U.S. patent application number 10/417510 was filed with the patent office on 2003-09-18 for plant folate biosynthetic genes.
Invention is credited to Harvell, Leslie T., Orozco, Emil M. JR., Weng, Zude.
Application Number | 20030177522 10/417510 |
Document ID | / |
Family ID | 43706136 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030177522 |
Kind Code |
A1 |
Orozco, Emil M. JR. ; et
al. |
September 18, 2003 |
Plant folate biosynthetic genes
Abstract
This invention relates to an isolated nucleic acid fragment
encoding a folate biosynthetic enzyme. The invention also relates
to the construction of a chimeric gene encoding all or a portion of
the folate biosynthetic enzyme, in sense or antisense orientation,
wherein expression of the chimeric gene results in production of
altered levels of the folate biosynthetic enzyme in a transformed
host cell.
Inventors: |
Orozco, Emil M. JR.;
(Cochranville, PA) ; Weng, Zude; (Des Plaines,
IL) ; Harvell, Leslie T.; (Newark, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY
LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
43706136 |
Appl. No.: |
10/417510 |
Filed: |
April 17, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10417510 |
Apr 17, 2003 |
|
|
|
09465559 |
Dec 17, 1999 |
|
|
|
60112735 |
Dec 18, 1998 |
|
|
|
Current U.S.
Class: |
800/278 ;
435/193; 435/419; 435/468; 536/23.2 |
Current CPC
Class: |
C12N 9/1085 20130101;
C12N 9/88 20130101; C12N 9/93 20130101; C12N 15/821 20130101; C12N
15/8243 20130101 |
Class at
Publication: |
800/278 ;
435/193; 435/419; 536/23.2; 435/468 |
International
Class: |
A01H 001/00; C07H
021/04; C12N 009/10; C12N 015/82; C12N 005/04 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide having tetrahydrofolylpolyglutamate
synthase/folylpolyglutamate synthase activity, wherein the amino
acid sequence of the polypeptide and the amino acid sequence of SEQ
ID NO:8 have at least 80% sequence identity based on the Clustal
alignment method, or (b) the complement of the nucleotide
sequence.
2. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:8 have
at least 85% sequence identity based on the Clustal alignment
method.
3. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:8 have
at least 90% sequence identity based on the Clustal alignment
method.
4. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:8 have
at least 95% sequence identity based on the Clustal alignment
method.
5. The polynucleotide of claim 1, wherein the nucleotide sequence
comprises the nucleotide sequence of SEQ ID NO:7.
6. The polynucleotide of claim 1, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO:8.
7. A chimeric gene recombinant DNA construct comprising the
polynucleotide of claim 1 operably linked to a regulatory
sequence.
8. A method for transforming a cell comprising transforming a plant
cell with the polynucleotide of claim 1.
9. A cell comprising the chimeric gene recombinant DNA construct of
claim 7.
10. A method for producing a plant comprising transforming a plant
cell with the polynucleotide of claim 1 and regenerating a plant
from the transformed plant cell.
11. A plant comprising the chimeric gene recombinant DNA construct
of claim 7.
12. A seed comprising the chimeric gene recombinant DNA construct
of claim 7.
Description
[0001] This application is a divisional of U.S. Application Ser.
No. 09/465,559, filed Dec. 17, 1999, which claims the benefit of
U.S. Provisional Application No. 60/112,735, filed Dec. 18,
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 folate biosynthetic enzymes in plants and
seeds.
BACKGROUND OF THE INVENTION
[0003] Tetrahydrofolic acid and its derivatives
N.sup.5,N.sup.10-methylene- tetrahydrofolate,
N.sup.5,N.sup.10-methenyltetrahydrofolate,
N.sup.10-formyltetrahydrofolate and N.sup.5-methyl-tetrahydrofolate
are biologically active forms of folic acid. The tetrahydrofolates
are coenzymes that function in a variety of enzyme catalyzed
reactions as specialized cosubstrates for one-carbon metabolism.
For example, tetrahydrofolate plays an important role in nucleic
acid biosynthesis by serving as the immediate source of one-carbon
units in purine and pyrimidine biosynthesis. The cellular
tetrahydrofolate coenzyme pool must be maintained at specific
levels to assure one-carbon metabolism operates efficiently. Thus,
one of the most important reactions of the cell is the reduction of
dihydrofolate to tetrahydrofolate by dihydrofolate reductase. The
importance of this reaction in mammalian cells can be shown by the
fact that methorexate, a very effective chemotherapy drug, is a
potent inhibitor of dihydrofolate reductase (Zubay, G. (1983)
Biochemistry, Addison-Wesley Publishing Co. Reading, Mass.). Other
enzymes involved in the folic acid biosynthetic pathway to maintain
the tetrahydrofolate coenzyme pool are tetrahydrofolypolyglutamate
synthase, dihydropteroate synthase and dihydroneopterin
aldolase.
[0004] There is a great deal of interest in identifying the genes
that encode proteins required for tetrahydrofolate biosynthesis in
plants. These genes may be used in plant cells to alter the
tetrahydrofolate coenzyme pool concentration and modulate
one-carbon metabolism. Accordingly, the availability of nucleic
acid sequences encoding all or a portion of the
tetrahydrofolypolyglutamate synthase, dihydropteroate synthase and
dihydroneopterin aldolase enzymes would facilitate studies to
better understand one-carbon metabolism in plants, provide genetic
tools to one-carbon metabolism. The tetrahydrofolate biosynthetic
enzymes may also provide targets to facilitate design and/or
identification of inhibitors of cell cycle that may be useful as
herbicides.
SUMMARY OF THE INVENTION
[0005] The present invention relates to isolated polynucleotides
comprising a nucleotide sequence encoding a polypeptide of at least
131 amino acids that has at least 80% identity based on the Clustal
method of alignment when compared to a polypeptide selected from
the group consisting of a corn dihydroneopterin aldolase
polypeptide of SEQ ID NO:2, a soybean dihydroneopterin aldolase
polypeptide of SEQ ID NO:4 and a wheat dihydroneopterin aldolase
polypeptide of SEQ ID NO:6. The present invention also relates to
an isolated polynucleotide comprising the complement of the
nucleotide sequences described above.
[0006] The present invention relates to isolated polynucleotides
comprising a nucleotide sequence encoding a polypeptide of at least
75 amino acids that has at least 80% identity based on the Clustal
method of alignment when compared to a polypeptide selected from
the group consisting of a corn dihydropteroate
synthase/dihydropteroate pyrophosphorylase polypeptide of SEQ ID
NO:8, a rice dihydropteroate synthase/dihydropteroate
pyrophosphorylase polypeptide of SEQ ID NO: 10 and a soybean
dihydropteroate synthase/dihydropteroate pyrophosphorylase
polypeptide of SEQ ID NO: 12. The present invention also relates to
an isolated polynucleotide comprising the complement of the
nucleotide sequences described above.
[0007] The present invention relates to isolated polynucleotides
comprising a nucleotide sequence encoding a polypeptide of at least
553 amino acids that has at least 80% identity based on the Clustal
method of alignment when compared to a corn
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
polypeptide of SEQ ID NO: 14. The present invention also relates to
an isolated polynucleotide comprising the complement of the
nucleotide sequences described above.
[0008] The present invention relates to isolated polynucleotides
comprising a nucleotide sequence encoding a polypeptide of at least
133 amino acids that has at least 80% identity based on the Clustal
method of alignment when compared to a polypeptide selected from
the group consisting of a corn tetrahydrofolypolyglutamate
synthase/folylpolyglutam- ate synthase polypeptide of SEQ ID NO: 16
and a soybean tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase polypeptide of SEQ ID NO: 18.
The present invention also relates to an isolated polynucleotide
comprising the complement of the nucleotide sequences described
above.
[0009] It is preferred that the isolated polynucleotides of the
claimed invention consists of a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29 and 31 that codes for the polypeptide
selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32. The present
invention also relates to an isolated polynucleotide comprising a
nucleotide sequences of at least one of 60 (preferably at least one
of 40, most preferably at least one of 30) contiguous nucleotides
derived from a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31 and the complement of such nucleotide
sequences.
[0010] The present invention relates to a chimeric gene comprising
an isolated polynucleotide of the present invention operably linked
to suitable regulatory sequences.
[0011] The present invention relates to an isolated host cell
comprising a chimeric gene of the present invention or an isolated
polynucleotide of the present invention. The host cell may be
eukaryotic, such as a yeast or a plant cell, or prokaryotic, such
as a bacterial cell. The present invention also relates to a virus,
preferably a baculovirus, comprising an isolated polynucleotide of
the present invention or a chimeric gene of the present
invention.
[0012] The present invention relates to a process for producing an
isolated host cell comprising a chimeric gene of the present
invention or an isolated polynucleotide of the present invention,
the process comprising either transforming or transfecting an
isolated compatible host cell with a chimeric gene or isolated
polynucleotide of the present invention.
[0013] The present invention relates to a dihydroneopterin aldolase
polypeptide of at least 131 amino acids comprising at least 80%
homology based on the Clustal method of alignment compared to a
polypeptide selected from the group consisting of SEQ ID NOs:2, 4
and 6.
[0014] The present invention relates to a dihydropteroate
synthase/dihydropteroate pyrophosphorylase polypeptide of at least
75 amino acids comprising at least 80% homology based on the
Clustal method of alignment compared to a polypeptide selected from
the group consisting of SEQ ID NOs:8, 10 and 12.
[0015] The present invention relates to a
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
polypeptide of at least 553 amino acids comprising at least 80%
homology based on the Clustal method of alignment compared to a
polypeptide of SEQ ID NO: 14.
[0016] The present invention relates to a
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
polypeptide of at least 133 amino acids comprising at least 80%
homology based on the Clustal method of alignment compared to a
polypeptide selected from the group consisting of SEQ ID NOs:16 and
18.
