U.S. patent application number 10/322149 was filed with the patent office on 2003-06-26 for plant histidinol-phosphate aminotransferase homologs.
Invention is credited to Allen, Stephen M., Rafalski, J. Antoni.
Application Number | 20030121072 10/322149 |
Document ID | / |
Family ID | 26804598 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030121072 |
Kind Code |
A1 |
Allen, Stephen M. ; et
al. |
June 26, 2003 |
Plant histidinol-phosphate aminotransferase homologs
Abstract
This invention relates to an isolated nucleic acid fragment
encoding a histidine biosynthetic enzyme. The invention also
relates to the construction of a chimeric gene encoding all or a
portion of the histidine biosynthetic enzyme, in sense or antisense
orientation, wherein expression of the chimeric gene results in
production of altered levels of the histidine biosynthetic enzyme
in a transformed host cell.
Inventors: |
Allen, Stephen M.;
(Wilmington, DE) ; Rafalski, J. Antoni;
(Wilmington, 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: |
26804598 |
Appl. No.: |
10/322149 |
Filed: |
December 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10322149 |
Dec 18, 2002 |
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09433241 |
Nov 4, 1999 |
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6525244 |
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60107273 |
Nov 5, 1998 |
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Current U.S.
Class: |
800/278 ;
435/193; 435/419; 435/6.18; 536/23.2 |
Current CPC
Class: |
C12N 9/1096
20130101 |
Class at
Publication: |
800/278 ; 435/6;
435/419; 435/193; 536/23.2 |
International
Class: |
A01H 005/00; C12Q
001/68; C07H 021/04; C12N 009/10; C12N 005/04; C12N 015/82 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide having histidinol-phosphate aminotransferase
activity, wherein the amino acid sequence of the polypeptide and
the amino acid sequence of SEQ ID NO: 14 have at least 80% sequence
identity based on the Clustal alignment method, or (b) the
complement of the nucleotide sequence, wherein the complement and
the nucleotide sequence contain the same number of nucleotides and
are 100% complementary.
2. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO: 14
have at least 85% sequence identity.
3. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO: 14
have at least 90% sequence identity.
4. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO: 14
have at least 95% sequence identity.
5. The polynucleotide of claim 1, wherein the nucleotide sequence
comprises the nucleotide sequence of SEQ ID NO: 13.
6. The polynucleotide of claim 1, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO: 14.
7. A 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 cell
with the polynucleotide of claim 1.
9. A cell comprising the 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 recombinant DNA construct of claim
7.
12. A seed comprising the recombinant DNA construct of claim 7.
13. A vector comprising the polynucleotide of claim 1.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/433,241, filed Nov. 4, 1999, allowed, which claims the
benefit of U.S. Provisional Application No. 60/107,273, filed Nov.
5, 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 a histidine biosynthetic enzyme in plants and
seeds.
BACKGROUND OF THE INVENTION
[0003] Histidine biosynthesis begins with condensation of ATP with
phosphoribosyl pyrophosphate (PRPP) to form
N.sup.1-(5'-phosphoribosyl)-A- TP. Imidazole glycerol phosphate
(IGP) synthase (also known as glutamine amidotransferase), a
heterodimeric enzyme consisting of the hisF and hisH gene products,
catalyzes the fifth step of histidine biosynthesis, wherein
phosphoribulosyl formimino-5-aminoimidazole-4-carboxamide
ribonucleotide (PRFAR) and glutamine are transformed into
glutamate, IGP and 5-aminoimidazole-4-carboxamide ribonucleotide
(AICAR). This reaction is of the glutamine amidotransferase class.
AICAR is a purine biosynthetic intermediate; thus there is a
linkage between the purine and histidine biosynthetic pathways such
that the purine ring removed in the first step of the histidine
pathway is replenished by the couple between the reaction catalyzed
by IGP synthase and the purine biosynthetic pathway.
[0004] It has been shown in a number of systems that missense
mutations that decrease but do not eliminate the catalytic
efficiency of the fourth step (formation of PRFAR from
Pro-phoshporibosyl formimino-5-aminoimidazo- le-4-carboxamide
ribonucleotide or 5'-ProFAR, catalyzed by 5'-ProFAR isomerase, the
product of the hisA gene) or fifth step of histidine biosynthesis
result in a biosynthetic limitation that is overcome by (a)
histidine, (b) adenine or (c) a false feedback inhibitor of the
first step the histidine pathway (Hartman, P. E. et al. (1960) J.
Gen Microbiol. 22:323; Shedlovsky and Magasanik (1962) J. Biol.
Chem 237:3725; Shedlovsky and Magasanik (1962) J. Biol. Chem
237:3731; Galloway and Taylor (1980) J. Bacteriol. 144:1068; Shioi
et al. (1982) J. Biol. Chem. 257:7969; Burton (1955) Biochem. J.
61:473; Burton (1957) Biochem. J. 66:488; Stougaard and Kennedy
(1988) J. Bacteriol. 170:250). This result indicates that a high
level flux through the partially blocked histidine biosynthetic
pathway results in an ATP (energy) drain. Such blockage has been
shown to have unique, deleterious pleiotropic effects upon a
diversity of energy-intensive microbial processes including
chemotaxis (Galloway and Taylor (1980) J Bacteriol. 144:1068), DNA
replication (Burton (1955) Biochem. J. 61:473; Burton (1957)
Biochem. J. 66:488) and nitrogen fixation (Stougaard and Kennedy
(1988) J. Bacteriol. 170:250). In each interrupted process,
activity is restored by (a) histidine, (b) adenine or (c) a false
feedback inhibitor of the first step in histidine biosynthesis.
[0005] These studies strongly suggest that enzymes encoded by the
hisA, hisF or hisH genes will be useful for discovering herbicides
and fungicides. The discovery of homologous biosynthetic pathways
and corresponding enzymes in plants and fungi indicates that
inhibition of such enzymes would be viable strategies for
herbicidal control of unwanted vegetation and fungicidal control of
plant disease For example, inhibition of the fourth and fifth steps
of histidine biosynthesis will result in the specific draining of
the ATP pool to levels significantly lower than normal (Johnson and
Taylor (1993) Applied Environ. Microbiol. 59:3509). This specific
drain is achieved by having the histidine synthetic pathway
operating at a high, near maximal rate through the relief from
allosteric feedback inhibition of the hisG encoded enzyme, ATP
phosphoribosyl transferase. By preventing the release of AICAR by
the IGP synthase, the adenylate pool is drained. Although energy
homeostasis can be maintained by simply rephosporylation of the
adenylate to a high energy state, inhibition of the hisHF or hisA
encoded enzymes traps the adenylate as histidine biosynthetic
intermiates. Accordingly, lowered flux through the enzymes encoded
by hisA and hisHF will cripple the cell's ability to carry out
necessary metabolic processes.
