U.S. patent application number 10/185591 was filed with the patent office on 2003-09-04 for plant histidine biosynthetic enzymes.
Invention is credited to Allen, Stephen M., Hitz, William D..
Application Number | 20030167509 10/185591 |
Document ID | / |
Family ID | 22305684 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030167509 |
Kind Code |
A1 |
Allen, Stephen M. ; et
al. |
September 4, 2003 |
Plant histidine biosynthetic enzymes
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) ; Hitz, William D.; (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: |
22305684 |
Appl. No.: |
10/185591 |
Filed: |
June 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10185591 |
Jun 27, 2002 |
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09425578 |
Oct 22, 1999 |
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6441271 |
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60105409 |
Oct 23, 1998 |
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Current U.S.
Class: |
800/278 ;
435/191; 435/193; 435/320.1; 435/419; 536/23.2 |
Current CPC
Class: |
C12N 9/88 20130101; C12N
15/8251 20130101 |
Class at
Publication: |
800/278 ;
435/193; 435/191; 435/419; 435/320.1; 536/23.2 |
International
Class: |
A01H 005/00; C07H
021/04; C12N 009/06; C12N 009/10; C12N 005/04 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide having imidazoleglycerol-phosphate
dehydratase activity, wherein the amino acid sequence of the
polypeptide and the amino acid sequence of SEQ ID NO:4 have at
least 96% sequence identity based on the Clustal alignment method,
or (b) the complement of the nucleotide sequence, wherein the
complement contains the same number of nucleotides as the
nucleotide sequence, and the complement and the nucleotide sequence
are 100% complementary.
2. The polynucleotide of claim 1 comprising the nucleotide sequence
of SEQ ID NO:3.
3. The polynucleotide of claim 1, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO:4.
4. A recombinant DNA construct comprising the polynucleotide of
claim 1 operably linked to a regulatory sequence.
5. A vector comprising the polynucleotide of claim 1.
6. A method for transforming a cell comprising transforming a cell
with the polynucleotide of claim 1.
7. A cell comprising the recombinant DNA construct of claim 4.
8. 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.
9. A plant comprising the recombinant DNA construct of claim 4.
10. A seed comprising the recombinant DNA construct of claim 4.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/105,409, filed Oct. 23, 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 histidine biosynthetic enzymes 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, 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-carb- oxamide 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). These results indicate 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
and thereby have a detrimental effect on plant growth and
development. The enzyme encoded by hisB,
imidazoleglycerol-phosphate dehydratase 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)gl- ycerol 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. Finally, 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
modem 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 210 amino acids that has at least 96% identity based on the
Clustal method of alignment when compared to a polypeptide selected
from the group consisting of a corn polypeptide of SEQ ID NO:2, a
rice polypeptide of SEQ ID NO:4, and a soybean polypeptide of SEQ
ID NO:6. The present invention also relates to an isolated
polynucleotide comprising the complement of the nucleotide
sequences described above.
[0009] It is preferred that the isolated polynucleotides of the
claimed invention consists of a nucleic acid sequence selected from
the group consisting of SEQ ID NOs:1, 3 and 5 that codes for the
polypeptide selected from the group consisting of SEQ ID NOs:2, 4
and 6. 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 and 5 and the complement of such nucleotide
sequences.
[0010] The present invention relates to a chimeric gene comprising
an isolated polynucleotide of the present invention operably linked
to suitable regulatory sequences.
[0011] The present invention relates to an isolated host cell
comprising a chimeric gene of the present invention or an isolated
polynucleotide of the present invention. The host cell may be
eukaryotic, such as a yeast or a plant cell, or prokaryotic, such
as a bacterial cell or a virus. If the host cell is a virus, it is
preferably a baculovirus. A baculovirus comprising an isolated
polynucleotide of the present invention or a chimeric gene of the
present invention is most preferred.
[0012] The present invention relates to a process for producing an
isolated host cell comprising a chimeric gene of the present
invention or an isolated polynucleotide of the present invention,
the process comprising either transforming or transfecting an
isolated compatible host cell with a chimeric gene or isolated
polynucleotide of the present invention.
[0013] The present invention relates to a
imidazoleglycerol-phosphate dehydratase polypeptide of at least 266
amino acids comprising at least 96% homology based on the Clustal
method of alignment compared to a polypeptide selected from the
group consisting of SEQ ID NOs:2, 4 and 6.
