altered levels" refers to the production of gene product(s)
in transgenic organisms in amounts or proportions that differ from
that of normal or non-transformed organisms.
[0033] "Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present
in the primary translation product have been removed. "Precursor"
protein refers to the primary product of translation of mRNA; i.e.,
with pre- and propeptides still present. Pre- and propeptides may
be but are not limited to intracellular localization signals.
[0034] A "chloroplast transit peptide" is an amino acid sequence
which is translated in conjunction with a protein and directs the
protein to the chloroplast or other plastid types present in the
cell in which the protein is made. "Chloroplast transit sequence"
refers to a nucleotide sequence that encodes a chloroplast transit
peptide. A "signal peptide" is an amino acid sequence which is
translated in conjunction with a protein and directs the protein to
the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant
Mol. Biol. 42:21-53). If the protein is to be directed to a
vacuole, a vacuolar targeting signal (supra) can further be added,
or if to the endoplasmic reticulum, an endoplasmic reticulum
retention signal (supra) may be added. If the protein is to be
directed to the nucleus, any signal peptide present should be
removed and instead a nuclear localization signal included (Raikhel
(1992) Plant Phys. 100:1627-1632).
[0035] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference).
[0036] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989
(hereinafter "Maniatis").
[0037] Nucleic acid fragments encoding at least a portion of
several aninoacyl-tRNA synthetases have been isolated and
identified by comparison of random plant cDNA sequences to public
databases containing nucleotide and protein sequences using the
BLAST algorithms well known to those skilled in the art. The
nucleic acid fragments of the instant invention may be used to
isolate cDNAs and genes encoding homologous proteins from the same
or other plant species. Isolation of homologous genes using
sequence-dependent protocols is well known in the art. Examples of
sequence-dependent protocols include, but are not limited to,
methods of nucleic acid hybridization, and methods of DNA and RNA
amplification as exemplified by various uses of nucleic acid
amplification technologies (e.g., polymerase chain reaction, ligase
chain reaction).
[0038] For example, genes encoding other aspartyl-tRNA synthetase,
cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase or
tyrosyl-tRNA synthetase enzymes, either as cDNAs or genomic DNAs,
could be isolated directly by using all or a portion of the instant
nucleic acid fragments as DNA hybridization probes to screen
libraries from any desired plant employing methodology well known
to those skilled in the art. Specific oligonucleotide probes based
upon the instant nucleic acid sequences can be designed and
synthesized by methods known in the art (Maniatis). Moreover, the
entire sequences can be used directly to synthesize DNA probes by
methods known to the skilled artisan such as random primer DNA
labeling, nick translation, or end-labeling techniques, or RNA
probes using available in vitro transcription systems. In addition,
specific primers can be designed and used to amplify a part or all
of the instant sequences. The resulting amplification products can
be labeled directly during amplification reactions or labeled after
amplification reactions, and used as probes to isolate full length
cDNA or genomic fragments under conditions of appropriate
stringency.
[0039] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA
85:8998) to generate cDNAs by using PCR to amplify copies of the
region between a single point in the transcript and the 3' or 5'
end. Primers oriented in the 3' and 5' directions can be designed
from the instant sequences. Using commercially available 3' RACE or
5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be
isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673;
Loh et al. (1989) Science 243:217). Products generated by the 3'
and 5' RACE procedures can be combined to generate full-length
cDNAs (Frohman and Martin (1989) Techniques 1:165).
[0040] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1; Maniatis).
[0041] The nucleic acid fragments of the instant invention may be
used to create transgenic plants in which the disclosed
polypeptides are present at higher or lower levels than normal or
in cell types or developmental stages in which they are not
normally found. This would have the effect of altering the level of
aminoacyl-tRNA synthetase activity in those cells.
[0042] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. For reasons of convenience, the chimeric gene may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals may also be provided. The instant
chimeric gene may also comprise one or more introns in order to
facilitate gene expression.
[0043] Plasmid vectors comprising the instant chimeric gene can
then constructed. The choice of plasmid vector is dependent upon
the method that will be used to transform host plants. The skilled
artisan is well aware of the genetic elements that must be present
on the plasmid vector in order to successfully transform, select
and propagate host cells containing the chimeric gene. The skilled
artisan will also recognize that different independent
transformation events will result in different levels and patterns
of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida
et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple
events must be screened in order to obtain lines displaying the
desired expression level and pattern. Such screening may be
accomplished by Southern analysis of DNA, Northern analysis of mRNA
expression, Western analysis of protein expression, or phenotypic
analysis.
[0044] For some applications it may be useful to direct the instant
polypeptides to different cellular compartments, or to facilitate
its secretion from the cell. It is thus envisioned that the
chimeric gene described above may be further supplemented by
altering the coding sequence to encode the instant polypeptides
with appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys. 100:1627-1632)
added and/or with targeting sequences that are already present
removed. While the references cited give examples of each of these,
the list is not exhaustive and more targeting signals of utility
may be discovered in the future.
[0045] It may also be desirable to reduce or eliminate expression
of genes encoding the instant polypeptides in plants for some
applications. In order to accomplish this, a chimeric gene designed
for co-suppression of the instant polypeptide can be constructed by
linking a gene or gene fragment encoding that polypeptide to plant
promoter sequences. Alternatively, a chimeric gene designed to
express antisense RNA for all or part of the instant nucleic acid
fragment can be constructed by linking the gene or gene fragment in
reverse orientation to plant promoter sequences. Either the
co-suppression or antisense chimeric genes could be introduced into
plants via transformation wherein expression of the corresponding
endogenous genes are reduced or eliminated.
[0046] Molecular genetic solutions to the generation of plants with
altered gene expression have a decided advantage over more
traditional plant breeding approaches. Changes in plant phenotypes
can be produced by specifically inhibiting expression of one or
more genes by antisense inhibition or cosuppression (U.S. Pat. Nos.
5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression
construct would act as a dominant negative regulator of gene
activity. While conventional mutations can yield negative
regulation of gene activity these effects are most likely
recessive. The dominant negative regulation available with a
transgenic approach may be advantageous from a breeding
perspective. In addition, the ability to restrict the expression of
specific phenotype to the reproductive tissues of the plant by the
use of tissue specific promoters may confer agronomic advantages
relative to conventional mutations which may have an effect in all
tissues in which a mutant gene is ordinarily expressed.
[0047] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppresion technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense genes may require the use of different chimeric
genes utilizing different regulatory elements known to the skilled
artisan. Once transgenic plants are obtained by one of the methods
described above, it will be necessary to screen individual
transgenics for those that most effectively display the desired
phenotype. Accordingly, the skilled artisan will develop methods
for screening large numbers of transformants. The nature of these
screens will generally be chosen on practical grounds, and is not
an inherent part of the invention. For example, one can screen by
looking for changes in gene expression by using antibodies specific
for the protein encoded by the gene being suppressed, or one could
establish assays that specifically measure enzyme activity. A
preferred method will be one which allows large numbers of samples
to be processed rapidly, since it will be expected that a large
number of transformants will be negative for the desired
phenotype.
[0048] The instant polypeptides (or portions thereof) may be
produced in heterologous host cells, particularly in the cells of
microbial hosts, and can be used to prepare antibodies to the these
proteins by methods well known to those skilled in the art. The
antibodies are useful for detecting the polypeptides of the instant
invention in situ in cells or in vitro in cell extracts. Preferred
heterologous host cells for production of the instant polypeptides
are microbial hosts. Microbial expression systems and expression
vectors containing regulatory sequences that direct high level
expression of foreign proteins are well known to those skilled in
the art. Any of these could be used to construct a chimeric gene
for production of the instant polypeptides. This chimeric gene
could then be introduced into appropriate microorganisms via
transformation to provide high level expression of the encoded
aninoacyl-tRNA synthetase. An example of a vector for high level
expression of the instant polypeptides in a bacterial host is
provided (Example 9).
[0049] Additionally, the instant polypeptides can be used as a
targets to facilitate design and/or identification of inhibitors of
those enzymes that may be useful as herbicides. This is desirable
because the polypeptides described herein catalyze various steps in
protein synthesis. Accordingly, inhibition of the activity of one
or more of the enzymes described herein could lead to inhibition
plant growth. Thus, the instant polypeptides could be appropriate
for new herbicide discovery and design.
[0050] All or a substantial portion of the nucleic acid fragments
of the instant invention may also be used as probes for genetically
and physically mapping the genes that they are a part of, and as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid fragments may be
used as restriction fragment length polymorphism (RFLP) markers.
Southern blots (Maniatis) of restriction-digested plant genomic DNA
may be probed with the nucleic acid fragments of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al. (1987) Genomics 1:174-181) in order to construct a genetic
map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et
al. (1980) Am. J Hum. Genet. 32:314-331).
[0051] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bematzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications
describe genetic mapping of specific cDNA clones using the
methodology outlined above or variations thereof. For example, F2
intercross populations, backcross populations, randomly mated
populations, near isogenic lines, and other sets of individuals may
be used for mapping. Such methodologies are well known to those
skilled in the art.
[0052] Nucleic acid probes derived from the instant nucleic acid
sequences may also be used for physical mapping (i.e., placement of
sequences on physical maps; see Hoheisel et al. In: Nonmammalian
Genomic Analysis: A Practical Guide, Academic press 1996, pp.
319-346, and references cited therein).
[0053] In another embodiment, nucleic acid probes derived from the
instant nucleic acid sequences may be used in direct fluorescence
in situ hybridization (FISH) mapping (Trask (1991) Trends Genet.
7:149-154). Although current methods of FISH mapping favor use of
large clones (several to several hundred KB; see Laan et al. (1995)
Genome Research 5:13-20), improvements in sensitivity may allow
performance of FISH mapping using shorter probes.
[0054] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J. Lab. Clin. Med. 114(2):95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nature Genetics 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 1 7:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0055] Loss of function mutant phenotypes may be identified for the
instant cDNA clones either by targeted gene disruption protocols or
by identifying specific mutants for these genes contained in a
maize population carrying mutations in all possible genes
(Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402;
Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al.
(1995) Plant Cell 7:75). The latter approach may be accomplished in
two ways. First, short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols in
conjunction with a mutation tag sequence primer on DNAs prepared
from a population of plants in which Mutator transposons or some
other mutation-causing DNA element has been introduced (see Bensen,
supra). The amplification of a specific DNA fragment with these
primers indicates the insertion of the mutation tag element in or
near the plant gene encoding the instant polypeptides.
Alternatively, the instant nucleic acid fragment may be used as a
hybridization probe against PCR amplification products generated
from the mutation population using the mutation tag sequence primer
in conjunction with an arbitrary genomic site primer, such as that
for a restriction enzyme site-anchored synthetic adaptor. With
either method, a plant containing a mutation in the endogenous gene
encoding the instant polypeptides can be identified and obtained.
This mutant plant can then be used to determine or confirm the
natural function of the instant polypeptides disclosed herein.
EXAMPLES
[0056] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions.
Example 1
Composition of cDNA Libraries; Isolation and Sequencing of cDNA
Clones
[0057] cDNA libraries representing mRNAs from various corn, rice,
soybean and wheat tissues were prepared. The characteristics of the
libraries are described below.
2TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library
Tissue Clone cs1 Corn leaf sheath from 5 week old plant
cs1.pk0035.d2 p0094 Corn ear leaf sheath, 2-3 p0094.cssth73r weeks
after pollen shed* p0118 Corn pooled stem tissue from
p0118.chsbl87r the 4-5 internodes subtending the tassel, V8-V12
stages* p0119 Corn ear shoot/w husk: V-12 stage* p0119.cmtmt52r
rl0n Rice 15 day old leaf* rl0n.pk0015.g11 rsl1n Rice 15 day old
seedling* rsl1n.pk016.p18 sdp4c Soybean developing embryo (9-11 mm)
sdp4c.pk033.n11 sfl1 Soybean immature flower sfl1.pk0013.f9
sfl1.pk0046.e8 wle1n Wheat leaf from 7 day wle1n.pk0021.e6 old
etiolated seedling* wlm4 Wheat seedlings 4 hours after
wlm4.pk0013.c12 treatment with a fungicide** *These libraries were
normalized essentially as described in U.S. Pat. No. 5,482,845,
incorporated herein by reference. **Fungicide: Application of
6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone synthesis and methods
of using this compound are described in USSN 08/545, 827,
incorporated herein by reference.
[0058] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP* XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP* XR libraries are
converted into plasmid libraries according to the protocol provided
by Stratagene. Upon conversion, cDNA inserts will be contained in
the plasmid vector pBluescript. In addition, the cDNAs may be
introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651). The resulting ESTs are analyzed using a Perkin
Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0059] cDNA clones encoding aninoacyl-tRNA synthetases were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, the last major release of
the SWISS-PROT protein sequence database, EMBL, and DDBJ
databases). The cDNA sequences obtained in Example 1 were analyzed
for similarity to all publicly available DNA sequences contained in
the "nr" database using the BLASTN algorithm provided by the
National Center for Biotechnology Information (NCBI). The DNA
sequences were translated in all reading frames and compared for
similarity to all publicly available protein sequences contained in
the "nr" database using the BLASTX algorithm (Gish and States
(1993) Nature Genetics 3:266-272) provided by the NCBI. For
convenience, the P-value (probability) of observing a match of a
cDNA sequence to a sequence contained in the searched databases
merely by chance as calculated by BLAST are reported herein as
"pLog" values, which represent the negative of the logarithm of the
reported P-value. Accordingly, the greater the pLog value, the
greater the likelihood that the cDNA sequence and the BLAST "hit"
represent homologous proteins.
Example 3
Characterization of cDNA Clones Encoding Aspartyl-tRNA
Synthetase
[0060] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to aspartyl-tRNA synthetase from Drosophila melanogaster
(NCBI Identifier No. gi 4512034), Rattus norvegicus (NCBI
Identifier No. gi 135099) and Homo sapiens (NCBI Identifier no. gi
4557513). Shown in Table 3 are the BLAST results for individual
ESTs ("EST"), the sequences of the entire cDNA inserts comprising
the indicated cDNA clones ("FIS"), or contigs assembled from two or
more ESTs ("Contig"):
3TABLE 3 BLAST Results for Sequences Encoding Polypeptides
Homologous to Drosophila melanogaster, Rattus norvegicus and Homo
sapiens Aspartyl-tRNA Synthetase Clone Status BLAST pLog Score
p0094.cssth73r FIS 134.00 (gi 4512034) rl0n.pk0015.g11 FIS 51.15
(gi 135099) sfl1.pk0046.e8 FIS 102.00 (gi 4557513) wle1n.pk0021.e6
FIS 21.40 (gi 4557513)
[0061] The data in Table 4 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs: 2, 4,
6 and 8 and the Drosophila melanogaster, Rattus norvegicus and Homo
sapiens aspartyl-tRNA synthetase sequences (SEQ ID NOs: 23, 24 and
25 respectively).
4TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Drosophila melanogaster, Rattus norvegicus and Homo
sapiens Aspartyl-tRNA Synthetase SEQ ID NO. Percent Identity to 2
51% (gi 4512034) 4 65% (gi 135099) 6 51% (gi 4557513) 8 52% (gi
4557513)
[0062] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151 -153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of an aspartyl-tRNA
synthetase. These sequences represent the first corn, rice, soybean
and wheat sequences encoding aspartyl-tRNA synthetase.
Example 4
Characterization of cDNA Clones Encoding Cysteinyl-tRNA
Synthetase
[0063] The BLASTX search using the EST sequences from clones listed
in Table 5 revealed similarity of the polypeptides encoded by the
cDNAs to cysteinyl-tRNA synthetase from Haemophilus influenzae
(NCBI Identifier No. gi 1174501) and Escherichia coli (NCBI
Identifier No. gi 41203). Shown in Table 5 are the BLAST results
for individual ESTs ("EST"), the sequences of the entire cDNA
inserts comprising the indicated cDNA clones ("FIS"), or contigs
assembled from two or more ESTs ("Contig"):
5TABLE 5 BLAST Results for Sequences Encoding Polypeptides
Homologous to Haemophilus influenzae and Escherichia coli
Cysteinyl-tRNA Synthetase Clone Status BLAST pLog Score
p0119.cmtmt52r FIS 104.00 (gi 1174501) rsl1n.pk016.p18 FIS 108.00
(gi 41203) sfl1.pk0013.f9 FIS 117.00 (gi 1174501)
[0064] The data in Table 6 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs: 10,
12 and 14 and the Haemophilus influenzae and Escherichia coli
sequences (SEQ ID NOs: 26 and 27 respectively).
6TABLE 6 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Haemophilus influenzae and Escherichia coli
Cysteinyl-tRNA Synthetase SEQ ID NO. Percent Identity to 10 43% (gi
1174501) 12 44% (gi 41203) 14 44% (gi 1174501)
[0065] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of a cysteinyl-tRNA
synthetase. These sequences represent the first corn, rice and
soybean sequences encoding cysteinyl-tRNA synthetase.
Example 5
Characterization of cDNA Clones Encoding Tryptophanyl-tRNA
Synthetase
[0066] The BLASTX search using the EST sequences from clones listed
in Table 7 revealed similarity of the polypeptides encoded by the
cDNAs to tryptophanyl-tRNA synthetase from Synechocystis sp. (NCBI
Identifier No. gi 2501072). Shown in Table 7 are the BLAST results
for individual ESTs ("EST"), the sequences of the entire cDNA
inserts comprising the indicated cDNA clones ("FIS"), or contigs
assembled from two or more ESTs ("Contig"):
7TABLE 7 BLAST Results for Sequences Encoding Polypeptides
Homologous to Synechocystis sp. Tryptophanyl-tRNA Synthetase BLAST
pLog Score Clone Status to (gi 2501072) p0118.chsbl87r EST 104.00
sdp4c.pk033.n11 FIS 103.00 wlm4.pk0013.c12 FIS 43.22
[0067] The data in Table 8 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs: 16,
18 and 24 and the Synechocystis sp. sequence (SEQ ID NO: 28).
8TABLE 8 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Synechocystis sp. Tryptophanyl-tRNA Synthetase
Percent Identity to SEQ ID NO. (gi 2501072) 16 49% 18 50% 20
51%
[0068] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of a tryptophanyl-tRNA
synthetase. These sequences represent the first corn, soybean and
wheat sequences encoding tryptophanyl-tRNA synthetase.
Example 6
Characterization of cDNA Clones Encoding Tyrosyl-tRNA
Synthetase
[0069] The BLASTX search using the EST sequence from the clone
listed in Table 9 revealed similarity of the polypeptide encoded by
the cDNA to tyrosyl-tRNA synthetase from Bacillus caldotenax (NCBI
Identifier No. gi 135196). Shown in Table 9 are the BLAST results
for the sequence of the entire cDNA insert comprising the indicated
cDNA clone ("FIS"):
9TABLE 9 BLAST Results for Sequence Encoding Polypeptide Homologous
to Bacillus caldotenax Tyrosyl-tRNA Synthetase BLAST pLog Score to
Clone Status (gi 135196) cs1.pk0035.d2 FIS 62.52
[0070] The data in Table 10 represents a calculation of the percent
identity of the amino acid sequence set forth in SEQ ID NO:22 the
Bacillus caldotenax sequence (SEQ ID NO: 29).
10TABLE 10 Percent Identity of Amino Acid Sequence Deduced From the
Nucleotide Sequence of cDNA Clone Encoding Polypeptide Homologous
to Bacillus caldotenax Tyrosyl-tRNA Synthetase Percent Identity to
SEQ ID NO. (gi 135196) 22 52%
[0071] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of a tyrosyl-tRNA
synthetase. This sequence represent the first corn sequence
encoding tyrosyl-tRNA synthetase.
Example 7
Expression of Chimeric Genes in Monocot Cells
[0072] A chimeric gene comprising a cDNA encoding the instant
polypeptides in sense orientation with respect to the maize 27 kD
zein promoter that is located 5' to the cDNA fragment, and the 10
kD zein 3' end that is located 3' to the cDNA fragment, can be
constructed. The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites (NcoI or SmaI) can be
incorporated into the oligonucleotides to provide proper
orientation of the DNA fragment when inserted into the digested
vector pML103 as described below. Amplification is then performed
in a standard PCR. The amplified DNA is then digested with
restriction enzymes NcoI and SmaI and fractionated on an agarose
gel. The appropriate band can be isolated from the gel and combined
with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid
pML103 has been deposited under the terms of the Budapest Treaty at
ATCC (American Type Culture Collection, 10801 University Blvd.,
Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter
fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI
fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at
15.degree. C. overnight, essentially as described (Maniatis). The
ligated DNA may then be used to transform E. coli XL1-Blue
(Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial transformants
can be screened by restriction enzyme digestion of plasmid DNA and
limited nucleotide sequence analysis using the dideoxy chain
termination method (Sequenase.TM. DNA Sequencing Kit; U.S.
Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD
zein promoter, a cDNA fragment encoding the instant polypeptides,
and the 10 kD zein 3' region.
[0073] The chimeric gene described above can then be introduced
into corn cells by the following procedure. Immature corn embryos
can be dissected from developing caryopses derived from crosses of
the inbred corn lines H99 and LH132. The embryos are isolated 10 to
11 days after pollination when they are 1.0 to 1.5 mm long. The
embryos are then placed with the axis-side facing down and in
contact with agarose-solidified N6 medium (Chu et al. (1975) Sci.
Sin. Peking 18:659-668). The embryos are kept in the dark at
27.degree. C. Friable embryogenic callus consisting of
undifferentiated masses of cells with somatic proembryoids and
embryoids borne on suspensor structures proliferates from the
scutellum of these immature embryos. The embryogenic callus
isolated from the primary explant can be cultured on N6 medium and
sub-cultured on this medium every 2 to 3 weeks.
[0074] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst
Ag, Frankfurt, Germany) may be used in transformation experiments
in order to provide for a selectable marker. This plasmid contains
the Pat gene (see European Patent Publication 0 242 236) which
encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT
confers resistance to herbicidal glutamine synthetase inhibitors
such as phosphinothricin. The pat gene in p35 S/Ac is under the
control of the 35S promoter from Cauliflower Mosaic Virus (Odell et
al. (1985) Nature 313:810-812) and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens.
[0075] The particle bombardment method (Klein et al. (1987) Nature
327:70-73) may be used to transfer genes to the callus culture
cells. According to this method, gold particles (1 .mu.m in
diameter) are coated with DNA using the following technique. Ten
.mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold
particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M
solution) and spermidine free base (20 .mu.L of a 1.0 M solution)
are added to the particles. The suspension is vortexed during the
addition of these solutions. After 10 minutes, the tubes are
briefly centrifuged (5 sec at 15,000 rpm) and the supernatant
removed. The particles are resuspended in 200 .mu.L of absolute
ethanol, centrifuged again and the supernatant removed. The ethanol
rinse is performed again and the particles resuspended in a final
volume of 30 .mu.L of ethanol. An aliquot (5 .mu.L) of the
DNA-coated gold particles can be placed in the center of a
Kapton.TM. flying disc (Bio-Rad Labs). The particles are then
accelerated into the corn tissue with a Biolistic.TM. PDS-1000/He
(Bio-Rad Instruments, Hercules Calif.), using a helium pressure of
1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0
cm.
[0076] For bombardment, the embryogenic tissue is placed on filter
paper over agarose-solidified N6 medium. The tissue is arranged as
a thin lawn and covered a circular area of about 5 cm in diameter.
The petri dish containing the tissue can be placed in the chamber
of the PDS-1000/He approximately 8 cm from the stopping screen. The
air in the chamber is then evacuated to a vacuum of 28 inches of
Hg. The macrocarrier is accelerated with a helium shock wave using
a rupture membrane that bursts when the He pressure in the shock
tube reaches 1000 psi.
[0077] Seven days after bombardment the tissue can be transferred
to N6 medium that contains gluphosinate (2 mg per liter) and lacks
casein or proline. The tissue continues to grow slowly on this
medium. After an additional 2 weeks the tissue can be transferred
to fresh N6 medium containing gluphosinate. After 6 weeks, areas of
about 1 cm in diameter of actively growing callus can be identified
on some of the plates containing the glufosinate-supplemented
medium. These calli may continue to grow when sub-cultured on the
selective medium.
[0078] Plants can be regenerated from the transgenic callus by
first transferring clusters of tissue to N6 medium supplemented
with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be
transferred to regeneration medium (Fromm et al. (1990)
Bio/Technology 8:833-839).
Example 8
Expression of Chimeric Genes in Dicot Cells
[0079] A seed-specific expression cassette composed of the promoter
and transcription terminator from the gene encoding the .beta.
subunit of the seed storage protein phaseolin from the bean
Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem.
261:9228-9238) can be used for expression of the instant
polypeptides in transformed soybean. The phaseolin cassette
includes about 500 nucleotides upstream (5' ) from the translation
initiation codon and about 1650 nucleotides downstream (3' ) from
the translation stop codon of phaseolin. Between the 5' and 3'
regions are the unique restriction endonuclease sites Nco I (which
includes the ATG translation initiation codon), Sma I, Kpn I and
Xba I. The entire cassette is flanked by Hind III sites.
[0080] The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the expression vector. Amplification is then
performed as described above, and the isolated fragment is inserted
into a pUC18 vector carrying the seed expression cassette.
[0081] Soybean embroys may then be transformed with the expression
vector comprising sequences encoding the instant polypeptides. To
induce somatic embryos, cotyledons, 3-5 mm in length dissected from
surface sterilized, immature seeds of the soybean cultivar A2872,
can be cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0082] Soybean embryogenic suspension cultures can maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0083] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70, U.S. Pat. No. 4,945,050). A DuPont
Biolistic.TM. PDS 1000/HE instrument (helium retrofit) can be used
for these transformations.
[0084] A selectable marker gene which can be used to facilitate
soybean transformation is a chimeric gene composed of the 35S
promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The seed expression cassette
comprising the phaseolin 5' region, the fragment encoding the
instant polypeptides and the phaseolin 3' region can be isolated as
a restriction fragment. This fragment can then be inserted into a
unique restriction site of the vector carrying the marker gene.
[0085] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.l
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
.mu.L of the DNA-coated gold particles are then loaded on each
macro carrier disk.
[0086] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0087] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media can be refreshed weekly. Seven to eight weeks post
bombardment, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
is removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line may be treated as an independent transformation
event. These suspensions can then be subcultured and maintained as
clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
Example 9
Expression of Chimeric Genes in Microbial Cells
[0088] The cDNAs' encoding the instant polypeptides can be inserted
into the T7 E. coli expression vector pBT430. This vector is a
derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135)
which employs the bacteriophage T7 RNA polymerase/T7 promoter
system. Plasmid pBT430 was constructed by first destroying the EcoR
I and Hind III sites in pET-3a at their original positions. An
oligonucleotide adaptor containing EcoR I and Hind III sites was
inserted at the BamH I site of pET-3a. This created pET-3aM with
additional unique cloning sites for insertion of genes into the
expression vector. Then, the Nde I site at the position of
translation initiation was converted to an Nco I site using
oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM
in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT
430.
[0089] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% NuSieve GTG.TM. low melting
agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium
bromide for visualization of the DNA fragment. The fragment can
then be purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20
.mu.L of water. Appropriate oligonucleotide adapters may be ligated
to the fragment using T4 DNA ligase (New England Biolabs, Beverly,
Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the instant polypeptides are then screened for the
correct orientation with respect to the T7 promoter by restriction
enzyme analysis.
[0090] For high level expression, a plasmid clone with the cDNA
insert in the correct orientation relative to the T7 promoter can
be transformed into E. coli strain BL21 (DE3) (Studier et al.
(1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 nm of approximately 1, IPTG
(isopropylthio-.beta.-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3
h at 25.degree.. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
Example 10
Evaluating Compounds for Their Ability to Inhibit the Activity of
Aminoacyl-tRNA Synthetase
[0091] The polypeptides described herein may be produced using any
number of methods known to those skilled in the art. Such methods
include, but are not limited to, expression in bacteria as
described in Example 9, or expression in eukaryotic cell culture,
in planta, and using viral expression systems in suitably infected
organisms or cell lines. The instant polypeptides may be expressed
either as mature forms of the proteins as observed in vivo or as
fusion proteins by covalent attachment to a variety of enzymes,
proteins or affinity tags. Common fusion protein partners include
glutathione S-transferase ("GST"), thioredoxin ("Trx"), maltose
binding protein, and C- and/or N-terminal hexahistidine polypeptide
("(His).sub.6"). The fusion proteins may be engineered with a
protease recognition site at the fusion point so that fusion
partners can be separated by protease digestion to yield intact
mature enzyme. Examples of such proteases include thrombin,
enterokinase and factor Xa. However, any protease can be used which
specifically cleaves the peptide connecting the fusion protein and
the enzyme.
[0092] Purification of the instant polypeptides, if desired, may
utilize any number of separation technologies familiar to those
skilled in the art of protein purification. Examples of such
methods include, but are not limited to, homogenization,
filtration, centrifugation, heat denaturation, ammonium sulfate
precipitation, desalting, pH precipitation, ion exchange
chromatography, hydrophobic interaction chromatography and affinity
chromatography, wherein the affinity ligand represents a substrate,
substrate analog or inhibitor. When the instant polypeptides are
expressed as fusion proteins, the purification protocol may include
the use of an affinity resin which is specific for the fusion
protein tag attached to the expressed enzyme or an affinity resin
containing ligands which are specific for the enzyme. For example,
the instant polypeptides may be expressed as a fusion protein
coupled to the C-terminus of thioredoxin. In addition, a
(His).sub.6 peptide may be engineered into the N-terminus of the
fused thioredoxin moiety to afford additional opportunities for
affinity purification. Other suitable affinity resins could be
synthesized by linking the appropriate ligands to any suitable
resin such as Sepharose-4B. In an alternate embodiment, a
thioredoxin fusion protein may be eluted using dithiothreitol;
however, elution may be accomplished using other reagents which
interact to displace the thioredoxin from the resin. These reagents
include .beta.-mercaptoethanol or other reduced thiol. The eluted
fusion protein may be subjected to further purification by
traditional means as stated above, if desired. Proteolytic cleavage
of the thioredoxin fusion protein and the enzyme may be
accomplished after the fusion protein is purified or while the
protein is still bound to the ThioBond.TM. affinity resin or other
resin.