[0006] Moreover, interruption of other steps in the histidine
biosynthetic pathway in plants may also result in plant growth
inhibition or death. For example, decrease or elimination of
histidinol-phosphate aminotransferase encoded by a plant homolog of
hisC may inhibit conversion of glutamate to .alpha.-ketoglutarate
(seventh step of the histidine biosynthetic pathway) and thereby
have a detrimental effect on plant growth and development. The
enzyme encoded by hisB is in part responsible for catalyzing the
seventh and ninth steps of the histidine biosynthetic pathway. In
the seventh step of the pathway D-erythro-1-(imidazol-4-yl)glycerol
3-phosphate is converted to 3-(imidazol-4-yl)-2oxopropyl phosphate
by HisB. In the ninth step of the pathway histidinol phosphate is
converted to histidinol by the action of HisB. Very little is know
about HisB activity in plants; however, because this enzyme
catalyzes two steps in the pathway, interruption of HisB activity
could severely alter normal histidine biosynthesis. Lastly,
interruption of histidinol dehydrogenase activity (encoded by a
homolog of the hisD gene), the enzyme that catalyzes the final step
in the pathway, would prevent the formation of histidine. Since the
biosynthesis of histidine is energetically costly to the cell,
inhibition of transformations at the later steps in the pathway
would consume significant cellular energy resources without the
formation of the expected end product, thus placing the affected
cell at a disadvantage.
[0007] Thus, availability of the genes and their encoded enzymes
has utility for herbicide and fungicide discovery via the design
and implementation of cell-based screening and assay methodologies,
enzyme-based screening and assay methodologies, rationale inhibitor
design, x-ray crystallography, combinatorial chemistry and other
modern biochemical and biotechnological methods.
SUMMARY OF THE INVENTION
[0008] The present invention relates to isolated polynucleotides
comprising a nucleotide sequence encoding a first polypeptide of at
least 182 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 histidinol-phosphate
aminotransferase polypeptide of SEQ ID NO: 10, a rice
histidinol-phosphate aminotransferase polypeptide of SEQ ID NO: 12,
a soybean histidinol-phosphate aminotransferase polypeptide of SEQ
ID NO: 14, a wheat histidinol-phosphate aminotransferase
polypeptide of SEQ ID NO: 16. 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: 9, 11, 13 and 15 that codes for
the polypeptide selected from the group consisting of SEQ ID NOs:
10, 12, 14 and 16. The present invention also relates to an
isolated polynucleotide comprising a nucleotide sequences of at
least one of 40 (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 and 15 and
the complement of such nucleotide sequences.
[0010] An isolated polynucleotide comprising a nucleotide sequence
encoding a first polypeptide of at least 25 amino acids having 80%
identity based on the Clustal method of alignment when compared to
a polypeptide selected from the group consisting of histidinol
phosphate aminotransferase polypeptides of SEQ ID NOs: 1, 3, 5 and
7; or an isolated polynucleotide comprising the complement of the
nucleotide sequences.
[0011] The present invention relates to a chimeric gene comprising
an isolated polynucleotide of the present invention operably linked
to suitable regulatory sequences.
[0012] 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.
[0013] 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. The present invention
relates to a histidinol-phosphate aminotransferase polypeptide of
at least 182 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: 10, 12, 14 and 16.
[0014] The present invention relates to a histidinol-phosphate
aminotransferase polypeptide of at least 25 amino acids having at
least 80% identity based on the Clustal method of alignment
compared to a polypeptide selected from the group consisting of SEQ
ID NOs: 2, 4, 6 and 8.
[0015] The present invention relates to a method of selecting an
isolated polynucleotide that affects the level of expression of a
histidinol-phosphate aminotransferase polypeptide in a host cell,
preferably a plant cell, the method comprising the steps of:
[0016] constructing an isolated polynucleotide of the present
invention or an isolated chimeric gene of the present
invention;
[0017] introducing the isolated polynucleotide or the isolated
chimeric gene into a host cell;
[0018] measuring the level a histidinol-phosphate aminotransferase
polypeptide in the host cell containing the isolated
polynucleotide; and
[0019] comparing the level of a histidinol-phosphate
aminotransferase polypeptide in the host cell containing the
isolated polynucleotide with the level of a histidinol-phosphate
aminotransferase polypeptide in a host cell that does not contain
the isolated polynucleotide.
[0020] The present invention relates to a method of obtaining a
nucleic acid fragment encoding a substantial portion of a
histidinol-phosphate aminotransferase polypeptide gene, preferably
a plant histidinol-phosphate aminotransferase polypeptide gene,
comprising the steps of: synthesizing an oligonucleotide primer
comprising a nucleotide sequence of at least one of 40 (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 and 15 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 histidinol-phosphate
aminotransferase amino acid sequence.
[0021] 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 histidinol-phosphate
aminotransferase 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.
[0022] 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 histidinol-phosphate aminotransferase, the method
comprising the steps of: (a) transforming a host cell with a
chimeric gene comprising a nucleic acid fragment encoding a
histidinol-phosphate aminotransferase, 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
histidinol-phosphate aminotransferase in the transformed host cell;
(c) optionally purifying the histidinol-phosphate aminotransferase
expressed by the transformed host cell; (d) treating the
histidinol-phosphate aminotransferase with a compound to be tested;
and (e) comparing the activity of the histidinol-phosphate
aminotransferase that has been treated with a test compound to the
activity of an untreated histidinol-phosphate aminotransferase,
thereby selecting compounds with potential for inhibitory
activity.
[0023] The present invention relates to a composition comprising an
isolated polynucleotide of the present invention.
[0024] The present invention relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a first polypeptide of at
least 25 amino acids having 80% identity based on the Clustal
method of alignment when compared to a polypeptide selected from
the group consisting of histidinol-phosphate aminotransferase
polypeptides of SEQ ID NOs: 1, 3, 5 and 7; or an isolated
polynucleotide comprising the complement of the nucleotide
sequence.
[0025] The present invention relates to an isolated polynucleotide
comprising at least one of 30 contiguous nucleic acid sequences
selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 14 and 15 and the complement of such sequences.
[0026] The present invention relates to an expression cassette
comprising an isolated polynucleotide of the present invention
operably linked to a promoter.
[0027] The present invention relates to a method for positive
selection of a transformed cell comprising:
[0028] (a) transforming a plant cell, preferably a monocot such as
corn, with a chimeric gene of the present invention or an
expression cassette of the present invention; and
[0029] (b) growing the transformed plant cell under conditions
allowing expression of the polynucleotide in an amount sufficient
to complement a histidine biosynthetic auxotroph.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
[0030] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0031] 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: 9, 11, 13 and 15 and amino acid sequences SEQ ID NOs: 10, 12,
14 and 16 were determined by further sequence analysis of cDNA
clones encoding the amino acid sequences set forth in SEQ ID NOs:
2, 4, 6 and 8. Nucleotide SEQ ID NOs: 1, 3, 5 and 7 and amino acid
SEQ ID NOs: 2, 4, 6 and 8 were presented in a U.S. Provisional
Application No. 60/107,273, filed Nov. 5, 1998.