[0017] The present invention relates to a method of selecting an
isolated polynucleotide that affects the level of expression of a
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase polypeptide in a host cell,
preferably a plant cell, the method comprising the steps of: (a)
constructing an isolated polynucleotide of the present invention or
an isolated chimeric gene of the present invention; (b) introducing
the isolated polynucleotide or the isolated chimeric gene into a
host cell; (c) measuring the level a dihydroneopterin aldolase,
dihydropteroate synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutam- ate synthase
polypeptide in the host cell containing the isolated
polynucleotide; and (d) comparing the level of a dihydroneopterin
aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase polypeptide in the host cell
containing the isolated polynucleotide with the level of a
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase polypeptide in the host cell
that does not contain the isolated polynucleotide.
[0018] The present invention relates to a method of obtaining a
nucleic acid fragment encoding a substantial portion of a
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase polypeptide gene, preferably a
plant dihydroneopterin aldolase, dihydropteroate
synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
polypeptide gene, comprising the steps of: synthesizing an
oligonucleotide primer comprising a nucleotide sequence of at least
one of 60 (preferably at least one of 40, most preferably at least
one of 30) contiguous nucleotides derived from a nucleotide
sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement
of such nucleotide sequences; and amplifying a nucleic acid
fragment (preferably a cDNA inserted in a cloning vector) using the
oligonucleotide primer. The amplified nucleic acid fragment
preferably will encode a portion of a dihydroneopterin aldolase,
dihydropteroate synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
amino acid sequence.
[0019] The present invention also relates to a method of obtaining
a nucleic acid fragment encoding all or a substantial portion of
the amino acid sequence encoding a dihydroneopterin aldolase,
dihydropteroate synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
polypeptide comprising the steps of: probing a cDNA or genomic
library with an isolated polynucleotide of the present invention;
identifying a DNA clone that hybridizes with an isolated
polynucleotide of the present invention; isolating the identified
DNA clone; and sequencing the cDNA or genomic fragment that
comprises the isolated DNA clone.
[0020] A further embodiment of the instant invention is a method
for evaluating at least one compound for its ability to inhibit the
activity of a dihydroneopterin aldolase, dihydropteroate
synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutam- ate synthase,
the method comprising the steps of: (a) transforming a host cell
with a chimeric gene comprising a nucleic acid fragment encoding a
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutam- ate synthase, 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
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase in the transformed host cell;
(c) optionally purifying the dihydroneopterin aldolase,
dihydropteroate synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
expressed by the transformed host cell; (d) treating the
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase with a compound to be tested;
and (e) comparing the activity of the dihydroneopterin aldolase,
dihydropteroate synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase
that has been treated with a test compound to the activity of an
untreated dihydroneopterin aldolase, dihydropteroate
synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutam- ate synthase,
thereby selecting compounds with potential for inhibitory
activity.
[0021] The present invention relates to a composition, such as a
hybridization mixture, comprising an isolated polynucleotide or
polypeptide of the present invention.
[0022] The present invention relates to an isolated polynucleotide
of the present invention comprising at least one of 30 contiguous
nucleotides derived from a nucleic acid sequence selected from the
group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29 and 3 1.
[0023] The present invention relates to an expression cassette
comprising an isolated polynucleotide of the present invention
operably linked to a promoter.
[0024] The present invention relates to a method for positive
selection of a transformed cell comprising: (a) transforming a host
cell with the chimeric gene of the present invention or an
expression cassette of the present invention; and (b) growing the
transformed host cell, preferably plant cell, such as a monocot or
a dicot, under conditions which allow expression of the
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophos-phorylase or tetrahydrofolypolyglutamat- e
synthase/folylpolyglutamate synthase polynucleotide in an amount
sufficient to complement a null mutant and folic acid biosynthesis
auxotroph to provide a positive selection means.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
[0025] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0026] Table 1 lists the polypeptides that are described herein,
the designation of the cDNA clones that comprise the nucleic acid
fragments encoding polypeptides representing all or a substantial
portion of these polypeptides, and the corresponding identifier
(SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also
identifies the cDNA clones as individual ESTs ("EST"), the
sequences of the entire cDNA inserts comprising the indicated cDNA
clones ("FIS"), contigs assembled from two or more ESTs ("Contig"),
contigs assembled from an FIS and one or more ESTs ("Contig*"), or
sequences encoding the entire protein derived from an FIS, a
contig, or an FIS and PCR ("CGS"). Nucleotide sequences, SEQ ID
NOs:1, 3, 5, 7, 9, 13 and 17 and amino acid sequences SEQ ID NOs:2,
4, 6, 8, 10, 14, 16 and 18 were determined by further sequence
analysis of cDNA clones encoding the amino acid sequences set forth
in SEQ ID NOs:20, 22, 24, 26, 28, 30 and 32. Nucleotide SEQ ID
NOs:19, 21, 23, 25, 27, 29 and 31 and amino acid SEQ ID 5 NOs:20,
22, 24, 26, 28, 30 and 32 were presented in a U.S. Provisional
Application No. 60/112,735, filed Dec. 18, 1998.
[0027] 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 Folate Biosynthetic Enzymes SEQ ID NO: (Nucleo- (Amino
Protein Clone Designation tide) Acid) Dihydroneopterin
cco1n.pk075.j3 (FIS) 1 2 aldolase Dihydroneopterin sdp3c.pk002.o16
(FIS) 3 4 aldolase Dihydroneopterin wdk1c.pk013.k22 (FIS) 5 6
aldolase Dihydropteroate cr1n.pk0057.a10 (FIS) 7 8 synthase/
Dihydropteroate pyrophosphorylase Dihydropteroate r10n.pk0041.c3
(FIS) 9 10 synthase/ Dihydropteroate pyrophosphorylase
Dihydropteroate sdp4c.pk034.b11 (EST) 11 12 synthase/
Dihydropteroate pyrophosphorylase Tetrahydrofolylpoly-
cco1n.pk061.l16 (FIS) 13 14 glutamate synthase/ Folylpolyglutamate
synthase Tetrahydrofolylpoly- p0006.cbysj94r (EST) 15 16 glutamate
synthase/ Folylpolyglutamate synthase Tetrahydrofolylpoly-
s12.pk123.k13 (EST) 17 18 glutamate synthase/ Folylpolyglutamate
synthase Dihydroneopterin cco1n.pk075.j3 (EST) 19 20 aldolase
Dihydroneopterin sdp3c.pk002.o16 (EST) 21 22 aldolase
Dihydroneopterin wdk1c.pk013.k22 (FIS) 23 24 aldolase
Dihydropteroate cr1n.pk0057.a10 (Contig) 25 26 synthase/
Dihydropteroate pyrophosphorylase Dihydropteroate r10n.pk0041.c3
(EST) 27 28 synthase/ Dihydropteroate pyrophosphorylase
Dihydropteroate sdp4c.pk034.b11 (EST) 29 30 synthase/
Dihydropteroate pyrophosphorylase Tetrahydrofolylpoly-
s12.pk123.k13 (EST) 31 32 glutamate synthase/ Folylpolyglutamate
synthase
[0028] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Res. 13:3021-3030 (1985) and in the
Biochemical J. 5 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn.1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0029] In the context of this disclosure, a number of terms shall
be utilized. As used herein, a "polynucleotide" is a nucleotide
sequence such as a nucleic acid fragment. A polynucleotide may be a
polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. An isolated polynucleotide of the present
invention may include at least one of 60 contiguous nucleotides,
preferably at least one of 40 contiguous nucleotides, most
preferably one of at least 30 contiguous nucleotides derived from
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31 or the complement of such sequences.
[0030] 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.
[0031] Substantially similar nucleic acid fragments may be selected
by screening nucleic acid fragments representing subfragments or
modifications of the nucleic acid fragments of the instant
invention, wherein one or more nucleotides are substituted, deleted
and/or inserted, for their ability to affect the level of the
polypeptide encoded by the unmodified nucleic acid fragment in a
plant or plant cell. For example, a substantially similar nucleic
acid fragment representing at least one of 30 contiguous
nucleotides derived from the instant nucleic acid fragment can be
constructed and introduced into a plant or plant cell. The level of
the polypeptide encoded by the unmodified nucleic acid fragment
present in a plant or plant cell exposed to the substantially
similar nucleic fragment can then be compared to the level of the
polypeptide in a plant or plant cell that is not exposed to the
substantially similar nucleic acid fragment.
[0032] 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.
Consequently, an isolated polynucleotide comprising a nucleotide
sequence of at least one of 60 (preferably at least one of 40, most
preferably at least one of 30) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and
the complement of such nucleotide sequences may be used in methods
of selecting an isolated polynucleotide that affects the expression
of a polypeptide (dihydroneopterin aldolase, dihydropteroate
synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase)
in a host cell. A method of selecting an isolated polynucleotide
that affects the level of expression of a polypeptide in a host
cell (eukaryotic, such as plant or yeast, prokaryotic such as
bacterial, or viral) may comprise the steps of: constructing an
isolated polynucleotide of the present invention or an isolated
chimeric gene of the present invention; introducing the isolated
polynucleotide or the isolated chimeric gene into a host cell;
measuring the level a polypeptide in the host cell containing the
isolated polynucleotide; and comparing the level of a polypeptide
in the host cell containing the isolated polynucleotide with the
level of a polypeptide in a host cell that does not contain the
isolated polynucleotide.
[0033] 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 6X SSC, 0.5% SDS at room temperature
for 15 min, then repeated with 2X SSC, 0.5% SDS at 45.degree. C.
for 30 min, and then repeated twice with 0.2X 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 0.2X SSC, 0.5% SDS was increased to 60.degree.
C. Another preferred set of highly stringent conditions uses two
final washes in 0.1X SSC, 0.1% SDS at 65.degree. C.
