U.S. patent application number 10/431293 was filed with the patent office on 2003-09-18 for gene involved in pyrimidine biosynthesis in plants.
Invention is credited to Falco, Saverio Carl, Rafalski, J. Antoni, Weng, Zude.
Application Number | 20030177524 10/431293 |
Document ID | / |
Family ID | 26853631 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030177524 |
Kind Code |
A1 |
Falco, Saverio Carl ; et
al. |
September 18, 2003 |
Gene involved in pyrimidine biosynthesis in plants
Abstract
This invention relates to an isolated nucleic acid fragment
encoding an OMP decarboxylase. The invention also relates to the
construction of a chimeric gene encoding all or a portion of the
OMP decarboxylase, in sense or antisense orientation, wherein
expression of the chimeric gene results in production of altered
levels of the OMP decarboxylase in a transformed host cell.
Inventors: |
Falco, Saverio Carl;
(Wilmington, DE) ; Rafalski, J. Antoni;
(Wilmington, DE) ; Weng, Zude; (Des Plaines,
IL) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
1220 N MARKET STREET
P O BOX 2207
WILMINGTON
DE
19899
|
Family ID: |
26853631 |
Appl. No.: |
10/431293 |
Filed: |
May 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10431293 |
May 7, 2003 |
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09675018 |
Sep 28, 2000 |
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6573426 |
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60156901 |
Sep 30, 1999 |
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Current U.S.
Class: |
800/278 ;
435/193; 435/320.1; 435/419; 435/5; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 15/8262 20130101;
Y02A 40/146 20180101; C12N 9/88 20130101; C12N 15/8261
20130101 |
Class at
Publication: |
800/278 ; 435/6;
435/69.1; 435/193; 435/320.1; 435/419; 536/23.2 |
International
Class: |
A01H 001/00; C12N
015/82; C12Q 001/68; C07H 021/04; C12N 009/10; C12N 005/04 |
Claims
What is claimed is:
1. An isolated polynucleotide that encodes a polypeptide of at
least 200 amino acids having a sequence identity of at least 85%
based on the Clustal method of alignment when compared to a
polypeptide selected from the group consisting of SEQ ID NOs:6, 8,
10, and 12.
2. A polynucleotide sequence of claim 1, wherein sequence identity
is at least 90%.
3. A polynucleotide sequence of claim 1, wherein sequence identity
is at least 95%.
4. The polynucleotide of claim 1 wherein the polynucleotide encodes
a polypeptide selected from the group consisting of SEQ ID NOs:6,
8, 10 and 12.
5. The polynucleotide of claim 1, wherein the polynucleotide
comprises a nucleotide sequence selected from the group consisting
of SEQ ID NO:5, 7, 9 and 11.
6. The polynucleotide of claim 1, wherein the polypeptide is an OMP
decarboxylase.
7. An isolated complement of the polynucleotide of claim 1, wherein
(a) the complement and the polynucleotide consist of the same
number of nucleotides, and (b) the nucleotide sequences of the
complement and the polynucleotide have 100% complementarity.
8. An isolated nucleic acid molecule that (1) comprises at least
800 nucleotides and (2) remains hybridized with the isolated
polynucleotide of claim 24 under a washing condition of
0.1.times.SSC, 0.1% SDS, and 65.degree. C.
9. A cell comprising the polynucleotide of claim 1.
10. The cell of claim 9, wherein the cell is selected from the
group consisting of a yeast cell, a bacterial cell and a plant
cell.
11. A transgenic plant comprising the polynucleotide of claim
1.
12. A method for transforming a cell comprising introducing into a
cell the polynucleotide of claim 1.
13. A method for producing a transgenic plant comprising (a)
transforming a plant cell with the polynucleotide of claim 1, and
(b) regenerating a plant from the transformed plant cell.
14. A method for producing a polynucleotide fragment comprising (a)
selecting a nucleotide sequence comprised by the polynucleotide of
claim 1, and (b) synthesizing a polynucleotide fragment containing
the nucleotide sequence.
15. The method of claim 14, wherein the fragment is produced in
vivo.
16. An isolated polypeptide comprising (a) at least 200 amino
acids, and (b) a first amino acid sequence, wherein the first amino
acid sequence and a second amino acid sequence have a sequence
identity of at least 85%, and wherein the second amino acid is
selected from the group consisting of SEQ ID NOs:6, 8, 10, and
12.
17. The polypeptide of claim 16, wherein the sequence identity is
at least 90%.
18. The polypeptide of claim 16, wherein the sequence identity is
at least 95%.
19. The polypeptide of claim 16 wherein the polypeptide has a
sequence selected from the group consisting of SEQ ID NOs:6, 8, 10,
and 12.
20. The polypeptide of claim 16, wherein the polypeptide is an OMP
decarboxylase.
21. A chimeric gene comprising the polynucleotide of claim 1
operably linked to at least one suitable regulatory sequence.
22. A method for altering the level of OMP decarboxylase.expression
in a host cell, the method comprising: (a) Transforming a host cell
with the chimeric gene of claim 21; and (b) Growing the transformed
cell in step (a) under condistions suitable for the expression of
the chimeric gene.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/156901, filed Sep. 30, 1999.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
molecules coding for an enzyme involved in pyrimidine biosynthesis
in plants, especially in seeds.
BACKGROUND OF THE INVENTION
[0003] Orotidine 5'-monophosphate decarboxylase (OMP decarboxylase)
catalyzes the final reaction in pyrimidine nucleotide biosynthesis,
converting OMP to uridine 5'-monophosphate (UMP). In eukaryotes,
this enzyme also performs the next-to-last step of linking
phosphoribosyl-pyrophosphate (PRPP) to orotate to form OMP (Reyes
and Guganig (1975) J Biol Chem 250:5097-108; Traut et al. (1980)
Biochemistry 19:6062-8). The enzyme is a target for feedback
inhibition wherein UTP and UMP both reduce its activity. In
prokaryotes, in contrast, there is no feedback inhibition, and and
the last two enzymatic reactions are not coupled.
[0004] Nucleotides are required for the synthesis of DNA and RNA,
and are indirectly responsible for protein synthesis, due to the
requirement for ribosomes and tRNAs in translation. Therefore,
pyrimidine biosynthesis is a key metabolic pathway in all
eukaryotes. Manipulation of this pathway is neverthelss possible
since mutations to key enzymes can be partially overcome by feeding
cells having potentially lethal mutations with CTP, UTP, or their
mono- or diphosphate derivatives. Inhibitors of OMP decarboxylase
have been identified which vary in their efficacy among different
organisms, implying that engineering of the active site may yield
enzymes that are more or less sensitive to inhibition (Shostak and
Jones (1992) Biochemistry 31:12155-61). It is believed that
overexpression or inhibition of OMP decarboxylase in plants may be
useful for enhancing growth rates, developing new herbicides,
developing new fungicides, developing new insecticides, or
selectively altering development of individual organs.
SUMMARY OF THE INVENTION
[0005] The present invention concerns an isolated polynucleotide
comprising a nucleotide sequence selected from the group consisting
of: (a) a first nucleotide sequence encoding a polypeptide of at
least 200 amino acids having at least 85% identity based on the
Clustal method of alignment when compared to a polypeptide selected
from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12, or
(b) a second nucleotide sequence comprising the complement of the
first nucleotide sequence.
[0006] In a second embodiment, it is preferred that the isolated
polynucleotide of the claimed invention comprises a first
nucleotide sequence which comprises a nucleic acid sequence
selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, and
11 that codes for the polypeptide selected from the group
consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12.
[0007] In a third embodiment, this invention concerns an isolated
polynucleotide comprising a nucleotide sequence of at least one of
800 (preferably at least one of 500, most preferably at least one
of 400) contiguous nucleotides derived from a nucleotide sequence
selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, and
11 and the complement of such nucleotide sequences.
[0008] In a fourth embodiment, this invention relates to a chimeric
gene comprising an isolated polynucleotide of the present invention
operably linked to at least one suitable regulatory sequence.
[0009] In a fifth embodiment, the present invention concerns 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.
[0010] In a sixth embodiment, the invention also 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.
[0011] In a seventh embodiment, the invention concerns an OMP
decarboxylase polypeptide of at least 200 amino acids comprising at
least 85% identity based on the Clustal method of alignment
compared to a polypeptide selected from the group consisting of SEQ
ID NOs:2, 4, 6, 8, 10, and 12.
[0012] In an eighth embodiment, the invention relates to a method
of selecting an isolated polynucleotide that affects the level of
expression of an OMP decarboxylase polypeptide or enzyme activity
in a host cell, preferably a plant cell, the method comprising the
steps of: (a) constructing an isolated polynucleotide of the
present invention or an isolated chimeric gene of the present
invention; (b) introducing the isolated polynucleotide or the
isolated chimeric gene into a host cell; (c) measuring the level of
the OMP decarboxylase polypeptide or enzyme activity in the host
cell containing the isolated polynucleotide; and (d) comparing the
level of the OMP decarboxylase polypeptide or enzyme activity in
the host cell containing the isolated polynucleotide with the level
of the OMP decarboxylase polypeptide or enzyme activity in the host
cell that does not contain the isolated polynucleotide.