[0014] The present invention relates to a method of selecting an
isolated polynucleotide that affects the level of expression of a
imidazoleglycerol-phosphate dehydratase polypeptide in a plant
cell, the method comprising the steps of:
[0015] constructing an isolated polynucleotide of the present
invention or an isolated chimeric gene of the present
invention;
[0016] introducing the isolated polynucleotide or the isolated
chimeric gene into a plant cell;
[0017] measuring the level an imidazoleglycerol-phosphate
dehydratase polypeptide in the plant cell containing the isolated
polynucleotide; and
[0018] comparing the level of an imidazoleglycerol-phosphate
dehydratase polypeptide in the plant cell containing the isolated
polynucleotide with the level of an imidazoleglycerol-phosphate
dehydratase polypeptide in a plant cell that does not contain the
isolated polynucleotide.
[0019] The present invention relates to a method of obtaining a
nucleic acid fragment encoding a substantial portion of an
imidazoleglycerol-phosphate dehydratase polypeptide gene,
preferably a plant imidazoleglycerol-phosphate dehydratase
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 and 5 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 an imidazoleglycerol-phosphate
dehydratase amino acid sequence.
[0020] The present invention also relates to a method of obtaining
a nucleic acid fragment encoding all or a subsantial portion of the
amino acid sequence encoding an imidazoleglycerol-phosphate
dehydratase 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.
[0021] A further embodiment of the instant invention is a method
for evaluating at least one compound for its ability to inhibit the
activity of an imidazoleglycerol-phosphate dehydratase, the method
comprising the steps of: (a) transforming a host cell with a
chimeric gene comprising a nucleic acid fragment encoding an
imidazoleglycerol-phosphate dehydratase, 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 imidazoleglycerol-phosphate dehydratase in the
transformed host cell; (c) optionally purifying the
imidazoleglycerol-phosphate dehydratase expressed by the
transformed host cell; (d) treating the imidazoleglycerol-phosphate
dehydratase with a compound to be tested; and (e) comparing the
activity of the imidazoleglycerol-phosphate dehydratase that has
been treated with a test compound to the activity of an untreated
imidazoleglycerol-phosphate dehydratase, thereby selecting
compounds with potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
[0022] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0023] Table 1 lists the polypeptides that are described herein,
the designation of the cDNA clones that comprise the nucleic acid
fragments encoding polypeptides representing all or a substantial
portion of these polypeptides, and the corresponding identifier
(SEQ ID NO:) as used in the attached Sequence Listing. The sequence
descriptions and Sequence Listing attached hereto comply with the
rules governing nucleotide and/or amino acid sequence disclosures
in patent applications as set forth in 37 C.F.R.
.sctn.1.821-1.825.
1TABLE 1 Histidine Biosynthetic Enzymes SEQ ID NO: Protein Clone
Designation (Nucleotide) (Amino Acid) Imidazoleglycerol-
cen3n.pk0093.c4 1 2 Phosphate Dehy- dratase, HisB
Imidazoleglycerol- rlr24.pk0016.b1 3 4 Phosphate Dehy- dratase,
HisB Imidazoleglycerol- ssm.pk0014.f1 5 6 Phosphate Dehy- dratase,
HisB
[0024] 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
[0025] 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 40 contiguous nucleotides, preferably
at least one of 30 contiguous nucleotides, most preferably one of
at least 15 contiguous nucleotides, of the nucleic acid sequence of
the SEQ ID NOs:1, 3 and 5.
[0026] 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.
[0027] 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 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 cell. The level of the polypeptide
encoded by the unmodified nucleic acid fragment present in a plant
cell exposed to the substantially similar nucleic fragment can then
be compared to the level of the polypeptide in a plant cell that is
not exposed to the substantially similar nucleic acid fragment. 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 40 (preferably at least one of 30, most preferably at
least one of 15) contiguous nucleotides derived from a nucleotide
sequence selected from the group consisting of SEQ ID NOs:1, 3 and
5 and the complement of such nucleotide sequences may be used in
methods of selecting an isolated polynucleotide that affects the
expression of a polypeptide in a plant 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
prokarotic such as yeast bacterial or virus) 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.
[0028] 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.
[0029] 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 80% identical to the amino acid sequences
reported herein. Preferred nucleic acid fragments encode amino acid
sequences that are 85% identical to the amino acid sequences
reported herein. More preferred nucleic acid fragments encode amino
acid sequences that are 90% identical to the amino acid sequences
reported herein. Most preferred are nucleic acid fragments that
encode amino acid sequences that are 95% identical to the amino
acid sequences reported herein. Suitable nucleic acid fragments not
only have the above homologies but typically encode a polypeptide
having at least 50 amino acids, preferably 100 amino acids, more
preferably 150 amino acids, still more preferably 200 amino acids,
and most preferably 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.