[0093] Crude, partially purified or purified enzyme, either alone
or as a fusion protein, may be utilized in assays for the
evaluation of compounds for their ability to inhibit enzymatic
activation of the instant polypeptides disclosed herein. Assays may
be conducted under well known experimental conditions which permit
optimal enzymatic activity. For example, assays for aminoacyl-tRNA
synthetases are presented by Zon et al. (1988) Phytochemistry
27(3):711-714 and Heacock et al. (1996) Bioorganic Chemistry
24(3):273-289.
Sequence CWU 1
1
29 1 1948 DNA Zea mays 1 cgcacgatag ccgccgccgt cgaccagagc
actcccccgt cgtcgccacg atgtcgtctg 60 agcctccacc cgcctcctct
gccgccgccg gagaggaact cgctgctgac ctttccgccg 120 ctaccctcag
caagaagcag cagaagaagg acgcgaggaa ggcggagaag gcagagcagc 180
gccagcgtca gcagcagcag cagcagcagc cggcggacgc cgaggacccg ttcgcggcca
240 actacggcga ggtccccgtc gaggagatcc agtcaaaggc catctccggc
cgctcgtggt 300 cccatgtcgg cgacctcgac gactccgctg cgggccgctc
cgtgcttatc cgcggagccg 360 cgcaggccat ccgtccggtc agcaagaaga
tggctttcgt cgtgctgcgc cagagtatg 420 gcaccgtgca gtgcgtgctc
gtcgccagcg ccgacgccgg cgtcagcacg cagatggtc 480 gcttcgccac
cgccctcagc aaggagtcca tcgtcgacgt tgagggcgtc gtctccccc 540
caaaggagcc cctcaaggcc accacacagc aggttgagat ccaagtgagg aagatcatt
600 gcatcaatag ggctattccg acccttccaa ttaaccttga agatgcggct
cggaggagg 660 cagattttga gaaggctgaa ttggctggag aaaagcttgt
tcgcgttggc caagtaccc 720 gcttgaacta cagagctatt gatctacgaa
caccctcgaa tcaagccata ttcggatcc 780 agtgtcaagt tgaaaacaaa
tttagagatt ttttgttgtc gaagaacttt gtgggatcc 840 acaccccaaa
attgatttct ggatctagtg aagggggtgc ggctgtattc agcttctgt 900
acaatggtca acctgcttgt ttggcacaat cccctcagtt atacaagcaa tggctatct
960 ctggtggttt tgagcgagta tttgaggtcg gccctgtgtt tagagcagaa
aattcaaaca 1020 cacacaggca tctatgtgag ttcgttggtc ttgatgctga
aatggagatt aaggagcatt 1080 attttgaggt ctgtgacatt atagatggct
tattcgtatc aatatttaaa cacttgtctg 1140 aaaactgcaa gaaagaactc
gaatcaataa acaggcagta tccatttgaa cctctgaagt 1200 atctagacaa
aacctttaag ctcacttatg aagaaggaat tcaaatgttg aaggaagccg 1260
gaacagaaat cgagcctatg ggtgacctca ataccgaagc tgagaaaaaa cttggtcggc
1320 ttgtcaggga aaagtatgac acagattttt tcatcctgta tcggtatcct
ttggctgtac 1380 gtccgttcta caccatgcct tgttatgaca acccagcgta
caccaattct tttgatgtct 1440 tcattcgagg cgaggagata atatctggag
cacaaaggat acacactcct gagctgctgg 1500 ccaagcgcgc gacagagtgt
ggaatcgacg tgagcactat ctcggcctac attgaatcct 1560 tcagctatgg
cgtgccgcca cacggcggtt tcggggtggg tttggagagg gtggtgatgc 1620
tgttctgtgc cctgaacaac atcaggaaga cctccctgtt cccgcgcgac ccgcagaggc
1680 tcgtgccgta agtttctgat tccaagcctg agtcttcgag tggtctacgg
agcagatccg 1740 atgttgttac catcagagtt gacttgcaat cttagctcct
gaacctggcg gttaccgtgg 1800 atcagagttc ctgttgaatt tcacaaaagc
ctacttgttc ctaatagatt gctgcaacca 1860 acaatattac gaccctttcg
ggcttttctt cccgcctcac gtgttattct ggtctatact 1920 tgtttttaag
tgcaagtatt gctcagtt 1948 2 546 PRT Zea mays 2 Met Ser Ser Glu Pro
Pro Pro Ala Ser Ser Ala Ala Ala Gly Glu Glu 1 5 10 15 Leu Ala Ala
Asp Leu Ser Ala Ala Thr Leu Ser Lys Lys Gln Gln Lys 20 25 30 Lys
Asp Ala Arg Lys Ala Glu Lys Ala Glu Gln Arg Gln Arg Gln Gln 35 40
45 Gln Gln Gln Gln Gln Pro Ala Asp Ala Glu Asp Pro Phe Ala Ala Asn
50 55 60 Tyr Gly Glu Val Pro Val Glu Glu Ile Gln Ser Lys Ala Ile
Ser Gly 65 70 75 80 Arg Ser Trp Ser His Val Gly Asp Leu Asp Asp Ser
Ala Ala Gly Arg 85 90 95 Ser Val Leu Ile Arg Gly Ala Ala Gln Ala
Ile Arg Pro Val Ser Lys 100 105 110 Lys Met Ala Phe Val Val Leu Arg
Gln Ser Met Ser Thr Val Gln Cys 115 120 125 Val Leu Val Ala Ser Ala
Asp Ala Gly Val Ser Thr Gln Met Val Arg 130 135 140 Phe Ala Thr Ala
Leu Ser Lys Glu Ser Ile Val Asp Val Glu Gly Val 145 150 155 160 Val
Ser Leu Pro Lys Glu Pro Leu Lys Ala Thr Thr Gln Gln Val Glu 165 170
175 Ile Gln Val Arg Lys Ile Tyr Cys Ile Asn Arg Ala Ile Pro Thr Leu
180 185 190 Pro Ile Asn Leu Glu Asp Ala Ala Arg Ser Glu Ala Asp Phe
Glu Lys 195 200 205 Ala Glu Leu Ala Gly Glu Lys Leu Val Arg Val Gly
Gln Asp Thr Arg 210 215 220 Leu Asn Tyr Arg Ala Ile Asp Leu Arg Thr
Pro Ser Asn Gln Ala Ile 225 230 235 240 Phe Arg Ile Gln Cys Gln Val
Glu Asn Lys Phe Arg Asp Phe Leu Leu 245 250 255 Ser Lys Asn Phe Val
Gly Ile His Thr Pro Lys Leu Ile Ser Gly Ser 260 265 270 Ser Glu Gly
Gly Ala Ala Val Phe Lys Leu Leu Tyr Asn Gly Gln Pro 275 280 285 Ala
Cys Leu Ala Gln Ser Pro Gln Leu Tyr Lys Gln Met Ala Ile Ser 290 295
300 Gly Gly Phe Glu Arg Val Phe Glu Val Gly Pro Val Phe Arg Ala Glu
305 310 315 320 Asn Ser Asn Thr His Arg His Leu Cys Glu Phe Val Gly
Leu Asp Ala 325 330 335 Glu Met Glu Ile Lys Glu His Tyr Phe Glu Val
Cys Asp Ile Ile Asp 340 345 350 Gly Leu Phe Val Ser Ile Phe Lys His
Leu Ser Glu Asn Cys Lys Lys 355 360 365 Glu Leu Glu Ser Ile Asn Arg
Gln Tyr Pro Phe Glu Pro Leu Lys Tyr 370 375 380 Leu Asp Lys Thr Phe
Lys Leu Thr Tyr Glu Glu Gly Ile Gln Met Leu 385 390 395 400 Lys Glu
Ala Gly Thr Glu Ile Glu Pro Met Gly Asp Leu Asn Thr Glu 405 410 415
Ala Glu Lys Lys Leu Gly Arg Leu Val Arg Glu Lys Tyr Asp Thr Asp 420
425 430 Phe Phe Ile Leu Tyr Arg Tyr Pro Leu Ala Val Arg Pro Phe Tyr
Thr 435 440 445 Met Pro Cys Tyr Asp Asn Pro Ala Tyr Thr Asn Ser Phe
Asp Val Phe 450 455 460 Ile Arg Gly Glu Glu Ile Ile Ser Gly Ala Gln
Arg Ile His Thr Pro 465 470 475 480 Glu Leu Leu Ala Lys Arg Ala Thr
Glu Cys Gly Ile Asp Val Ser Thr 485 490 495 Ile Ser Ala Tyr Ile Glu
Ser Phe Ser Tyr Gly Val Pro Pro His Gly 500 505 510 Gly Phe Gly Val
Gly Leu Glu Arg Val Val Met Leu Phe Cys Ala Leu 515 520 525 Asn Asn
Ile Arg Lys Thr Ser Leu Phe Pro Arg Asp Pro Gln Arg Leu 530 535 540
Val Pro 545 3 730 DNA Oryza sativa 3 gcacgagctt acacggcacg
agcttacagg aattcaaatg ctgaaggaag ctggaacaga 60 aatcgaaccc
atgggtgacc tcaacactga agctgagaaa aaactaggcc ggcttgttaa 120
ggagaagtat ggaacagaat ttttcatcct ctatcggtat cctttggctg tgcgtccctt
180 ctacaccatg ccttgttatg acaacccagc ttacagtaac tcttttgatg
tctttattcg 240 aggagaggaa ataatatctg gagcacaaag aatacattta
ccagagctat tgacgaaacg 300 tgcaacagag tgtggaattg atgcgagtac
tatttcatca tatatcgaat cgttcagcta 360 tggtgcacct cctcatggtg
gttttggtgt cggcctggag agggtggtaa tgctgttctg 420 cgccctaaac
aacatcagga agacatcact tttccctcgc gatccacaaa ggctggtgcc 480
ataatttgct ttttttccca agagcaaggt ttggactcag tacggactgg gcagttttcc
540 tcggctggtt tttttacctg gacattattt tcgtatttat taatgtgctg
tactgcaaaa 600 gctgctcctt tccacaacat ttggaatagt tgccgataca
tttggaatag ggctcaacgt 660 tggcgttgtg atttcgttga tgatcccgct
attcgtaaca aaaaaaaaaa aaaaaaaaaa 720 aaaaaaaaaa 730 4 148 PRT Oryza
sativa 4 Met Leu Lys Glu Ala Gly Thr Glu Ile Glu Pro Met Gly Asp
Leu Asn 1 5 10 15 Thr Glu Ala Glu Lys Lys Leu Gly Arg Leu Val Lys
Glu Lys Tyr Gly 20 25 30 Thr Glu Phe Phe Ile Leu Tyr Arg Tyr Pro
Leu Ala Val Arg Pro Phe 35 40 45 Tyr Thr Met Pro Cys Tyr Asp Asn
Pro Ala Tyr Ser Asn Ser Phe Asp 50 55 60 Val Phe Ile Arg Gly Glu
Glu Ile Ile Ser Gly Ala Gln Arg Ile His 65 70 75 80 Leu Pro Glu Leu
Leu Thr Lys Arg Ala Thr Glu Cys Gly Ile Asp Ala 85 90 95 Ser Thr
Ile Ser Ser Tyr Ile Glu Ser Phe Ser Tyr Gly Ala Pro Pro 100 105 110
His Gly Gly Phe Gly Val Gly Leu Glu Arg Val Val Met Leu Phe Cys 115
120 125 Ala Leu Asn Asn Ile Arg Lys Thr Ser Leu Phe Pro Arg Asp Pro
Gln 130 135 140 Arg Leu Val Pro 145 5 1109 DNA Glycine max 5
gcacgaggtc atcagagaga atggcttcac cgttcaatgc ttggtgcagg cgcaggccga
60 tacggtgagc ccgcagatgg tgaagttcgc cgctgcactc agccgcgagt
ccatcgtcga 120 tgtcgaaggc gttgtttcga tcccctccgc tcccatcaaa
ggcgccacac aacaggtgga 180 aattcaagtg aggaagttgt attgtgtcag
tagggctgta cctactctgc ctattaatct 240 tgaggatgct gctcgaagtg
aagttgaaat cgagacggct cttcaggctg gtgagcaact 300 tgttcgtgtt
aatcaggata cacgtctgaa ctttagggtg cttgatgtgc gaacgccagc 360
taatcaaggg attttccgca ttcagtctca agttggaaat gcgtttagac aattcttatt
420 atctgaaggt ttttgtgaaa tccacactcc aaagttgata gctggatcta
gtgagggagg 480 agctgctgtt tttagactgg actacaaagg tcaacctgca
tgcctggccc agtcacctca 540 gcttcacaag caaatgtcta tttgtggaga
ttttggccgt gtttttgaga ttggtcctgt 600 gtttagagca gaagattcct
acactcacag gcatctgtgt gagtttacag gtcttgatgt 660 tgaaatggag
attaagaagc attactttga ggttatggat atagtcgata gattgtttgt 720
cgcaatgttt gacagtttga accagaattg taagaaggat ctggaagctg tcgggtctca
780 gtatccattt gaacctttga agtatctgcg gacgacacta cggcttacat
atgaagaagg 840 gattcagatg ctcaaggatg ttggagtaga aattgaacct
tatggtgact tgaatactga 900 agcggaaagg aaattgggtc agctagtctc
agagaaatat ggcacagagt tctatatcct 960 tcaccggtac cctttggctg
taaggccatt ctatacaatg ccttgctacg acaatcctgc 1020 atacagcaac
tcgtttgatg tctttattcg aggtgaggag ataatttcag gagctcagcg 1080
tgttcatgtg ccagaatttt tggaacaag 1109 6 369 PRT Glycine max 6 His
Glu Val Ile Arg Glu Asn Gly Phe Thr Val Gln Cys Leu Val Gln 1 5 10
15 Ala Gln Ala Asp Thr Val Ser Pro Gln Met Val Lys Phe Ala Ala Ala
20 25 30 Leu Ser Arg Glu Ser Ile Val Asp Val Glu Gly Val Val Ser
Ile Pro 35 40 45 Ser Ala Pro Ile Lys Gly Ala Thr Gln Gln Val Glu
Ile Gln Val Arg 50 55 60 Lys Leu Tyr Cys Val Ser Arg Ala Val Pro
Thr Leu Pro Ile Asn Leu 65 70 75 80 Glu Asp Ala Ala Arg Ser Glu Val
Glu Ile Glu Thr Ala Leu Gln Ala 85 90 95 Gly Glu Gln Leu Val Arg