[0013] In a ninth embodiment, the invention concerns a method of
obtaining a nucleic acid fragment encoding a substantial portion of
an OMP decarboxylase polypeptide, preferably a plant OMP
decarboxylase polypeptide, comprising the steps of: synthesizing an
oligonucleotide primer comprising a nucleotide sequence of at least
one of 800 (preferably at least one of 500, most preferably at
least one of 400) contiguous nucleotides. derived from a nucleotide
sequence selected from the group consisting of SEQ ID NOs:1, 3, 5,
7, 9, and 11, 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 substantial portion
of an OMP decarboxylase amino acid sequence.
[0014] In a tenth embodiment, this invention relates to a method of
obtaining a nucleic acid fragment encoding all or a substantial
portion of the amino acid sequence encoding an OMP decarboxylase
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.
[0015] In an eleventh embodiment, this invention concerns a
composition, such as a hybridization mixture, comprising an
isolated polynucleotide of the present invention.
[0016] In a twelfth embodiment, this invention concerns a method
for positive selection of a transformed cell comprising: (a)
transforming a host cell with the chimeric gene of the present
invention or an expression cassette of the present invention; and
(b) growing the transformed host cell, preferably a plant cell,
such as a monocot or a dicot, under conditions which allow
expression of the OMP decarboxylase polynucleotide in an amount
sufficient to complement a null mutant to provide a positive
selection means.
[0017] In a thirteenth embodiment, this invention relates to a
method of altering the level of expression of an OMP decarboxylase
in a host cell comprising: (a) transforming a host cell with a
chimeric gene of the present invention; and (b) growing the
transformed host cell under conditions that are suitable for
expression of the chimeric gene wherein expression of the chimeric
gene results in production of altered levels of the OMP
decarboxylase in the transformed host cell.
[0018] 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 enzyrne involved in primidine biosynthesis, the
method comprising the steps of: (a) transforming a host cell with a
chimeric gene comprising a nucleic acid fragment encoding an OMP
decarboxylase polypeptide, 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 an OMP
decarboxylase in the transformed host cell; (c) optionally
purifying the OMP decarboxylase polypeptide expressed by the
transformed host cell; (d) treating the OMP decarboxylase
polypeptide with a compound to be tested; and (e) comparing the
activity of the OMP decarboxylase polypeptide that has been treated
with a test compound to the activity of an untreated OMP
decarboxylase polypeptide, thereby selecting compounds with
potential for inhibitory activity.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0019] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing which form a part of this application.
[0020] FIG. 1 shows a comparison of the amino acid sequences set
forth in SEQ ID NOs:6, 8, 10, and 12, and the Arabidopsis thaliana
and Nicotiana tabacum (NCBI General Identifier No. gi 2499945 and
gi 2499946, respectively) sequences (SEQ ID NOs:13 and 14,
respectively).
[0021] 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 Orotidine-5-Phospbate Decarboxylase SEQ ID NO: Protein
Clone Designation (Nucleotide) (Amino Acid) Corn OMP p0127.cntbe57r
1 2 Decarboxylase Rice OMP rsl1n.pk004.j19 3 4 Decarboxylase
Soybean OMP sfl1.pk135.i17 5 6 Decarboxylase Wheat OMP
wl1n.pk0029.c7 7 8 Decarboxylase Corn OMP p0127.cntbe57r:fis 9 10
Decarboxylase Rice OMP rsl1n.pk004.j19:fis 11 12 Decarboxylase
[0022] 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
[0023] In the context of this disclosure, a number of terms shall
be utilized. The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", and "nucleic acid fragment"/"isolated
nucleic acid fragment" are used interchangeably herein. These terms
encompass nucleotide sequences and the like. A polynucleotide may
be a polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. An isolated polynucleotide of the present
invention may include at least one of 800 contiguous nucleotides,
preferably at least one of 40 contiguous nucleotides, most
preferably one of at least 30 contiguous nucleotides derived from
SEQ ID NOs:1, 3, 5, 7, 9, and 11, or the complement of such
sequences.
[0024] The term "isolated" polynucleotide refers to a
polynucleotide that is substantially free from other nucleic acid
sequences, such as other chromosomal and extrachromosomal DNA and
RNA, that normally accompany or interact with it as found in its
naturally occurring environment. Isolated polynucleotides may be
purified from a host cell in which they naturally occur.
Conventional nucleic acid purification methods known to skilled
artisans may be used to obtain isolated polynucleotides. The term
also embraces recombinant polynucleotides and chemically
synthesized polynucleotides.
[0025] The term "recombinant" means, for example, that a nucleic
acid sequence is made by an artificial combination of two otherwise
separated segments of sequence, e.g., by chemical synthesis or by
the manipulation of isolated nucleic acids by genetic engineering
techniques.
[0026] 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.
[0027] 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 The terms "substantially similar" and
"corresponding substantially" are used interchangeably herein.
[0028] 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 800 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.
[0029] 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 using 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 800 (preferably at least one of 500,
most preferably at least one of 400) contiguous nucleotides derived
from a nucleotide sequence selected from the group consisting of
SEQ ID NOs:1, 3, 5, 7, 9, and 11, and the complement of such
nucleotide sequences may be used in methods of selecting an
isolated polynucleotide that affects the expression of an OMP
decarboxylase polypeptide in a host cell. A method of selecting an
isolated polynucleotide that affects the level of expression of a
polypeptide in a virus or in a host cell (eukaryotic, such as plant
or yeast, prokaryotic such as bacterial) 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 of a polypeptide or enzyme activity in
the host cell containing the isolated polynucleotide; and comparing
the level of a polypeptide or enzyme activity in the host cell
containing the isolated polynucleotide with the level of a
polypeptide or enzyme activity in a host cell that does not contain
the isolated polynucleotide.
[0030] 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.
[0031] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Suitable nucleic acid fragments
(isolated polynucleotides of the present invention) encode
polypeptides that are at least about 70% identical, preferably at
least about 80% identical to the amino acid sequences reported
herein. Preferred nucleic acid fragments encode amino acid
sequences that are about 85% identical to the amino acid sequences
reported herein. More preferred nucleic acid fragments encode amino
acid sequences that are at least about 90% identical to the amino
acid sequences reported herein. Most preferred are nucleic acid
fragments that encode amino acid sequences that are at least about
95% identical to the amino acid sequences disclosed herein.
Suitable nucleic acid fragments not only have the above identities
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.
[0032] 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.
[0033] "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.
[0034] "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 a 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 the 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.
[0035] "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.
[0036] "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.
[0037] "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 may be composed of different
elements derived from different promoters found in nature, or may
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.
[0038] "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).
[0039] "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.
[0040] "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 polypeptides by the cell. "cDNA" refers to DNA that
is complementary to and derived from an mRNA template. The cDNA can
be single-stranded or converted to double stranded form using, for
example, the Kienow fragment of DNA polymerase I. "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.
[0041] The term "operably linked" refers to the association of two
or more nucleic acid fragments on a single polynucleotide 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.
[0042] 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).
[0043] A "protein" or "polypeptide" is a chain of amino acids
arranged in a specific order determined by the coding sequence in a
polynucleotide encoding the polypeptide. Each protein or
polypeptide has a unique function.
[0044] "Altered levels" or "altered expression" 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.
[0045] "Null mutant" refers here to a host cell which either lacks
the expression of a certain polypeptide or expresses a polypeptide
which is inactive or does not have any detectable expected
enzymatic function.
[0046] "Mature protein" or the term "mature" when used in
describing a 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" or the term "precursor" when used in describing a 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.
[0047] 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).
[0048] "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). Thus, isolated polynucleotides of the present invention
can be incorporated into recombinant constructs, typically DNA
constructs, capable of introduction into and replication in a host
cell. Such a construct can be a vector that includes a replication
system and sequences that are capable of transcription and
translation of a polypeptide-encoding sequence in a given host
cell. A number of vectors suitable for stable transfection of plant
cells or for the establishment of transgenic plants have been
described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory
Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for
Plant Molecular Biology, Academic Press, 1989; and Flevin et al.,
Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990.
Typically, plant expression vectors include, for example, one or
more cloned plant genes under the transcriptional control of 5' and
3' regulatory sequences and a dominant selectable marker. Such
plant expression vectors also can contain a promoter regulatory
region (e.g., a regulatory region controlling inducible or
constitutive, environmentally- or developmentally-regulated, or
cell- or tissue-specific expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0049] 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").
[0050] "PCR" or "polymerase chain reaction" is well known by those
skilled in the art as a technique used for the amplification of
specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).