[0030] 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.
[0031] "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.
[0032] "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.
[0033] "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.
[0034] "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.
[0035] "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.
[0036] 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).
[0037] 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.
[0038] "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.
[0039] 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.
[0040] 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).
[0041] "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.
[0042] "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.
[0043] 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).
[0044] "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).
[0045] 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").
[0046] 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).
[0047] For example, genes encoding other
imidazoleglycerol-phosphate dehydratase, 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.
[0048] 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 nuclcic 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 40 (preferably one of at least 30, most
preferably one of at least 15) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs:1, 3 and 5 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 imidazoleglycerol-phosphate dehydratase) 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 40 (preferably at least one
of 30, most preferably at least one of 15) contiguous nucleotides
derived from a nucleotide sequence selected from the group
consisting of SEQ ID NOs:1, 3 and 5, 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.
[0049] 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).
[0050] 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
altering histidine biosynthesis in those cells.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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 enzymes. An example of a vector for high
level expression of the instant polypeptides in a bacterial host is
provided (Example 6).
[0058] 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.
[0059] 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).
[0060] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky 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.
[0061] 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).
[0062] 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.
[0063] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0064] 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
[0065] 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
[0066] cDNA libraries representing mRNAs from various corn, rice
and soybean tissues were prepared. The characteristics of the
libraries are described below.
2TABLE 2 cDNA Libraries from Corn, Rice and Soybean Library Tissue
Clone cen3n Corn endosperm 20 days after pollination*
cen3n.pk0093.c4 rlr24 Rice leaf 15 days after germination, 24 hours
rlr24.pk0016.b1 after infection of strain Magaporthe grisea
(4360-R-67) ssm Soybean shoot meristem ssm.pk0014.f1 *This library
was normalized essentially as described in U.S. Pat. No. 5,482,845,
incorporated herein by reference.
[0067] 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
[0068] 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
Imidazoleglycerol-Phosphate Dehydratase
[0069] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to imidazoleglycerol-phosphate dehydratase from Triticum
aestivum (NCBI Identifier No. gi 551331) and Pisum sativum (NCBI
Identifier No. gi 1381086). 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 Triticum aestivum and Pisum sativum
Imidazoleglycerol- Phosphate Dehydratase Clone Status BLAST pLog
Score cen3n.pk0093.c4 FIS 103.00 (gi 551331) rlr24.pk0016.b1 FIS
104.00 (gi 551331) ssm.pk0014.f1 FIS 117.00 (gi 1381086)
[0070] The data in Table 4 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs:2, 4
and 6 and the Triticum aestivum and Pisum sativum sequences.
4TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Triticum aestivum and Pisum sativum
Imidazoleglycerol- Phosphate Dehydratase SEQ ID NO. Percent
Identity to 2 94% (gi 551331) 4 95% (gi 551331) 6 80% (gi
1381086)
[0071] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of an
imidazoleglycerol-phosphate dehydratase. These sequences represent
the first corn, rice and soybean sequences encoding
imidazoleglycerol-phospha- te dehydratase.
Example 4
Expression of Chimeric Genes in Monocot Cells
[0072] A chimeric gene comprising a cDNA encoding the instant
polypeptides in sense orientation with respect to the maize 27 kD
zein promoter that is located 5' to the cDNA fragment, and the 10
kD zein 3' end that is located 3' to the cDNA fragment, can be
constructed. The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites (NcoI or SmaI) can be
incorporated into the oligonucleotides to provide proper
orientation of the DNA fragment when inserted into the digested
vector pML103 as described below. Amplification is then performed
in a standard PCR. The amplified DNA is then digested with
restriction enzymes NcoI and SmaI and fractionated on an agarose
gel. The appropriate band can be isolated from the gel and combined
with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid
pML103 has been deposited under the terms of the Budapest Treaty at
ATCC (American Type Culture Collection, 10801 University Blvd.,
Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter
fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI
fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at
15.degree. C. overnight, essentially as described (Maniatis). The
ligated DNA may then be used to transform E. coli XL1-Blue
(Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial transformants
can be screened by restriction enzyme digestion of plasmid DNA and
limited nucleotide sequence analysis using the dideoxy chain
termination method (Sequenase.TM. DNA Sequencing Kit; U.S.
Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD
zein promoter, a cDNA fragment encoding the instant polypeptides,
and the 10 kD zein 3' region.
[0073] The chimeric gene described above can then be introduced
into corn cells by the following procedure. Immature corn embryos
can be dissected from developing caryopses derived from crosses of
the inbred corn lines H99 and LH132. The embryos are isolated 10 to
11 days after pollination when they are 1.0 to 1.5 mm long. The
embryos are then placed with the axis-side facing down and in
contact with agarose-solidified N6 medium (Chu et al. (1975) Sci.