Val Asn Gln Asp Thr Arg Leu Asn Phe Arg 100 105 110 Val Leu Asp Val
Arg Thr Pro Ala Asn Gln Gly Ile Phe Arg Ile Gln 115 120 125 Ser Gln
Val Gly Asn Ala Phe Arg Gln Phe Leu Leu Ser Glu Gly Phe 130 135 140
Cys Glu Ile His Thr Pro Lys Leu Ile Ala Gly Ser Ser Glu Gly Gly 145
150 155 160 Ala Ala Val Phe Arg Leu Asp Tyr Lys Gly Gln Pro Ala Cys
Leu Ala 165 170 175 Gln Ser Pro Gln Leu His Lys Gln Met Ser Ile Cys
Gly Asp Phe Gly 180 185 190 Arg Val Phe Glu Ile Gly Pro Val Phe Arg
Ala Glu Asp Ser Tyr Thr 195 200 205 His Arg His Leu Cys Glu Phe Thr
Gly Leu Asp Val Glu Met Glu Ile 210 215 220 Lys Lys His Tyr Phe Glu
Val Met Asp Ile Val Asp Arg Leu Phe Val 225 230 235 240 Ala Met Phe
Asp Ser Leu Asn Gln Asn Cys Lys Lys Asp Leu Glu Ala 245 250 255 Val
Gly Ser Gln Tyr Pro Phe Glu Pro Leu Lys Tyr Leu Arg Thr Thr 260 265
270 Leu Arg Leu Thr Tyr Glu Glu Gly Ile Gln Met Leu Lys Asp Val Gly
275 280 285 Val Glu Ile Glu Pro Tyr Gly Asp Leu Asn Thr Glu Ala Glu
Arg Lys 290 295 300 Leu Gly Gln Leu Val Ser Glu Lys Tyr Gly Thr Glu
Phe Tyr Ile Leu 305 310 315 320 His Arg Tyr Pro Leu Ala Val Arg Pro
Phe Tyr Thr Met Pro Cys Tyr 325 330 335 Asp Asn Pro Ala Tyr Ser Asn
Ser Phe Asp Val Phe Ile Arg Gly Glu 340 345 350 Glu Ile Ile Ser Gly
Ala Gln Arg Val His Val Pro Glu Phe Leu Glu 355 360 365 Gln 7 836
DNA Triticum aestivum 7 tacacatgca gactttcagt gagtttttgt tctcggactt
gggatccaca gtccaaagtt 60 gattggtgga tcaagtgaac ttggtgcatc
tccattcaag ctggcgtaca attaccaacc 120 tgcttattta gcgcagtctc
tacaatcata caagcaaatg agcatctgtg gtggctttgg 180 gcgcgtgttt
gaggctggtc cggtatttag atcagaaaaa tcaaacactc acaggcatct 240
atgtgagttt attgggttgg atgcagaaat ggagattaag gagcactact ttgaggtttg
300 tgatatcata gattgctaat tgtagcaata ttcaaacacc caaatgaaaa
ttgtcagaag 360 gaactcgaga caataaatag gcagtatcca tttgaacctc
tgaagtacct agagaaaacg 420 ttgaagctaa cgtacgagga agggattaaa
atgctcaagg tttcattctg gaatcctcta 480 ggcagggtgc ttgcaatccc
ctacatctcg gctgcaacaa aaaagaccca acgaggctgt 540 tgtttcaagc
tcagaccctc ttcattgcac gcggtgctag aaggagaact gggttgtggt 600
gctgttgctg gtcgttttcc tttttacttt tgcactttgg ccgtcataaa cgatacatgc
660 ttgctccctg gatggatctc tttctctccc tggatctttt aaacaggtgt
tgtgattaaa 720 attgtgataa atcagtgttc atcactaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 780 aatctcgagg gggggcccgg tactgttcac
cgcgtggcgc cgggctagag actagt 836 8 98 PRT Triticum aestivum 8 Val
Phe Val Leu Gly Leu Gly Ile His Ser Pro Lys Leu Ile Gly Gly 1 5 10
15 Ser Ser Glu Leu Gly Ala Ser Pro Phe Lys Leu Ala Tyr Asn Tyr Gln
20 25 30 Pro Ala Tyr Leu Ala Gln Ser Leu Gln Ser Tyr Lys Gln Met
Ser Ile 35 40 45 Cys Gly Gly Phe Gly Arg Val Phe Glu Ala Gly Pro
Val Phe Arg Ser 50 55 60 Glu Lys Ser Asn Thr His Arg His Leu Cys
Glu Phe Ile Gly Leu Asp 65 70 75 80 Ala Glu Met Glu Ile Lys Glu His
Tyr Phe Glu Val Cys Asp Ile Ile 85 90 95 Asp Cys 9 2085 DNA Zea
mays 9 ggaaaccgtg tttcgacggg ccgcagtggg cagtggcttg gcccatcgaa
cccacttgcc 60 actcacttcc acctgaactt tgccctgcct tctctcgacg
actcccctgt ccccgccgcc 120 gccgccgccg caaatcccct tccgcgtctg
tctggcctct ggggcttcta ggttagcgcg 180 tgcgaccacc atggccgagg
aggtccaggc tccactttcc gccaccatgg cgaaggaggc 240 ccagtcgccg
ccgtccgcaa ccatagcgga ggcgacggcg ccgccgcagc tcttattatt 300
taactccttt acgaagaggg aggagccatt ccagccccgg gtagagggga aggtagggat
360 gtacgtctgt ggcgtcactc cctacgactt tagccacatc ggccacgcgc
gtgcctacgt 420 cgccttcgac gtcctctaca ggtaccttaa attcttgggg
tatgaagttg aatatgtccg 480 taatttcacg gatattgatg acaagattat
taagcgtgcc aatgaacgcg gtgaaacagt 540 aacaagcttg agtagccagt
ttatcaatga atttcttctt gacatgactg agctccagtg 600 cttgcctcct
acctgcgagc cacgggtaac agaacacatt gagcatatta taaagttgat 660
aacacagata atggagaatg gcaaagccta tgctattgaa ggagatgttt acttttcagt
720 tgaaagtttt cctgaatatc tcagtttatc tggaagaaaa tttgatcaaa
atcaggcagg 780 tgcacgggtt gcttttgata caagaaagcg taatcctgca
gacttcgcac tctggaaagc 840 tgcaaaggag ggtgaacctt tttgggatag
cccttggggc cgtggaagac caggttggca 900 tattgaatgc agcgcaatga
gtgctcacta tttaggacat gtattcgata ttcatggtgg 960 ggggaaagat
ttgatttttc ctcatcatga gaatgagctt gcacaaagcc gcgcagctta 1020
tcctgatagc gaggtcaaat gctggatgca caatggcttt gttaacaagg atgataaaaa
1080 aatggcaaaa tcagataata actttttcac gattagagat atcattgctc
tttaccatcc 1140 aatggcttta agatttttct tgatgcgcac acattataga
tcagatgtta accattctga 1200 tcaagcgctt gagattgcat ctgatcgtgt
ctactacatt tatcagactc tatatgactg 1260 tgaggaagtg ttagctacat
atcgtgaaga gggtacctct ctcccagtgc cgtctgagga 1320 gcaaaatctg
attggtaagc accattcaga attcttgaaa catatgtcga atgatcttaa 1380
aaccacagat gttctggacc gttgcttcat ggagctgctg aaggccataa acagcagtct
1440 gaatgatttg aagaaactgc agcaaaaaat agaacagcaa aagaagaaac
agcaacagca 1500 gaagaagcag caacagcaga agcagcagca acagaagcaa
cagcaattgc aaaaacagcc 1560 agaagattat attcaagctc tgattgcact
ggaaacagaa cttaaaaaca aattgtctat 1620 acttggtctg atgccatctt
catctttggc agaggtactg aagcaattga aggacaaatc 1680 attaaagcga
gcagggctga ctgaagaaca attgcaagag cagattgagc agagaaatgt 1740
cgcaaggaag aataagcagt ttgagatatc tgatggaatc aggaaaaacc ttgctaccaa
1800 aggcatcgcc ctgatggacg aaccttctgg tacagtatgg agaccatgcg
aaccagagcg 1860 gtctgaagag tcatgattag ctcactgact caacaagtga
tggcggtgta aaatgagatt 1920 tttgcctgag ggcagttatc gcattttgaa
gactaacaaa aatcgccatc tctggatgtg 1980 gtattctaca gggtaggggt
tccaggttga ctcaccagtt aaaacatgca tttctggttg 2040 tataacaagc
aatgaacccc atatatatac ttgacagttg actcc 2085 10 599 PRT Zea mays 10
Thr Leu Pro Cys Leu Leu Ser Thr Thr Pro Leu Ser Pro Pro Pro Pro 1 5
10 15 Pro Pro Gln Ile Pro Phe Arg Val Cys Leu Ala Ser Gly Ala Ser
Arg 20 25 30 Leu Ala Arg Ala Thr Thr Met Ala Glu Glu Val Gln Ala
Pro Leu Ser 35 40 45 Ala Thr Met Ala Lys Glu Ala Gln Ser Pro Pro
Ser Ala Thr Ile Ala 50 55 60 Glu Ala Thr Ala Pro Pro Gln Leu Leu
Leu Phe Asn Ser Phe Thr Lys 65 70 75 80 Arg Glu Glu Pro Phe Gln Pro
Arg Val Glu Gly Lys Val Gly Met Tyr 85 90 95 Val Cys Gly Val Thr
Pro Tyr Asp Phe Ser His Ile Gly His Ala Arg 100 105 110 Ala Tyr Val
Ala Phe Asp Val Leu Tyr Arg Tyr Leu Lys Phe Leu Gly 115 120 125 Tyr
Glu Val Glu Tyr Val Arg Asn Phe Thr Asp Ile Asp Asp Lys Ile 130 135
140 Ile
Lys Arg Ala Asn Glu Arg Gly Glu Thr Val Thr Ser Leu Ser Ser 145 150
155 160 Gln Phe Ile Asn Glu Phe Leu Leu Asp Met Thr Glu Leu Gln Cys
Leu 165 170 175 Pro Pro Thr Cys Glu Pro Arg Val Thr Glu His Ile Glu
His Ile Ile 180 185 190 Lys Leu Ile Thr Gln Ile Met Glu Asn Gly Lys
Ala Tyr Ala Ile Glu 195 200 205 Gly Asp Val Tyr Phe Ser Val Glu Ser
Phe Pro Glu Tyr Leu Ser Leu 210 215 220 Ser Gly Arg Lys Phe Asp Gln
Asn Gln Ala Gly Ala Arg Val Ala Phe 225 230 235 240 Asp Thr Arg Lys
Arg Asn Pro Ala Asp Phe Ala Leu Trp Lys Ala Ala 245 250 255 Lys Glu
Gly Glu Pro Phe Trp Asp Ser Pro Trp Gly Arg Gly Arg Pro 260 265 270
Gly Trp His Ile Glu Cys Ser Ala Met Ser Ala His Tyr Leu Gly His 275
280 285 Val Phe Asp Ile His Gly Gly Gly Lys Asp Leu Ile Phe Pro His
His 290 295 300 Glu Asn Glu Leu Ala Gln Ser Arg Ala Ala Tyr Pro Asp
Ser Glu Val 305 310 315 320 Lys Cys Trp Met His Asn Gly Phe Val Asn
Lys Asp Asp Lys Lys Met 325 330 335 Ala Lys Ser Asp Asn Asn Phe Phe
Thr Ile Arg Asp Ile Ile Ala Leu 340 345 350 Tyr His Pro Met Ala Leu
Arg Phe Phe Leu Met Arg Thr His Tyr Arg 355 360 365 Ser Asp Val Asn
His Ser Asp Gln Ala Leu Glu Ile Ala Ser Asp Arg 370 375 380 Val Tyr
Tyr Ile Tyr Gln Thr Leu Tyr Asp Cys Glu Glu Val Leu Ala 385 390 395
400 Thr Tyr Arg Glu Glu Gly Thr Ser Leu Pro Val Pro Ser Glu Glu Gln
405 410 415 Asn Leu Ile Gly Lys His His Ser Glu Phe Leu Lys His Met
Ser Asn 420 425 430 Asp Leu Lys Thr Thr Asp Val Leu Asp Arg Cys Phe
Met Glu Leu Leu 435 440 445 Lys Ala Ile Asn Ser Ser Leu Asn Asp Leu
Lys Lys Leu Gln Gln Lys 450 455 460 Ile Glu Gln Gln Lys Lys Lys Gln
Gln Gln Gln Lys Lys Gln Gln Gln 465 470 475 480 Gln Lys Gln Gln Gln
Gln Lys Gln Gln Gln Leu Gln Lys Gln Pro Glu 485 490 495 Asp Tyr Ile
Gln Ala Leu Ile Ala Leu Glu Thr Glu Leu Lys Asn Lys 500 505 510 Leu
Ser Ile Leu Gly Leu Met Pro Ser Ser Ser Leu Ala Glu Val Leu 515 520
525 Lys Gln Leu Lys Asp Lys Ser Leu Lys Arg Ala Gly Leu Thr Glu Glu
530 535 540 Gln Leu Gln Glu Gln Ile Glu Gln Arg Asn Val Ala Arg Lys
Asn Lys 545 550 555 560 Gln Phe Glu Ile Ser Asp Gly Ile Arg Lys Asn
Leu Ala Thr Lys Gly 565 570 575 Ile Ala Leu Met Asp Glu Pro Ser Gly
Thr Val Trp Arg Pro Cys Glu 580 585 590 Pro Glu Arg Ser Glu Glu Ser
595 11 1957 DNA Oryza sativa 11 cgccagttct agggttagct cgtcggcgtc
cagccctctc actctccccc tccgctctca 60 cgatggcgga gagcgcgaag
ccgacgccgc agctggagct cttcaactcg atgacgaaga 120 agaaggagct
cttcgagccg cttgtggagg ggaaggtccg catgtatgtg tgcggcgtca 180
cgccctacga cttcagccac atcggccacg cccgcgccta cgtcgccttc gacgtcctct
240 acaggtatct taaattcttg gggtacgagg tcgaatatgt gcgcaacttc
actgatattg 300 atgacaagat tatcaaacga gcaaatgaag ctggtgaaac
tgtaactagc ttgagcagcc 360 ggtttattaa tgaattcctt ctcgatatgg
ctcagctcca gtgcttaccc ccaacttgtg 420 agccacgtgt gacggatcac
attgaacata ttatagagtt gataaccaag ataatggaga 480 atgggaaagc
ctatgctatg gaaggagatg tttacttttc agttgatact ttccctgagt 540
atctcagttt atctggaagg aagttagatc ataatcttgc tggttcgcgg gttgctgtcg