[0051] The present invention concerns an isolated polynucleotide
comprising a nucleotide sequence selected from the group consisting
of: (a) first nucleotide sequence encoding a polypeptide of at
least 200 amino acids having at least 85% identity based on the
Clustal method of alignment when compared to a polypeptide selected
from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12, or
(b) a second nucleotide sequence comprising the complement of the
first nucleotide sequence.
[0052] Preferably, the first nucleotide sequence comprises a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs:1, 3, 5, 7, 9, and 11, that codes for the polypeptide selected
from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, and 12.
[0053] Nucleic acid fragments encoding at least a portion of
several OMP decarboxylase 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).
[0054] For example, genes encoding other OMP decarboxylase, 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, an entire sequence can be used directly
to synthesize DNA probes by methods known to the skilled artisan
such as random primer DNA labeling, nick translation, 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.
[0055] 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 800 (preferably one of at least 500,
most preferably one of at least 400) contiguous nucleotides derived
from a nucleotide sequence selected from the group consisting of
SEQ ID NOs:1, 3, 5, 7, 9, and 11 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.
[0056] The present invention relates to a method of obtaining a
nucleic acid fragment encoding a substantial portion of an OMP
decarboxylase polypeptide, preferably a substantial portion of a
plant OMP decarboxylase polypeptide, comprising the steps of:
synthesizing an oligonucleotide primer comprising a nucleotide
sequence of at least one of 800 (preferably at least one of 500,
most preferably at least one of 400) contiguous nucleotides derived
from a nucleotide sequence selected from the group consisting of
SEQ ID NOs:1, 3, 5, 7, 9, and 11, 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 OMP decarboxylase
polypeptide.
[0057] 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 (Lemer (1984) Adv. Immunol. 36:1-34; Maniatis).
[0058] In another embodiment, this invention concerns viruses and
host cells comprising either the chirneric genes of the invention
as described herein or an isolated polynucleotide of the invention
as described herein. Examples of host cells which can be used to
practice the invention include, but are not limited to, yeast,
bacteria, and plants.
[0059] As was noted above, 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 pyrimidine biosynthesis or metabolism in those cells.
[0060] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. The chimeric gene may comprise promoter sequences and
translation leader sequences derived from the same genes. 3'
Non-coding sequences encoding transcription termination signals may
also be provided. The instant chimeric gene may also comprise one
or more introns in order to facilitate gene expression.
[0061] Plasmid vectors comprising the instant isolated
polynucleotide (or chimeric gene) may be constructed. The choice of
plasmid vector is dependent upon the method that will be used to
transform host plants. The skilled artisan is well aware of the
genetic elements that must be present on the plasmid vector in
order to successfully transform, select and propagate host cells
containing the chimeric gene. The skilled artisan will also
recognize that different independent transformation events will
result in different levels and patterns of expression (Jones et al.
(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.
Genetics 218:78-86), and thus that multiple events must be screened
in order to obtain lines displaying the desired expression level
and pattern. Such screening may be accomplished by Southern
analysis of DNA, Northern analysis of mRNA expression, Western
analysis of protein expression, or phenotypic analysis.
[0062] For some applications it may be useful to direct the instant
polypeptides to different cellular compartments, or to facilitate
its secretion from the cell. It is thus envisioned that the
chimeric gene described above may be further supplemented by
directing the coding sequence to encode the instant polypeptides
with appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys. 100:1627-1632)
with or without removing targeting sequences that are already
present. While the references cited give examples of each of these,
the list is not exhaustive and more targeting signals of use may be
discovered in the future.
[0063] 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.
[0064] 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
a 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.
[0065] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppression technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense genes may require the use of different chimeric
genes utilizing different regulatory elements known to the skilled
artisan. Once transgenic plants are obtained by one of the methods
described above, it will be necessary to screen individual
transgenics for those that most effectively display the desired
phenotype. Accordingly, the skilled artisan will develop methods
for screening large numbers of transformants. The nature of these
screens will generally be chosen on practical grounds. For example,
one can screen by looking for changes in gene expression by using
antibodies specific for the protein encoded by the gene being
suppressed, or one could establish assays that specifically measure
enzyme activity. A preferred method will be one which allows large
numbers of samples to be processed rapidly, since it will be
expected that a large number of transformants will be negative for
the desired phenotype.
[0066] In another embodiment, the present invention concerns a
polypeptide of at least 200 amino acids that has at least 85%
identity based on the Clustal method of alignment when compared to
a polypeptide selected from the group consisting of SEQ ID NOs:2,
4, 6, 8, 10, and 12.
[0067] 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 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 OMP
decarboxylase. An example of a vector for high level expression of
the instant polypeptides in a bacterial host is provided (Example
6).
[0068] Additionally, the instant polypeptides can be used as a
target 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 a key step in
pyrimidine 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.
[0069] All or a substantial portion of the polynucleotides of the
instant invention may also be used as probes for genetically and
physically mapping the genes that they are a part of, and used as
markers for traits linked to those genes. Such information may be
usefull 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).
[0070] 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.
[0071] 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).
[0072] 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.
[0073] 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.
[0074] 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
[0075] The present invention is further defined in the following
Examples, in which 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. Thus, various modifications of the invention
in addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0076] The disclosure of each reference set forth herein is
incorporated herein by reference in its entirety.
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 p0127 Nucellus tissue, 5 days after silking,
p0127.cntbe57r screened 1 rsl1n Rice (Oryza sativa, YM) 15 day old
rsl1n.pk004.j19 seedling normalized* sfl1 Soybean Immature Flower
sfl1.pk135.i17 wl1n Wheat Leaf From 7 Day Old Seedling*
wl1n.pk0029.c7 p0127 Nucellus tissue, 5 days after silking,
p0127.cntbe57r:fis screened 1 rsl1n Rice (Oryza sativa, YM) 15 day
old rsl1n.pk004.j19:fis seedling normalized sfl1 Soybean Immature
Flower sfl1.pk135.i17:fis *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.
[0079] Full-insert sequence (FIS) data is generated utilizing a
modified transposition protocol. Clones identified for FIS are
recovered from archived glycerol stocks as single colonies, and
plasmid DNAs are isolated via alkaline lysis. Isolated DNA
templates are reacted with vector primed M13 forward and reverse
oligonucleotides in a PCR-based sequencing reaction and loaded onto
automated sequencers. Confirmation of clone identification is
performed by sequence alignment to the original EST sequence from
which the FIS request is made.
[0080] Confirmed templates are transposed via the Primer Island
transposition kit (PE Applied Biosystems, Foster City, Calif.)
which is based upon the Saccharomyces cerevisiae Ty1 transposable
element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772).
The in vitro transposition system places unique binding sites
randomly throughout a population of large DNA molecules. The
transposed DNA is then used to transform DH10B electro-competent
cells (Gibco BRL/Life Technologies, Rockville, Md.) via
electroporation. The transposable element contains an additional
selectable marker (named DHFR; Fling and Richards (1983) Nucleic
Acids Res. 11:5147-5158), allowing for dual selection on agar
plates of only those subclones containing the integrated
transposon. Multiple subclones are randomly selected from each
transposition reaction, plasmid DNAs are prepared via alkaline
lysis, and templates are sequenced (ABI Prism dye-terminator
ReadyReaction mix) outward from the transposition event site,
utilizing unique primers specific to the binding sites within the
transposon.
[0081] Sequence data is collected (ABI Prism Collections) and
assembled using Phred/Phrap (P. Green, University of Washington,
Seattle). Phrep/Phrap is a public domain software program which
re-reads the ABI sequence data, re-calls the bases, assigns quality
values, and writes the base calls and quality values into editable
output files. The Phrap sequence assembly program uses these
quality values to increase the accuracy of the assembled sequence
contigs. Assemblies are viewed by the Consed sequence editor (D.
Gordon, University of Washington, Seattle).
EXAMPLE 2
Identification of cDNA Clones
[0082] cDNA clones encoding OMP decarboxylase 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.
[0083] ESTs submitted for analysis are compared to the genbank
database as described above. ESTs that contain sequences more 5- or
3-prime can be found by using the BLASTn algorithm (Altschul et al
(1997) Nucleic Acids Res. 25:3389-3402.) against the Du Pont
proprietary database comparing nucleotide sequences that share
common or overlapping regions of sequence homology. Where common or
overlapping sequences exist between two or more nucleic acid
fragments, the sequences can be assembled into a single contiguous
nucleotide sequence, thus extending the original fragment in either
the 5 or 3 prime direction. Once the most 5-prime EST is
identified, its complete sequence can be determined by Full Insert
Sequencing as described in Example 1. Homologous genes belonging to
different species can be found by comparing the amino acid sequence
of a known gene (from either a proprietary source or a public
database) against an EST database using the tBLASTn algorithm. The
tBLASTn algorithm searches an amino acid query against a nucleotide
database that is translated in all 6 reading frames. This search
allows for differences in nucleotide codon usage between different
species, and for codon degeneracy.