Sin. Peking 18:659-668). The embryos are kept in the dark at
27.degree. C. Friable embryogenic callus consisting of
undifferentiated masses of cells with somatic proembryoids and
embryoids borne on suspensor structures proliferates from the
scutellum of these immature embryos. The embryogenic callus
isolated from the primary explant can be cultured on N6 medium and
sub-cultured on this medium every 2 to 3 weeks.
[0074] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst
Ag, Frankfurt, Germany) may be used in transformation experiments
in order to provide for a selectable marker. This plasmid contains
the Pat gene (see European Patent Publication 0 242 236) which
encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT
confers resistance to herbicidal glutamine synthetase inhibitors
such as phosphinothricin. The pat gene in 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.
[0075] The particle bombardment method (Klein et al. (1987) Nature
327:70-73) may be used to transfer genes to the callus culture
cells. According to this method, gold particles (1 .mu.m in
diameter) are coated with DNA using the following technique. Ten
.mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold
particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M
solution) and spermidine free base (20 .mu.L of a 1.0 M solution)
are added to the particles. The suspension is vortexed during the
addition of these solutions. After 10 minutes, the tubes are
briefly centrifuged (5 sec at 15,000 rpm) and the supernatant
removed. The particles are resuspended in 200 .mu.L of absolute
ethanol, centrifuged again and the supernatant removed. The ethanol
rinse is performed again and the particles resuspended in a final
volume of 30 .mu.L of ethanol. An aliquot (5 .mu.L) of the
DNA-coated gold particles can be placed in the center of a
Kapton.TM. flying disc (Bio-Rad Labs). The particles are then
accelerated into the corn tissue with a Biolistic.TM. PDS-1000/He
(Bio-Rad Instruments, Hercules Calif.), using a helium pressure of
1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0
cm.
[0076] For bombardment, the embryogenic tissue is placed on filter
paper over agarose-solidified N6 medium. The tissue is arranged as
a thin lawn and covered a circular area of about 5 cm in diameter.
The petri dish containing the tissue can be placed in the chamber
of the PDS-1000/He approximately 8 cm from the stopping screen. The
air in the chamber is then evacuated to a vacuum of 28 inches of
Hg. The macrocarrier is accelerated with a helium shock wave using
a rupture membrane that bursts when the He pressure in the shock
tube reaches 1000 psi.
[0077] Seven days after bombardment the tissue can be transferred
to N6 medium that contains gluphosinate (2 mg per liter) and lacks
casein or proline. The tissue continues to grow slowly on this
medium. After an additional 2 weeks the tissue can be transferred
to fresh N6 medium containing gluphosinate. After 6 weeks, areas of
about 1 cm in diameter of actively growing callus can be identified
on some of the plates containing the glufosinate-supplemented
medium. These calli may continue to grow when sub-cultured on the
selective medium.
[0078] Plants can be regenerated from the transgenic callus by
first transferring clusters of tissue to N6 medium supplemented
with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be
transferred to regeneration medium (Fromm et al. (1990)
Bio/Technology 8:833-839).
Example 5
Expression of Chimeric Genes in Dicot Cells
[0079] A seed-specific expression cassette composed of the promoter
and transcription terminator from the gene encoding the .beta.
subunit of the seed storage protein phaseolin from the bean
Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem.
261:9228-9238) can be used for expression of the instant
polypeptides in transformed soybean. The phaseolin cassette
includes about 500 nucleotides upstream (5') from the translation
initiation codon and about 1650 nucleotides downstream (3') from
the translation stop codon of phaseolin. Between the 5' and 3'
regions are the unique restriction endonuclease sites Nco I (which
includes the ATG translation initiation codon), Sma I, Kpn I and
Xba I. The entire cassette is flanked by Hind III sites.
[0080] The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the expression vector. Amplification is then
performed as described above, and the isolated fragment is inserted
into a pUC18 vector carrying the seed expression cassette.
[0081] Soybean embroys may then be transformed with the expression
vector comprising sequences encoding the instant polypeptides. To
induce somatic embryos, cotyledons, 3-5 mm in length dissected from
surface sterilized, immature seeds of the soybean cultivar A2872,
can be cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0082] Soybean embryogenic suspension cultures can maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0083] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic.TM. PDS 1000/HE instrument (helium retrofit) can
be used for these transformations.