600 atacaagaaa gcggaaccct gcagactttg cgctgtggaa ggctgctaag
gaaggcgaac 660 ctttctggga tagcccatgg ggccgtggta gaccaggatg
gcatattgaa tgcagtgcaa 720 tgagtgctca ttatttagga catgtgtttg
atatccatgg tggagggaaa gatctgatat 780 ttcctcatca tgagaatgag
cttgctcaga gccgggcagc ttatccagaa agtgaggtca 840 aatgttggat
gcacaatggg tttgttaaca aggatgatca gaaaatgtca aagtcagata 900
aaaatttctt cacaatccga gatattattg atctgtacca tcccatggct ttgaggtttt
960 tcctgatgcg cacacattac agaggagatg tgaatcactc tgacaaagca
cttgagatag 1020 catctgatcg tgtctactac atatatcaga ctttatatga
ctgtgaggaa gtgttgtctc 1080 aatatcgtgg agagaatatc tctgtcccgg
tccctgttga ggaacaagat atggttaaca 1140 agcaccattc agaattcttg
gaatctatgg cggatgatct tagaacaaca gatgttctgg 1200 atggctttac
tgacttgctg aaggcaatta acagcaattt gaatgatttt aagaagttgc 1260
aacagaagct agagcagcaa aagaagaaac aacaacagca gaagcagcag aagcaaaagc
1320 agcagcaggc acagaaacaa ccagaagaat atattcaagc tatgtttgca
cttgagacag 1380 aaattaaaaa taaaatatct atccttggtc tgatgccacc
ttcttccttg gcagaggcac 1440 tgaagcaact taaggataaa gctttgaaga
gagcagggtt gactgaagaa ctgttgcagg 1500 agcaaattga gcagagaact
gctgcaagga aaaacaagca gtttgatgtg tctgaccaaa 1560 tcaggaaaca
gctaggcagc aaaggcatag ccctcatgga tgaacctact ggtacagtat 1620
ggagaccatg cgagccagag tctgaatagt cacatgattg atttgtgctt tggttaacag
1680 gtgatggtac aaactggaaa atttaaccaa gcacatctgc tgaattggtg
taaattgatg 1740 cagatcaaca tttttttttg taattttgta ggggtttaag
ttcactggcc aactgaaact 1800 tgcgtttctc gtggtgtaag aagcaaaacc
ccatatactg atatactcga ggactccctt 1860 gttggatgtt atgctttgga
tttgaatatt gaagtcaaat cataattaca tttgcatgat 1920 caaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaa 1957 12 548 PRT Oryza sativa 12 Pro
Val Leu Gly Leu Ala Arg Arg Arg Pro Ala Leu Ser Leu Ser Pro 1 5 10
15 Ser Ala Leu Thr Met Ala Glu Ser Ala Lys Pro Thr Pro Gln Leu Glu
20 25 30 Leu Phe Asn Ser Met Thr Lys Lys Lys Glu Leu Phe Glu Pro
Leu Val 35 40 45 Glu Gly Lys Val Arg Met Tyr Val Cys Gly Val Thr
Pro Tyr Asp Phe 50 55 60 Ser His Ile Gly His Ala Arg Ala Tyr Val
Ala Phe Asp Val Leu Tyr 65 70 75 80 Arg Tyr Leu Lys Phe Leu Gly Tyr
Glu Val Glu Tyr Val Arg Asn Phe 85 90 95 Thr Asp Ile Asp Asp Lys
Ile Ile Lys Arg Ala Asn Glu Ala Gly Glu 100 105 110 Thr Val Thr Ser
Leu Ser Ser Arg Phe Ile Asn Glu Phe Leu Leu Asp 115 120 125 Met Ala
Gln Leu Gln Cys Leu Pro Pro Thr Cys Glu Pro Arg Val Thr 130 135 140
Asp His Ile Glu His Ile Ile Glu Leu Ile Thr Lys Ile Met Glu Asn 145
150 155 160 Gly Lys Ala Tyr Ala Met Glu Gly Asp Val Tyr Phe Ser Val
Asp Thr 165 170 175 Phe Pro Glu Tyr Leu Ser Leu Ser Gly Arg Lys Leu
Asp His Asn Leu 180 185 190 Ala Gly Ser Arg Val Ala Val Asp Thr Arg
Lys Arg Asn Pro Ala Asp 195 200 205 Phe Ala Leu Trp Lys Ala Ala Lys
Glu Gly Glu Pro Phe Trp Asp Ser 210 215 220 Pro Trp Gly Arg Gly Arg
Pro Gly Trp His Ile Glu Cys Ser Ala Met 225 230 235 240 Ser Ala His
Tyr Leu Gly His Val Phe Asp Ile His Gly Gly Gly Lys 245 250 255 Asp
Leu Ile Phe Pro His His Glu Asn Glu Leu Ala Gln Ser Arg Ala 260 265
270 Ala Tyr Pro Glu Ser Glu Val Lys Cys Trp Met His Asn Gly Phe Val
275 280 285 Asn Lys Asp Asp Gln Lys Met Ser Lys Ser Asp Lys Asn Phe
Phe Thr 290 295 300 Ile Arg Asp Ile Ile Asp Leu Tyr His Pro Met Ala
Leu Arg Phe Phe 305 310 315 320 Leu Met Arg Thr His Tyr Arg Gly Asp
Val Asn His Ser Asp Lys Ala 325 330 335 Leu Glu Ile Ala Ser Asp Arg
Val Tyr Tyr Ile Tyr Gln Thr Leu Tyr 340 345 350 Asp Cys Glu Glu Val
Leu Ser Gln Tyr Arg Gly Glu Asn Ile Ser Val 355 360 365 Pro Val Pro
Val Glu Glu Gln Asp Met Val Asn Lys His His Ser Glu 370 375 380 Phe
Leu Glu Ser Met Ala Asp Asp Leu Arg Thr Thr Asp Val Leu Asp 385 390
395 400 Gly Phe Thr Asp Leu Leu Lys Ala Ile Asn Ser Asn Leu Asn Asp
Phe 405 410 415 Lys Lys Leu Gln Gln Lys Leu Glu Gln Gln Lys Lys Lys
Gln Gln Gln 420 425 430 Gln Lys Gln Gln Lys Gln Lys Gln Gln Gln Ala
Gln Lys Gln Pro Glu 435 440 445 Glu Tyr Ile Gln Ala Met Phe Ala Leu
Glu Thr Glu Ile Lys Asn Lys 450 455 460 Ile Ser Ile Leu Gly Leu Met
Pro Pro Ser Ser Leu Ala Glu Ala Leu 465 470 475 480 Lys Gln Leu Lys
Asp Lys Ala Leu Lys Arg Ala Gly Leu Thr Glu Glu 485 490 495 Leu Leu
Gln Glu Gln Ile Glu Gln Arg Thr Ala Ala Arg Lys Asn Lys 500 505 510
Gln Phe Asp Val Ser Asp Gln Ile Arg Lys Gln Leu Gly Ser Lys Gly 515
520 525 Ile Ala Leu Met Asp Glu Pro Thr Gly Thr Val Trp Arg Pro Cys
Glu 530 535 540 Pro Glu Ser Glu 545 13 2183 DNA Glycine max 13
gcacgagata aacgataacg ttatttggct gtgaatttgg gatgagctgg tccggtgcaa
60 aaatgggtac ggtgtctctt ctcaagtgct acagaccctt tttctctatg
cttttccctc 120 actccgctcc acccagactc cacgccgcca tcttcaggag
caaaaacttt tctttttgcg 180 ccacctcgtc cccgccgttg acggcggaga
agggttgcgg caaatccgac gccgagtgtc 240 ccaccttgcc ggaggtgtgg
ctgcacaaca ccatgagtag gacgaaggaa ctcttcaaac 300 ccaaagtgga
atccaaagtg ggaatgtacg tgtgcggcgt caccgcttat gatcttagcc 360
atattggaca cgctcgcgta tacgtcaatt tcgaccttct ttacagatac tttaagcatt
420 tgggatttga agtctgttat gttcgcaatt tcactgacgt agatgacaag
ataattgcta 480 gagcaaagga gttaggagaa gatccaatca gtttgagctg
gcgctattgt gaagagttct 540 gtcaagacat ggtaactctt aattgtctgt
ctccctctgt ggaaccaaag gtctcagagc 600 acatgcccca aatcattgat
atgattgaga agatccttaa taatgggtat gcctacattg 660 ttgatgggga
tgtgtacttt aatgtagaaa aatttccaga atatgggaaa ctatctagtc 720
gagatctaga agataatcga gctggtgaga gggttgcagt tgattctaga aagaaaaatc
780 ctgctgattt tgctctttgg aagtctgcaa agccagggga gccattttgg
gagagtccct 840 ggggtcctgg aagacctggg tggcatattg aatgcagtgc
catgagtgca gcttatcttg 900 gttactcttt tgatatccat ggtggaggaa
tcgaccttgt gtttcctcac catgagaatg 960 aaattgctca gagttgtgct
gcatgtaaga aaagtgatat aagtatatgg atgcacaatg 1020 gttttgtcac
cattgactct gtgaaaatgt caaaatcttt ggggaatttt ttcacaatac 1080
gtcaggttat agacgtttac catccactgg ccttgagata ttttttgatg agcgcacatt
1140 atcgatctcc tattaactac tcaaatatac agctcgaaag tgcttcagac
cgtgtttttt 1200 atatatatga gacattacat gaatgtgaaa gctttttgaa
tcagcatgat cagaggaagg 1260 attccacccc accggatact ttggatatta
ttgataagtt ccacgatgtt tttttgacct 1320 caatgtcgga tgatcttcac
actccagttg tattggctgg aatgtctgat ccattaaaat 1380 caatcaatga
tttgctgcat gctcgtaagg ggaaaaaaca acaatttcga atcgaatcac 1440
tatcagcttt ggagaagagc gtcagggatg tccttactgt tttaggactt atgcctgcaa
1500 gttactctga ggttttgcag cagcttaagg taaaagcttt aaaacgtgca
aactttacgg 1560 aagaagaagt cttgcagaaa attgaagaac gggctactgc
tagaatgcaa aaggagtatg 1620 ctaaatcgga tgcaatcagg aaggatttgg
ctgtacttgg tattactctt atggacagtc 1680 caaatggcac aacttggagg
cctgccattc ctcttccact tcaagagctg ctctaagtca 1740 agagttgttc
aacatctcca aagcaaaacc aagaaatgta agttactagg ttctggtata 1800
tggaaatcaa ttataaggga tgccacgggt gtatctcgct atcaacttct cagaatgata
1860 aaggcgaccc cttcttaact cttgatgccg taaaaacatg gattacaatt
tacgttttga 1920 tagagatgtg cttagtgtag ttgtcttggt gaccaatatt
gaattttttt tttttcttca 1980 tataccgggc ttttaacccc tagagtattc
atagtttcaa cgaatttgag tttcagatta 2040 atattaaaat aaatagtcgc
actatcacta gagtagtgtt atgtttctac tttctagagt 2100 agcttcggtt
taatattgag aaagacattt tttttgtggt gataatgaat tttctgttgt 2160
tttttaaaaa aaaaaaaaaa aaa 2183 14 574 PRT Glycine max 14 Thr Ile
Thr Leu Phe Gly Cys Glu Phe Gly Met Ser Trp Ser Gly Ala 1 5 10 15
Lys Met Gly Thr Val Ser Leu Leu Lys Cys Tyr Arg Pro Phe Phe Ser 20
25 30 Met Leu Phe Pro His Ser Ala Pro Pro Arg Leu His Ala Ala Ile
Phe 35 40 45 Arg Ser Lys Asn Phe Ser Phe Cys Ala Thr Ser Ser Pro
Pro Leu Thr 50 55 60 Ala Glu Lys Gly Cys Gly Lys Ser Asp Ala Glu
Cys Pro Thr Leu Pro 65 70 75 80 Glu Val Trp Leu His Asn Thr Met Ser
Arg Thr Lys Glu Leu Phe Lys 85 90 95 Pro Lys Val Glu Ser Lys Val
Gly Met Tyr Val Cys Gly Val Thr Ala 100 105 110 Tyr Asp Leu Ser His
Ile Gly His Ala Arg Val Tyr Val Asn Phe Asp 115 120 125 Leu Leu Tyr
Arg Tyr Phe Lys His Leu Gly Phe Glu Val Cys Tyr Val 130 135 140 Arg
Asn Phe Thr Asp Val Asp Asp Lys Ile Ile Ala Arg Ala Lys Glu 145 150
155 160 Leu Gly Glu Asp Pro Ile Ser Leu Ser Trp Arg Tyr Cys Glu Glu
Phe 165 170 175 Cys Gln Asp Met Val Thr Leu Asn Cys Leu Ser Pro Ser
Val Glu Pro 180 185 190 Lys Val Ser Glu His Met Pro Gln Ile Ile Asp
Met Ile Glu Lys Ile 195 200 205 Leu Asn Asn Gly Tyr Ala Tyr Ile Val
Asp Gly Asp Val Tyr Phe Asn 210 215 220 Val Glu Lys Phe Pro Glu Tyr
Gly Lys Leu Ser Ser Arg Asp Leu Glu 225 230 235 240 Asp Asn Arg Ala
Gly Glu Arg Val Ala Val Asp Ser Arg Lys Lys Asn 245 250 255 Pro Ala
Asp Phe Ala Leu Trp Lys Ser Ala Lys Pro Gly Glu Pro Phe 260 265 270
Trp Glu Ser Pro Trp Gly Pro Gly Arg Pro Gly Trp His Ile Glu Cys 275
280 285 Ser Ala Met Ser Ala Ala Tyr Leu Gly Tyr Ser Phe Asp Ile His
Gly 290 295 300 Gly Gly Ile Asp Leu Val Phe Pro His His Glu Asn Glu
Ile Ala Gln 305 310 315 320 Ser Cys Ala Ala Cys Lys Lys Ser Asp Ile
Ser Ile Trp Met His Asn 325 330 335 Gly Phe Val Thr Ile Asp Ser Val
Lys Met Ser Lys Ser Leu Gly Asn 340 345 350 Phe Phe Thr Ile Arg Gln
Val Ile Asp Val Tyr His Pro Leu Ala Leu 355 360 365 Arg Tyr Phe Leu
Met Ser Ala His Tyr Arg Ser Pro Ile Asn Tyr Ser 370 375 380 Asn Ile
Gln Leu Glu Ser Ala Ser Asp Arg Val Phe Tyr Ile Tyr Glu 385 390 395
400 Thr Leu His Glu Cys Glu Ser Phe Leu Asn Gln His Asp Gln Arg Lys