EXAMPLE 3
Characterization of cDNA Clones Encoding OMP Decarboxylase
[0084] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to OMP decarboxylase from Arabidopsis thaliana and Nicotiana
tabacum (NCBI General Identifier No. gi 2499945 and gi 2499946,
respectively). 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"), the sequences of
contigs assembled from two or more ESTs ("Contig"), sequences of
contigs assembled from an FIS and one or more ESTs ("Contig*"), or
sequences encoding an entire protein derived from an FIS, a contig,
or an FIS and PCR ("CGS"):
3TABLE 3 BLAST Results for Sequences Encoding Polypeptides
Homologous to OMP Decarboxylase BLAST pLog Score Clone Status
gi2599945 gi2499946 p0127.cntbe57r EST 25.70 rsl1n.pk004.j19 EST
37.70 sfl1.pk135.i17 EST >180.00 wl1n.pk0029.c7 FIS 167.00
[0085] The sequence of the entire cDNA insert in the clones listed
in Table 3 was determined. Further sequencing and searching of the
DuPont proprietary database allowed the identification of other
corn, rice, soybean and/or wheat clones encoding OMP decarboxylase.
The BLASTX search using the EST sequences from clones listed in
Table 4 revealed similarity of the polypeptides encoded by the
cDNAs to OMP decarboxylase from Arabidopsis thaliana (NCBI General
Identifier No. gi 2599945). Shown in Table 4 are the BLAST results
for individual ESTs ("EST"), the sequences of the entire cDNA
inserts comprising the indicated cDNA clones ("FIS"), sequences of
contigs assembled from two or more ESTs ("Contig"), sequences of
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"):
4TABLE 4 BLAST Results for Sequences Encoding Polypeptides
Homologous to OMP Decarboxylase BLAST pLog Score Clone Status
gi2599945 p0127.cntbe57r:fis FIS >180.00 rsl1n.pk004.j19:fis CGS
172.00
[0086] FIG. 1 presents an alignment of the amino acid sequences set
forth in SEQ ID NOs:6, 8, 10, and 12, and the Arabidopsis thaliana
and Nicotiana tabacum (NCBI General Identifier No. gi 2499945 and
gi 2499946, respectively) sequences (SEQ ID NOs:13 and 14,
respectively). The data in Table 5 represents a calculation of the
percent identity of the amino acid sequences set forth in SEQ ID
NOs:2, 4, 6, 8, 10, and 12, and the Arabidopsis thaliana and
Nicotiana tabacum (NCBI General Identifier No. gi 2499945 and gi
2499946, respectively) sequences (SEQ ID NOs:13 and 14,
respectively).
5TABLE 5 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to OMP Decarboxylase Percent Identity to SEQ ID NO.
gi2599945 gi2599946 2 52.6% 4 79.5% 6 75.6% 8 69.2% 10 67.0% 12
65.7%
[0087] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of a OMP decarboxylase.
These sequences represent the first corn, soybean, and wheat
sequences encoding OMP decarboxylase known to Applicant.
EXAMPLE 4
Expression of Chimeric Genes in Monocot Cells
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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
[0095] 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.
[0096] 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.
[0097] 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.
[0098] Soybean embryogenic suspension cultures can be 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.
[0099] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0100] 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 phosphotranferase 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 5 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.
[0101] 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.
[0102] 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.
[0103] 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
[0104] 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.
[0105] 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% low melting agarose gel.
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 GELasem (Epicentre
Technologies, Madison, Wis.) 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 (NEB),
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.
[0106] 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
OMP Decarboxylase
[0107] 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,
inplanta, 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.
[0108] 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.
[0109] 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 OMP
decarboxylase are presented by Strych et al. (1994) Curr Microbiol
29:353-9; Seymour et al. (1994) Biochemistry 33:5268-74; Shostak
and Jones (1992) Biochemistry 31:12155-61; and Traut et al. (1980)
Biochemistry 19:6062-8.
Sequence CWU 1
1
14 1 510 DNA Zea mays unsure (9) n = A, C, G or T 1 gtacaaagnc
acttcctgct ctcgctccgc cgccgccgcc tccctcccca gtcgatcacc 60
aaacctcagt ccaaactcca aacccccgcc gcatcagaaa aaaaccctag gccatggacg
120 ccgcggcgct ggagtcgctc atcctggacc tccacgccat cgaggtcgtg
aagctgggct 180 ccttcacgct caagtccggc atcaaatcgc ccatctacct
cgacctccgc gcgctcgnct 240 cccacccgcg cctgctctcc gncgtcgcct
cgctccttca cgcgctcccg gccacgcgcc 300 cctacggcct cgtctgcggt
gncccctaca ccgcgctccc catcgccgnc gncctctccg 360 tcgaccgctc
aatccccatg ctcatgcgcc gcaaggaggt caaggnccac ggaccgcaag 420
tccatcgagg gctcttcagc cggggacacc gnctatatng agactcgcac agtggnnctc
480 ggctcngacg cgccgtcggc gagggctgcg 510 2 97 PRT Zea mays UNSURE
(39) Xaa = ANY AMINO ACID 2 Ala Ala Leu Glu Ser Leu Ile Leu Asp Leu
His Ala Ile Glu Val Val 1 5 10 15 Lys Leu Gly Ser Phe Thr Leu Lys
Ser Gly Ile Lys Ser Pro Ile Tyr 20 25 30 Leu Asp Leu Arg Ala Leu
Xaa Ser His Pro Arg Leu Leu Ser Xaa Val 35 40 45 Ala Ser Leu Leu
His Ala Leu Pro Ala Thr Arg Pro Tyr Gly Leu Val 50 55 60 Cys Gly
Xaa Pro Tyr Thr Ala Leu Pro Ile Ala Xaa Xaa Leu Ser Val 65 70 75 80
Asp Arg Ser Ile Pro Met Leu Met Arg Arg Lys Glu Val Lys Xaa His 85
90 95 Gly 97 3 514 DNA Oryza sativa unsure (376) n = A, C, G or T 3