[0034] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Suitable nucleic acid fragments
(isolated polynucleotides of the present invention) encode
polypeptides that are at least about 70% identical, preferably at
least about 80% identical to the amino acid sequences reported
herein. Preferred nucleic acid fragments encode amino acid
sequences that are about 85% identical to the amino acid sequences
reported herein. More preferred nucleic acid fragments encode amino
acid sequences that are at least about 90% identical to the amino
acid sequences reported herein. Most preferred are nucleic acid
fragments that encode amino acid sequences that are at least about
95% identical to the amino acid sequences reported herein. Suitable
nucleic acid fragments not only have the above homologies but
typically encode a polypeptide having at least about 50 amino
acids, preferably at least about 100 amino acids, more preferably
at least about 150 amino acids, still more preferably at least
about 200 amino acids, and most preferably at least about 250 amino
acids. Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the 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.
[0035] 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.
[0036] "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.
[0037] "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.
[0038] "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.
[0039] "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.
[0040] "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.
[0041] 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) Mol. Biotechnol.
3:225-236).
[0042] 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.
[0043] "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.
[0044] 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.
[0045] 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).
[0046] "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.
[0047] "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.
[0048] 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).
[0049] "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. Pa. No. 4,945,050, incorporated herein by
reference).
[0050] 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").
[0051] Nucleic acid fragments encoding at least a portion of
several folate biosynthetic enzymes 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).
[0052] For example, genes encoding other dihydroneopterin aldolase,
dihydropteroate synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase,
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.
[0053] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA
85:8998-9002) to generate cDNAs by using PCR to amplify copies of
the region between a single point in the transcript and the 3' or
5' end. Primers oriented in the 3' and 5' directions can be
designed from the instant sequences. Using commercially available
3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments
can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA
86:5673-5677; Loh et al. (1989) Science 243:217-220). Products
generated by the 3' and 5' RACE procedures can be combined to
generate full-length cDNAs (Frohman and Martin (1989) Techniques
1:165). Consequently, a polynucleotide comprising a nucleotide
sequence of at least one of 60 (preferably one of at least 40, most
preferably one of at least 30) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and
the complement of such nucleotide sequences may be used in such
methods to obtain a nucleic acid fragment encoding a substantial
portion of an amino acid sequence of a polypeptide. The present
invention relates to a method of obtaining a nucleic acid fragment
encoding a substantial portion of a polypeptide of a gene (such as
dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate
pyrophosphorylase or tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase) preferably a substantial
portion of a plant polypeptide of a gene, comprising the steps of:
synthesizing an oligonucleotide primer comprising a nucleotide
sequence of at least one of 60 (preferably at least one of 40, most
preferably at least one of 30) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and
the complement of such nucleotide sequences; and amplifying a
nucleic acid fragment (preferably a cDNA inserted in a cloning
vector) using the oligonucleotide primer. The amplified nucleic
acid fragment preferably will encode a portion of a polypeptide
(dihydroneopterin aldolase, dihydropteroate
synthase/dihydropteroate pyrophosphorylase or
tetrahydrofolypolyglutamate synthase/folylpolyglutam- ate
synthase).
[0054] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).
[0055] 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
folic acid in those cells.
[0056] 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. 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.
[0057] Plasmid vectors comprising the isolated polynucleotide (or
chimeric gene) may be constructed. The choice of plasmid vector is
dependent upon the method that will be used to transform host
plants. The skilled artisan is well aware of the genetic elements
that must be present on the plasmid vector in order to successfully
transform, select and propagate host cells containing the 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.
[0058] 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
directing the coding sequence to encode the instant polypeptides
with appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys. 00:1627-1632) with
or without removing targeting sequences that are already present.
While the references cited give examples of each of these, the list
is not exhaustive and more targeting signals of use may be
discovered in the future.
[0059] 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.
[0060] 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.
[0061] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppression technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense genes may require the use of different 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. 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.
[0062] 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
folate biosynthetic enzyme. An example of a vector for high level
expression of the instant polypeptides in a bacterial host is
provided (Example 8).
[0063] 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
folic acid biosynthesis. Accordingly, inhibition of the activity of
one or more of the enzymes described herein could lead to
inhibition of plant growth. Thus, the instant polypeptides could be
appropriate for new herbicide discovery and design.
[0064] 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).
[0065] 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 4:37-41. 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.
[0066] 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).
[0067] 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 Res. 5:13-20), improvements in sensitivity may allow
performance of FISH mapping using shorter probes.
[0068] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0069] Loss of function mutant phenotypes may be identified for the
instant cDNA clones either by targeted gene disruption protocols or
by identifying specific mutants for these genes contained in a
maize population carrying mutations in all possible genes
(Ballinger and Benzer (1989) Proc. Natl Acad. Sci USA 86:9402-9406;
Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen
et al. (1995) Plant Cell 7:75-84). The latter approach may be
accomplished in two ways. First, short segments of the instant
nucleic acid fragments may be used in polymerase chain reaction
protocols in conjunction with a mutation tag sequence primer on
DNAs prepared from a population of plants in which Mutator
transposons or some other mutation-causing DNA element has been
introduced (see Bensen, supra). The amplification of a specific DNA
fragment with these primers indicates the insertion of the mutation
tag element in or near the plant gene encoding 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
[0070] 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
[0071] 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 cco1n Corn Cob of 67 Day Old Plants Grown in
cco1n.pk075.j3 Green House* cco1n.pk061.l16 cr1n Corn Root From 7
Day Old Seedlings* cr1n.pk0057.a10 p0006 Corn Young Shoot
p0006.cbysj94r r10n Rice 15 Day Old Leaf* r10n.pk0041.c3 sdp4c
Soybean Developing Pods (10-12 mm) sdp4c.pk034.b11 sdp3c Soybean
Developing Pods (8-9 mm) sdp3c.pk002.o16 s12 Soybean Two-Week-Old
Developing s12.pk123.k13 Seedlings Treated With 2.5 ppm chlorimuron
wdk1c Wheat Developing Kernel, 3 Days After wdk1c.pk013.k22
Anthesis *These libraries were normalized essentially as described
in U.S. Pat. No. 5,482,845, incorporated herein by reference.
[0072] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651-1656). The resulting ESTs are analyzed using a
Perkin Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0073] cDNA clones encoding folate biosynthetic enzymes 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) Nat. Genet. 3:266-272) provided by the NCBI. For
convenience, the P-value (probability) of observing a match of a
cDNA sequence to a sequence contained in the searched databases
merely by chance as calculated by BLAST are reported herein as
"pLog" values, which represent the negative of the logarithm of the
reported P-value. Accordingly, the greater the pLog value, the
greater the likelihood that the cDNA sequence and the BLAST "hit"
represent homologous proteins.
Example 3
Characterization of cDNA Clones Encoding Dihydroneopterin
Aldolase
[0074] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to dihydroneopterin aldolase from Bacillus subtilis (NCBI
Identifier No. gi 141435). 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"), contigs
assembled from two or more ESTs ("Contig"), contigs assembled from
an FIS and one or more ESTs ("Contig*"), or sequences encoding the
entire protein derived from an FIS, a contig, or an FIS and PCR
("CGS"):
3TABLE 3 BLAST Results for Sequences Encoding Polypeptides
Homologous to Bacillus subtilis Dihydroneopterin Aldolase Clone
Status BLAST pLog Score to (gi 141435) cco1n.pk075.j3 (FIS) 21.70
sdp3c.pk002.o16 (FIS) 20.70 wdk1c.pk013.k22 (FIS) 22.04
[0075] 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
and 6 and the Bacillus subtilis sequence.
4TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Bacillus subtilis Dihydroneopterin Aldolase SEQ ID
NO. Percent Identity to 2 41% (gi 141435) 4 38% (gi 141435) 6 41%
(gi 141435)
[0076] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the 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 dihydroneopterin
aldolase. These sequences represent the first corn, soybean and
wheat sequences encoding dihydroneopterin aldolase.
Example 4
Characterization of cDNA Clones Encoding Dihydropteroate
Synthase/Dihydropteroate Pyrophosphorylase
[0077] The BLASTX search using the EST sequences from clones listed
in Table 5 revealed similarity of the polypeptides encoded by the
cDNAs to dihydropteroate synthase/dihydropteroate pyrophosphorylase
from Pisum sativum (NCBI Identifier No. gi 1934972) and Arabidopsis
thaliana (NCBI Identifier No. gi 4938476). 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"),
contigs assembled from two or more ESTs ("Contig"), contigs
assembled from an FIS and one or more ESTs ("Contig*"), or
sequences encoding the entire protein derived from an FIS, a
contig, or an FIS and PCR ("CGS"):
5TABLE 5 BLAST Results for Sequences Encoding Polypeptides
Homologous to Pisum sativum and Arabidosis thaliana Dihydroteroate
Synthase/Dihydroteroate Pyrophoshorylase Clone Status BLAST pLog
Score cr1n.pk0057.a10 FIS 157.00 (gi 1934972) r10n.pk0041.c3 FIS
20.52 (gi 4938476) sdp4c.pk034.b11 FIS 57.30 (gi 1934972)
[0078] The data in Table 6 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs:8, 10
and 12 and the Pisum sativum and Arabidopsis thaliana
sequences.
6TABLE 6 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Pisum sativum and Arabidopsis thaliana
Dihydropteroate Synthase/Dihydroteroate Pyrophosphorylase SEQ ID
NO. Percent Identity to 8 58% (gi 1934972) 10 59% (gi 4938476) 12
71% (gi 1934972)
[0079] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the 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 dihydropteroate
synthase/dihydropteroate pyrophosphorylase. These sequences
represent the first corn, rice and soybean sequences encoding
dihydropteroate synthase/dihydropteroate pyrophosphorylase.