405 410 415 Asp Ser Thr Pro Pro Asp Thr Leu Asp Ile Ile Asp Lys Phe
His Asp 420 425 430 Val Phe Leu Thr Ser Met Ser Asp Asp Leu His Thr
Pro Val Val Leu 435 440 445 Ala Gly Met Ser Asp Pro Leu Lys Ser Ile
Asn Asp Leu Leu His Ala 450 455 460 Arg Lys Gly Lys Lys Gln Gln Phe
Arg Ile Glu Ser Leu Ser Ala Leu 465 470 475 480 Glu Lys Ser Val Arg
Asp Val Leu Thr Val Leu Gly Leu Met Pro Ala 485 490 495 Ser Tyr Ser
Glu Val Leu Gln Gln Leu Lys Val Lys Ala Leu Lys Arg 500 505 510 Ala
Asn Phe Thr Glu Glu Glu Val Leu Gln Lys Ile Glu Glu Arg Ala 515 520
525 Thr Ala Arg Met Gln Lys Glu Tyr Ala Lys Ser Asp Ala Ile Arg Lys
530 535 540 Asp Leu Ala Val Leu Gly Ile Thr Leu Met Asp Ser Pro Asn
Gly Thr 545 550 555 560 Thr Trp Arg Pro Ala Ile Pro Leu Pro Leu Gln
Glu Leu Leu 565 570 15 633 DNA Zea mays 15 gcacacacgt cggtccaaac
acgcgccgtc cgctcgcggc ttctccaacc aaagccgtgc 60 agccaaatcc
gaagggtagc gtagcacggg gacgacgcca tgagccgcgc gctcctctcc 120
cacgtcctcc accgtccgcc gcacttcgcg tacacctgct taaggagtgg cgttggtgcc
180 cgaggaggag tgctcgcttc tggcatccac ccactccgtc gtctcaattg
cagcgcggtt 240 gaagccgttc ccggccccac cgaggaggcg cctgctcctc
aggcaaggaa gaaaagagta 300 gtttctggtg tacagccaac aggatcggtt
caccttggaa attatctagg ggcaattaag 360 aattgggttg cacttcagga
ttcatatgag acattctttt tcatcgtgga tcttcatgca 420 attactttac
catatgaggc gccactgctt tctaaagcaa caagaagcac tgctgcaata 480
tatcttgcat gtggcgtcga cagctccaag gcttctatct ttgtacagtc tcatgtccgt
540 gctcatgttg agttgatgtg gctattgagt tcttctactc ctattggctg
gctgaataga 600 atgatccagt tcaaagagaa gtctcgcaag gcg 633 16 410 PRT
Zea mays 16 His Gly Asp Asp Ala Met Ser Arg Ala Leu Leu Ser His Val
Leu His 1 5 10 15 Arg Pro Pro His Phe Ala Tyr Thr Cys Leu Arg Ser
Gly Val Gly Ala 20 25 30 Arg Gly Gly Val Leu Ala Ser Gly Ile His
Pro Leu Arg Arg Leu Asn 35 40 45 Cys Ser Ala Val Glu Ala Val Pro
Gly Pro Thr Glu Glu Ala Pro Ala 50 55
60 Pro Gln Ala Arg Lys Lys Arg Val Val Ser Gly Val Gln Pro Thr Gly
65 70 75 80 Ser Val His Leu Gly Asn Tyr Leu Gly Ala Ile Lys Asn Trp
Val Ala 85 90 95 Leu Gln Asp Ser Tyr Glu Thr Phe Phe Phe Ile Val
Asp Leu His Ala 100 105 110 Ile Thr Leu Pro Tyr Glu Ala Pro Leu Leu
Ser Lys Ala Thr Arg Ser 115 120 125 Thr Ala Ala Ile Tyr Leu Ala Cys
Gly Val Asp Ser Ser Lys Ala Ser 130 135 140 Ile Phe Val Gln Ser His
Val Arg Ala His Val Glu Leu Met Trp Leu 145 150 155 160 Leu Ser Ser
Ser Thr Pro Ile Gly Trp Leu Asn Arg Met Ile Gln Phe 165 170 175 Lys
Glu Lys Ser Arg Lys Ala Gly Asp Glu Asn Val Gly Val Ala Leu 180 185
190 Leu Thr Tyr Pro Val Leu Met Ala Ser Asp Ile Leu Leu Tyr Gln Ser
195 200 205 Asp Leu Val Pro Val Gly Glu Asp Gln Thr Gln His Leu Glu
Leu Thr 210 215 220 Arg Glu Ile Ala Glu Arg Val Asn Asn Leu Tyr Gly
Gly Arg Lys Trp 225 230 235 240 Lys Lys Leu Gly Gly Arg Gly Gly Leu
Leu Phe Lys Val Pro Glu Ala 245 250 255 Leu Ile Pro Pro Ala Gly Ala
Arg Val Met Ser Leu Thr Asp Gly Leu 260 265 270 Ser Lys Met Ser Lys
Ser Ala Pro Ser Asp Gln Ser Arg Ile Asn Leu 275 280 285 Leu Asp Pro
Lys Asp Val Ile Ala Asn Lys Ile Lys Arg Cys Lys Thr 290 295 300 Asp
Ser Phe Pro Gly Met Glu Phe Asp Asn Pro Glu Arg Pro Glu Cys 305 310
315 320 Arg Asn Leu Leu Ser Ile Tyr Gln Ile Ile Thr Glu Lys Thr Lys
Glu 325 330 335 Glu Val Val Ser Glu Cys Gln His Met Asn Trp Gly Thr
Phe Lys Thr 340 345 350 Thr Leu Thr Glu Ala Leu Ile Asp His Leu Gln
Pro Ile Gln Val Arg 355 360 365 Tyr Glu Glu Ile Met Ser Asp Pro Ala
Tyr Leu Asp Asn Val Leu Leu 370 375 380 Glu Gly Ala Val Lys Ala Ala
Glu Ile Ala Asp Ile Thr Leu Asn Asn 385 390 395 400 Val Tyr Gln Ala
Met Gly Phe Leu Arg Arg 405 410 17 1536 DNA Glycine max 17
gcacgaggga agatgagcgt ttcacatttc gcggttctat cgtcgtgttg ttgtccacgc
60 ttggcccctt ctctgtcgcg tgcttcaacc cttcgttctc gcatccggtg
ttgtactact 120 ctcactgcta cttcttcaga gactcccact ccaaccttcg
tgaagaaacg agtagtgtcg 180 ggggttcagc ccacgggctc aattcacctc
ggaaactatt ttggcgccat caagaattgg 240 gttgcccttc agaatgtgta
tgatacactt ttcttcattg tggacctgca cgcgattaca 300 ttaccatatg
acacccaaca attatctaag gctacaaggt caactgctgc tatttaccta 360
gcatgtggag tggatccttc aaaggcttca gtatttgtac agtctcatgt tcgggcacat
420 gtagaattga tgtggctgct aagttccaca acaccaattg gttggctgaa
caaaatgata 480 caatttaaag agaaatctcg caaggcggga gatgaagaag
ttggggttgc ccttttgact 540 tatcctgttc tgatggcttc tgatatactt
ctatatcagt ctgattttgt ccctgttggt 600 gaagatcaaa agcagcactt
ggagttgact cgtgacttgg ctgaacgggt taataattta 660 tatggaggaa
gaaagtggaa gaaattaggc ggttatgaca gccgaggtgg tactatattt 720
aaggttccag agccccttat acctccagcc ggagcccgga taatgtccct aactgatggc
780 ctgtccaaga tgtcaaagtc tgcaccttct gatcaatcca gaatcaatat
tcttgatcct 840 aaagatctca tagcaaacaa gatcaaacgt tgcaaaactg
attcatttcc tggcttggaa 900 tttgacaact ctgagaggcc tgaatgtaac
aatcttgttt ccatatacca gcttatttca 960 ggaaagacga aagaggaagt
tgtgcaggaa tgccaaaaca tgaactgggg cacattcaaa 1020 cctcttttaa
cagatgcctt gattgatcat ttgcatccca ttcaggttcg ctatgaggaa 1080
atcatgtccg attcaggtta tttagatgga gttttagcac aaggtgctag aaatgcagca
1140 gatatagcag attctacact taataatatt taccaagcaa tgggattttt
taagagacag 1200 tgataattga tgccaaataa attaaagatt ggcgagacgt
caacttaaaa gctaacttct 1260 ggatgattca tgatgggcct caaaattttg
gagtaatctt atggacatat acttgactac 1320 tggaaatgga aagattattg
atgcaaagcc taaaggtccc attagttctt gatgcaatgg 1380 gctttgtatc
tccttcattt ttctccgagt atggtcgttg ccttcatttt atattttatt 1440
gtttcaatct ctttcattat ttacttgtat tttataatga attcagcata ttgataaatt
1500 gttccgccat tgtatttaaa aaaaaaaaaa aaaaaa 1536 18 400 PRT
Glycine max 18 Ala Arg Gly Lys Met Ser Val Ser His Phe Ala Val Leu
Ser Ser Cys 1 5 10 15 Cys Cys Pro Arg Leu Ala Pro Ser Leu Ser Arg
Ala Ser Thr Leu Arg 20 25 30 Ser Arg Ile Arg Cys Cys Thr Thr Leu
Thr Ala Thr Ser Ser Glu Thr 35 40 45 Pro Thr Pro Thr Phe Val Lys
Lys Arg Val Val Ser Gly Val Gln Pro 50 55 60 Thr Gly Ser Ile His
Leu Gly Asn Tyr Phe Gly Ala Ile Lys Asn Trp 65 70 75 80 Val Ala Leu
Gln Asn Val Tyr Asp Thr Leu Phe Phe Ile Val Asp Leu 85 90 95 His
Ala Ile Thr Leu Pro Tyr Asp Thr Gln Gln Leu Ser Lys Ala Thr 100 105
110 Arg Ser Thr Ala Ala Ile Tyr Leu Ala Cys Gly Val Asp Pro Ser Lys
115 120 125 Ala Ser Val Phe Val Gln Ser His Val Arg Ala His Val Glu
Leu Met 130 135 140 Trp Leu Leu Ser Ser Thr Thr Pro Ile Gly Trp Leu
Asn Lys Met Ile 145 150 155 160 Gln Phe Lys Glu Lys Ser Arg Lys Ala
Gly Asp Glu Glu Val Gly Val 165 170 175 Ala Leu Leu Thr Tyr Pro Val
Leu Met Ala Ser Asp Ile Leu Leu Tyr 180 185 190 Gln Ser Asp Phe Val
Pro Val Gly Glu Asp Gln Lys Gln His Leu Glu 195 200 205 Leu Thr Arg
Asp Leu Ala Glu Arg Val Asn Asn Leu Tyr Gly Gly Arg 210 215 220 Lys
Trp Lys Lys Leu Gly Gly Tyr Asp Ser Arg Gly Gly Thr Ile Phe 225 230
235 240 Lys Val Pro Glu Pro Leu Ile Pro Pro Ala Gly Ala Arg Ile Met
Ser 245 250 255 Leu Thr Asp Gly Leu Ser Lys Met Ser Lys Ser Ala Pro
Ser Asp Gln 260 265 270 Ser Arg Ile Asn Ile Leu Asp Pro Lys Asp Leu
Ile Ala Asn Lys Ile 275 280 285 Lys Arg Cys Lys Thr Asp Ser Phe Pro
Gly Leu Glu Phe Asp Asn Ser 290 295 300 Glu Arg Pro Glu Cys Asn Asn
Leu Val Ser Ile Tyr Gln Leu Ile Ser 305 310 315 320 Gly Lys Thr Lys
Glu Glu Val Val Gln Glu Cys Gln Asn Met Asn Trp 325 330 335 Gly Thr
Phe Lys Pro Leu Leu Thr Asp Ala Leu Ile Asp His Leu His 340 345 350
Pro Ile Gln Val Arg Tyr Glu Glu Ile Met Ser Asp Ser Gly Tyr Leu 355
360 365 Asp Gly Val Leu Ala Gln Gly Ala Arg Asn Ala Ala Asp Ile Ala
Asp 370 375 380 Ser Thr Leu Asn Asn Ile Tyr Gln Ala Met Gly Phe Phe
Lys Arg Gln 385 390 395 400 19 725 DNA Triticum aestivum 19
ctcgtgccga attcggcacg aggcggttca ttatttaagg ttcctgaagc ccttatccct
60 ccagcagggg cccgtgtgat gtccttaact gatggcctct ccaagatgtc
gaagtctgct 120 ccttcagatt tgtctcgcat taaccttctt gacccaaatg
atgtgattgt gaacaaaatc 180 aaacgctgca aaactgactc gctccctggc
ttggaattcg acaacccaga gaggccggaa 240 tgcaaaaatc ttctctcagt
ctaccagatc atcactggaa aaacgaaaga ggaagttgtt 300 agtgaatgcc
aagatatgaa ctgggggacg ttcaaggtta cccttacgga tgccttaatt 360
gatcatctgc aacctattca ggttcgatac gaggagatca tgtctgatcc aggttatttg
420 gacaatgttc tgctaaatgg ggcagggaaa gcttctgaga tagcagacgc
caccctcaac 480 aacgtctacc aagccatggg tttcttgcgc agatagcata
tgtagaacat tttttataac 540 tgcacaatgc tagttttgca cttgttggcc
tttctgctag tggtactgat aagcgttttg 600 tttgatatgc ttggattagc
cttttgttcc tggttattat ggacactgtt aataggtatt 660 aaaaggatta
tttactgaaa aaaaaaaaaa aaaaaaaaaa attaaaaggg ggcgcgcgta 720 ccata
725 20 171 PRT Triticum aestivum 20 Leu Val Pro Asn Ser Ala Arg Gly
Gly Ser Leu Phe Lys Val Pro Glu 1 5 10 15 Ala Leu Ile Pro Pro Ala
Gly Ala Arg Val Met Ser Leu Thr Asp Gly 20 25 30 Leu Ser Lys Met
Ser Lys Ser Ala Pro Ser Asp Leu Ser Arg Ile Asn 35 40 45 Leu Leu
Asp Pro Asn Asp Val Ile Val Asn Lys Ile Lys Arg Cys Lys 50 55 60
Thr Asp Ser Leu Pro Gly Leu Glu Phe Asp Asn Pro Glu Arg Pro Glu 65
70 75 80 Cys Lys Asn Leu Leu Ser Val Tyr Gln Ile Ile Thr Gly Lys
Thr Lys 85 90 95 Glu Glu Val Val Ser Glu Cys Gln Asp Met Asn Trp
Gly Thr Phe Lys 100 105 110 Val Thr Leu Thr Asp Ala Leu Ile Asp His
Leu Gln Pro Ile Gln Val 115 120 125 Arg Tyr Glu Glu Ile Met Ser Asp
Pro Gly Tyr Leu Asp Asn Val Leu 130 135 140 Leu Asn Gly Ala Gly Lys