ctgcttttgc ttgctgaaat gagctcggct ggcaaccttg ctcatggaga gtacactgct
60 gcagctgtaa agattgctga gcaacattct gattttgtaa ttggatttat
atccgttaat 120 ccagcatctt ggtcagttgc gccatcaagt ccagcattta
tccatgccac tcctggagtg 180 cagatggttt ctggaggaga tgctcttggt
caacagtaca atacccctca ttctgttata 240 aacgacaaga ggcaagtgac
ataattatag tccggacgag ggattataaa ggcgaagtaa 300 tccagcccga
gaccgcgagg gaagtaccgc atccaagggt gggggagcaa aacaatccag 360
ctttgccatg agaaantgag aatngtgttt aggcaatggt tggttcnagc ttatgattta
420 ttataaccaa gaataattaa gccangattg cnnataaagc cgggattaat
antnaagctg 480 ccatanaaat aaactgtgna gttggttgnt ttgg 514 4 83 PRT
Oryza sativa 4 Leu Leu Leu Leu Ala Glu Met Ser Ser Ala Gly Asn Leu
Ala His Gly 1 5 10 15 Glu Tyr Thr Ala Ala Ala Val Lys Ile Ala Glu
Gln His Ser Asp Phe 20 25 30 Val Ile Gly Phe Ile Ser Val Asn Pro
Ala Ser Trp Ser Val Ala Pro 35 40 45 Ser Ser Pro Ala Phe Ile His
Ala Thr Pro Gly Val Gln Met Val Ser 50 55 60 Gly Gly Asp Ala Leu
Gly Gln Gln Tyr Asn Thr Pro His Ser Val Ile 65 70 75 80 Asn Asp Lys
83 5 1730 DNA Glycine max 5 gcacgaggtt ttcctttact ggtaagttgt
aaccctataa gccgttgctc ccaccgccgc 60 cgcgcagatc tccgtttcaa
cttgggttat ctctaaggtc ctaaacaatc ctctttcaaa 120 aacataccga
gaaaagtgtg gaaatgacga caccatcatt ggtagagtct ctagttcttc 180
aactccatga gatctcagct gtcaaatttg gcaacttcaa gctcaaatct ggcatcttct
240 caccaatcta catagacctc cgcctcatca tatcttaccc ttctctcctc
caacagatct 300 ctcaaaccct tatttcttca gtctcttcca cttcctttga
cctcgtatgc ggtgtccctt 360 acactgcctt acccattgct acatgtgtct
ctcttgctca gaacattccc atggtcatgc 420 gccgcaaaga aatcaaagat
tatggcactg ctaaagctat tgaaggcgat ttcaagcctg 480 gccaaagttg
cttaatcatt gaggatttgg ttaccagtgg cacgtcagtt ttggaaactg 540
cggcgccatt gcgtgctgtg ggattaaaga tcagtgatgc tgttgtgttg atcgatagag
600 agcaaggtgg cagagaaaac ttggaggaga atggcatcaa gctgcatgca
attattaaat 660 tgactgaaat ggtgaaaatt ttgggcaatc acgggaagct
tgatgaagag atggtagggg 720 ttgttacgaa gttcttagag gataatcgta
aggttgctgc tttggcaaag gtggagaagc 780 ctgtaactaa ggtcaaagct
ttgccatttg gggagagggc taagctgtcg aagaatccaa 840 tgggaaagag
gttgtttgag ataatggctg agaaggagag taatctatgt ttggctgctg 900
atgttggaac tgcagctgaa ttgcttgaaa ttgctgagaa ggttggacct gagatatgct
960 tgctgaagac tcatgtggat atttttccag attttactgc tgattttggc
tctaagcttc 1020 tctcgattgc agaaaaacat aacttcttaa tctttgagga
tcgtaaattt gctgatattg 1080 gcaacacagt gaccatgcaa tatgaaggag
gggtttttcg tatattggat tgggctcata 1140 tagtaaatgc tcacataatc
tcaggtcctg gaattgttga tggattaaaa ttgaagggtt 1200 tacctcgtgg
taggggtcta ttactgcttg ctgaaatgag ctcagctggt aaccttgcca 1260
agggagatta tacaacttct gcagtaaaaa ttgctgagga tcattctgac tttgtaattg
1320 gcttcatctc agtcaatcct gcatcatggc caggggcacc aataaatcct
tctttcattc 1380 aagcaacccc tggagttcaa atggtaactg gtggcgatgc
tttagggcag caatataaca 1440 ctccatattc tgtgatccat gataggggca
gtgacatcat catcgtggga cgtggcatca 1500 tcaaagcagc aaaccatgct
gagatagctc gtgaatatcg tcttcaagga tggaatgcat 1560 atttggctaa
atgtaattga tgcctgcatt cctagaataa aattatgagc ttaaattatg 1620
ttttaatggg acatctgatc tcactgtaac ccagatgaat aaggtcttgg ggtacaatat
1680 gaagacattt ttcggttgga atattgaaaa aaaaaaaaaa aaaaaaaaaa 1730 6
478 PRT Glycine max 6 Met Thr Thr Pro Ser Leu Val Glu Ser Leu Val
Leu Gln Leu His Glu 1 5 10 15 Ile Ser Ala Val Lys Phe Gly Asn Phe
Lys Leu Lys Ser Gly Ile Phe 20 25 30 Ser Pro Ile Tyr Ile Asp Leu
Arg Leu Ile Ile Ser Tyr Pro Ser Leu 35 40 45 Leu Gln Gln Ile Ser
Gln Thr Leu Ile Ser Ser Val Ser Ser Thr Ser 50 55 60 Phe Asp Leu
Val Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Thr 65 70 75 80 Cys
Val Ser Leu Ala Gln Asn Ile Pro Met Val Met Arg Arg Lys Glu 85 90
95 Ile Lys Asp Tyr Gly Thr Ala Lys Ala Ile Glu Gly Asp Phe Lys Pro
100 105 110 Gly Gln Ser Cys Leu Ile Ile Glu Asp Leu Val Thr Ser Gly
Thr Ser 115 120 125 Val Leu Glu Thr Ala Ala Pro Leu Arg Ala Val Gly
Leu Lys Ile Ser 130 135 140 Asp Ala Val Val Leu Ile Asp Arg Glu Gln
Gly Gly Arg Glu Asn Leu 145 150 155 160 Glu Glu Asn Gly Ile Lys Leu
His Ala Ile Ile Lys Leu Thr Glu Met 165 170 175 Val Lys Ile Leu Gly
Asn His Gly Lys Leu Asp Glu Glu Met Val Gly 180 185 190 Val Val Thr
Lys Phe Leu Glu Asp Asn Arg Lys Val Ala Ala Leu Ala 195 200 205 Lys
Val Glu Lys Pro Val Thr Lys Val Lys Ala Leu Pro Phe Gly Glu 210 215
220 Arg Ala Lys Leu Ser Lys Asn Pro Met Gly Lys Arg Leu Phe Glu Ile
225 230 235 240 Met Ala Glu Lys Glu Ser Asn Leu Cys Leu Ala Ala Asp
Val Gly Thr 245 250 255 Ala Ala Glu Leu Leu Glu Ile Ala Glu Lys Val
Gly Pro Glu Ile Cys 260 265 270 Leu Leu Lys Thr His Val Asp Ile Phe
Pro Asp Phe Thr Ala Asp Phe 275 280 285 Gly Ser Lys Leu Leu Ser Ile
Ala Glu Lys His Asn Phe Leu Ile Phe 290 295 300 Glu Asp Arg Lys Phe
Ala Asp Ile Gly Asn Thr Val Thr Met Gln Tyr 305 310 315 320 Glu Gly
Gly Val Phe Arg Ile Leu Asp Trp Ala His Ile Val Asn Ala 325 330 335
His Ile Ile Ser Gly Pro Gly Ile Val Asp Gly Leu Lys Leu Lys Gly 340
345 350 Leu Pro Arg Gly Arg Gly Leu Leu Leu Leu Ala Glu Met Ser Ser
Ala 355 360 365 Gly Asn Leu Ala Lys Gly Asp Tyr Thr Thr Ser Ala Val
Lys Ile Ala 370 375 380 Glu Asp His Ser Asp Phe Val Ile Gly Phe Ile
Ser Val Asn Pro Ala 385 390 395 400 Ser Trp Pro Gly Ala Pro Ile Asn
Pro Ser Phe Ile Gln Ala Thr Pro 405 410 415 Gly Val Gln Met Val Thr
Gly Gly Asp Ala Leu Gly Gln Gln Tyr Asn 420 425 430 Thr Pro Tyr Ser
Val Ile His Asp Arg Gly Ser Asp Ile Ile Ile Val 435 440 445 Gly Arg
Gly Ile Ile Lys Ala Ala Asn Pro Ala Glu Ile Ala Arg Glu 450 455 460
Tyr Arg Leu Gln Gly Trp Asn Ala Tyr Leu Ala Lys Cys Asn 465 470 475
7 1781 DNA Triticum aestivum 7 gcacgagtct cgctccgccg ccgccgcctc
cctccccagt cgatcaccaa acctcagtcc 60 aaactccaaa cccccgccgc
atcagaaaaa aaccctaggc catggacgcc gcggcgctgg 120 agtcgctcat
cctggacctc cacgccatcg aggtcgtgaa gctgggctcc ttcacgctca 180
agtccggcat caaatcgccc atctacctcg acctccgcgc gctcgtctcc cacccgcgcc
240 tgctctccgc cgtcgcctcg ctccttcacg cgctcccggc cacgcgcccc
tacggcctcg 300 tctgcggtgt cccctacacc gcgctcccca tcgccgccgt