[0032] 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 Histidine Biosynthetic Enzymes SEQ ID NO: (Nucleo- (Amino
Protein Clone Desigination tide) Acid) Histidinol-phosphate
cr1.pk0012.c7 (EST) 1 2 aminotransferase Histidinol-phosphate
rl0n.pk093.g16 (EST) 3 4 aminotransferase Histidinol-phosphate
se1.pk0022.f4 (EST) 5 6 aminotransferase Histidinol-phosphate
wdr1.pk0006.a4 (EST) 7 8 aminotransferase Histidinol-phosphate
Contig composed of (ESTs): 9 10 aminotransferase cco1n.pk0039.c8
cr1.pk0012.c7 ctn1c.pk001.n24 p0006.cbyvt93r p0021.cperd48r
p0031.ccmai09r p0040.cftac80r p0128.cpidb67r Histidinol-phosphate
rl0n.pk093.g16 (FIS) 11 12 aminotransferase Histidinol-phosphate
se1.pk0022.f4 (FIS) 13 14 aminotransferase Histidinol-phosphate
Contig* composed of: 15 16 aminotransferase wdr1.pk0006.a4 (FIS)
wkm2n.pk005.n12 (EST)
[0033] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Res. 13:3021-3030 (1985) and in the
Biochemical J. 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn.1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0034] 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, or
synthetic DNA. 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, of the nucleic acid sequence of
the SEQ ID NOs: 9, 11,13 and 15.
[0035] As used herein, "contig" refers to a nucleotide sequence
that is assembled from two or more constituent nucleotide sequences
that share common or overlapping regions of sequence homology. For
example, the nucleotide sequences of two or more nucleic acid
fragments can be compared and aligned in order to identify common
or overlapping sequences. Where common or overlapping sequences
exist between two or more nucleic acid fragments, the sequences
(and thus their corresponding nucleic acid fragments) can be
assembled into a single contiguous nucleotide sequence.
[0036] 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.
[0037] 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.
[0038] 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: 9, 11, 13 and 15 and the complement of such nucleotide
sequences may be used in methods of selecting an isolated
polynucleotide that affects the expression of a polypeptide (such
as histidinol-phosphate aminotransferase) 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.
[0039] Moreover, substantially similar nucleic acid fragments may
also be characterized by their ability to hybridize. Estimates of
such homology are provided by either DNA-DNA or DNA-RNA
hybridization under conditions of stringency as is well understood
by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic
Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions
can be adjusted to screen for moderately similar fragments, such as
homologous sequences from distantly related organisms, to highly
similar fragments, such as genes that duplicate functional enzymes
from closely related organisms. Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a
series of washes starting with 6.times.SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at
45.degree. C. for 30 min, and then repeated twice with
0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 min. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times.SSC, 0.1%
SDS at 65.degree. C.
[0040] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Suitable nucleic acid fragments
(isolated polynucleotides of the present invention) encode
polypeptides that are at least 70% identical, preferably at least
80% identical to the amino acid sequences reported herein.
Preferred nucleic acid fragments encode amino acid sequences that
are at least 85% identical to the amino acid sequences reported
herein. More preferred nucleic acid fragments encode amino acid
sequences that are at least 90% identical to the amino acid
sequences reported herein. Most preferred are nucleic acid
fragments that encode amino acid sequences that are at least 95%
identical to the amino acid sequences reported herein. Suitable
nucleic acid fragments not only have the above homologies but
typically encode a polypeptide having at least 50 amino acids,
preferably at least 100 amino acids, more preferably at least 150
amino acids, still more preferably at least 200 amino acids, and
most preferably at least 250 amino acids. 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.
[0041] 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.
[0042] "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.
[0043] "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.
[0044] "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.
[0045] "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.
[0046] "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.
[0047] 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).
[0048] 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.
[0049] "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.
[0050] 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.
[0051] 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).
[0052] "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.
[0053] "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.
[0054] 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).
[0055] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference).
[0056] 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").
[0057] Nucleic acid fragments encoding at least a portion of
several histidine 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).
[0058] For example, genes encoding other histidine biosynthetic
enzymes, either as cDNAs or genomic DNAs, could be isolated
directly by using all or a portion of the instant nucleic acid
fragments as DNA hybridization probes to screen libraries from any
desired plant employing methodology well known to those skilled in
the art. Specific oligonucleotide probes based upon the instant
nucleic acid sequences can be designed and synthesized by methods
known in the art (Maniatis). Moreover, the entire sequences can be
used directly to synthesize DNA probes by methods known to the
skilled artisan such as random primer DNA labeling, nick
translation, or end-labeling techniques, or RNA probes using
available in vitro transcription systems. In addition, specific
primers can be designed and used to amplify a part or all of the
instant sequences. The resulting amplification products can be
labeled directly during amplification reactions or labeled after
amplification reactions, and used as probes to isolate full length
cDNA or genomic fragments under conditions of appropriate
stringency.
[0059] 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: 9, 11, 13 and 15 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 histidinol-phosphate
aminotransferase) 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: 9, 11,
13 and 15 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.
[0060] 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).
[0061] 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
histidine biosynthesis in those cells.
[0062] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. For reasons of convenience, the chimeric gene may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals may also be provided. The instant
chimeric gene may also comprise one or more introns in order to
facilitate gene expression.
[0063] Plasmid vectors comprising the instant chimeric gene can
then 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.
[0064] For some applications it may be useful to direct the instant
polypeptides to different cellular compartments, or to facilitate
its secretion from the cell. It is thus envisioned that the
chimeric gene described above may be further supplemented by
altering the coding sequence to encode the instant polypeptides
with appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys. 100: 1627-1632)
added and/or with targeting sequences that are already present
removed. While the references cited give examples of each of these,
the list is not exhaustive and more targeting signals of utility
may be discovered in the future.
[0065] 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.
[0066] 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.
[0067] 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, and is not
an inherent part of the invention. For example, one can screen by
looking for changes in gene expression by using antibodies specific
for the protein encoded by the gene being suppressed, or one could
establish assays that specifically measure enzyme activity. A
preferred method will be one which allows large numbers of samples
to be processed rapidly, since it will be expected that a large
number of transformants will be negative for the desired
phenotype.
[0068] 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
histidine biosynthetic enzyme. An example of a vector for high
level expression of the instant polypeptides in a bacterial host is
provided (Example 6).
[0069] 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
histidine 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.
[0070] 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).
[0071] 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.
[0072] 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).
[0073] 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.