Example 5
Characterization of cDNA Clones Encoding
Tetrahydrofolypolyglutamate Synthase/Folylpolyglutamate
Synthase
[0080] The BLASTX search using the EST sequences from clones listed
in Table 7 revealed similarity of the polypeptides encoded by the
cDNAs to tetrahydrofolypolyglutamate synthase/folylpolyglutamate
synthase from Arabidopsis thaliana (NCBI Identifier No. gi
6143861), Homo sapiens (NCBI Identifier No. gi 4826728) and Homo
sapiens (NCBI Identifier No. gi 1709377). 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"),
contigs assembled from two or more ESTs ("Contig"), contigs
assembled from an FIS and one or more ESTs ("Contig*"), or
sequences encoding the entire protein derived from an FIS, a
contig, or an FIS and PCR ("CGS"):
7TABLE 7 BLAST Results for Sequences Encoding Polypeptides
Homologous to Arabidopsis thaliana and Homo sapiens
Tetrahydrofolypolyglutamate Synthase/Folylpolyglutamate Synthase
Clone Status BLAST pLog Score cco1n.pk061.l16 FIS 116.00 (gi
6143861) p0006.cbysj94r EST 7.52 (gi 1709377) s12.pk123.k13 EST
31.70 (gi 6143861)
[0081] The data in Table 8 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs: 14,
16 and 18 and the Arabidopsis thaliana and Homo sapiens
sequences.
8TABLE 8 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Arabidopsis thaliana and Homo sapiens
Tetrahydrofolypolyglutamate Synthase/Folylpolyglutamate Synthase
SEQ ID NO. Percent Identity to 14 46% (gi 6143861) 16 21% (gi
1709377) 18 37% (gi 6143861)
[0082] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the 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
tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase.
These sequences represent the first corn, rice and soybean
sequences encoding tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase.
Example 6
Expression of Chimeric Genes in Monocot Cells
[0083] 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 pML 103. Plasmid
pML 103 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.
[0084] 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.
[0085] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst
Ag, Frankfurt, Germany) may be used in transformation experiments
in order to provide for a selectable marker. This plasmid contains
the Pat gene (see European Patent Publication 0 242 236) which
encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT
confers resistance to herbicidal glutamine synthetase inhibitors
such as phosphinothricin. The pat gene in p35S/Ac is under the
control of the 35S promoter from Cauliflower Mosaic Virus (Odell et
al. (1985) Nature 313:810-812) and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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 7
Expression of Chimeric Genes in Dicot Cells
[0090] 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.
[0091] 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 pUC 18 vector carrying the seed expression cassette.
[0092] Soybean embryos may then be transformed with the expression
vector comprising sequences encoding the instant polypeptides. To
induce somatic embryos, cotyledons, 3-5 mm in length dissected from
surface sterilized, immature seeds of the soybean cultivar A2872,
can be cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0093] 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.
[0094] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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 8
Expression of Chimeric Genes in Microbial Cells
[0099] 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
pBT430.
[0100] 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 GTGTM 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 electro-competent 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.
[0101] 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 BL2 1 (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 9
Evaluating Compounds for Their Ability to Inhibit the Activity of
Folate Biosynthetic Enzymes
[0102] 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 8, 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.
[0103] 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.
[0104] 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
dihydroneopterin aldolase are presented by Hennig et al. 1998 Nat.
Struct. Biol. 5(5):357-362. Assays for dihydropteroate
synthase/dihydropteroate pyrophosphorylase are presented by Rebeile
et al. EMBO J. 16(5):947-957 and tetrahydrofolypolyglutamate
synthase/folylpolyglutamate synthase are presented by Shane et al.
1987 Biochemistry 26(2):504-512.
[0105] Various modifications of the invention in addition to those
shown and described herein will be apparent to those skilled in the
art from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
[0106] The disclosure of each reference set forth above is
incorporated herein by reference in its entirety.
Sequence CWU 1
1
32 1 658 DNA Zea mays 1 gcacgagggc ggcggctacg tggggtggcg acgacaagct
cattctgcgc ggccttcagt 60 tccatggctt ccacggtgtc ctgcaggagg
agaagacgtt gggacagaag ttcgtggttg 120 acatcgacgc ctggatagac
ctcgccgctg ccggcgagtc cgactgcatt gctgacaccg 180 tcagctacac
cgatatctac agcattgcaa aggatgttgt cgagggcacg ccacgcaacc 240
tcttggagtc ggtagctcac tcgatcgcag aggccacgct gctcaagttc cctcagatct
300 ccgcagtccg agtgaaggtt ggcaagcctc acgtcgcggt gcgaggcgtt
ctggactacc 360 tgggcgtgga gataacgagg cacagaaaga aagaatgaga
tgctgtacac atgtggtgat 420 ggggagccag ttcaatgctg atggcactgc
ggccataacc ataatccacg cacgcttgtt 480 gcttgttggc aactaggcat
atccctttca cctctgaact gttggaatat cgggaatctg 540 ttcccctagt
tgctttatta cgaattcaga tcatatctgg ctagtaagat caaccttctt 600
ctggtctgta acaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 658
2 131 PRT Zea mays 2 Thr Arg Ala Ala Ala Thr Trp Gly Gly Asp Asp
Lys Leu Ile Leu Arg 1 5 10 15 Gly Leu Gln Phe His Gly Phe His Gly
Val Leu Gln Glu Glu Lys Thr 20 25 30 Leu Gly Gln Lys Phe Val Val
Asp Ile Asp Ala Trp Ile Asp Leu Ala 35 40 45 Ala Ala Gly Glu Ser
Asp Cys Ile Ala Asp Thr Val Ser Tyr Thr Asp 50 55 60 Ile Tyr Ser
Ile Ala Lys Asp Val Val Glu Gly Thr Pro Arg Asn Leu 65 70 75 80 Leu
Glu Ser Val Ala His Ser Ile Ala Glu Ala Thr Leu Leu Lys Phe 85 90
95 Pro Gln Ile Ser Ala Val Arg Val Lys Val Gly Lys Pro His Val Ala
100 105 110 Val Arg Gly Val Leu Asp Tyr Leu Gly Val Glu Ile Thr Arg
His Arg 115 120 125 Lys Lys Glu 130 3 705 DNA Glycine max 3
gcacgagcgg agaggcgagg gagtgaggga ctagcacaga aagatattgt ttggtgtacg
60 gtggtgagtg tcgacgctgc cactctcgcc tgtgtctgtg ataaatggaa
