U.S. patent application number 14/174014 was filed with the patent office on 2014-06-05 for plant diacylglycerol acyltransferases.
This patent application is currently assigned to E I DU PONT DE NEMOURS AND COMPANY. The applicant listed for this patent is E I DU PONT DE NEMOURS AND COMPANY. Invention is credited to Karlene H Butler, Edgar Benjamin Cahoon, Rebecca E Cahoon, Anthony J Kinney.
Application Number | 20140154720 14/174014 |
Document ID | / |
Family ID | 26808207 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154720 |
Kind Code |
A1 |
Butler; Karlene H ; et
al. |
June 5, 2014 |
PLANT DIACYLGLYCEROL ACYLTRANSFERASES
Abstract
This invention relates to an isolated nucleic acid fragment
encoding a diacylglycerol acyltransferase. The invention also
relates to the construction of a chimeric gene encoding all or a
portion of the diacylglycerol acyltransferase, in sense or
antisense orientation, wherein expression of the chimeric gene
results in production of altered levels of the diacylglycerol
acyltransferase in a transformed host cell.
Inventors: |
Butler; Karlene H; (Newark,
DE) ; Cahoon; Edgar Benjamin; (Lincoln, NE) ;
Cahoon; Rebecca E; (Lincoln, NE) ; Kinney; Anthony
J; (Wilmington, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
E I DU PONT DE NEMOURS AND COMPANY |
Wilmington |
DE |
US |
|
|
Assignee: |
E I DU PONT DE NEMOURS AND
COMPANY
Wilmington
DE
|
Family ID: |
26808207 |
Appl. No.: |
14/174014 |
Filed: |
February 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13906846 |
May 31, 2013 |
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14174014 |
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12975791 |
Dec 22, 2010 |
8497362 |
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13906846 |
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12338607 |
Dec 18, 2008 |
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12975791 |
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10690994 |
Oct 21, 2003 |
7524945 |
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12338607 |
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09856018 |
May 16, 2001 |
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PCT/US99/28354 |
Dec 1, 1999 |
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10690994 |
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60127111 |
Mar 31, 1999 |
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60110602 |
Dec 2, 1998 |
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Current U.S.
Class: |
435/15 ;
435/235.1; 435/252.3; 435/254.2; 435/320.1; 435/419; 536/23.2 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12N 9/1029 20130101 |
Class at
Publication: |
435/15 ;
536/23.2; 435/320.1; 435/254.2; 435/252.3; 435/419; 435/235.1 |
International
Class: |
C12N 9/10 20060101
C12N009/10 |
Claims
1. An isolated polynucleotide comprising a nucleotide sequence
encoding a first polypeptide of at least 50 amino acids that has at
least 60% identity based on the Clustal method of alignment when
compared to a polypeptide selected from the group consisting of SEQ
ID NOs:4, 6, 8, 10, 14, 20 and 22, or an isolated polynucleotide
comprising the complement of the nucleotide sequence.
2. The isolated polynucleotide of claim 1, wherein the isolated
nucleotide sequence consists of a nucleic acid sequence selected
from the group consisting of SEQ ID NOs:1, 7, 13, 15, and 21 that
codes for the polypeptide selected from the group consisting of SEQ
ID NOs:2, 8, 14, 16, and 22.
3. The isolated polynucleotide of claim 1 wherein the isolated
polynucleotide is DNA.
4. The isolated polynucleotide of claim 1 wherein the isolated
polynucleotide is RNA.
5. A chimeric gene comprising the isolated polynucleotide of claim
1 operably linked to suitable regulatory sequences.
6. An isolated host cell comprising the chimeric gene of claim
5.
7. An isolated host cell comprising the isolated polynucleotide of
claim 1.
8. The isolated host cell of claim 7 wherein the isolated host is
selected from the group consisting of yeast, bacteria, plant, and
virus.
9. A virus comprising the isolated polynucleotide of claim 1.
10. A method of selecting an isolated polynucleotide that affects
the level of expression of a diacylglycerol acyltransferase
polypeptide in a plant cell, the method comprising the steps of:
(a) constructing an isolated polynucleotide comprising a nucleotide
sequence of at least one of 30 contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, and the complement of such
nucleotide sequences; (b) introducing the isolated polynucleotide
into a plant cell; (c) measuring the level of a polypeptide in the
plant cell containing the polynucleotide; and (d) comparing the
level of polypeptide in the plant cell containing the isolated
polynucleotide with the level of polypeptide in a plant cell that
does not contain the isolated polynucleotide.