[0074] 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. 1 7:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0075] 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
[0076] 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
[0077] 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.pk0039.c8 Green House* cr1 Corn Root From 7 Day Old Seedlings
cr1.pk0012.c7 ctn1c Corn Tassel, Night Harvested ctn1c.pk001.n24
p0006 Corn Young Shoot p0006.cbyvt93r p0021 Corn Pericarp 11 Days
After Pollination p0021.cperd48r p0031 Corn Shoot Culture
p0031.ccmai09r p0040 Corn Tassel: Apical Meristem > Floral
p0040.cftac80r Transition p0128 Corn Primary and Secondary Immature
p0128.cpidb67r Ear rl0n Rice 15 Day Old Leaf* rl0n.pk093.g16 se1
Soybean Embryo, 6 to 10 Days After se1.pk0022.f4 Flowering wdr1
Wheat Developing Root and Leaf wdr1.pk0006.a4 wkm2n Wheat Kernel
Malted 175 Hours at 4 wkm2n.pk005.n12 Degrees Celsius* *These
libraries were normalized essentially as described in U.S. Pat. No.
5,482,845, incorporated herein by reference.
[0078] 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
[0079] cDNA clones encoding histidine 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 Histidinol-Phosphate
Aminotransferase
[0080] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to histidinol-phosphate aminotransferase from Nicotiana
tabacum (NCBI Identifier No. gi 3355626). 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 Nicotiana tabacum Histidinol-Phosphate
Aminotransferase BLAST pLog Score Clone Status to gi 3355626
cco1n.pk0039.c8 Contig >254.00 cr1.pk0012.c7 ctn1c.pk001.n24
p0006.cbyvt93r p0021.cperd48r p0031.ccmai09r p0040.cftac80r
p0128.cpidb67r rl0n.pk093.g16 FIS 178.00 se1.pk0022.f4 FIS
>254.00 wdr1.pk0006.a4 Contig* 94.70 wkm2n.pk005.n12
[0081] The data in Table 4 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs: 10,
12, 14 and 16 and the Nicotiana tabacum sequence.
4TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Nicotiana tabacum Histidinol-Phosphate
Aminotransferase Percent Identity to SEQ ID NO. gi 3355626 10 74%
12 73% 14 78% 16 78%
[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 KTLPLE 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 histidinol-phosphate
aminotransferase. These sequences represent the first corn, rice,
soybean and wheat sequences encoding histidinol-phosphate
aminotransferase.
Example 4
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 pML103. Plasmid
pML103 has been deposited under the terms of the Budapest Treaty at
ATCC (American Type Culture Collection, 10801 University Blvd.,
Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
The DNA segment from pML1 03 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 5
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 D 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 pUC18 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. PDS 1000/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
pJm25 (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 6
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 GTG.TM. low melting
agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium
bromide for visualization of the DNA fragment. The fragment can
then be purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20
.mu.L of water. Appropriate oligonucleotide adapters may be ligated
to the fragment using T4 DNA ligase (New England Biolabs, Beverly,
Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the instant polypeptides are then screened for the
correct orientation with respect to the T7 promoter by restriction
enzyme analysis.
[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 BL21(DE3) (Studier et al. (1986)
J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 mn of approximately 1, IPTG
(isopropylthio-.beta.-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3
h at 25.degree.. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
Example 7
Evaluating Compounds for Their Ability to Inhibit the Activity of
Histidine Biosynthetic Enzyme
[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 6, 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
histidinol-phosphate aminotransferase are presented by Malki et
al., (1998) Plant Mol. Biol. 37(6):1013-1022.
Sequence CWU 1
1
16 1 431 DNA Zea mays 1 cgccgcttcg ttgatggatt ggtccacccg gttccgcatc
cgaaacccgt cgccggccgc 60 gtcccacttt gccggccaca ggcggagtca
agcggatagg gtatttctcc ggaccatggc 120 gtcggcggcc ccggtggagg