Ala Ser Glu Ile Ala Asp Ala Thr Leu Asn 145 150 155 160 Asn Val Tyr
Gln Ala Met Gly Phe Leu Arg Arg 165 170 21 1062 DNA Zea mays 21
gcacgaggga catcacgctg ctggatttcc tgagagaggt gggccgtttt gcacgcgtgg
60 gtacaatgat cgccaaggag agcgtcaaga agcgtcttgc gtcggaagac
gggatgagct 120 acaccgagtt tacctaccag ctgctgcagg gctacgactt
cctttacatg ttcaagaata 180 tgggtgtcaa tgtgcagatc gggggcagcg
atcagtgggg gaacatcaca gcgggaactg 240 agttgatcag aaaaatcttg
caggttgaag gggcgcatgg actcacattc ccacttctgc 300 tgaagagcga
cggtaccaaa tttggaaaga cggaggatgg ggcaatctgg ctctcttcga 360
agatgctttc tccttacaag ttctatcagt acttctttgc ggtgccagac atcgatgtca
420 tcaggtttat gaagatcctg acgttcctga gcttggatga gattctggag
ctagaagact 480 cgatgaagaa gcctggctat gtgccaaaca ctgttcagaa
gaggcttgca gaagaggtga 540 cgcgatttgt tcatggcgag gagggattgg
aggaggcatt gaaggcaacc gaggccttga 600 gacctggtgc tcagacacaa
ttggatgcac aaacaattga ggggatagca gatgatgtgc 660 cttcatgctc
tttagcttat gatcaagtgt tcaagtctcc acttattgat ttggctgttt 720
ccacaggttt gctcactagt aagtcagcag ttaagcggct tattaagcaa ggtggtctgt
780 acttgaataa cgtgaggatt gatagtgagg ataagctggt tgaggaaggt
gatatagttg 840 atgggaaggt gctcttgttg tctgctggaa agaagaacaa
gatggttgtg aggatatctt 900 gactactctt atttgttctt tataacttat
tttagccatt gaggagaaaa gtaacggtgt 960 tgtgtcttca aaactcaaat
gagctgtcta tgagcataca gattgttata ttggagaggt 1020 tgaacacacc
tttttttttg ctctaaaaaa aaaaaaaaaa aa 1062 22 299 PRT Zea mays 22 Thr
Arg Asp Ile Thr Leu Leu Asp Phe Leu Arg Glu Val Gly Arg Phe 1 5 10
15 Ala Arg Val Gly Thr Met Ile Ala Lys Glu Ser Val Lys Lys Arg Leu
20 25 30 Ala Ser Glu Asp Gly Met Ser Tyr Thr Glu Phe Thr Tyr Gln
Leu Leu 35 40 45 Gln Gly Tyr Asp Phe Leu Tyr Met Phe Lys Asn Met
Gly Val Asn Val 50 55 60 Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn
Ile Thr Ala Gly Thr Glu 65 70 75 80 Leu Ile Arg Lys Ile Leu Gln Val
Glu Gly Ala His Gly Leu Thr Phe 85 90 95 Pro Leu Leu Leu Lys Ser
Asp Gly Thr Lys Phe Gly Lys Thr Glu Asp 100 105 110 Gly Ala Ile Trp
Leu Ser Ser Lys Met Leu Ser Pro Tyr Lys Phe Tyr 115 120 125 Gln Tyr
Phe Phe Ala Val Pro Asp Ile Asp Val Ile Arg Phe Met Lys 130 135 140
Ile Leu Thr Phe Leu Ser Leu Asp Glu Ile Leu Glu Leu Glu Asp Ser 145
150 155 160 Met Lys Lys Pro Gly Tyr Val Pro Asn Thr Val Gln Lys Arg
Leu Ala 165 170 175 Glu Glu Val Thr Arg Phe Val His Gly Glu Glu Gly
Leu Glu Glu Ala 180 185 190 Leu Lys Ala Thr Glu Ala Leu Arg Pro Gly
Ala Gln Thr Gln Leu Asp 195 200 205 Ala Gln Thr Ile Glu Gly Ile Ala
Asp Asp Val Pro Ser Cys Ser Leu 210 215 220 Ala Tyr Asp Gln Val Phe
Lys Ser Pro Leu Ile Asp Leu Ala Val Ser 225 230 235 240 Thr Gly Leu
Leu Thr Ser Lys Ser Ala Val Lys Arg Leu Ile Lys Gln 245 250 255 Gly
Gly Leu Tyr Leu Asn Asn Val Arg Ile Asp Ser Glu Asp Lys Leu 260 265
270 Val Glu Glu Gly Asp Ile Val Asp Gly Lys Val Leu Leu Leu Ser Ala
275 280 285 Gly Lys Lys Asn Lys Met Val Val Arg Ile Ser 290 295 23
346 PRT Drosophila melanogaster 23 Met Val Asp Lys Val Ala Asn Gly
Val Ser Lys Lys Gly Ala Lys Lys 1 5 10 15 Ala Lys Ala Ala Lys Lys
Ala Lys Ala Asn Ala Ser Thr Ala Ala Ala 20 25 30 Asn Asn Ser Gly
Gly Asp Ser Ala Asp His Ala Ala Gly Arg Tyr Gly 35 40 45 Ser Met
Ser Lys Asp Lys Arg Ser Arg Asn Val Val Ser Ser Gly Val 50 55 60
Gly Lys Gly Val Trp Val Arg Gly Arg Val His Thr Ser Arg Ala Lys 65
70 75 80 Gly Lys Cys Arg Ser Ser Thr Val Cys Ala Val Gly Asp Val
Ser Lys 85 90 95 Met Val Lys Ala Gly Asn Lys Ser Asp Ala Lys Val
Ala Val Ser Ser 100 105 110 Lys Ser Cys Thr Ser Ser Val Val Ser Ala
Lys Ala Asp Ala Ser Arg 115 120 125 Asn Ala Asp Asp Ala Gly Asn Arg
Val Asn Asp Thr Arg Asp Asn Arg 130 135 140 Val Asp Arg Thr Ala Asn
Ala Arg Ala Gly Val Cys Arg Arg Asp Thr 145 150 155 160 Gly Thr His
Thr Lys Ser Ala Ala Ser Gly Gly Ala Asn Val Thr Val 165 170 175 Ser
Tyr Lys Asp Ser Ala Tyr Ala Ser Tyr Lys Met Ala Ala Ala Asp 180 185
190 Asp Lys Val Tyr Thr Val Gly Ala Val Arg Ala Asp Ser Asn Thr His
195 200 205 Arg His Thr Val Gly Asp Met Ala Lys Tyr His Tyr His Val
His Thr 210 215 220 Gly Asn Thr Thr Ser Lys Gly Arg Asp Lys Tyr Ala
Lys Ser Val Gly 225 230 235 240 Tyr Lys Val Asp Ala Lys Ala Asp Gly
Val Ala Met Arg Ala Gly Val 245 250 255 Thr Gly Asp Asp Ser Thr Asn
Lys Gly Arg Val Lys Ala Lys Tyr Asp 260 265 270 Thr Asp Tyr Asp Lys
Ala Arg Tyr Thr Met Asp Asn Asn Val Tyr Ser 275 280 285 Asn Ser Tyr
Asp Met Met Arg Gly Ser Gly Ala Arg His Asp Tyr Arg 290 295 300 Ala
Lys His His Gly Asp Thr Ser Lys Ala Ala Tyr Ser Arg Tyr Gly 305 310
315 320 Cys His Ala Gly Gly Gly Gly Met Arg Val Val Met Tyr Gly Asp
Asn 325 330 335 Arg Lys Thr Ser Met Arg Asp Lys Arg Thr 340 345 24
501 PRT Rattus norvegicus 24 Met Pro Ser Ala Asn Ala Ser Arg Lys
Gly Gln Glu Lys Pro Arg Glu 1 5 10 15 Ile Val Asp Ala Ala Glu Asp
Tyr Ala Lys Glu Arg Tyr Gly Val Ser 20 25 30 Ser Met Ile Gln Ser
Gln Glu Lys Pro Asp Arg Val Leu Val Arg Val 35 40 45 Lys Asp Leu
Thr Val Gln Lys Ala Asp Glu Val Val Trp Val Arg Ala 50 55 60 Arg
Val His Thr Ser Arg Ala Lys Gly Lys Gln Cys Phe Leu Val Leu 65 70
75 80 Arg Gln Gln Gln Phe Asn Val Gln Ala Leu Val Ala Val Gly Asp
His 85 90 95 Ala Ser Lys Gln Met Val Lys Phe Ala Ala Asn Ile Asn
Lys Glu Ser 100 105 110 Ile Ile Asp Val Glu Gly Ile Val Arg Lys Val
Asn Gln Lys Ile Gly 115 120 125 Ser Cys Thr Gln Gln Asp Val Glu Leu
His Val Gln Lys Ile Tyr Val 130 135 140 Ile Ser Leu Ala Glu Pro Arg
Leu Pro Leu Gln Leu Asp Asp Ala Ile 145 150 155 160 Arg Pro Glu Val
Glu Gly Glu Glu Asp Gly Arg Ala Thr Val Asn Gln 165 170 175 Asp Thr
Arg Leu Asp Asn Arg Ile Ile Asp Leu Arg Thr Ser Thr Ser 180 185 190
Gln Ala Ile Phe His Leu Gln Ser Gly Ile Cys His Leu Phe Arg Glu 195
200 205 Thr Leu Ile Asn Lys Gly Phe Val Glu Ile Gln Thr Pro Lys Ile
Ile 210 215 220 Ser Ala Ala Ser Glu Gly Gly Ala Asn Val Phe Thr Val
Ser Tyr Phe 225 230 235 240 Lys Ser Asn Ala Tyr Leu Ala Gln Ser Pro
Gln Leu Tyr Lys Gln Met 245 250 255 Cys Ile Cys Ala Asp Phe Glu Lys
Val Phe Cys Ile Gly Pro Val Phe 260 265 270 Arg Ala Glu Asp Ser Asn
Thr His Arg His Leu Thr Glu Phe Val Gly 275 280 285 Leu Asp Ile Glu
Met Ala Phe Asn Tyr His Tyr His Glu Val
Val Glu 290 295 300 Glu Ile Ala Asp Thr Leu Val Gln Ile Phe Lys Gly
Leu Gln Glu Arg 305 310 315 320 Phe Gln Thr Glu Ile Gln Thr Val Asn
Lys Gln Phe Pro Cys Glu Pro 325 330 335 Phe Lys Phe Leu Glu Pro Thr
Leu Arg Leu Glu Tyr Cys Glu Ala Leu 340 345 350 Ala Met Leu Arg Glu
Ala Gly Val Glu Met Asp Asp Glu Glu Asp Leu 355 360 365 Ser Thr Pro
Asn Glu Lys Leu Leu Gly Arg Leu Val Lys Glu Lys Tyr 370 375 380 Asp
Thr Asp Phe Tyr Val Leu Asp Lys Tyr Pro Leu Ala Val Arg Pro 385 390
395 400 Phe Tyr Thr Met Pro Asp Pro Arg Asn Pro Lys Gln Ser Asn Ser
Tyr 405 410 415 Asp Met Phe Met Arg Gly Glu Glu Ile Leu Ser Gly Ala
Gln Arg Ile 420 425 430 His Asp Pro Gln Leu Leu Thr Glu Arg Ala Leu
His His Gly Ile Asp 435 440 445 Leu Glu Lys Ile Lys Ala Tyr Ile Asp
Ser Phe Arg Phe Gly Ala Pro 450 455 460 Pro His Ala Gly Gly Gly Ile
Gly Leu Glu Arg Val Thr Met Leu Phe 465 470 475 480 Leu Gly Leu His
Asn Val Arg Gln Thr Ser Met Phe Pro Arg Asp Pro 485 490 495 Lys Arg
Leu Thr Pro 500 25 500 PRT Homo sapiens 25 Met Pro Ser Ala Thr Gln
Arg Lys Ser Gln Glu Lys Pro Arg Glu Ile 1 5 10 15 Met Asp Ala Ala
Glu Asp Tyr Ala Lys Glu Arg Tyr Gly Ile Ser Ser 20 25 30 Met Ile
Gln Ser Gln Glu Lys Pro Asp Arg Val Leu Val Arg Val Arg 35 40 45
Asp Leu Thr Ile Gln Lys Ala Asp Glu Val Val Trp Val Arg Ala Arg 50
55 60 Val His Thr Ser Arg Ala Lys Gly Lys Gln Cys Phe Leu Val Leu
Arg 65 70 75 80 Gln Gln Gln Phe Asn Val Gln Ala Leu Val Ala Val Gly
Asp His Ala 85 90 95 Ser Lys Gln Met Val Lys Phe Ala Ala Asn Ile
Asn Lys Glu Ser Ile 100 105 110 Val Asp Val Glu Gly Val Val Arg Lys
Val Asn Gln Lys Ile Gly Ser 115 120 125 Cys Thr Gln Gln Asp Val Glu
Leu His Val Gln Lys Ile Tyr Val Ile 130 135 140 Ser Leu Ala Glu Pro
Arg Leu Pro Leu Gln Leu Asp Asp Ala Val Arg 145 150 155 160 Pro Glu
Gln Glu Gly Glu Glu Glu Gly Arg Ala Thr Val Asn Gln Asp 165 170 175
Thr Arg Leu Asp Asn Arg Val Ile Asp Leu Arg Thr Ser Thr Ser Gln 180
185 190 Ala Val Phe Arg Leu Gln Ser Gly Ile Cys His Leu Phe Arg Glu
Thr 195 200 205 Leu Ile Asn Lys Gly Phe Val Glu Ile Gln Thr Pro Lys
Ile Ile Ser 210 215 220 Ala Ala Ser Glu Gly Gly Ala Asn Val Phe Thr
Val Ser Tyr Phe Lys 225 230 235 240 Asn Asn Ala Tyr Leu Ala Gln Ser
Pro Gln Leu Tyr Lys Gln Met Cys 245 250 255 Ile Cys Ala Asp Phe Glu
Lys Val Phe Ser Ile Gly Pro Val Phe Arg 260 265 270 Ala Glu Asp Ser
Asn Thr His Arg His Leu Thr Glu Phe Val Gly Leu 275 280 285 Asp Ile
Glu Met Ala Phe Asn Tyr His Tyr His Glu Val Met Glu Glu 290 295 300
Ile Ala Asp Thr Met Val Gln Ile Phe Lys Gly Leu Gln Glu Arg Phe 305
310 315 320 Gln Thr Glu Ile Gln Thr Val Asn Lys Gln Phe Pro Cys Glu
Pro Phe 325 330 335 Lys Phe Leu Glu Pro Thr Leu Arg Leu Glu Tyr Cys
Glu Ala Leu Ala 340 345 350 Met Leu Arg Glu Ala Gly Val Glu Met Gly
Asp Glu Asp Asp Leu Ser 355 360 365 Thr Pro Asn Glu Lys Leu Leu Gly
His Leu Val Lys Glu Lys Tyr Asp 370 375 380 Thr Asp Phe Tyr Ile Leu
Asp Lys Tyr Pro Leu Ala Val Arg Pro Phe 385 390 395 400 Tyr Thr Met