tctgatgcac 120 cgacatgggg agacaaactc atgttgaggg gattgtcatt
ccatggtttt catggagcaa 180 agcctgaaga aaggacactg ggccagaagt
tcttcataga tatagatgct tggatggatc 240 tcaaagcagc tggcaaatct
gatcacttat cagattctgt tagttacaca gaaatatatg 300 atatagctaa
ggatgttctt gaagggtcac ctcacaatct tctggagtca gtggcccaaa 360
aaattgcaat cactactctt acaaatcata aagaaatatc tgctgtccga gtgaaggttg
420 gaaagcctca tgtggcagtt cggggtccag ttgattactt aggcgttgag
attcttagac 480 gcagaagcga cttgtcaggc tagaaatttc atatttattg
ctgcacaatt tttatatttt 540 cacattccac ttgatacaaa agtaatgtaa
ctctttcctt catgccccat tagtcttttc 600 tctcttaagc aatcttgcta
atgaaattaa aagatcaaag ttaggcatat taaaggaact 660 atacaattaa
tttggattct ccaaaacaaa aaaaaaaaaa aaaaa 705 4 132 PRT Glycine max 4
Met Glu Ser Asp Ala Pro Thr Trp Gly Asp Lys Leu Met Leu Arg Gly 1 5
10 15 Leu Ser Phe His Gly Phe His Gly Ala Lys Pro Glu Glu Arg Thr
Leu 20 25 30 Gly Gln Lys Phe Phe Ile Asp Ile Asp Ala Trp Met Asp
Leu Lys Ala 35 40 45 Ala Gly Lys Ser Asp His Leu Ser Asp Ser Val
Ser Tyr Thr Glu Ile 50 55 60 Tyr Asp Ile Ala Lys Asp Val Leu Glu
Gly Ser Pro His Asn Leu Leu 65 70 75 80 Glu Ser Val Ala Gln Lys Ile
Ala Ile Thr Thr Leu Thr Asn His Lys 85 90 95 Glu Ile Ser Ala Val
Arg Val Lys Val Gly Lys Pro His Val Ala Val 100 105 110 Arg Gly Pro
Val Asp Tyr Leu Gly Val Glu Ile Leu Arg Arg Arg Ser 115 120 125 Asp
Leu Ser Gly 130 5 759 DNA Triticum aestivum 5 gcacgagcca ggttccactc
cacccaccca cctgcgccgc cagctctaaa ggaggcggcg 60 tcggccggcg
ggcgagcgca cgcccaggcc caatcgatcg atcccagctc tagaggggag 120
ggagcaacca tggcggggga cggggaggac gaggtgccgg cgatgggcgg agacaagctg
180 atcctgcggg ggctgcagtt ccacggcttc cacggcgtga agcaggagga
gaagaagctg 240 ggccagaagt tcgtggtcga cgtggacgcc tggatggacc
tcgccgccgc cggggactcc 300 gacgacatcg cccacaccgt cagctacacc
gacatctaca ggatagccaa gggcgtggtg 360 gaaggcccgt cgcggaacct
cctggagtcg gtggcgcagt cgatcgccgg caccacgctg 420 ctcgagtttc
cccagatctc cgccgtccgg gtgaaggtcg ggaagcccca cgtcgcggtg 480
cagggcgtcg tcgactacct cggggtggag atactgagga ggcgcagaga ggcatgagca
540 caagaaccgg agtacctcat atgagaagcc tgaacagagt tgatctcagt
tgagcccatc 600 gatccctgtg tcttatatct atcaatctat gtatgtatgg
acatgatgtt tgtctgcgct 660 caataatttc tgaattggga atcatgttct
tgccaaaaaa aaaaaaaaaa aaaaaaaaaa 720 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaa 759 6 178 PRT Triticum aestivum 6 Ala Arg Ala
Arg Phe His Ser Thr His Pro Pro Ala Pro Pro Ala Leu 1 5 10 15 Lys
Glu Ala Ala Ser Ala Gly Gly Arg Ala His Ala Gln Ala Gln Ser 20 25
30 Ile Asp Pro Ser Ser Arg Gly Glu Gly Ala Thr Met Ala Gly Asp Gly
35 40 45 Glu Asp Glu Val Pro Ala Met Gly Gly Asp Lys Leu Ile Leu
Arg Gly 50 55 60 Leu Gln Phe His Gly Phe His Gly Val Lys Gln Glu
Glu Lys Lys Leu 65 70 75 80 Gly Gln Lys Phe Val Val Asp Val Asp Ala
Trp Met Asp Leu Ala Ala 85 90 95 Ala Gly Asp Ser Asp Asp Ile Ala
His Thr Val Ser Tyr Thr Asp Ile 100 105 110 Tyr Arg Ile Ala Lys Gly
Val Val Glu Gly Pro Ser Arg Asn Leu Leu 115 120 125 Glu Ser Val Ala
Gln Ser Ile Ala Gly Thr Thr Leu Leu Glu Phe Pro 130 135 140 Gln Ile
Ser Ala Val Arg Val Lys Val Gly Lys Pro His Val Ala Val 145 150 155
160 Gln Gly Val Val Asp Tyr Leu Gly Val Glu Ile Leu Arg Arg Arg Arg
165 170 175 Glu Ala 7 1824 DNA Zea mays 7 gcacgagcct cgaacgaggg
ccgtacctag cgcctctgtc cttcgtcggc cgtcgcactg 60 tgctcccgtc
cgcctccggc ctccgccaac ccgcgtccgc ccacgactag gcggctctgg 120
gcaggtcctt ccacaaagat gtgaaggatt aaagctcatg tgaaagattc taagactaca
180 attggtatca agcggttgct ttcttatttc tcatacgctc aaccatgctc
ctgcatgcta 240 aggattcagt taggaagatg cattcagttg ctaagaacta
ctttgtgtct gatcttactc 300 atcctccaag atccttgaac agagcttcca
gacatgttgt tccattcaag acccgtttct 360 ttacgcattg ctcacttgag
agccgttcag ttgaccaaga gattgtgatt gctatgggaa 420 gcaatgtagg
cgatagagtc agtacattca acagggcatt gcagctgatg aaaagctctg 480
acgtgaacat cactaggcat gcctgtctct atgagaccgc ccctgcttat ttgactgatc
540 agccgcggtt tcttaactct gccattcggg gcacaactag gctcaggcca
catgagcttc 600 ttaaactgct aaaggaaatt gagaaggata ttggccgcac
tggcggaata aggtgcatct 660 agtgacaacg gtatcgaaac aagttggcac
tctctctcaa agtgtagtgg aggtttcttt 720 gagttatgga ataaccttgg
gggtgaatct ataattggaa cagaaagcat taaaagggta 780 ttacctgttg
gggatcgttt gttggattgg tgtgagagga ctcttgtcat gggggtcctt 840
aatttgacac cagacagctt tagtgatgga ggtaagtttc tagaagtggg agctgccatt
900 tctcaggcta agtcattaat ctcagaaggt gcagatatca ttgatattgg
tgctcaatct 960 accaggccct ttgcaaaaag attatctcca aatgaggagc
ttgagaggtt ggttcctgtt 1020 ctggatgaga ttacaaaaat ccctgagatg
gagggcaagt tactctcagt ggatacattc 1080 tatgcagaag ttgccagtga
agctgtgaaa agaggagctc acatgatcaa cgatgtatcc 1140 agtggacagc
ttgatccaat aattcttaaa gtggcagctg aacttggagt tccatatgtt 1200
gcaatgcaca tgaggggaga tccgtcaact atgcaaagcg aacaaaatgt tcactatgat
1260 aatgtctgca aggaagttgc tttggagcta tacacacagg tgagagaagc
agagttatct 1320 gggattccat tgtggaggct ggttcttgat cctggcattg
gcttctccaa gaaatctgaa 1380 cataaccttg aagtaattat gggattggaa
tccattagga gggagatggg taaaatgagt 1440 ataggtgctt cacatgtgcc
aatattactg ggaccttcaa ggaaaagctt tttgggtgaa 1500 atatgcaatc
gtgccaatcc agttgagaga gatgttgcta ctgttgcagc cgtgacagct 1560
gggattttga atggtgctaa cattgtaaga gtccataatg ctggatatgg tgtagacgcc
1620 gcaaaggttt gtgatgcatt gcgtaagcgt aagggaagtt gcagaaactg
aactatcgct 1680 ccagttttat acaagaaaaa agtgatgtcg aaaaatgtga
tttgtgaagt atcgttgttg 1740 taatgaacca gagataatgc tttttcttgt
gtcaccaagg aataaagtca agaagctgct 1800 actcaaaaaa aaaaaaaaaa aaaa
1824 8 481 PRT Zea mays 8 Met Leu Leu His Ala Lys Asp Ser Val Arg
Lys Met His Ser Val Ala 1 5 10 15 Lys Asn Tyr Phe Val Ser Asp Leu
Thr His Pro Pro Arg Ser Leu Asn 20 25 30 Arg Ala Ser Arg His Val
Val Pro Phe Lys Thr Arg Phe Phe Thr His 35 40 45 Cys Ser Leu Glu
Ser Arg Ser Val Asp Gln Glu Ile Val Ile Ala Met 50 55 60 Gly Ser
Asn Val Gly Asp Arg Val Ser Thr Phe Asn Arg Ala Leu Gln 65 70 75 80
Leu Met Lys Ser Ser Asp Val Asn Ile Thr Arg His Ala Cys Leu Tyr 85
90 95 Glu Thr Ala Pro Ala Tyr Leu Thr Asp Gln Pro Arg Phe Leu Asn
Ser 100 105 110 Ala Ile Arg Gly Thr Thr Arg Leu Arg Pro His Glu Leu
Leu Lys Leu 115 120 125 Leu Lys Glu Ile Glu Lys Asp Ile Gly Arg Thr
Gly Gly Ile Arg Cys 130 135 140 Thr Ser Asp Asn Gly Ile Glu Thr Ser
Trp His Ser Leu Ser Lys Cys 145 150 155 160 Ser Gly Gly Phe Phe Glu
Leu Trp Asn Asn Leu Gly Gly Glu Ser Ile 165 170 175 Ile Gly Thr Glu
Ser Ile Lys Arg Val Leu Pro Val Gly Asp Arg Leu 180 185 190 Leu Asp
Trp Cys Glu Arg Thr Leu Val Met Gly Val Leu Asn Leu Thr 195 200 205
Pro Asp Ser Phe Ser Asp Gly Gly Lys Phe Leu Glu Val Gly Ala Ala 210
215 220 Ile Ser Gln Ala Lys Ser Leu Ile Ser Glu Gly Ala Asp Ile Ile
Asp 225 230 235 240 Ile Gly Ala Gln Ser Thr Arg Pro Phe Ala Lys Arg