agccgacggc ggcggccgag gcgaaggggc ggctgaccgg 180 tgactccttc
atccggcgcc acctcaggac cctcgccccg tatcagccca tcctgccctt 240
tgaggtgtta tctgctcgcc ttgggcgtag accagaggac ataatcaagt tggatgcaaa
300 tgagaatcca tatggtccac ccccggaggt cgctgcagca ctaggtagtc
tcaagttccc 360 ctatgtgtac cctgatcctg aaagccgcca attgcgtgct
gcccttgctg aagattctgg 420 acttgaatct g 431 2 139 PRT Zea mays
UNSURE (47) Xaa = ANY AMINO ACID 2 Met Asp Trp Ser Thr Arg Phe Arg
Ile Arg Asn Pro Ser Pro Ala Ala 1 5 10 15 Ser His Phe Ala Gly His
Arg Arg Ser Gln Ala Asp Arg Val Phe Leu 20 25 30 Arg Thr Met Ala
Ser Ala Ala Pro Val Glu Glu Pro Thr Ala Xaa Ala 35 40 45 Glu Ala
Lys Gly Arg Leu Thr Gly Asp Ser Phe Ile Arg Arg His Leu 50 55 60
Arg Thr Leu Ala Pro Tyr Gln Pro Ile Leu Pro Phe Glu Val Leu Ser 65
70 75 80 Ala Arg Leu Gly Arg Arg Pro Glu Asp Ile Ile Lys Leu Asp
Ala Asn 85 90 95 Glu Asn Pro Tyr Gly Pro Pro Pro Glu Val Ala Ala
Ala Leu Gly Ser 100 105 110 Leu Lys Phe Pro Tyr Val Tyr Pro Asp Pro
Glu Ser Arg Gln Leu Arg 115 120 125 Ala Ala Leu Ala Glu Asp Ser Gly
Leu Glu Ser 130 135 3 501 DNA Oryza sp. 3 cttacatgta agctcgtgcc
gaattcggca cgagcttaca cgagctcgca tccagagccc 60 gcaccggtcg
gccgcccact tcgtcgccgg cgagggggga cgccgccgcc cggcaacgtc 120
cagggtatcc ttccgctcca tggcgtcggc cgcttccgtg gaggagcctg ccgctgctgc
180 ggcggcggcg gctgagacga agaggggacc gagcggcgcc tccttcatcc
gggaacacct 240 caggagtctc gccccgtacc aagcccatcc tgcccttcga
ggtgttgtcc gctcggcttg 300 ggcgtaaacc agaggatata atcaagttgg
atgcaaatga aaatccatat ggtccacctc 360 cggaggtagc taaagcatta
ggaaatttga agtttcccta tgtgtacctg atctgaaagc 420 cgtcagttgc
gtgctgctct tgctgaagat tctggtcttg aatctgagta catacttgct 480
ggatgttggt gcaaatgaat t 501 4 162 PRT Oryza sp. UNSURE (88) Xaa =
ANY AMINO ACID 4 Leu His Val Ser Ser Cys Arg Ile Arg His Glu Leu
Thr Arg Ala Arg 1 5 10 15 Ile Gln Ser Pro His Arg Ser Ala Ala His
Phe Val Ala Gly Glu Gly 20 25 30 Gly Arg Arg Arg Pro Ala Thr Ser
Arg Val Ser Phe Arg Ser Met Ala 35 40 45 Ser Ala Ala Ser Val Glu
Glu Pro Ala Ala Ala Ala Ala Ala Ala Ala 50 55 60 Glu Thr Lys Arg
Gly Pro Ser Gly Ala Ser Phe Ile Arg Glu His Leu 65 70 75 80 Arg Ser
Leu Ala Pro Tyr Gln Xaa Ile Leu Pro Phe Glu Val Leu Ser 85 90 95
Ala Arg Leu Gly Arg Lys Pro Glu Asp Ile Ile Lys Leu Asp Ala Asn 100
105 110 Glu Asn Pro Tyr Gly Pro Pro Pro Glu Val Ala Lys Ala Leu Gly
Asn 115 120 125 Leu Lys Phe Pro Tyr Val Tyr Xaa Xaa Xaa Glu Ser Arg
Gln Leu Arg 130 135 140 Ala Ala Leu Ala Glu Asp Ser Gly Leu Glu Ser
Glu Tyr Ile Leu Ala 145 150 155 160 Gly Cys 5 529 DNA Glycine max
unsure (206) n=a,c,g or t 5 ccagcaacct ctgccaatct ttaatgggtg
tgattgattt ctacaacact ggtgctttgt 60 gctgggttaa gtccaacgcc
aatctgaagc agcaagtggg tttggcacca agacccattt 120 gttcatttga
ggggaataat cagaggaagt ttgtggcaat ggcttctacc gttcctgtgg 180
agcaagtcaa caatggcccc ctgcangtga caggtgactc cttcatcaga gagcatctga
240 ggaagttggc tccttatcag cccattttgc cctttgaggt tttatcagct
cgccttggac 300 gtaancctga ggatatcgtg aagttagang ctaatgaaaa
tcnttanggt ccccctccag 360 agtcatggaa agccctagga tcaatgnaat
tccccanatg tctatcctga acccagagag 420 ncngcnagat tgcgcgaagt
cttggcccat gaattcaggg ccttgaagct gaataatatt 480 cttgcagggt
gtngtgaaga nngngcctaa tgaatnngaa cangcgtaa 529 6 131 PRT Glycine
max UNSURE (68) Xaa = ANY AMINO ACID 6 Ser Asn Leu Cys Gln Ser Leu
Met Gly Val Ile Asp Phe Tyr Asn Thr 1 5 10 15 Gly Ala Leu Cys Trp
Val Lys Ser Asn Ala Asn Leu Lys Gln Gln Val 20 25 30 Gly Leu Ala
Pro Arg Pro Ile Cys Ser Phe Glu Gly Asn Asn Gln Arg 35 40 45 Lys
Phe Val Ala Met Ala Ser Thr Val Pro Val Glu Gln Val Asn Asn 50 55
60 Gly Pro Leu Xaa Val Thr Gly Asp Ser Phe Ile Arg Glu His Leu Arg
65 70 75 80 Lys Leu Ala Pro Tyr Gln Pro Ile Leu Pro Phe Glu Val Leu
Ser Ala 85 90 95 Arg Leu Gly Arg Xaa Pro Glu Asp Ile Val Lys Leu
Xaa Ala Asn Glu 100 105 110 Asn Xaa Xaa Gly Pro Pro Pro Glu Ser Trp
Lys Ala Leu Gly Ser Met 115 120 125 Xaa Phe Pro 130 7 151 DNA
Triticum sp. unsure (35) n=a,c,g or t 7 gggttatgga gcatttcctc
taagcattat tgagnactta tggcggncca agcagcctta 60 taatntttct
ntngcagcag aagtctctgc atgtgctgcc ttnnagaacc cagtctantt 120
gganagcgtg caaaatctgc tactacaaga g 151 8 50 PRT Triticum sp. UNSURE
(12) Xaa = ANY AMINO ACID 8 Gly Tyr Gly Ala Phe Pro Leu Ser Ile Ile
Glu Xaa Leu Trp Arg Xaa 1 5 10 15 Lys Gln Pro Tyr Asn Xaa Ser Xaa
Ala Ala Glu Val Ser Ala Cys Ala 20 25 30 Ala Xaa Xaa Asn Pro Val
Xaa Leu Xaa Ser Val Gln Asn Leu Leu Leu 35 40 45 Gln Glu 50 9 1338
DNA Zea mays unsure (1099) n=a,c,g or t 9 cgagtggcag cctcacgctc
actttaacga ccctttgcga cgccaaccgg ccaaagctcc 60 cggctcggcg