Pro Asp Pro Arg Asn Pro Lys Gln Ser Lys Ser Tyr Asp 405 410 415 Met
Phe Met Arg Gly Glu Glu Ile Leu Ser Gly Ala Gln Arg Ile His 420 425
430 Asp Pro Gln Leu Leu Thr Glu Arg Ala Leu His His Gly Asn Asp Leu
435 440 445 Glu Lys Ile Lys Ala Tyr Ile Asp Ser Phe Arg Phe Gly Ala
Pro Pro 450 455 460 His Ala Gly Gly Gly Ile Gly Leu Glu Arg Val Thr
Met Leu Phe Leu 465 470 475 480 Gly Leu His Asn Val Arg Gln Thr Ser
Met Phe Pro Arg Asp Pro Lys 485 490 495 Arg Leu Thr Pro 500 26 459
PRT Haemophilus influenzae Rd 26 Met Leu Lys Ile Phe Asn Thr Leu
Thr Arg Glu Lys Glu Ile Phe Lys 1 5 10 15 Pro Ile His Glu Asn Lys
Val Gly Met Tyr Val Cys Gly Val Thr Val 20 25 30 Tyr Asp Leu Cys
His Ile Gly His Gly Arg Thr Phe Val Cys Phe Asp 35 40 45 Val Ile
Ala Arg Tyr Leu Arg Ser Leu Gly Tyr Asp Leu Thr Tyr Val 50 55 60
Arg Asn Ile Thr Asp Val Asp Asp Lys Ile Ile Lys Arg Ala Leu Glu 65
70 75 80 Asn Lys Glu Thr Cys Asp Gln Leu Val Asp Arg Met Val Gln
Glu Met 85 90 95 Tyr Lys Asp Phe Asp Ala Leu Asn Val Leu Arg Pro
Asp Phe Glu Pro 100 105 110 Arg Ala Thr His His Ile Pro Glu Ile Ile
Glu Ile Val Glu Lys Leu 115 120 125 Ile Lys Arg Gly His Ala Tyr Val
Ala Asp Asn Gly Asp Val Met Phe 130 135 140 Asp Val Glu Ser Phe Lys
Glu Tyr Gly Lys Leu Ser Arg Gln Asp Leu 145 150 155 160 Glu Gln Leu
Gln Ala Gly Ala Arg Ile Glu Ile Asn Glu Ile Lys Lys 165 170 175 Asn
Pro Met Asp Phe Val Leu Trp Lys Met Ser Lys Glu Asn Glu Pro 180 185
190 Ser Trp Ala Ser Pro Trp Gly Ala Gly Arg Pro Gly Trp His Ile Glu
195 200 205 Cys Ser Ala Met Asn Cys Lys Gln Leu Gly Glu Tyr Phe Asp
Ile His 210 215 220 Gly Gly Gly Ser Asp Leu Met Phe Pro His His Glu
Asn Glu Ile Ala 225 230 235 240 Gln Ser Cys Cys Ala His Gly Gly Gln
Tyr Val Asn Tyr Trp Ile His 245 250 255 Ser Gly Met Ile Met Val Asp
Lys Glu Lys Met Ser Lys Ser Leu Gly 260 265 270 Asn Phe Phe Thr Ile
Arg Asp Val Leu Asn His Tyr Asn Ala Glu Ala 275 280 285 Val Arg Tyr
Phe Leu Leu Thr Ala His Tyr Arg Ser Gln Leu Asn Tyr 290 295 300 Ser
Glu Glu Asn Leu Asn Leu Ala Gln Gly Ala Leu Glu Arg Leu Tyr 305 310
315 320 Thr Ala Leu Arg Gly Thr Asp Gln Ser Ala Val Ala Phe Gly Gly
Glu 325 330 335 Asn Phe Val Ala Thr Phe Arg Glu Ala Met Asp Asp Asp
Phe Asn Thr 340 345 350 Pro Asn Ala Leu Ser Val Leu Phe Glu Met Ala
Arg Glu Ile Asn Lys 355 360 365 Leu Lys Thr Glu Asp Val Glu Lys Ala
Asn Gly Leu Ala Ala Arg Leu 370 375 380 Arg Glu Leu Gly Ala Ile Leu
Gly Leu Leu Gln Gln Glu Pro Glu Lys 385 390 395 400 Phe Leu Gln Ala
Gly Ser Asn Asp Asp Glu Val Ala Lys Ile Glu Ala 405 410 415 Leu Ile
Lys Gln Arg Asn Glu Ala Arg Thr Ala Lys Asp Trp Ser Ala 420 425 430
Ala Asp Ser Ala Arg Asn Glu Leu Thr Ala Met Gly Ile Val Leu Glu 435
440 445 Asp Gly Pro Asn Gly Thr Thr Trp Arg Lys Gln 450 455 27 461
PRT Escherichia coli 27 Met Leu Lys Ile Phe Asn Thr Leu Thr Arg Gln
Lys Glu Glu Phe Lys 1 5 10 15 Pro Ile His Ala Gly Glu Val Gly Met
Tyr Val Cys Gly Ile Thr Val 20 25 30 Tyr Asp Leu Cys His Ile Gly
His Gly Arg Thr Phe Val Ala Phe Asp 35 40 45 Val Val Ala Arg Tyr
Leu Arg Phe Leu Gly Tyr Lys Leu Lys Tyr Val 50 55 60 Arg Asn Ile
Thr Asp Ile Asp Asp Lys Ile Ile Lys Arg Ala Asn Glu 65 70 75 80 Asn
Gly Glu Ser Phe Val Ala Met Val Asp Arg Met Ile Ala Glu Met 85 90
95 His Lys Asp Phe Asp Ala Leu Asn Ile Leu Arg Pro Asp Met Glu Pro
100 105 110 Arg Ala Thr His His Ile Ala Glu Ile Ile Glu Leu Thr Glu
Gln Leu 115 120 125 Ile Ala Lys Gly His Ala Tyr Val Ala Asp Asn Gly
Asp Val Met Phe 130 135 140 Asp Val Pro Thr Asp Pro Thr Tyr Gly Val
Leu Ser Arg Gln Asp Leu 145 150 155 160 Asp Gln Leu Gln Ala Gly Ala
Arg Val Asp Val Val Asp Asp Lys Arg 165 170 175 Asn Pro Met Asp Phe
Val Leu Trp Lys Met Ser Lys Glu Gly Glu Pro 180 185 190 Ser Trp Pro
Ser Pro Trp Gly Ala Gly Arg Pro Gly Trp His Ile Glu 195 200 205 Cys
Ser Ala Met Asn Cys Lys Gln Leu Gly Asn His Phe Asp Ile His 210 215
220 Gly Gly Gly Ser Asp Leu Met Phe Pro His His Glu Asn Glu Ile Ala
225 230 235 240 Gln Ser Thr Cys Ala His Asp Gly Gln Tyr Val Asn Tyr
Trp Met His 245 250 255 Ser Gly Met Val Met Val Asp Arg Glu Lys Met
Ser Lys Ser Leu Gly 260 265 270 Asn Phe Phe Thr Val Arg Asp Val Leu
Lys Tyr Tyr Asp Ala Glu Thr 275 280 285 Val Arg Tyr Phe Leu Met Ser
Gly His Tyr Arg Ser Gln Leu Asn Tyr 290 295 300 Ser Glu Glu Asn Leu
Lys Gln Ala Arg Ala Ala Val Glu Arg Leu Tyr 305 310 315 320 Thr Ala
Leu Arg Gly Thr Asp Lys Thr Val Ala Pro Ala Gly Gly Glu 325 330 335
Ala Phe Glu Ala Arg Phe Ile Glu Ala Met Asp Asp Asp Phe Asn Thr 340
345 350 Pro Glu Ala Tyr Ser Val Leu Phe Asp Met Ala Arg Glu Val Asn
Arg 355 360 365 Leu Lys Ala Glu Asp Met Ala Ala Ala Asn Ala Met Ala
Ser His Leu 370 375 380 Arg Lys Leu Ser Ala Val Leu Gly Leu Leu Glu
Gln Glu Pro Glu Ala 385 390 395 400 Phe Leu Gln Ser Gly Ala Gln Ala
Asp Asp Ser Glu Val Ala Glu Ile 405 410 415 Glu Ala Leu Ile Gln Gln
Arg Leu Asp Ala Arg Lys Ala Lys Asp Trp 420 425 430 Ala Ala Ala Asp
Ala Ala Arg Asp Arg Leu Asn Glu Met Gly Ile Val 435 440 445 Leu Glu
Asp Gly Pro Gln Gly Thr Thr Trp Arg Arg Lys 450 455 460 28 377 PRT
Synechocystis sp. 28 Met Lys Asn Cys Glu Asn Asp His Arg Phe Thr
Thr Val Ser Ser Gly 1 5 10 15 Lys Ala Trp Gly Gln Leu His Arg Phe
Pro Ser Leu Ile Lys Phe Asn 20 25 30 Phe Ala His Arg Ser Thr Thr
Ala Met Asp Lys Pro Arg Ile Leu Ser 35 40 45 Gly Val Gln Pro Thr
Gly Asn Leu His Leu Gly Asn Tyr Leu Gly Ala 50 55 60 Ile Arg Ser
Trp Val Glu Gln Gln Gln His Tyr Asp Asn Phe Phe Cys 65 70 75 80 Val
Val Asp Leu His Ala Ile Thr Val Pro His Asn Pro Gln Thr Leu 85 90
95 Ala Gln Asp Thr Leu Thr Ile Ala Ala Leu Tyr Leu Ala Cys Gly Ile
100 105 110 Asp Leu Gln Tyr Ser Thr Ile Phe Val Gln Ser His Val Ala
Ala His 115 120 125 Ser Glu Leu Ala Trp Leu Leu Asn Cys Val Thr Pro
Leu Asn Trp Leu 130 135 140 Glu Arg Met Ile Gln Phe Lys Glu Lys Ala
Val Lys Gln Gly Glu Asn 145 150 155 160 Val Ser Val Gly Leu Leu Asp
Tyr Pro Val Leu Met Ala Ala Asp Ile 165 170 175 Leu Leu Tyr Asp Ala
Asp Lys Val Pro Val Gly Glu Asp Gln Lys Gln 180 185 190 His Leu Glu
Leu Thr Arg Asp Ile Val Ile Arg Ile Asn Asp Lys Phe 195 200 205 Gly
Arg Glu Asp Ala Pro Val Leu Lys Leu Pro Glu Pro Leu Ile Arg 210 215
220 Lys Glu Gly Ala Arg Val Met Ser Leu Ala Asp Gly Thr Lys Lys Met
225 230 235 240 Ser Lys Ser Asp Glu Ser Glu Leu Ser Arg Ile Asn Leu
Leu Asp Pro 245 250 255 Pro Glu Met Ile Lys Lys Lys Val Lys Lys Cys
Lys Thr Asp Pro Gln 260 265 270 Arg Gly Leu Trp Phe Asp Asp Pro Glu
Arg Pro Glu Cys His Asn Leu 275 280 285 Leu Thr Leu Tyr Thr Leu Leu
Ser Asn Gln Thr Lys Glu Ala Val Ala 290 295 300 Gln Glu Cys Ala Glu
Met Gly Trp Gly Gln Phe Lys Pro Leu Leu Thr 305 310 315 320 Glu Thr
Ala Ile Ala Ala Leu Glu Pro Ile Gln Ala Lys Tyr Ala Glu 325 330 335
Ile Leu Ala Asp Arg Gly Glu Leu Asp Arg Ile Ile Gln Ala Gly Asn 340
345 350 Ala Lys Ala Ser Gln Thr Ala Gln Gln Thr Leu Ala Arg Val Arg
Asp 355 360 365 Ala Leu Gly Phe Leu Ala Pro Pro Tyr 370 375 29 419
PRT Bacillus caldotenax 29 Met Asp Leu Leu Ala Glu Leu Gln Trp Arg
Gly Leu Val Asn Gln Thr 1 5 10 15 Thr Asp Glu Asp Gly Leu Arg Lys
Leu Leu Asn Glu Glu Arg Val Thr 20 25 30 Leu Tyr Cys Gly Phe Asp
Pro Thr Ala Asp Ser Leu His Ile Gly Asn 35 40 45 Leu Ala Ala Ile
Leu Thr Leu Arg Arg Phe Gln Gln Ala Gly His Arg 50 55 60 Pro Ile
Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly Asp Pro Ser 65 70 75 80
Gly Lys Lys Ser Glu Arg Thr Leu Asn Ala Lys Glu Thr Val Glu Ala 85
90 95 Trp Ser Ala Arg Ile Lys Glu Gln Leu Gly Arg Phe Leu Asp Phe
Glu 100 105 110 Ala Asp Gly Asn Pro Ala Lys Ile Lys Asn Asn Tyr Asp
Trp Ile Gly 115 120 125 Pro Leu Asp Val Ile Thr Phe Leu Arg Asp Val
Gly Lys His Phe Ser 130 135 140 Val Asn Tyr Met Met Ala Lys Glu Ser
Val Gln Ser Arg Ile Glu Thr 145 150 155 160 Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Met Met Leu Gln Ala Tyr Asp 165 170 175 Phe Leu Arg Leu
Tyr Glu Thr Glu Gly Cys Arg Leu Gln Ile Gly Gly 180 185 190 Ser Asp
Gln Trp Gly Asn Ile Thr Ala Gly Leu Glu Leu Ile Arg Lys 195 200 205
Thr Lys Gly Glu Ala Arg Ala Phe Gly Leu Thr Ile Pro Leu Val Thr 210
215 220 Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu Ser Gly Thr Ile
Trp 225 230 235 240 Leu Asp Lys Glu Lys Thr Ser Pro Tyr Glu Phe Tyr
Gln Phe Trp Ile 245 250 255 Asn Thr Asp Asp Arg Asp Val Ile Arg Tyr
Leu Lys Tyr Phe Thr Phe 260 265 270 Leu Ser Lys Glu Glu Ile Glu Ala
Leu Glu Gln Glu Leu Arg Glu Ala 275 280 285 Pro Glu Lys Arg Ala Ala
Gln Lys Ala Leu Ala Glu Glu Val Thr Lys 290 295 300 Leu Val His Gly
Glu Glu Ala Leu Arg Gln Ala Ile Arg Ile Ser Glu 305 310 315 320 Ala
Leu Phe Ser Gly Asp Ile Ala Asn Leu Thr Ala Ala Glu Ile Glu 325 330
335 Gln Gly Phe Lys Asp Val Pro Ser Phe Val His Glu Gly Gly Asp Val
340 345 350 Pro Leu Val Glu Leu Leu Val Ser Ala Gly Ile Ser Pro Ser
Lys Arg 355 360 365 Gln Ala Arg Glu Asp Ile Gln Asn Gly Ala Ile Tyr
Val Asn Gly Glu 370 375 380 Arg Leu Gln Asp Val Gly Ala Ile Leu Thr
Ala Glu His Arg Leu Glu 385 390 395 400 Gly Arg Phe Thr Val Ile Arg
Arg Gly Lys Lys Lys Tyr Tyr Leu Ile 405 410 415 Arg Tyr Ala
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References