Leu Ser Pro Asn 245 250 255 Glu Glu Leu Glu Arg Leu Val Pro Val Leu
Asp Glu Ile Thr Lys Ile 260 265 270 Pro Glu Met Glu Gly Lys Leu Leu
Ser Val Asp Thr Phe Tyr Ala Glu 275 280 285 Val Ala Ser Glu Ala Val
Lys Arg Gly Ala His Met Ile Asn Asp Val 290 295 300 Ser Ser Gly Gln
Leu Asp Pro Ile Ile Leu Lys Val Ala Ala Glu Leu 305 310 315 320 Gly
Val Pro Tyr Val Ala Met His Met Arg Gly Asp Pro Ser Thr Met 325 330
335 Gln Ser Glu Gln Asn Val His Tyr Asp Asn Val Cys Lys Glu Val Ala
340 345 350 Leu Glu Leu Tyr Thr Gln Val Arg Glu Ala Glu Leu Ser Gly
Ile Pro 355 360 365 Leu Trp Arg Leu Val Leu Asp Pro Gly Ile Gly Phe
Ser Lys Lys Ser 370 375 380 Glu His Asn Leu Glu Val Ile Met Gly Leu
Glu Ser Ile Arg Arg Glu 385 390 395 400 Met Gly Lys Met Ser Ile Gly
Ala Ser His Val Pro Ile Leu Leu Gly 405 410 415 Pro Ser Arg Lys Ser
Phe Leu Gly Glu Ile Cys Asn Arg Ala Asn Pro 420 425 430 Val Glu Arg
Asp Val Ala Thr Val Ala Ala Val Thr Ala Gly Ile Leu 435 440 445 Asn
Gly Ala Asn Ile Val Arg Val His Asn Ala Gly Tyr Gly Val Asp 450 455
460 Ala Ala Lys Val Cys Asp Ala Leu Arg Lys Arg Lys Gly Ser Cys Arg
465 470 475 480 Asn 9 589 DNA Oryza sativa 9 gcacgagctt acagtttagg
tgcttcacat gtgccaattt tacttggacc ctcaaggaaa 60 agatttttag
gtgaaatatg caatcgtgtc aatcccactg agagagatgc tgctaccatg 120
gtcgttgcta ctgctgggat attgaatggt gctaatatag taagggtgca taatgttaaa
180 tatggcgtgg atactgcaaa ggtctctgat gcattgagca aaggcagaag
atgattatac 240 caccttcgga aaatagatca tactccagtt ttgtactaga
aaataatgat caataatagt 300 aactcggcca taatgttggc ttctcagata
ataccatagg gcgagtatca tcatagaaag 360 catgtgcaca caactgttat
gtgagcttga gatggaattt ttctttttgt cacatcattt 420 caataatctt
ctgaggtaac ggttatacag atctctagag ttttgacctt tcaggattca 480
caaattttct acaggtctga tttgtttgga actttgggcc ataacttgaa gttattctcc
540 atgtaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 589 10 75
PRT Oryza sativa 10 Ala Arg Ala Tyr Ser Leu Gly Ala Ser His Val Pro
Ile Leu Leu Gly 1 5 10 15 Pro Ser Arg Lys Arg Phe Leu Gly Glu Ile
Cys Asn Arg Val Asn Pro 20 25 30 Thr Glu Arg Asp Ala Ala Thr Met
Val Val Ala Thr Ala Gly Ile Leu 35 40 45 Asn Gly Ala Asn Ile Val
Arg Val His Asn Val Lys Tyr Gly Val Asp 50 55 60 Thr Ala Lys Val
Ser Asp Ala Leu Ser Lys Gly 65 70 75 11 493 DNA Glycine max unsure
(376)..(377) unsure (421) unsure (441)..(442)..(443) unsure (456)
unsure (458) unsure (462) unsure (467) unsure (473)..(474) unsure
(477) unsure (485) unsure (491) 11 cttgactggt cgcggagaac ttccgtcatg
gggatcctta atgtgactcc agatagtttt 60 agtgatgggg gaaatttcaa
gtctgtggag tctgctgttt atcaggttcg gttaatgatt 120 tcagaaggag
cagatatgat tgatatcggg gctcagtcta ctcggccaac ggcctctagg 180
atctctgctg cagaagaatt aggtagatta atccctgtcc tggaagctgt agtgtcaatg
240 cctgaggtag aaggaaagct catttctgtg gatactttct actctgaagt
tgcttcacaa 300 gcagtgagta aaggggctca tcttataaat gatgtatcct
gcctggacag ttggatagta 360 acatgtttaa agtccnnggg ctggatcttg
atgttcttaa tgttgcaaat ggcacaatga 420 nggggggaac catccttaca
nnngcaagaa taagtngnaa antctgnaaa tannggnaat 480 tgttntgtta naa 493
12 139 PRT Glycine max UNSURE (126) 12 Leu Asp Trp Ser Arg Arg Thr
Ser Val Met Gly Ile Leu Asn Val Thr 1 5 10 15 Pro Asp Ser Phe Ser
Asp Gly Gly Asn Phe Lys Ser Val Glu Ser Ala 20 25 30 Val Tyr Gln
Val Arg Leu Met Ile Ser Glu Gly Ala Asp Met Ile Asp 35 40 45 Ile
Gly Ala Gln Ser Thr Arg Pro Thr Ala Ser Arg Ile Ser Ala Ala 50 55
60 Glu Glu Leu Gly Arg Leu Ile Pro Val Leu Glu Ala Val Val Ser Met
65 70 75 80 Pro Glu Val Glu Gly Lys Leu Ile Ser Val Asp Thr Phe Tyr
Ser Glu 85 90 95 Val Ala Ser Gln Ala Val Ser Lys Gly Ala His Leu
Ile Asn Asp Val 100 105 110 Ser Cys Leu Asp Ser Trp Ile Val Thr Cys
Leu Lys Ser Xaa Gly Trp 115 120 125 Ile Leu Met Phe Leu Met Leu Gln
Met Ala Gln 130 135 13 2041 DNA Zea mays 13 gcacgagccc tcgcctgctc
cacgacgctt atgcgctcgc gtcccgctct cgccgcccac 60 ctccggcgcc
tgctcctcct ctctccctcc gcccacctca tcatcatccg ccgcgccatg 120
gcatccgccg ccgccgcgca ggcgcagcca ggtggcgccc cgccggcgac cgcggagtac
180 gaggaggtgc tggggcggct ctcctcgctc atcacgcaga aggtgcgcgc
gaacagcgcc 240 aaccgcggca accagtggga cctcatggag cactacgtca
agattctgga gctggaggag 300 tcgatcgcgc ggatgaaagt gattcacgtc
gcggggacca aggggaaggg ttccacatgc 360 acattcaccg agtcaatcct
gcgatcgtgt ggcttccata ctgggctgtt cacctcacca 420 catttgatgg
atgttagaga gcgatttcag ctagatgggg ttaatatttc tgaagagaaa 480
tttttgaagt acttctggtg gtgctggaac aagttgaagg agaagactga tgatgatatt
540 cccatgccag cctatttcag gttcctcgcg ttgctcgcat tcaagatatt
ttctgctgag 600 caggtagatg ttgctgttct cgaggttggc ctaggaggga
agtttgatgc aactaatgtg 660 gttaaagcac ctgtagtttg tggcatatct
tcccttggat atgatcatat ggaaattctt 720 gggaatacac ttggagaaat
cgcaggagag aaggctggga ttttcaagaa aggagttccg 780 gcctatactg
ctccacaacc agaagaggca atgactgctc tcaaacaaag agcttcggaa 840
ttgggtatct ctctccaagt cgttgatcct ttggagcccc atcacctaaa agatcagcat
900 cttgggctgc atggagaaca tcaatatata aatgctggcc ttgcagttgc
tttggctagt 960 acgtggcttg agaagcaggg acataaagat acgttaccac
tcaatcgtac tgatccctta 1020 ccagatcatt ttattagagg gctatcaagt
gcttctttgc aaggccgagc acagattgtt 1080 ccagattcac aagtgaattc
agaagagaag gacaaaaatt gttctcttgt tttttatttg 1140 gatggagcgc
acagtcctga aagcatggaa gtatgtggca agtggttttc ccatgtcaca 1200
aaggatgata caaggctacc atcttctgtg gagcagtctc atacatctat gtctcaaaag
1260 atccttctgt tcaactgcat gtctgtgaga gatccgatga gattgcttcc
ttgtctcctg 1320 gatgcatcaa ctcaaaatgg agtccacttt gacctggccc
tatttgtgcc gaaccaatca 1380 caacacacga agcttggttc taacacttca
gcaccagcgg agcctgagca aatcgatttg 1440 tcatggcagc tgtcacttca
aacagtgtgg gagaagttac ttcaggataa aggtataaat 1500 actacaaaat
ccagtgatac tagtaaagtt tttgattcgc ttccaatcgc aatcgagtgg 1560
ctaaggagaa atgcccgaga aaaccaatct acttctttcc aggtgctggt tactggctcc
1620 ctgcatctcg ttggggatgt cttgagaata atcaagaagt gatacgccgc
ctcgaaatcc 1680 aaactggaac tggactatga tctatggtct ctcccaaggc
taacatgatt agcaagggga 1740 gacatttgaa cggtgcttgc ttattggtgc
caaccaagct gcgagcttct tgtgtttttt 1800 ttgtgtggcc acggtcgcct
gcctaccact cgggaaaccg ccgcgcccgt tcttgtgaag 1860 gcatgaaata
ggatgatcgt gccaccatag aacataactg ggaaatgaat tcgacatgga 1920
actgggacag tctgtatact cacaaaataa gatcgcatgg ggttttcttg ttcaagtgca
1980 aagaaaccag tcaattctta tccagagtag caagaattca ttcaaaaaaa
aaaaaaaaaa 2040 a 2041 14 553 PRT Zea mays 14 Ala Arg Ala Leu Ala
Cys Ser Thr Thr Leu Met Arg Ser Arg Pro Ala 1 5 10 15 Leu Ala Ala
His Leu Arg Arg Leu Leu Leu Leu Ser Pro Ser Ala His 20 25 30 Leu
Ile Ile Ile Arg Arg Ala Met Ala Ser Ala Ala Ala Ala Gln Ala 35 40
45 Gln Pro Gly Gly Ala Pro Pro Ala Thr Ala Glu Tyr Glu Glu Val Leu
50 55
60 Gly Arg Leu Ser Ser Leu Ile Thr Gln Lys Val Arg Ala Asn Ser Ala