[0084] A selectable marker gene which can be used to facilitate
soybean transformation is a chimeric gene composed of the 35S
promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The seed expression cassette
comprising the phaseolin 5' region, the fragment encoding the
instant polypeptides and the phaseolin 3' region can be isolated as
a restriction fragment. This fragment can then be inserted into a
unique restriction site of the vector carrying the marker gene.
[0085] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.l
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
.mu.L of the DNA-coated gold particles are then loaded on each
macro carrier disk.
[0086] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0087] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media can be refreshed weekly. Seven to eight weeks post
bombardment, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
is removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line may be treated as an independent transformation
event. These suspensions can then be subcultured and maintained as
clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
Example 6
Expression of Chimeric Genes in Microbial Cells
[0088] The cDNAs encoding the instant polypeptides can be inserted
into the T7 E. coli expression vector pBT430. This vector is a
derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135)
which employs the bacteriophage T7 RNA polymerase/T7 promoter
system. Plasmid pBT430 was constructed by first destroying the EcoR
I and Hind III sites in pET-3a at their original positions. An
oligonucleotide adaptor containing EcoR I and Hind III sites was
inserted at the BamH I site of pET-3a. This created pET-3aM with
additional unique cloning sites for insertion of genes into the
expression vector. Then, the Nde I site at the position of
translation initiation was converted to an Nco I site using
oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM
in this region, 5'-CATATGG, was converted to 5'-CCCATGG in
pBT430.
[0089] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% NuSieve GTG.TM. low melting
agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium
bromide for visualization of the DNA fragment. The fragment can
then be purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20
.mu.L of water. Appropriate oligonucleotide adapters may be ligated
to the fragment using T4 DNA ligase (New England Biolabs, Beverly,
Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the instant polypeptides are then screened for the
correct orientation with respect to the T7 promoter by restriction
enzyme analysis.
[0090] For high level expression, a plasmid clone with the cDNA
insert in the correct orientation relative to the T7 promoter can
be transformed into E. coli strain BL21(DE3) (Studier et al. (1986)
J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 nm of approximately 1, IPTG
(isopropylthio-.beta.-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3
h at 25.degree.. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
Example 7
Evaluating Compounds for Their Ability to Inhibit the Activity of
Histidine Biosynthetic Enzymes
[0091] The polypeptides described herein may be produced using any
number of methods known to those skilled in the art. Such methods
include, but are not limited to, expression in bacteria as
described in Example 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.
[0092] Purification of the instant polypeptides, if desired, may
utilize any number of separation technologies familiar to those
skilled in the art of protein purification. Examples of such
methods include, but are not limited to, homogenization,
filtration, centrifugation, heat denaturation, ammonium sulfate
precipitation, desalting, pH precipitation, ion exchange
chromatography, hydrophobic interaction chromatography and affinity
chromatography, wherein the affinity ligand represents a substrate,
substrate analog or inhibitor. When the instant polypeptides are
expressed as fusion proteins, the purification protocol may include
the use of an affinity resin which is specific for the fusion
protein tag attached to the expressed enzyme or an affinity resin
containing ligands which are specific for the enzyme. For example,
the instant polypeptides may be expressed as a fusion protein
coupled to the C-terminus of thioredoxin. In addition, a
(His).sub.6 peptide may be engineered into the N-terminus of the
fused thioredoxin moiety to afford additional opportunities for
affinity purification. Other suitable affinity resins could be
synthesized by linking the appropriate ligands to any suitable
resin such as Sepharose-4B. In an alternate embodiment, a
thioredoxin fusion protein may be eluted using dithiothreitol;
however, elution may be accomplished using other reagents which
interact to displace the thioredoxin from the resin. These reagents
include .beta.-mercaptoethanol or other reduced thiol. The eluted
fusion protein may be subjected to further purification by
traditional means as stated above, if desired. Proteolytic cleavage
of the thioredoxin fusion protein and the enzyme may be
accomplished after the fusion protein is purified or while the
protein is still bound to the ThioBond.TM. affinity resin or other
resin.
[0093] Crude, partially purified or purified enzyme, either alone
or as a fusion protein, may be utilized in assays for the
evaluation of compounds for their ability to inhibit enzymatic
activation of the instant polypeptides disclosed herein. Assays may
be conducted under well known experimental conditions which permit
optimal enzymatic activity.