cctctccgtc gaccgctcaa 360 tccccatgct catgcgccgc aaggaggtca
aggcccacgg caccgccaag tccatcgagg 420 gctccttcag ccccggggac
accgtcctca tcatcgagga cctcgtcacc agtggcgcct 480 ccgtgctcga
gaccgccgcc ccgctccgcg ccgaggggct cgtcgtcgcc gacgccgtag 540
tcgtcgtcga ccgcgagcag ggtggcaggg agaacctcgc cgctaatggg atcacgctgc
600 actcgctcat gaccctcacg gaggtgctgg ccgtgctgct caagcacggg
aaggtgaccg 660 aggagaaggc gcaggaggtg aggcagttcc tcgacgccaa
caggaaggtg gcggtgcctg 720 gggcagcacc tgttacaccc agggtgctca
gaaagacatt ttcggagagg gcgaatcttg 780 ccaccaaccc tatggggaag
aagctcttcg agctgatgga gaccaagcag accaacctgt 840 gtgttgccgc
tgatgtcggg acaacaaagg aactccttga gctggctgac aaggtcggcc 900
ctcaaatttg tatgttgaaa acccatgtgg atatattatc tgattttacc ccagattttg
960 gctctaagct ccgctcgatt gctgagaagc acaacttttt gatcttcgaa
gaccgcaagt 1020 ttgctgacat tggaaataca gtaaccatgc aatatgaagg
aggaatattc cgcatattgg 1080 attgggccga tattgttaat gcgcatatag
tacctggacc tggaatcgta gatggcttga 1140 agctgaaggg tttgcctaaa
ggaagagggc tacttctgct cgctgagatg agctctgccg 1200 gcaaccttgc
ccatggagat tacactgctg ctgccgtaaa gattgctgag caacattctg 1260
attttgtgat gggatttata tcagtaaatc ctgagtcttg gtcagtaaaa ccatcaagcc
1320 ctgcatttat ccatgccacg cctggagttc agatggtcgc aggaggagat
gatcttgggc 1380 aacaatacaa cactcccgaa tctgtgataa actacagggg
cagtgacata atcatagttg 1440 gccgtgggat tataaaggcg agcgatccta
tgaagaaggc gtgggagtac cgcttgcaag 1500 ggtggcaggc atacaagaac
agcttgctat gaaggaaggg gggcgccatg agcatcccca 1560 agtataaggg
cgaatccagt cagtttggcg aaataagcgc atgcggaaag gttttcctgc 1620
agttgagtca ggacctaatt gacatcagat tcactgcaga ggagactcat gccccatcat
1680 cgtttctgtt acaataattt cctctcggtt taccctgttc ttgctggttg
agttaggcac 1740 gttgtgatgc ctgtgcgcgg ttaaaaaaaa aaaaaaaaaa a 1781
8 476 PRT Triticum aestivum 8 Met Asp Ala Ala Ala Leu Glu Ser Leu
Ile Leu Asp Leu His Ala Ile 1 5 10 15 Glu Val Val Lys Leu Gly Ser
Phe Thr Leu Lys Ser Gly Ile Lys Ser 20 25 30 Pro Ile Tyr Leu Asp
Leu Arg Ala Leu Val Ser His Pro Arg Leu Leu 35 40 45 Ser Ala Val
Ala Ser Leu Leu His Ala Leu Pro Ala Thr Arg Pro Tyr 50 55 60 Gly
Leu Val Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Ala Val 65 70
75 80 Leu Ser Val Asp Arg Ser Ile Pro Met Leu Met Arg Arg Lys Glu
Val 85 90 95 Lys Ala His Gly Thr Ala Lys Ser Ile Glu Gly Ser Phe
Ser Pro Gly 100 105 110 Asp Thr Val Leu Ile Ile Glu Asp Leu Val Thr
Ser Gly Ala Ser Val 115 120 125 Leu Glu Thr Ala Ala Pro Leu Arg Ala
Glu Gly Leu Val Val Ala Asp 130 135 140 Ala Val Val Val Val Asp Arg
Glu Gln Gly Gly Arg Glu Asn Leu Ala 145 150 155 160 Ala Asn Gly Ile
Thr Leu His Ser Leu Met Thr Leu Thr Glu Val Leu 165 170 175 Ala Val
Leu Leu Lys His Gly Lys Val Thr Glu Glu Lys Ala Gln Glu 180 185 190
Val Arg Gln Phe Leu Asp Ala Asn Arg Lys Val Ala Val Pro Gly Ala 195
200 205 Ala Pro Val Thr Pro Arg Val Leu Arg Lys Thr Phe Ser Glu Arg
Ala 210 215 220 Asn Leu Ala Thr Asn Pro Met Gly Lys Lys Leu Phe Glu
Leu Met Glu 225 230 235 240 Thr Lys Gln Thr Asn Leu Cys Val Ala Ala
Asp Val Gly Thr Thr Lys 245 250 255 Glu Leu Leu Glu Leu Ala Asp Lys
Val Gly Pro Gln Ile Cys Met Leu 260 265 270 Lys Thr His Val Asp Ile
Leu Ser Asp Phe Thr Pro Asp Phe Gly Ser 275 280 285 Lys Leu Arg Ser
Ile Ala Glu Lys His Asn Phe Leu Ile Phe Glu Asp 290 295 300 Arg Lys
Phe Ala Asp Ile Gly Asn Thr Val Thr Met Gln Tyr Glu Gly 305 310 315
320 Gly Ile Phe Arg Ile Leu Asp Trp Ala Asp Ile Val Asn Ala His Ile
325 330 335 Val Pro Gly Pro Gly Ile Val Asp Gly Leu Lys Leu Lys Gly
Leu Pro 340 345 350 Lys Gly Arg Gly Leu Leu Leu Leu Ala Glu Met Ser
Ser Ala Gly Asn 355 360 365 Leu Ala His Gly Asp Tyr Thr Ala Ala Ala
Val Lys Ile Ala Glu Gln 370 375 380 His Ser Asp Phe Val Met Gly Phe
Ile Ser Val Asn Pro Glu Ser Trp 385 390 395 400 Ser Val Lys Pro Ser
Ser Pro Ala Phe Ile His Ala Thr Pro Gly Val 405 410 415 Gln Met Val
Ala Gly Gly Asp Asp Leu Gly Gln Gln Tyr Asn Thr Pro 420 425 430 Glu
Ser Val Ile Asn Tyr Arg Gly Ser Asp Ile Ile Ile Val Gly Arg 435 440
445 Gly Ile Ile Lys Ala Ser Asp Pro Met Lys Lys Ala Trp Glu Tyr Arg
450 455 460 Leu Gln Gly Trp Gln Ala Tyr Lys Asn Ser Leu Leu 465 470
475 9 1889 DNA Zea mays 9 ccacgcgtcc gtacaaagcc acttcctgct
ctcgctccgc cgccgccgcc tccctcccca 60 gtcgatcacc aaacctcagt
ccaaactcca aacccccgcc gcatcagaaa aaaaccctag 120 gccatggacg
ccgcggcgct ggagtcgctc atcctggacc tccacgccat cgaggtcgtg 180
aagctgggct ccttcacgct caagtccggc atcaaatcgc ccatctacct cgacctccgc
240 gcgctcgtct cccacccgcg cctgctctcc gccgtcgcct cgctccttca
cgcgctcccg 300 gccacgcgcc cctacggcct cgtctgcggt gtcccctaca
ccgcgctccc catcgccgcc 360 gtcctctccg tcgaccgctc aatccccatg
ctcatgcgcc gcaaggaggt caaggcccac 420 ggcaccgcca agtccatcga
gggctccttc agccccgggg acaccgtcct catcatcgag 480 gacctcgtca
ccagtggcgc ctccgtgctc gagaccgccg ccccgctccg cgccgagggg 540
ctcgtcgtcg ccgacgccgt agtcgtcgtc gaccgcgagc agggtggcag ggagaacctc
600 gccgctaatg ggatcacgct gcactcgctc atgaccctca cggaggtgct
ggccgtgctg 660 ctcaagcacg ggaaggtgac cgaggagaag gcgcaggagg
tgaggcagtt cctcgacgcc 720 aacaggaagg tggcggtgcc tggggcagca
cctgttacac ccagggtgct cagaaagaca 780 ttttcggaga gggcgaatct
tgccaccaac cctatgggga agaagctctt cgagctgatg 840 gagaccaagc
agaccaacct gtgtgttgcc gctgatgtcg ggacaacaaa ggaactcctt 900
gagctggctg acaaggtcgg ccctcaaatt tgtatgttga aaacccatgt ggatatatta
960 tctgatttta ccccagattt tggctctaag ctccgctcga ttgctgagaa
gcacaacttt 1020 ttgatcttcg aagaccgcaa gtttgctgac attggaaata
cagtaaccat gcaatatgaa 1080 ggaggaatat tccgcatatt ggattgggcc
gatattgtta atgcgcatat agtacctgga 1140 cctggaatcg tagatggctt
gaagctgaag ggtttgccta aaggaagagg gctacttctg 1200 ctcgctgaga
tgagctctgc cggcaacctt gcccatggag attacactgc tgctgccgta 1260
aagattgctg agcaacattc tgattttgtg atgggattta tatcagtaaa tcctgagtct
1320 tggtcagtaa aaccatcaag ccctgcattt atccatgcca cgcctggagt
tcagatggtc 1380 gcaggaggag atgatcttgg gcaacaatac aacactcccg
aatctgtgat aaactacagg 1440 ggcagtgaca taatcatagt tggccgtggg