gcgccgcttc gttgatggat tggtccaccc ggttccgcat ccgaaacccg 120
tcgccggccg cgtcccactt tgccggccac aggcggagtc aagcggatag ggtatttctc
180 cggaccatgg cgtcggcggc cccggtggag gagccgacgg cggcggccga
ggcgaagggg 240 cggctgaccg gtgactcctt catccggcgc cacctcagga
ccctcgcccc gtatcagccc 300 atcctgccct ttgaggtgtt atctgctcgc
cttgggcgta gaccagagga cataatcaag 360 ttggatgcaa atgagaatcc
atatggtcca cccccggagg tcgctgcagc actaggtagt 420 ctcaagttcc
cctatgtgta ccctgatcct gaaagccgcc aattgcgtgc tgcccttgct 480
gaagattctg gacttgaatc tgattacata cttgctggat gtggcgcaga tgaactaatt
540 gatttaatta tgagatgtgt gcttgaacca ggcgacaaaa ttgttgattg
ccctccaaca 600 ttcacaatgt atgagttcga cgcttcagtc aatggtgcac
ttgttatcaa ggttccaaga 660 ctgcccgatt tttccctaga tgttgatctc
attgtcgaag tggttgaaca ggaaatgcca 720 aaatgcatat ttctgacatc
cccaaataat ccagatggca gtgtaatcaa tgatgaggat 780 cttttaaaga
tacttgatct cccaatactt gtagtgctgg atgaagctta tattgagttt 840
tcaagccttc agtcaagaat ggcatgggtt aagaagcatg ataatttgat tgttctccga
900 acatttagca aacgggcagg tttagctggt cttcgtgtgg gttatggtgc
atttcctctg 960 agcattatcg agtatttgtg gcgggccaag cagccctata
atgtttctgt ggccgcagaa 1020 gtttcagcat gtgcagcttt acagaatcca
acttatctgg agaatgtgaa aaatttactg 1080 gtaaaagaaa gggagaggnt
gtttaatctt ctcaagggaa taccattcct gaagccattt 1140 cccagtcatt
ctaacttcat tctctgcgag gtcacgtcag gaaaggatgc aaagaaaata 1200
aaggaagacc ttgcgaagat gggagtgatg atccgccact atgacaagaa ggaactgaaa
1260 ggctatattc gtatctcggt tgggaaaccc gagcacactg atgcactaat
gaagggcctg 1320 aatgcacttc gattgtga 1338 10 417 PRT Zea mays UNSURE
(339) Xaa = ANY AMINO ACID 10 Met Asp Trp Ser Thr Arg Phe Arg Ile
Arg Asn Pro Ser Pro Ala Ala 1 5 10 15 Ser His Phe Ala Gly His Arg
Arg Ser Gln Ala Asp Arg Val Phe Leu 20 25 30 Arg Thr Met Ala Ser
Ala Ala Pro Val Glu Glu Pro Thr Ala Ala Ala 35 40 45 Glu Ala Lys
Gly Arg Leu Thr Gly Asp Ser Phe Ile Arg Arg His Leu 50 55 60 Arg
Thr Leu Ala Pro Tyr Gln Pro Ile Leu Pro Phe Glu Val Leu Ser 65 70
75 80 Ala Arg Leu Gly Arg Arg Pro Glu Asp Ile Ile Lys Leu Asp Ala
Asn 85 90 95 Glu Asn Pro Tyr Gly Pro Pro Pro Glu Val Ala Ala Ala
Leu Gly Ser 100 105 110 Leu Lys Phe Pro Tyr Val Tyr Pro Asp Pro Glu
Ser Arg Gln Leu Arg 115 120 125 Ala Ala Leu Ala Glu Asp Ser Gly Leu
Glu Ser Asp Tyr Ile Leu Ala 130 135 140 Gly Cys Gly Ala Asp Glu Leu
Ile Asp Leu Ile Met Arg Cys Val Leu 145 150 155 160 Glu Pro Gly Asp
Lys Ile Val Asp Cys Pro Pro Thr Phe Thr Met Tyr 165 170 175 Glu Phe
Asp Ala Ser Val Asn Gly Ala Leu Val Ile Lys Val Pro Arg 180 185 190
Leu Pro Asp Phe Ser Leu Asp Val Asp Leu Ile Val Glu Val Val Glu 195
200 205 Gln Glu Met Pro Lys Cys Ile Phe Leu Thr Ser Pro Asn Asn Pro
Asp 210 215 220 Gly Ser Val Ile Asn Asp Glu Asp Leu Leu Lys Ile Leu
Asp Leu Pro 225 230 235 240 Ile Leu Val Val Leu Asp Glu Ala Tyr Ile
Glu Phe Ser Ser Leu Gln 245 250 255 Ser Arg Met Ala Trp Val Lys Lys
His Asp Asn Leu Ile Val Leu Arg 260 265 270 Thr Phe Ser Lys Arg Ala
Gly Leu Ala Gly Leu Arg Val Gly Tyr Gly 275 280 285 Ala Phe Pro Leu
Ser Ile Ile Glu Tyr Leu Trp Arg Ala Lys Gln Pro 290 295 300 Tyr Asn
Val Ser Val Ala Ala Glu Val Ser Ala Cys Ala Ala Leu Gln 305 310 315
320 Asn Pro Thr Tyr Leu Glu Asn Val Lys Asn Leu Leu Val Lys Glu Arg
325 330 335 Glu Arg Xaa Phe Asn Leu Leu Lys Gly Ile Pro Phe Leu Lys
Pro Phe 340 345 350 Pro Ser His Ser Asn Phe Ile Leu Cys Glu Val Thr
Ser Gly Lys Asp 355 360 365 Ala Lys Lys Ile Lys Glu Asp Leu Ala Lys
Met Gly Val Met Ile Arg 370 375 380 His Tyr Asp Lys Lys Glu Leu Lys
Gly Tyr Ile Arg Ile Ser Val Gly 385 390 395 400 Lys Pro Glu His Thr
Asp Ala Leu Met Lys Gly Leu Asn Ala Leu Arg 405 410 415 Leu 11 1605
DNA Oryza sativa 11 gcacgagctt acatgtaagc tcgtgccgaa ttcggcacga
gcttacacga gctcgcatcc 60 agagcccgca ccggtcggcc gcccacttcg
tcgccggcga ggggggacgc cgccgcccgg 120 caacgtccag ggtatccttc
cgctccatgg cgtcggccgc ttccgtggag gagcctgccg 180 ctgctgcggc
ggcggcggct gagacgaaga ggggaccgag cggcgcctcc ttcatccggg 240
aacacctcag gagtctcgcc ccgtaccagc ccatcctgcc cttcgaggtg ttgtccgctc
300 ggcttgggcg taaaccagag gatataatca agttggatgc aaatgaaaat
ccatatggtc 360 cacctccgga ggtagctaaa gcattaggaa atttgaagtt
tccctatgtg taccctgatc 420 ctgaaagccg tcagttgcgt gctgctcttg
ctgaagattc tggtcttgaa tctgagtaca 480 tacttgctgg atgtggtgca
gatgaattaa ttgatttaat aatgagatgt gtactcgaac 540 caggtgacaa
aattgttgat tgccctccaa cttttacgat gtatgagttt gatgcgtcag 600
tcaatggtgc acttgtgatc aaggtaccga gacttcctga tttttctcta gacgttgcac
660 agattgtcaa agtggttgaa caggaaaagc caaaatccat atttctgaca
tctccgaaca 720 acccagatgg cagcataatc aatgatgagg atcttttaaa