Sequence CWU 1
1
6 1 1294 DNA Zea mays 1 gcacgagccg ttgtccactg cctccaggca aaccggcagc
gccgccgccg gcgatgacca 60 ccgcgcggct cgtctcaaca tcccccgccc
gcctaccctc cacgtcggct tccccgattc 120 ccagaacctc gctgtgtgtt
gtcggtcgtg cccctgcttt ctccgctagg gcgctcggct 180 tctcgcttcg
tctcaagccg tcgcctgcca tggccgccgc cggcccccct accgcgcatg 240
gagactcaat aggatcttct aggattggag aggttaagag ggtgaccaaa gagacaaatg
300 tgcacgtgaa gatcaacctt gatggcactg gtgtcgccga gtgtagcaca
gggatactgt 360 tcttggatca catgctcgat cagttggcat cacatggact
ctttgatgtc tacgttaaag 420 caataggcga cacccatatt gatgatcatc
actcaaatga ggacattgct ttggcaattg 480 gaacggcact acttgaagca
cttggtgatc gaaagggaat taaccgattt ggtcatttta 540 cagcaccgct
tgatgaggca gcggttgagg ttatactgga tctgtctggt cgccctcatt 600
taagctgtgg cttagatatt cctactcaaa gagttggcac atatgataca cagctggttg
660 agcacttctt ccaatctgtg gtgaacacat ctgggatgac tcttcacatc
cgtcagcttg 720 ctgggaaaaa ctcacaccat atcatcgagg catctttcaa
agcgtttgct cgggcccttc 780 gacaggcaac agagtatgac ctgcgccgtc
aaggcactgt ccccagctcc aaaggtgtct 840 tgtcgagatc atagcatcgc
agtttggacg aacgcatatc ttccactgca gcgcacatca 900 atgagaggaa
aacctgcaga gtagacagct tggtttttgc tgctcacgtt gggagcctgc 960
gttagcctct gaacattgct aatgggataa tcaagaacca tctagcaact agcaagcacc
1020 acttattatc tgccttgtga cagtggcata gcgcgtctgc tcgctactgc
aactctatta 1080 tctgctcgct ggagttacag gatatctgaa gattgaaggt
ctagacatgc tgtgagcctg 1140 tgactttgta agctacaccg gtcaggaata
atgctgcgtc tggatcatcc gcagaaacat 1200 aggggtatgt aaattgtttg
gacattgaag ggaatgcatt gatctttgcg cgttcaaaaa 1260 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaa 1294 2 283 PRT Zea mays 2 Thr Ser Arg
Cys Pro Leu Pro Pro Gly Lys Pro Ala Ala Pro Pro Pro 1 5 10 15 Ala
Met Thr Thr Ala Arg Leu Val Ser Thr Ser Pro Ala Arg Leu Pro 20 25
30 Ser Thr Ser Ala Ser Pro Ile Pro Arg Thr Ser Leu Cys Val Val Gly
35 40 45 Arg Ala Pro Ala Phe Ser Ala Arg Ala Leu Gly Phe Ser Leu
Arg Leu 50 55 60 Lys Pro Ser Pro Ala Met Ala Ala Ala Gly Pro Pro
Thr Ala His Gly 65 70 75 80 Asp Ser Ile Gly Ser Ser Arg Ile Gly Glu
Val Lys Arg Val Thr Lys 85 90 95 Glu Thr Asn Val His Val Lys Ile
Asn Leu Asp Gly Thr Gly Val Ala 100 105 110 Glu Cys Ser Thr Gly Ile
Leu Phe Leu Asp His Met Leu Asp Gln Leu 115 120 125 Ala Ser His Gly
Leu Phe Asp Val Tyr Val Lys Ala Ile Gly Asp Thr 130 135 140 His Ile
Asp Asp His His Ser Asn Glu Asp Ile Ala Leu Ala Ile Gly 145 150 155
160 Thr Ala Leu Leu Glu Ala Leu Gly Asp Arg Lys Gly Ile Asn Arg Phe
165 170 175 Gly His Phe Thr Ala Pro Leu Asp Glu Ala Ala Val Glu Val
Ile Leu 180 185 190 Asp Leu Ser Gly Arg Pro His Leu Ser Cys Gly Leu
Asp Ile Pro Thr 195 200 205 Gln Arg Val Gly Thr Tyr Asp Thr Gln Leu
Val Glu His Phe Phe Gln 210 215 220 Ser Val Val Asn Thr Ser Gly Met
Thr Leu His Ile Arg Gln Leu Ala 225 230 235 240 Gly Lys Asn Ser His
His Ile Ile Glu Ala Ser Phe Lys Ala Phe Ala 245 250 255 Arg Ala Leu