attataaagg cgagcgatcc tatgaagaag 1500 gcgtgggagt accgcttgca
agggtggcag gcatacaaga acagcttgct atgaaggaag 1560 gggggcgcca
tgagcatccc caagtataag ggcgaatcca gtcagtttgg cgaaataagc 1620
gcatgcggaa aggttttcct gcagttgagt caggacctaa ttgacatcag attcactgca
1680 gaggagactc atgccccatc atcgtttctg ttacaataat ttcctctcgg
tttaccctgt 1740 tcttgctggt tgagttaggc acgttgtgat gcctgtgcgc
ggttaaatcg tcttactgcc 1800 atgccacttg aggtttggac tcttgagcaa
gcaattttat cgatgccgag aattgtatga 1860 aaaaaaaaaa aaaaaaaaaa
aaaaaaaag 1889 10 476 PRT Zea mays 10 Met Asp Ala Ala Ala Leu Glu
Ser Leu Ile Leu Asp Leu His Ala Ile 1 5 10 15 Glu Val Val Lys Leu
Gly Ser Phe Thr Leu Lys Ser Gly Ile Lys Ser 20 25 30 Pro Ile Tyr
Leu Asp Leu Arg Ala Leu Val Ser His Pro Arg Leu Leu 35 40 45 Ser
Ala Val Ala Ser Leu Leu His Ala Leu Pro Ala Thr Arg Pro Tyr 50 55
60 Gly Leu Val Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Ala Val
65 70 75 80 Leu Ser Val Asp Arg Ser Ile Pro Met Leu Met Arg Arg Lys
Glu Val 85 90 95 Lys Ala His Gly Thr Ala Lys Ser Ile Glu Gly Ser
Phe Ser Pro Gly 100 105 110 Asp Thr Val Leu Ile Ile Glu Asp Leu Val
Thr Ser Gly Ala Ser Val 115 120 125 Leu Glu Thr Ala Ala Pro Leu Arg
Ala Glu Gly Leu Val Val Ala Asp 130 135 140 Ala Val Val Val Val Asp
Arg Glu Gln Gly Gly Arg Glu Asn Leu Ala 145 150 155 160 Ala Asn Gly
Ile Thr Leu His Ser Leu Met Thr Leu Thr Glu Val Leu 165 170 175 Ala
Val Leu Leu Lys His Gly Lys Val Thr Glu Glu Lys Ala Gln Glu 180 185
190 Val Arg Gln Phe Leu Asp Ala Asn Arg Lys Val Ala Val Pro Gly Ala
195 200
205 Ala Pro Val Thr Pro Arg Val Leu Arg Lys Thr Phe Ser Glu Arg Ala
210 215 220 Asn Leu Ala Thr Asn Pro Met Gly Lys Lys Leu Phe Glu Leu
Met Glu 225 230 235 240 Thr Lys Gln Thr Asn Leu Cys Val Ala Ala Asp
Val Gly Thr Thr Lys 245 250 255 Glu Leu Leu Glu Leu Ala Asp Lys Val
Gly Pro Gln Ile Cys Met Leu 260 265 270 Lys Thr His Val Asp Ile Leu
Ser Asp Phe Thr Pro Asp Phe Gly Ser 275 280 285 Lys Leu Arg Ser Ile
Ala Glu Lys His Asn Phe Leu Ile Phe Glu Asp 290 295 300 Arg Lys Phe
Ala Asp Ile Gly Asn Thr Val Thr Met Gln Tyr Glu Gly 305 310 315 320
Gly Ile Phe Arg Ile Leu Asp Trp Ala Asp Ile Val Asn Ala His Ile 325
330 335 Val Pro Gly Pro Gly Ile Val Asp Gly Leu Lys Leu Lys Gly Leu
Pro 340 345 350 Lys Gly Arg Gly Leu Leu Leu Leu Ala Glu Met Ser Ser
Ala Gly Asn 355 360 365 Leu Ala His Gly Asp Tyr Thr Ala Ala Ala Val
Lys Ile Ala Glu Gln 370 375 380 His Ser Asp Phe Val Met Gly Phe Ile
Ser Val Asn Pro Glu Ser Trp 385 390 395 400 Ser Val Lys Pro Ser Ser
Pro Ala Phe Ile His Ala Thr Pro Gly Val 405 410 415 Gln Met Val Ala
Gly Gly Asp Asp Leu Gly Gln Gln Tyr Asn Thr Pro 420 425 430 Glu Ser
Val Ile Asn Tyr Arg Gly Ser Asp Ile Ile Ile Val Gly Arg 435 440 445
Gly Ile Ile Lys Ala Ser Asp Pro Met Lys Lys Ala Trp Glu Tyr Arg 450
455 460 Leu Gln Gly Trp Gln Ala Tyr Lys Asn Ser Leu Leu 465 470 475
11 1627 DNA Oryza sativa unsure (1489) n = A, C, G or T 11
gcacgagctt acacccgccc aaaaccctag ctaagcctag ccgccatgga cgccgccgcg
60 caggaatccc tcatcctgga gctccacgcc atcgaggcca tcaagttcgg
caccttcgtg 120 ctcaagtccg gcatcacctc cccgatctac ctcgacctcc
gcgcgctcgt ctcccacccg 180 ggcctcctct cctccatcgc caccctcctc
cacaccctcc cggcgacccg cccctacgac 240 ctcctctgcg gcgtccccta
caccgcgctc cccatcgcct ccgtcctctc cgtccaccgc 300 tccgtcccca
tggtcatgcg ccgcaaggag gccaaggccc acggcaccgc caagtccatc 360
gagggcgcct tccgcgccgg ggaggccgtg ctcatcatcg aggacctcgt caccagcggc
420 gcctccgttc tcgagaccgc cgcgccgctc cgcgaccagg ggctcgtcgt
cgccgacgcc 480 gtcgtcgtcg tcgaccgcaa gcagggcggg agggagaacc
ttgccgccaa tgggatcacg 540 ctgcactcgc tcatgaccct cacggaggtg
ctcgccgtgc tgctcaagca cgggaaggtg 600 acccaagaag agcgaggagg
taagcagttt cttgacgcca ataggaaggt gaccgttccc 660 ggagcggcgg
gcgccgttaa gcccaaagcg gtcaggaagg ggtttgctga gagggctgga 720
ttggccaaga acccgatggg gaagaggctt ttcgaggtga tggaggcaaa gcagagcaat
780 ttatgtgttg ctgccgatgt gggaactgca aaggagctcc ttgagcttgc
agagaaggtt 840 ggtccagaga tttgcatgct gaaaactcat gtggatatct
tgtctgactt tactccagat 900 tttggagcta agcttcgctc gattgccgag
aagcacaact ttttgatatt tgaagaccgc 960 aagtttgctg acattggaaa
cacagtgact atgcaatatg aaggaggaat atttcgcata 1020 ttagactggg
ctgatatcgt caatgcccat ataattcctg gacctggaat tgtggatggt 1080
ctgaagctta agggtttgcc aaaaggaaga gggctgcttt tgcttgctga aatgagctcg
1140 gctggcaacc ttgctcatgg agagtacact gctgcagctg taaagattgc
tgagcaacat 1200 tctgattttg taattggatt tatatccgtt aatccagcat
cttggtcagt tgcgccatca 1260 agtccagcat ttatccatgc cactcctgga
gtgcagatgg tttctggagg agatgctctt 1320 ggtcaacagt acaatacccc
tcattctgtt ataaacgaca agaggcaagt gacataatta 1380 tagtccggac
gagggattat aaaggcgaag taatccagcc cgagaccgcg agggaagtac 1440
cgcatccaag ggtgggggag caaaacaatc cagctttgcc atgagaaant gagaatngtg
1500 tttaggcaat ggttggttcn agcttatgat ttattataac caagaataat
taagccanga 1560 ttgcnnataa agccgggatt aatantnaag ctgccatana
aataaactgt gnagttggtt 1620 gntttgg 1627 12 443 PRT Oryza sativa 12
Met Asp Ala Ala Ala Gln Glu Ser Leu Ile Leu Glu Leu His Ala Ile 1 5
10 15 Glu Ala Ile Lys Phe Gly Thr Phe Val Leu Lys Ser Gly Ile Thr
Ser 20 25 30 Pro Ile Tyr Leu Asp Leu Arg Ala Leu Val Ser His Pro
Gly Leu Leu 35 40 45 Ser Ser Ile Ala Thr Leu Leu His Thr Leu Pro
Ala Thr Arg Pro Tyr 50 55 60 Asp Leu Leu Cys Gly Val Pro Tyr Thr
Ala Leu Pro Ile Ala Ser Val 65 70 75 80 Leu Ser Val His Arg Ser Val
Pro Met Val Met Arg Arg Lys Glu Ala 85 90 95 Lys Ala His Gly Thr
Ala Lys Ser Ile Glu Gly Ala Phe Arg Ala Gly 100 105 110 Glu Ala Val
Leu Ile Ile Glu Asp Leu Val Thr Ser Gly Ala Ser Val 115 120 125 Leu
Glu Thr Ala Ala Pro Leu Arg Asp Gln Gly Leu Val Val Ala Asp 130 135
140 Ala Val Val Val Val Asp Arg Lys Gln Gly Gly Arg Glu Asn Leu Ala
145 150 155 160 Ala Asn Gly Ile Thr Leu His Ser Leu Met Thr Leu Thr
Glu Val Leu 165 170 175 Ala Val Leu Leu Lys His Gly Lys Val Thr Gln
Glu Glu Arg Gly Gly 180 185 190 Lys Gln Phe Leu Asp Ala Asn Arg Lys
Val Thr Val Pro Gly Ala Ala 195 200 205 Gly Ala Val Lys Pro Lys Ala