gatccttgat cttccaatac 780 ttgtagtgct ggatgaagca tatattgagt
tttcgagtct tcaaacaagg atgtcatggg 840 ttaagaagca tgataatttg
attgttcttc ggacatttag caaacgagca ggtttagctg 900 gacttcgtgt
gggttacgga gcatttcctc taagcataat cgagtattta tggagggcta 960
agcagcccta taatgtttct gtagcagcag aagtttcagc ctgtgctgcc ttgcagaacc
1020 cgacttattt agaggaagtg aaaaatctgc tactacaaga gagggacagg
ctgtacgatc 1080 ttctcaaaga aataccattc ctaaagccat ttcccagcca
ctctaacttt attctctgcg 1140 aggtcacatc aggcaaagat gcaaagaaaa
taaaggaaga ccttgcgaag atgggagtaa 1200 tgatccgcca ctatgacaag
aaggaactaa agggatatat tcgtatttca gtgggcaagc 1260 cagagcatac
cgatgcacta atgaaaggcc tgaaagcact tcaactgtga tcatcccatc 1320
tgtttgacgg aagcactgaa gcacttgccc gtggtagtgc actagatgca gtctctcaat
1380 ggaggttgca tcaatctaac acaaataagg tgcatcctct agggtcgatt
atgtctcaat 1440 aatacactct tctgttttga ccagtggcgt tttgtccagc
atttttgtgt tggtcgactt 1500 gggtttcttc tcaaggtgat tgttcgaagc
aagaatttgt actgccgtgc cctgattgga 1560 ataaatatga gcgtaaaagt
atggcaaaaa aaaaaaaaaa aaaaa 1605 12 435 PRT Oryza sativa 12 Thr Ser
Leu His Val Ser Ser Cys Arg Ile Arg His Glu Leu Thr Arg 1 5 10 15
Ala Arg Ile Gln Ser Pro His Arg Ser Ala Ala His Phe Val Ala Gly 20
25 30 Glu Gly Gly Arg Arg Arg Pro Ala Thr Ser Arg Val Ser Phe Arg
Ser 35 40 45 Met Ala Ser Ala Ala Ser Val Glu Glu Pro Ala Ala Ala
Ala Ala Ala 50 55 60 Ala Ala Glu Thr Lys Arg Gly Pro Ser Gly Ala
Ser Phe Ile Arg Glu 65 70 75 80 His Leu Arg Ser Leu Ala Pro Tyr Gln
Pro Ile Leu Pro Phe Glu Val 85 90 95 Leu Ser Ala Arg Leu Gly Arg
Lys Pro Glu Asp Ile Ile Lys Leu Asp 100 105 110 Ala Asn Glu Asn Pro
Tyr Gly Pro Pro Pro Glu Val Ala Lys Ala Leu 115 120 125 Gly Asn Leu
Lys Phe Pro Tyr Val Tyr Pro Asp Pro Glu Ser Arg Gln 130 135 140 Leu
Arg Ala Ala Leu Ala Glu Asp Ser Gly Leu Glu Ser Glu Tyr Ile 145 150
155 160 Leu Ala Gly Cys Gly Ala Asp Glu Leu Ile Asp Leu Ile Met Arg
Cys 165 170 175 Val Leu Glu Pro Gly Asp Lys Ile Val Asp Cys Pro Pro
Thr Phe Thr 180 185 190 Met Tyr Glu Phe Asp Ala Ser Val Asn Gly Ala
Leu Val Ile Lys Val 195 200 205 Pro Arg Leu Pro Asp Phe Ser Leu Asp
Val Ala Gln Ile Val Lys Val 210 215 220 Val Glu Gln Glu Lys Pro Lys
Ser Ile Phe Leu Thr Ser Pro Asn Asn 225 230 235 240 Pro Asp Gly Ser
Ile Ile Asn Asp Glu Asp Leu Leu Lys Ile Leu Asp 245 250 255 Leu Pro
Ile Leu Val Val Leu Asp Glu Ala Tyr Ile Glu Phe Ser Ser 260 265 270
Leu Gln Thr Arg Met Ser Trp Val Lys Lys His Asp Asn Leu Ile Val 275
280 285 Leu Arg Thr Phe Ser Lys Arg Ala Gly Leu Ala Gly Leu Arg Val
Gly 290 295 300 Tyr Gly Ala Phe Pro Leu Ser Ile Ile Glu Tyr Leu Trp
Arg Ala Lys 305 310 315 320 Gln Pro Tyr Asn Val Ser Val Ala Ala Glu
Val Ser Ala Cys Ala Ala 325 330 335 Leu Gln Asn Pro Thr Tyr Leu Glu
Glu Val Lys Asn Leu Leu Leu Gln 340 345 350 Glu Arg Asp Arg Leu Tyr
Asp Leu Leu Lys Glu Ile Pro Phe Leu Lys 355 360 365 Pro Phe Pro Ser
His Ser Asn Phe Ile Leu Cys Glu Val Thr Ser Gly 370 375 380 Lys Asp
Ala Lys Lys Ile Lys Glu Asp Leu Ala Lys Met Gly Val Met 385 390 395
400 Ile Arg His Tyr Asp Lys Lys Glu Leu Lys Gly Tyr Ile Arg Ile Ser
405 410 415 Val Gly Lys Pro Glu His Thr Asp Ala Leu Met Lys Gly Leu
Lys Ala 420 425 430 Leu Gln Leu 435 13 1476 DNA Glycine max 13
gcacgagcca gcaacctctg ccaatcttta atgggtgtga ttgatttcta caacactggt
60 gctttgtgct gggttaagtc caacgccaat ctgaagcagc aagtgggttt
ggcaccaaga 120 cccatttgtt catttgaggg gaataatcag aggaagtttg
tggcaatggc ttctaccgtt 180 cctgtggagc aagtcaacaa tggccccctg
caggtgacag gtgactcctt catcagagag 240 catctgagga agttggctcc
ttatcagccc attttgccct ttgaggtttt atcagctcgc 300 cttggacgta
agcctgagga tatcgtgaag ttagatgcta atgaaaatcc ttatggtccc 360
cctccagagg tcatggaagc cctaggatca atgcaattcc catatgtcta tcctgaccca
420 gagagccgca gattgcgcgc agctcttgcc catgattcag gccttgaagc
tgaatatatt 480 cttgcagggt gtggtgcaga tgagcttatt gatttgatca
tgcgttgtgt gctggatcct 540 ggagacaaga ttgtggactg ccctccgacc
ttcacaatgt atgaatttga tgctgcggtt 600 aatggagcac ttgttatcaa
agttccaagg aggccagatt tcagcttgaa tgttgaacaa 660 attgctgaag
ttgttaaaca agagaagccc aaatgcatat ttttaacatc tccaaataat 720
ccagatggaa gtataattga tgacgaagtt ctcttaaaaa tactcgagct tcctatattg
780 gtgatactgg atgaagcata cattgagttt tcagcaattg aatcaaggat
gagttgggtg 840 aagaaacatg ataatttgat tgttcttcgg acatttagca
aaagagctgg tttagctgga 900 cttcgagtgg gatatggagc ttttcctttg
agtataattg agtatctttg gagagcaaag 960 cagccgtata atgtatctgt
tgctgctgaa atttctgcat gtgcagcatt gcaaaatcct 1020 acctatctag
agaatgtaaa aaatgctttg ttgaaagaaa gagggagact ttatgacctt 1080
ttgaaagaag ttccattcct ccggccattt ccaagccatt ctaacttcat tctttgtgag