Arg Gln Ala Thr Glu Tyr Asp Leu Arg Arg Gln Gly Thr 260 265 270 Val
Pro Ser Ser Lys Gly Val Leu Ser Arg Ser 275 280 3 1309 DNA Oryza
sativa 3 gcaagagggc gtacccgaat cccctccact cccccgcggt caccggcggc
gccggcggcg 60 agctctcgcc gacgccgatg accaccgcgc ggttcgtctc
cccctccctc tcgcgcgtgt 120 ccccctcgcc ggccggccga gtctccgggt
cgtcgtggtt gtccagggct ggggtcgccc 180 tccccgctcg gccgcacggt
ctctcgctcc acctgaggcc gcctgctatg gcctccgccg 240 ccgccgccgg
gaacggctct ccttccgcgc cggaggactc cacagcgttg tccaggatag 300
gagaagtcaa gagggtgacc aaggagacga atgtgcatgt gaagatcaac ctggatggca
360 ctggtgttgc cgattgcagc acagggatac ctttcttgga tcacatgctc
gatcaactgg 420 catctcatgg gctctttgac gtatgtgtta aggcaaaggg
tgacactcac atcgatgacc 480 atcactcaaa cgaagacatc gctttggcaa
ttggaacggc actacttgaa gcacttggag 540 atcgaaaagg aattaatcgg
tttgggcatt ttacagcacc acttgatgag gcagcggttg 600 aggttatact
ggatctatct ggtcgtcctc atttgagttg cggcttaagt attccgactg 660
agagagttgg cacatacgat actcagctag ttgagcactt tttccaatct cttgtgaata
720 catctgggat gacacttcac atccgtcagc ttgctggaaa aaactcacac
catattattg 780 aggcaacttt caaagcattt gctagagccc ttcgacaagc
aacagaatat gacttgcgcc 840 gacgcggcac tgtccccagc tcaaaaggtg
tgttgtcaag gtcatagtgt tgcaggtttg 900 gaaaacaagg ggtgcatgct
actgttcctt gtagattgca atattcaaag gaaacaaatg 960 gaatggcctc
tacagctcct cgagtcctac ttaccctggg aatctccgtt ggttgcgata 1020
acagaacatg aagccattgc taactagatc accaaaaaag gaacaaattt agcactgttc
1080 tcatgatcta ttcttctgta acatttagca ttcgaaataa acatgtccgg
ccgttattgc 1140 aattttggat gctagatgtc ttgagttgtc atctgtgttg
tccatcaaac ggcgtgtcta 1200 gcagctcagt ttcactctgt atgagttcag
agttggtcat gttgctgaac ctctgtattt 1260 tcaagggaat aaataagttt
tgcacctaaa aaaaaaaaaa aaaaaaaaa 1309 4 294 PRT Oryza sativa 4 Lys
Arg Ala Tyr Pro Asn Pro Leu His Ser Pro Ala Val Thr Gly Gly 1 5 10
15 Ala Gly Gly Glu Leu Ser Pro Thr Pro Met Thr Thr Ala Arg Phe Val
20 25 30 Ser Pro Ser Leu Ser Arg Val Ser Pro Ser Pro Ala Gly Arg
Val Ser 35 40 45 Gly Ser Ser Trp Leu Ser Arg Ala Gly Val Ala Leu
Pro Ala Arg Pro 50 55 60 His Gly Leu Ser Leu His Leu Arg Pro Pro
Ala Met Ala Ser Ala Ala 65 70 75 80 Ala Ala Gly Asn Gly Ser Pro Ser
Ala Pro Glu Asp Ser Thr Ala Leu 85 90 95 Ser Arg Ile Gly Glu Val
Lys Arg Val Thr Lys Glu Thr Asn Val His 100 105 110 Val Lys Ile Asn
Leu Asp Gly Thr Gly Val Ala Asp Cys Ser Thr Gly 115 120 125 Ile Pro
Phe Leu Asp His Met Leu Asp Gln Leu Ala Ser His Gly Leu 130 135 140
Phe Asp Val Cys Val Lys Ala Lys Gly Asp Thr His Ile Asp Asp His 145
150 155 160 His Ser Asn Glu Asp Ile Ala Leu Ala Ile Gly Thr Ala Leu
Leu Glu 165 170 175 Ala Leu Gly Asp Arg Lys Gly Ile Asn Arg Phe Gly
His Phe Thr Ala 180 185 190 Pro Leu Asp Glu Ala Ala Val Glu Val Ile
Leu Asp Leu Ser Gly Arg 195 200 205 Pro His Leu Ser Cys Gly Leu Ser
Ile Pro Thr Glu Arg Val Gly Thr 210 215 220 Tyr Asp Thr Gln Leu Val