Val Arg Lys Gly Phe Ala Glu Arg Ala 210 215 220 Gly Leu Ala Lys Asn
Pro Met Gly Lys Arg Leu Phe Glu Val Met Glu 225 230 235 240 Ala Lys
Gln Ser Asn Leu Cys Val Ala Ala Asp Val Gly Thr Ala Lys 245 250 255
Glu Leu Leu Glu Leu Ala Glu Lys Val Gly Pro Glu Ile Cys Met Leu 260
265 270 Lys Thr His Val Asp Ile Leu Ser Asp Phe Thr Pro Asp Phe Gly
Ala 275 280 285 Lys Leu Arg Ser Ile Ala Glu Lys His Asn Phe Leu Ile
Phe Glu Asp 290 295 300 Arg Lys Phe Ala Asp Ile Gly Asn Thr Val Thr
Met Gln Tyr Glu Gly 305 310 315 320 Gly Ile Phe Arg Ile Leu Asp Trp
Ala Asp Ile Val Asn Ala His Ile 325 330 335 Ile Pro Gly Pro Gly Ile
Val Asp Gly Leu Lys Leu Lys Gly Leu Pro 340 345 350 Lys Gly Arg Gly
Leu Leu Leu Leu Ala Glu Met Ser Ser Ala Gly Asn 355 360 365 Leu Ala
His Gly Glu Tyr Thr Ala Ala Ala Val Lys Ile Ala Glu Gln 370 375 380
His Ser Asp Phe Val Ile Gly Phe Ile Ser Val Asn Pro Ala Ser Trp 385
390 395 400 Ser Val Ala Pro Ser Ser Pro Ala Phe Ile His Ala Thr Pro
Gly Val 405 410 415 Gln Met Val Ser Gly Gly Asp Ala Leu Gly Gln Gln
Tyr Asn Thr Pro 420 425 430 His Ser Val Ile Asn Asp Lys Arg Gln Val
Thr 435 440 13 476 PRT Arabidopsis thaliana 13 Met Ser Ala Met Glu
Ala Leu Ile Leu Gln Leu His Glu Ile Gly Ala 1 5 10 15 Val Lys Phe
Gly Asn Phe Lys Leu Lys Ser Gly Ile Phe Ser Pro Val 20 25 30 Tyr
Ile Asp Leu Arg Leu Ile Val Ser Tyr Pro Ser Leu Leu Thr Gln 35 40
45 Ile Ser Gln Thr Leu Ile Ser Ser Leu Pro Pro Ser Ala Thr Phe Asp
50 55 60 Val Val Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Thr
Val Val 65 70 75 80 Ser Val Ser Asn Gly Ile Pro Met Leu Met Arg Arg
Lys Glu Ile Lys 85 90 95 Asp Tyr Gly Thr Ser Lys Ala Ile Glu Gly
Ile Phe Glu Lys Asp Gln 100 105 110 Thr Cys Leu Ile Ile Glu Asp Leu
Val Thr Ser Gly Ala Ser Val Leu 115 120 125 Glu Thr Ala Ala Pro Leu
Arg Ala Val Gly Leu Lys Val Ser Asp Ala 130 135 140 Val Val Leu Ile
Asp Arg Gln Gln Gly Gly Arg Glu Asn Leu Ala Glu 145 150 155 160 Asn
Gly Ile Lys Leu His Ser Met Ile Met Leu Thr Asp Met Val Arg 165 170
175 Val Leu Lys Glu Lys Gly Lys Ile Glu Glu Glu Val Glu Val Asn Leu
180 185 190 Leu Lys Phe Leu Glu Glu Asn Arg Arg Val Ser Val Pro Ser
Val Glu 195 200 205 Lys Pro Lys Pro Lys Pro Arg Val Leu Gly Phe Lys
Glu Arg Ser Glu 210 215 220 Leu Ser Lys Asn Pro Thr Gly Lys Lys Leu
Phe Asp Ile Met Leu Lys 225 230 235 240 Lys Glu Thr Asn Leu Cys Leu
Ala Ala Asp Val Gly Thr Ala Ala Glu 245 250 255 Leu Leu Asp Ile Ala
Asp Lys Val Gly Pro Glu Ile Cys Leu Leu Lys 260 265 270 Thr His Val
Asp Ile Leu Pro Asp Phe Thr Pro Asp Phe Gly Ser Lys 275 280 285 Leu
Arg Ala Ile Ala Asp Lys His Lys Phe Leu Ile Phe Glu Asp Arg 290 295
300 Lys Phe Ala Asp Ile Gly Asn Thr Val Thr Met Gln Tyr Glu Gly Gly
305 310 315 320 Ile Phe Lys Ile Leu Glu Trp Ala Asp Ile Ile Asn Ala
His Val Ile 325 330 335 Ser Gly Pro Gly Ile Val Asp Gly Leu Lys Leu
Lys Gly Met Pro Arg 340 345 350 Gly Arg Gly Leu Leu Leu Leu Ala Glu
Met Ser Ser Ala Gly Asn Leu 355 360 365 Ala Thr Gly Asp Tyr Thr Ala
Ala Ala Val Lys Ile Ala Asp Ala His 370 375 380 Ser Asp Phe Val Met
Gly Phe Ile Ser Val Asn Pro Ala Ser Trp Lys 385 390 395 400 Cys Gly
Tyr Val Tyr Pro Ser Met Ile His Ala Thr Pro Gly Val Gln 405 410 415
Met Val Lys Gly Gly Asp Ala Leu Gly Gln Gln Tyr Asn Thr Pro His 420
425 430 Ser Val Ile Thr Glu Arg Gly Ser Asp Ile Ile Ile Val Gly Arg
Gly 435 440 445 Ile Ile Lys Ala Glu Asn Pro Ala Glu Thr Ala His Glu
Tyr Arg Val 450 455 460 Gln Gly Trp Asn Ala Tyr Leu Glu Lys Cys Ser
Gln 465 470 475 14 476 PRT Nicotiana tabacum 14 Met Ser Ala Met Glu
Ala Leu Ile Leu Gln Leu His Glu Ile Gly Ala 1 5 10 15 Val Lys Phe
Gly Asn Phe Lys Leu Lys Ser Gly Ile Phe Ser Pro Val 20 25 30 Tyr
Ile Asp Leu Arg Leu Ile Val Ser Tyr Pro Ser Leu Leu Thr Gln 35 40
45 Ile Ser Gln Thr Leu Ile Ser Ser Leu Pro Pro Ser Ala Thr Phe Asp
50 55 60 Val Val Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Thr
Val Val 65 70 75 80 Ser Val Ser Asn Gly Ile Pro Met Leu Met Arg Arg
Lys Glu Ile Lys 85 90 95 Asp Tyr Gly Thr Ser Lys Ala Ile Glu Gly
Ile Phe Glu Lys Asp Gln 100 105 110 Thr Cys Leu Ile Ile Glu Asp Leu
Val Thr Ser Gly Ala Ser Val Leu 115 120 125 Glu Thr Ala Ala Pro Leu
Arg Ala Val Gly Leu Lys Val Ser Asp Ala 130 135 140 Val Val Leu Ile
Asp Arg Gln Gln Gly Gly Arg Glu Asn Leu Ala Glu 145 150 155 160 Asn
Gly Ile Lys Leu His Ser Met Ile Met Leu Thr Asp Met Val Arg 165 170
175 Val Leu Lys Glu Lys Gly Lys Ile Glu Glu Glu Val Glu Val Asn Leu
180 185 190 Leu Lys Phe Leu Glu Glu Asn Arg Arg Val Ser Val Pro Ser
Val Glu 195 200 205 Lys Pro Lys Pro Lys Pro Arg Val Leu Gly Phe Lys
Glu Arg Ser Glu 210 215 220 Leu Ser Lys Asn Pro Thr Gly Lys Lys Leu
Phe Asp Ile Met Leu Lys 225 230 235 240 Lys Glu Thr Asn Leu Cys Leu
Ala Ala Asp Val Gly Thr Ala Ala Glu 245 250 255 Leu Leu Asp Ile Ala
Asp Lys Val Gly Pro Glu Ile Cys Leu Leu Lys 260 265 270 Thr His Val
Asp Ile Leu Pro Asp Phe Thr Pro Asp Phe Gly Ser Lys 275 280 285 Leu
Arg Ala Ile Ala Asp Lys His Lys Phe Leu Ile Phe Glu Asp Arg 290 295
300 Lys Phe Ala Asp Ile Gly Asn Thr Val Thr Met Gln Tyr Glu Gly Gly
305 310 315 320 Ile Phe Lys Ile Leu Glu Trp Ala Asp Ile Ile Asn Ala
His Val Ile 325 330 335 Ser Gly Pro Gly Ile Val Asp Gly Leu Lys Leu
Lys Gly Met Pro Arg 340 345 350 Gly Arg Gly Leu Leu Leu Leu Ala Glu
Met Ser Ser Ala Gly Asn Leu 355 360 365 Ala Thr Gly Asp Tyr Thr Ala
Ala Ala Val Lys Ile Ala Asp Ala His 370 375 380 Ser Asp Phe Val Met
Gly Phe Ile Ser Val Asn Pro Ala Ser Trp Lys 385 390 395 400 Cys Gly
Tyr Val Tyr Pro Ser Met Ile His Ala Thr Pro Gly Val Gln 405 410 415
Met Val Lys Gly Gly Asp Ala Leu Gly Gln Gln Tyr Asn Thr Pro His 420
425 430 Ser Val Ile Thr Glu Arg Gly Ser Asp Ile Ile Ile Val Gly Arg
Gly 435 440 445 Ile Ile Lys Ala Glu Asn Pro Ala Glu Thr Ala His Glu
Tyr Arg Val 450 455 460 Gln Gly Trp Asn Ala Tyr Leu Glu Lys Cys Ser
Gln 465 470 475
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References