1140 gttacatcag gaaaggatgc aaagaagcta aaggaggacc tagcacaaat
gggtgtgatg 1200 attcgtcact atgacaagaa agagctgaaa gggtacgttc
gtgtgactgt tgggaagcct 1260 gaacaaacag atacacttat gaagtgcctc
aagagactct cgtaggagga aaatttgatg 1320 taataaatat tgtaacacgt
catgctaaac tcctcttagc taatctttat atagagccgt 1380 caaaattaga
agaaaatatg ttgattttgg caagggatgt ggatgtagct ttatatatta 1440
ttgacctaaa tctaccatga taaatattgt gttttg 1476 14 434 PRT Glycine max
14 Ala Arg Ala Ser Asn Leu Cys Gln Ser Leu Met Gly Val Ile Asp Phe
1 5 10 15 Tyr Asn Thr Gly Ala Leu Cys Trp Val Lys
Ser Asn Ala Asn Leu Lys 20 25 30 Gln Gln Val Gly Leu Ala Pro Arg
Pro Ile Cys Ser Phe Glu Gly Asn 35 40 45 Asn Gln Arg Lys Phe Val
Ala Met Ala Ser Thr Val Pro Val Glu Gln 50 55 60 Val Asn Asn Gly
Pro Leu Gln Val Thr Gly Asp Ser Phe Ile Arg Glu 65 70 75 80 His Leu
Arg Lys Leu Ala Pro Tyr Gln Pro Ile Leu Pro Phe Glu Val 85 90 95
Leu Ser Ala Arg Leu Gly Arg Lys Pro Glu Asp Ile Val Lys Leu Asp 100
105 110 Ala Asn Glu Asn Pro Tyr Gly Pro Pro Pro Glu Val Met Glu Ala
Leu 115 120 125 Gly Ser Met Gln Phe Pro Tyr Val Tyr Pro Asp Pro Glu
Ser Arg Arg 130 135 140 Leu Arg Ala Ala Leu Ala His Asp Ser Gly Leu
Glu Ala Glu Tyr Ile 145 150 155 160 Leu Ala Gly Cys Gly Ala Asp Glu
Leu Ile Asp Leu Ile Met Arg Cys 165 170 175 Val Leu Asp Pro Gly Asp
Lys Ile Val Asp Cys Pro Pro Thr Phe Thr 180 185 190 Met Tyr Glu Phe
Asp Ala Ala Val Asn Gly Ala Leu Val Ile Lys Val 195 200 205 Pro Arg
Arg Pro Asp Phe Ser Leu Asn Val Glu Gln Ile Ala Glu Val 210 215 220
Val Lys Gln Glu Lys Pro Lys Cys Ile Phe Leu Thr Ser Pro Asn Asn 225
230 235 240 Pro Asp Gly Ser Ile Ile Asp Asp Glu Val Leu Leu Lys Ile
Leu Glu 245 250 255 Leu Pro Ile Leu Val Ile Leu Asp Glu Ala Tyr Ile
Glu Phe Ser Ala 260 265 270 Ile Glu Ser Arg Met Ser Trp Val Lys Lys
His Asp Asn Leu Ile Val 275 280 285 Leu Arg Thr Phe Ser Lys Arg Ala
Gly Leu Ala Gly Leu Arg Val Gly 290 295 300 Tyr Gly Ala Phe Pro Leu
Ser Ile Ile Glu Tyr Leu Trp Arg Ala Lys 305 310 315 320 Gln Pro Tyr
Asn Val Ser Val Ala Ala Glu Ile Ser Ala Cys Ala Ala 325 330 335 Leu
Gln Asn Pro Thr Tyr Leu Glu Asn Val Lys Asn Ala Leu Leu Lys 340 345
350 Glu Arg Gly Arg Leu Tyr Asp Leu Leu Lys Glu Val Pro Phe Leu Arg
355 360 365 Pro Phe Pro Ser His Ser Asn Phe Ile Leu Cys Glu Val Thr
Ser Gly 370 375 380 Lys Asp Ala Lys Lys Leu Lys Glu Asp Leu Ala Gln
Met Gly Val Met 385 390 395 400 Ile Arg His Tyr Asp Lys Lys Glu Leu
Lys Gly Tyr Val Arg Val Thr 405 410 415 Val Gly Lys Pro Glu Gln Thr
Asp Thr Leu Met Lys Cys Leu Lys Arg 420 425 430 Leu Ser 15 845 DNA
Triticum aestivum 15 agatccttga ccttccggta cttgtagtgc tggacgaagc
ttatgttgaa ttttcgagcc 60 ttcaatcaag gatgacatgg gttaagaagc
atgataattt gattgtcctt cgaacattta 120 gcaaacgagc aggtttagct
gggcttcgtg tgggttatgg agcatttcct ctaagcatta 180 ttgagtactt
atggcgggcc aagcagcctt ataatgtttc tgtggcagca gaagtctctg 240
catgtgctgc cttgcagaac ccagtctatt tggagagcgt gaaaaatctg ctactacaag
300 agagggagag gctgtataat cttctcaaag gaatacctta cctgaaacca
tttcccagtc 360 atgctaactt cattctgtgt gaagtcacgt caggaaaaga
tgcaaagaaa ataaaggagg 420 atcttgcaaa gatgggagtg atgatccgcc
actacgacaa gaaggaactg aagggttata 480 ttcgtatttc agttggaaag
cctgagcaca ctgatgcact gatggaaggc ttcaaagcac 540 tcaaactttg
agaatttgcc atgatttact ttgatggaag cagtgaagag cttattgagt 600
atgtgtctac ccattactag gcttgtagta cactggatgc agtctatcaa ttagacactg
660 cttccctcca acatcggtaa agtgcattct tcagatttca agccaaccag
ggtcaattag 720 ttttgaataa aaatatctat gtttaactag tgctgtaggt
ccaaccattt agccataaac 780 tctgtgtcag caaagttact gtgcagagca
agactttttt taaaaaaaaa aaaaaaaaaa 840 aaaaa 845 16 182 PRT Triticum
aestivum 16 Ile Leu Asp Leu Pro Val Leu Val Val Leu Asp Glu Ala Tyr
Val Glu 1 5 10 15 Phe Ser Ser Leu Gln Ser Arg Met Thr Trp Val Lys
Lys His Asp Asn 20 25 30 Leu Ile Val Leu Arg Thr Phe Ser Lys Arg
Ala Gly Leu Ala Gly Leu 35 40 45 Arg Val Gly Tyr Gly Ala Phe Pro
Leu Ser Ile Ile Glu Tyr Leu Trp 50 55 60 Arg Ala Lys Gln Pro Tyr
Asn Val Ser Val Ala Ala Glu Val Ser Ala 65 70 75 80 Cys Ala Ala Leu
Gln Asn Pro Val Tyr Leu Glu Ser Val Lys Asn Leu 85 90 95 Leu Leu
Gln Glu Arg Glu Arg Leu Tyr Asn Leu Leu Lys Gly Ile Pro 100 105 110
Tyr Leu Lys Pro Phe Pro Ser His Ala Asn Phe Ile Leu Cys Glu Val 115
120 125 Thr Ser Gly Lys Asp Ala Lys Lys Ile Lys Glu Asp Leu Ala Lys
Met 130 135 140 Gly Val Met Ile Arg His Tyr Asp Lys Lys Glu Leu Lys
Gly Tyr Ile 145 150 155 160 Arg Ile Ser Val Gly Lys Pro Glu His Thr
Asp Ala Leu Met Glu Gly 165 170 175 Phe Lys Ala Leu Lys Leu 180
* * * * *
References