Glu His Phe Phe Gln Ser Leu Val Asn Thr 225 230 235 240 Ser Gly Met
Thr Leu His Ile Arg Gln Leu Ala Gly Lys Asn Ser His 245 250 255 His
Ile Ile Glu Ala Thr Phe Lys Ala Phe Ala Arg Ala Leu Arg Gln 260 265
270 Ala Thr Glu Tyr Asp Leu Arg Arg Arg Gly Thr Val Pro Ser Ser Lys
275 280 285 Gly Val Leu Ser Arg Ser 290 5 1020 DNA Glycine max 5
gcacgagcta agatgtcctt catccctgct tctgaattct agggttaggg ttttccatac
60 aatacccaac cccacacgcc cacctttcca agcttcaatc ttttcactca
accaacgcaa 120 tctcactcca atggatcttc ccagaaacgt ctcctccgct
gccttggtgg gagacaacgc 180 ttcggccacg accactttgc caattgactc
agatgctaga atcggagagg ttaaaagggt 240 caccaaggag accaatgtat
cagtcaaaat aaacttggat ggttctgggg tggctgatag 300 tagtactgga
attcccttcc tcgatcatat gcttgatcaa cttgcttcac atgggctgtt 360
agatgtacat gtaaaggcta caggtgacat acatattgat gatcatcaca caaatgaaga
420 cgttgccctt gctattggaa cagctttgct gcatgccctt ggtgatagga
agggtattaa 480 ccggtttggt aacttctctg ctcctcttga tgaagcattg
atacatgttt cactggattt 540 gtctggccga ccacatctaa gttataattt
ggacataccc actcagaggg ttgggacata 600 tgatactcag ttggtggagc
atttcttcca atccctggtg aacacgtctg gtatgacact 660 tcacattcag
cagcttgctg ggaaaaattc tcatcatatt attgaggcaa ccttcaaagc 720
ttttgctcgg gctcttcgac aagcaacaga gtatgatcca cgtcgccgtg gaactatacc
780 aagttcgaaa ggggttctgt cccgtagcta atatttttct ggcggtggac
cttcatggct 840 ccacaatcac agtccttaga tgctgtgtga tggataaatg
cttgtgcggg ggcagtattt 900 gtgatagaac actgacctct gtaaacaata
ttgttgtacg tatacttatt tctagaaata 960 aaaataaatt ttctagtgta
taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020 6 267 PRT Glycine
max 6 Leu Arg Cys Pro Ser Ser Leu Leu Leu Asn Ser Arg Val Arg Val
Phe 1 5 10 15 His Thr Ile Pro Asn Pro Thr Arg Pro Pro Phe Gln Ala
Ser Ile Phe 20 25 30 Ser Leu Asn Gln Arg Asn Leu Thr Pro Met Asp
Leu Pro Arg Asn Val 35 40 45 Ser Ser Ala Ala Leu Val Gly Asp Asn
Ala Ser Ala Thr Thr Thr Leu 50 55 60 Pro Ile Asp Ser Asp Ala Arg
Ile Gly Glu Val Lys Arg Val Thr Lys 65 70 75 80 Glu Thr Asn Val Ser
Val Lys Ile Asn Leu Asp Gly Ser Gly Val Ala 85 90 95 Asp Ser Ser
Thr Gly Ile Pro Phe Leu Asp His Met Leu Asp Gln Leu 100 105 110 Ala
Ser His Gly Leu Leu Asp Val His Val Lys Ala Thr Gly Asp Ile 115 120
125 His Ile Asp Asp His His Thr Asn Glu Asp Val Ala Leu Ala Ile Gly
130 135 140 Thr Ala Leu Leu His Ala Leu Gly Asp Arg Lys Gly Ile Asn
Arg Phe 145 150 155 160 Gly Asn Phe Ser Ala Pro Leu Asp Glu Ala Leu
Ile His Val Ser Leu 165 170 175 Asp Leu Ser Gly Arg Pro His Leu Ser
Tyr Asn Leu Asp Ile Pro Thr 180 185 190 Gln Arg Val Gly Thr Tyr Asp
Thr Gln Leu Val Glu His Phe Phe Gln 195 200 205 Ser Leu Val Asn Thr
Ser Gly Met Thr Leu His Ile Gln Gln Leu Ala 210 215 220 Gly Lys Asn
Ser His His Ile Ile Glu Ala Thr Phe Lys Ala Phe Ala 225 230 235 240
Arg Ala Leu Arg Gln Ala Thr Glu Tyr Asp Pro Arg Arg Arg Gly Thr 245
250 255 Ile Pro Ser Ser Lys Gly Val Leu Ser Arg Ser 260 265
* * * * *
References