65 70 75 80 Asn Arg Gly Asn Gln Trp Asp Leu Met Glu His Tyr Val Lys
Ile Leu 85 90 95 Glu Leu Glu Glu Ser Ile Ala Arg Met Lys Val Ile
His Val Ala Gly 100 105 110 Thr Lys Gly Lys Gly Ser Thr Cys Thr Phe
Thr Glu Ser Ile Leu Arg 115 120 125 Ser Cys Gly Phe His Thr Gly Leu
Phe Thr Ser Pro His Leu Met Asp 130 135 140 Val Arg Glu Arg Phe Gln
Leu Asp Gly Val Asn Ile Ser Glu Glu Lys 145 150 155 160 Phe Leu Lys
Tyr Phe Trp Trp Cys Trp Asn Lys Leu Lys Glu Lys Thr 165 170 175 Asp
Asp Asp Ile Pro Met Pro Ala Tyr Phe Arg Phe Leu Ala Leu Leu 180 185
190 Ala Phe Lys Ile Phe Ser Ala Glu Gln Val Asp Val Ala Val Leu Glu
195 200 205 Val Gly Leu Gly Gly Lys Phe Asp Ala Thr Asn Val Val Lys
Ala Pro 210 215 220 Val Val Cys Gly Ile Ser Ser Leu Gly Tyr Asp His
Met Glu Ile Leu 225 230 235 240 Gly Asn Thr Leu Gly Glu Ile Ala Gly
Glu Lys Ala Gly Ile Phe Lys 245 250 255 Lys Gly Val Pro Ala Tyr Thr
Ala Pro Gln Pro Glu Glu Ala Met Thr 260 265 270 Ala Leu Lys Gln Arg
Ala Ser Glu Leu Gly Ile Ser Leu Gln Val Val 275 280 285 Asp Pro Leu
Glu Pro His His Leu Lys Asp Gln His Leu Gly Leu His 290 295 300 Gly
Glu His Gln Tyr Ile Asn Ala Gly Leu Ala Val Ala Leu Ala Ser 305 310
315 320 Thr Trp Leu Glu Lys Gln Gly His Lys Asp Thr Leu Pro Leu Asn
Arg 325 330 335 Thr Asp Pro Leu Pro Asp His Phe Ile Arg Gly Leu Ser
Ser Ala Ser 340 345 350 Leu Gln Gly Arg Ala Gln Ile Val Pro Asp Ser
Gln Val Asn Ser Glu 355 360 365 Glu Lys Asp Lys Asn Cys Ser Leu Val
Phe Tyr Leu Asp Gly Ala His 370 375 380 Ser Pro Glu Ser Met Glu Val
Cys Gly Lys Trp Phe Ser His Val Thr 385 390 395 400 Lys Asp Asp Thr
Arg Leu Pro Ser Ser Val Glu Gln Ser His Thr Ser 405 410 415 Met Ser
Gln Lys Ile Leu Leu Phe Asn Cys Met Ser Val Arg Asp Pro 420 425 430
Met Arg Leu Leu Pro Cys Leu Leu Asp Ala Ser Thr Gln Asn Gly Val 435
440 445 His Phe Asp Leu Ala Leu Phe Val Pro Asn Gln Ser Gln His Thr
Lys 450 455 460 Leu Gly Ser Asn Thr Ser Ala Pro Ala Glu Pro Glu Gln
Ile Asp Leu 465 470 475 480 Ser Trp Gln Leu Ser Leu Gln Thr Val Trp
Glu Lys Leu Leu Gln Asp 485 490 495 Lys Gly Ile Asn Thr Thr Lys Ser
Ser Asp Thr Ser Lys Val Phe Asp 500 505 510 Ser Leu Pro Ile Ala Ile
Glu Trp Leu Arg Arg Asn Ala Arg Glu Asn 515 520 525 Gln Ser Thr Ser
Phe Gln Val Leu Val Thr Gly Ser Leu His Leu Val 530 535 540 Gly Asp
Val Leu Arg Ile Ile Lys Lys 545 550 15 534 DNA Zea mays unsure (8)
unsure (14)..(15) unsure (26) unsure (49) unsure (182) unsure (189)
unsure (271) unsure (285) unsure (321)..(322) unsure (342) unsure
(413) unsure (500) unsure (510) unsure (512) unsure (517) unsure
(529) 15 catggctncc aaannttcgg cactanacgt actgagaaga gctcgcgtnc
ccgctactcg 60 ccggcccacc tccgggcgcc tgctcctcct ctctccctcc
gcccacctca tcatcatccg 120 ccgcgccatg gcctccgccg ccgccgcgca
ggcgcagcag gtggcgcccc accggcgacc 180 gnggagtang aggaggtgct
ggggcggctc tcctcgctca tcacgcagaa ggtgcgcgcg 240 aacagcgcca
accgcggcaa ccagtgggac ntcatggagc actangtcaa gattctggag 300
ctggaggagt cgatcgcgcg nnatgaaagt gattcacgtc gnagggacca aggggaaggg
360 ttccacatgc acattcaccg agtcaatcct gcgatcgtgt ggcttccata
atnggctgtt 420 taactcacca acatttgatt ggatgttaga gagcgaattc
agctagattg gggttaataa 480 tttctgaaga gaaatttttn aagtactctn
gntgtgntgg aacaagttna agga 534 16 154 PRT Zea mays UNSURE (3)
UNSURE (5) UNSURE (9) UNSURE (61) UNSURE (63) UNSURE (95) UNSURE
(107) UNSURE (114) UNSURE (138) 16 Met Ala Xaa Lys Xaa Ser Ala Leu
Xaa Val Leu Arg Arg Ala Arg Val 1 5 10 15 Pro Ala Thr Arg Arg Pro
Thr Ser Gly Arg Leu Leu Leu Leu Ser Pro 20 25 30 Ser Ala His Leu
Ile Ile Ile Arg Arg Ala Met Ala Ser Ala Ala Ala 35 40 45 Ala Gln
Ala Gln Gln Val Ala Pro His Arg Arg Pro Xaa Ser Xaa Arg 50 55 60
Arg Cys Trp Gly Gly Ser Pro Arg Ser Ser Arg Arg Arg Cys Ala Arg 65
70 75 80 Thr Ala Pro Thr Ala Ala Thr Ser Gly Thr Ser Trp Ser Thr
Xaa Ser 85 90 95 Arg Phe Trp Ser Trp Arg Ser Arg Ser Arg Xaa Met
Lys Val Ile His 100 105 110 Val Xaa Gly Thr Lys Gly Lys Gly Ser Thr
Cys Thr Phe Thr Glu Ser 115 120 125 Ile Leu Arg Ser Cys Gly Phe His
Asn Xaa Leu Phe Asn Ser Pro Thr 130 135 140 Phe Asp Trp Met Leu Glu
Ser Glu Phe Ser 145 150 17 553 DNA Glycine max unsure (520) unsure
(549) 17 ggcttctcag ttaaatgtac ctcttcaagt ggtaacccca ttagatgcca
aattgctaaa 60 tggttcaaga ctagcgcttg gaggtgaaca ccaatatata
aatgctggtc ttgctattgc 120 attatgctct acgtggctga aaatgaatgg
gcatcttgaa gactcgtact tgaaacatat 180 acaacacact ttaccagaga
agttcataaa agggttaaca actgcaagtt tgcaaggaag 240 ggctcagatt
gttcctgatc agttcatcaa tgatgaaata ccaaatgaac ttgtcttctt 300
tttagatggg gctcatagtc ctgaaagcat ggaagcatgt gccaggtggt tttctcttgc
360 tattaaagat caagaccaga ttttgtttca tcaagaaact tgataattct
aacttctcaa 420 accaagtagt gaagatgcac aatggtgaaa ctgtacagaa
gaaatccaca cagattttgc 480 tgttcaattg tatgtctgag cgaaaccctc
aattgcttcn tccccacttg atgaaaacat 540 gtgctgatna agg 553 18 133 PRT
Glycine max 18 Ala Ser Gln Leu Asn Val Pro Leu Gln Val Val Thr Pro
Leu Asp Ala 1 5 10 15 Lys Leu Leu Asn Gly Ser Arg Leu Ala Leu Gly
Gly Glu His Gln Tyr 20 25 30 Ile Asn Ala Gly Leu Ala Ile Ala Leu
Cys Ser Thr Trp Leu Lys Met 35 40 45 Asn Gly His Leu Glu Asp Ser
Tyr Leu Lys His Ile Gln His Thr Leu 50 55 60 Pro Glu Lys Phe Ile
Lys Gly Leu Thr Thr Ala Ser Leu Gln Gly Arg 65 70 75 80 Ala Gln Ile
Val Pro Asp Gln Phe Ile Asn Asp Glu Ile Pro Asn Glu 85 90 95 Leu
Val Phe Phe Leu Asp Gly Ala His Ser Pro Glu Ser Met Glu Ala 100 105
110 Cys Ala Arg Trp Phe Ser Leu Ala Ile Lys Asp Gln Asp Gln Ile Leu
115 120 125 Phe His Gln Glu Thr 130 19 564 DNA Zea mays unsure
(257) unsure (371) unsure (402) unsure (433) unsure (441) unsure
(447) unsure (453) unsure (468) unsure (473) unsure (480) unsure
(483) unsure (496) unsure (498) unsure (500) unsure (519) unsure
(533) unsure (539) unsure (563) 19 ggcggcggct acgtggggtg gcgacgacaa
gctcattctg cgcggccttc agttccatgg 60 cttccacggt gtcctgcagg
aggagaagac gttgggacag aagttcgtgg ttgacatcga 120 cgcctggata
gacctcgccg ctgccggcga agtccgactg cattgctgac accgtcagct 180
acaccgatat ctacagcatt gcaaaggatg ttgtcgaggg cacgccacgc aacctcttgg
240 agtcggtagc tcactcnatc gcagaggcca cgctgctcaa gttccctcaa
atctccgcag 300 tccgagtgaa ggttggcaag cctcacctcg cggtgcgagg
cgttctggac taactgggcg 360 tggggataac naggcacaaa aagaaagaat
tgagattctg tncacatgtg gtgatggggg 420 aaccagttca atnctgatgg
nactgcnggc aanaccataa tccacccncc ccntgttgcn 480 tgntgggaac
taagcnantn cctttcacct ctgaactgnt gggaatatcg ggnaatctng 540
ttcccctaaa ttgctttatt acna 564 20 116 PRT Zea mays 20 Asp Lys Leu
Ile Leu Arg Gly Leu Gln Phe His Gly Phe His Gly Val 1 5 10 15 Leu
Gln Glu Glu Lys Thr Leu Gly Gln Lys Phe Val Val Asp Ile Asp 20 25
30 Ala Trp Thr Ser Pro Leu Pro Ala Lys Ser Asp Cys Ile Ala Asp Thr
35 40 45 Val Ser Tyr Thr Asp Ile Tyr Ser Ile Ala Lys Asp Val Val
Glu Gly 50 55 60 Thr Pro Arg Asn Leu Leu Glu Ser Val Ala His Ser
Ile Ala Glu Ala 65 70 75 80 Thr Leu Leu Lys Phe Pro Gln Ile Ser Ala
Val Arg Val Lys Val Gly 85 90 95 Lys Pro His Leu Ala Val Arg Gly
Val Leu Asp Leu Gly Val Gly Ile 100 105 110 Thr Arg His Lys 115 21
601 DNA Glycine max unsure (406) unsure (413) unsure (450) unsure
(479) unsure (491) unsure (493) unsure (514) unsure (520) unsure
(539) unsure (579) unsure (585) 21 cggagaggcg agggagtgag ggactagcac
agaaagatat tgtttggtgt acggtggtga 60 gtgtcgacgc tgccactctc
gcctgtgtct gtgataaatg gaatctgatg caccgacatg 120 gggagacaaa
ctcatgttga ggggattgtc attccatggt tttcatggag caaagcctga 180
agaaaggaca ctgggccaga agttcttcat agatatagat gcttggatgg atctcaaagc
240 agctgggcaa atctgatcac ttatcaaatt ctgttagtta cacagaaata
tatgatatag 300 ctaaggatgt tcttgaaggg tcacctcaca atcctctggg
agtcaagtgg gccaaaaaaa 360 ttgcaatcac tactcttaca aatcaaaaag
aaatatctgc tgtccnagtg aanggtggga 420 aaccccatgt ggcaattccg
ggtccaattn attacttaag cgtttgagaa tcctaaacnc 480 aaaaaccaac
ntnttcaagg ctaaaaaatt taanatttan tgctgcacaa attttatant 540
ttcaaaatcc accttgatac aaaaagtaaa ggtactccnt tcccntcaag gccccaatta
600 g 601 22 67 PRT Glycine max UNSURE (43)..(44) 22 Asp Lys Leu
Met Leu Arg Gly Leu Ser Phe His Gly Phe His Gly Ala 1 5 10 15 Lys
Pro Glu Glu Arg Thr Leu Gly Gln Lys Phe Phe Ile Asp Ile Asp 20 25
30 Ala Trp Met Asp Leu Lys Ala Ala Gly Gln Xaa Xaa His Leu Ser Asn
35 40 45 Ser Val Ser Tyr Thr Glu Ile Tyr Asp Ile Ala Lys Asp Val
Leu Glu 50 55 60 Gly Ser Pro 65 23 439 DNA Triticum aestivum 23
ccaggttcca ctccacccac ccacctgcgc cgccagctct aaaggaggcg gcgtcggccg
60 gcgggcgagc gcacgcccag gcccaatcga tcgatcccag ctctagaggg
gagggagcaa 120 ccatggcggg ggacggggag gacgaggtgc cggcgatggg
cggagacaag ctgatcctgc 180 gggggctgca gttccacggc ttccacggcg
tgaagcagga ggagaagaag ctgggccaga 240 agttcgtggt cgacgtggac
gcctggatgg acctcgccgc cgccggggac tccgacgaca 300 tcgcccacac
cgtcagctac accgacatct acaggatagc caagggcgtg gtggaaggcc 360
cgtcgcggaa acctcctgga gtcggtggcg cagtcgatcg ccggcaacaa cgctgctccg
420 aagtttcccc aaatctccg 439 24 65 PRT Triticum aestivum 24 Asp Lys
Leu Ile Leu Arg Gly Leu Gln Phe His Gly Phe His Gly Val 1 5 10 15
Lys Gln Glu Glu Lys Lys Leu Gly Gln Lys Phe Val Val Asp Val Asp 20
25 30 Ala Trp Met Asp Leu Ala Ala Ala Gly Asp Ser Asp Asp Ile Ala
His 35 40 45 Thr Val Ser Tyr Thr Asp Ile Tyr Arg Ile Ala Lys Gly
Val Val Glu 50 55 60 Gly 65 25 677 DNA Zea mays unsure (565) unsure
(643) unsure (656) unsure (676) 25 cctcgaacga gggccgtacc tagcgcctct
gtccttcgtc ggccgtcgca ctgtgctccc 60 gtccgcctcc ggcctccgcc
aacccgcgtc cgcccacgac taggcggctc tgggcaggtc 120 cttccacaaa
gatgtgaagg attaaagctc atgtgaaaga ttctaagact acaattggta 180
tcaagcggtt gctttcttat ttctcatacg ctcaaccatg ctcctgcatg ctaaggattc
240 agttaggaag atgcattcag ttgctaagaa ctactttgtg tctgatctta
ctcatcctcc 300 aagatccttg aacagagctt ccagacatgt tgttccattc
aagacccgtt tctttacgca 360 ttgctcactt gagagccgtt cagttgacca
agagattgtg attgctatgg gaagcaatgt 420 aggcgataga gtcagtacat
tcaacagggc attgcagctg atgaaaagct cagacgtgaa 480 catcactagg
catgcctgtc tctatgaaac cgcccctgct tatttgactg atcagccacg 540
gtttcttaac tctgccattc ggggnacaac taggctccag gccacatgag cttcttaaac
600 tggctaaagg aaattgagaa gggaattggc cgcactgggg ganataaagg
tacggnccaa 660 gacctatcga ttaagna 677 26 101 PRT Zea mays UNSURE
(69) UNSURE (92)..(93) 26 Ser Leu Glu Ser Arg Ser Val Asp Gln Glu
Ile Val Ile Ala Met Gly 1 5 10 15 Ser Asn Val Gly Asp Arg Val Ser
Thr Phe Asn Arg Ala Leu Gln Leu 20 25 30 Met Lys Ser Ser Asp Val
Asn Ile Thr Arg His Ala Cys Leu Tyr Glu 35 40 45 Thr Ala Pro Ala
Tyr Leu Thr Asp Gln Pro Arg Phe Leu Asn Ser Ala 50 55 60 Ile Arg
Gly Thr Xaa Ala Pro Gly His Met Ser Phe Leu Asn Trp Leu 65 70 75 80
Lys Glu Ile Glu Lys Gly Ile Gly Arg Thr Gly Xaa Xaa Arg Tyr Gly 85
90 95 Pro Arg Pro Ile Asp 100 27 227 DNA Oryza sativa unsure (125)
unsure (176) unsure (181) unsure (188) unsure (190) unsure (220)
unsure (222) 27 cttacagttt aggtgcttca catgtgccaa ttttacttgg
accctcaagg aaaagatttt 60 taggtgaaat atgcaatcgt gtcaatccca
ctgagagaga tgctgctacc atggtcgttg 120 ctacngctgg gatattgaat
ggtgctaata tagtaagggt gcataatgtt aaatanggct 180 nggatacngn
aaaggtctct aatcctttgc caaaggggan angtgtt 227 28 70 PRT Oryza sativa
UNSURE (57) UNSURE (59) UNSURE (62) 28 Ser Leu Gly Ala Ser His Val
Pro Ile Leu Leu Gly Pro Ser Arg Lys 1 5 10 15 Arg Phe Leu Gly Glu
Ile Cys Asn Arg Val Asn Pro Thr Glu Arg Asp 20 25 30 Ala Ala Thr
Met Val Val Ala Thr Ala Gly Ile Leu Asn Gly Ala Asn 35 40 45 Ile
Val Arg Val His Asn Val Lys Xaa Gly Xaa Asp Thr Xaa Lys Val 50 55
60 Ser Asn Pro Leu Pro Lys 65 70 29 534 DNA Zea mays unsure (8)
unsure (14)..(15) unsure (26) unsure (49) unsure (182) unsure (189)
unsure (271) unsure (285) unsure (321)..(322) unsure (342) unsure
(413) unsure (500) unsure (510) unsure (512) unsure (517) unsure
(529) 29 catggctncc aaannttcgg cactanacgt actgagaaga gctcgcgtnc
ccgctactcg 60 ccggcccacc tccgggcgcc tgctcctcct ctctccctcc
gcccacctca tcatcatccg 120 ccgcgccatg gcctccgccg ccgccgcgca
ggcgcagcag gtggcgcccc accggcgacc 180 gnggagtang aggaggtgct
ggggcggctc tcctcgctca tcacgcagaa ggtgcgcgcg 240 aacagcgcca
accgcggcaa ccagtgggac ntcatggagc actangtcaa gattctggag 300
ctggaggagt cgatcgcgcg nnatgaaagt gattcacgtc gnagggacca aggggaaggg
360 ttccacatgc acattcaccg agtcaatcct gcgatcgtgt ggcttccata
atnggctgtt 420 taactcacca acatttgatt ggatgttaga gagcgaattc
agctagattg gggttaataa 480 tttctgaaga gaaatttttn aagtactctn
gntgtgntgg aacaagttna agga 534 30 36 PRT Zea mays UNSURE (7) UNSURE
(31) 30 Met Lys Val Ile His Val Xaa Gly Thr Lys Gly Lys Gly Ser Thr
Cys 1 5 10 15 Thr Phe Thr Glu Ser Ile Leu Arg Ser Cys Gly Phe His
Asn Xaa Leu 20 25 30 Phe Asn Ser Pro 35 31 553 DNA Glycine max
unsure (520) unsure (549) 31 ggcttctcag ttaaatgtac ctcttcaagt
ggtaacccca ttagatgcca aattgctaaa 60 tggttcaaga ctagcgcttg
gaggtgaaca ccaatatata aatgctggtc ttgctattgc 120 attatgctct
acgtggctga aaatgaatgg gcatcttgaa gactcgtact tgaaacatat 180
acaacacact ttaccagaga agttcataaa agggttaaca actgcaagtt tgcaaggaag
240 ggctcagatt gttcctgatc agttcatcaa tgatgaaata ccaaatgaac
ttgtcttctt 300 tttagatggg gctcatagtc ctgaaagcat ggaagcatgt
gccaggtggt tttctcttgc 360 tattaaagat caagaccaga ttttgtttca
tcaagaaact tgataattct aacttctcaa 420 accaagtagt gaagatgcac
aatggtgaaa ctgtacagaa gaaatccaca cagattttgc 480 tgttcaattg
tatgtctgag cgaaaccctc aattgcttcn tccccacttg atgaaaacat 540
gtgctgatna agg 553 32 24 PRT Glycine max 32 Leu Ala Leu Gly Gly Glu
His Gln Tyr Ile Asn Ala Gly Leu Ala Ile 1 5 10 15 Ala Leu Cys Ser
Thr Trp Leu Lys 20
* * * * *
References