11. The method of claim 10 wherein the isolated polynucleotide
consists of a nucleotide sequence selected from the group
consisting of SEQ ID NOs:1, 3, 5, 7, 11, 13, 15, 17, 19 and 21 that
codes for the polypeptide selected from the group consisting of SEQ
ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22.
12. A method of selecting an isolated polynucleotide that affects
the level of expression of a diacylglycerol acyltransferase
polypeptide in a plant cell, the method comprising the steps of:
(a) constructing the isolated polynucleotide of claim 1; (b)
introducing the isolated polynucleotide into a plant cell; and (c)
measuring the level of diacylglycerol acyltransferase polypeptide
in the plant cell containing the polynucleotide.
13. A method for evaluating at least one compound for its ability
to inhibit the activity of a diacylglycerol acyltransferase, the
method comprising the steps of: (a) transforming a host cell with a
chimeric gene comprising a nucleic acid fragment encoding a
diacylglycerol acyltransferase, operably linked to suitable
regulatory sequences; (b) growing the transformed host cell under
conditions that are suitable for expression of the chimeric gene
wherein expression of the chimeric gene results in production of
the diacylglycerol acyltransferase encoded by the operably linked
nucleic acid fragment in the transformed host cell; (c) optionally
purifying the diacylglycerol acyltransferase expressed by the
transformed host cell; (d) treating the diacylglycerol
acyltransferase with a compound to be tested; and (e) comparing the
activity of the diacylglycerol acyltransferase that has been
treated with a test compound to the activity of an untreated
diacylglycerol acyltransferase, thereby selecting compounds with
potential for inhibitory activity.
14. A composition comprising the isolated polynucleotide of claim
1.
15. An expression cassette comprising the isolated polynucleotide
of claim 1 operably linked to a promoter.
16. A method for positive selection of a transformed cell
comprising: (a) transforming a plant cell with the expression
cassette of claim 15; and (b) growing the transformed plant cell
under conditions allowing expression of the polynucleotide in an
amount sufficient to modify oil content in the plant cell to
provide a positive selection means.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 13/906846 filed May 31, 2013, now pending, which is a
continuation of U.S. application Ser. No. 12/975,791, filed Dec.
22, 2010, now U.S. Pat. No. 8497362, which is a divisional of U.S.
application Ser. No. 12/338,607, filed Dec. 18, 2008, now
abandoned, which is a divisional of U.S. application Ser. No.
10/690,994, filed Oct. 21, 2003, now U.S. Pat. No. 7,524,945, which
is a continuation of U.S. application Ser. No. 09/856,018, filed
May 16, 2001, now abandoned, which is a National Stage Application
of PCT/US99/28354, filed Dec. 1, 1999, now expired, which claims
the benefit of U.S. Provisional Application No. 60/127,111, filed
Mar. 31, 1999, now expired, and U.S. Provisional Application No.
60/110,602, filed Dec. 2, 1998, now expired.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
fragments encoding diacylglycerol acyltransferase in plants and
seeds.
BACKGROUND OF THE INVENTION
[0003] In eukaryotic cells triacylglycerols are quantitatively the
most important storage form of energy. Acyl CoA:diacylglycerol
acyltransferase (DGAT, EC 2.3.1.20) uses fatty acyl CoA and
diacylglycerol as substrates to catalyze the only committed step in
triacylglycerol synthesis. DGAT plays a fundamental role in the
metabolism of cellular glycerolipids. Because it is an integral
membrane protein, DGAT has yet to be purified to homogeneity. A
mouse cDNA encoding a protein with DGAT activity has been isolated
by using a sequence tag clone sharing regions of similarity with an
acyl Co A cholesterol acyltransferase. This mouse DGAT has been
cloned, sequenced and expressed in insect cells and its activity
characterized (Cases, S. et al. (1998) Proc. Natl. Acad. Sci. USA
95:13018-13023).
[0004] DGAT is important for the generation of seed oils, thus
overexpression of DGAT may be useful for increasing oil content of
oilseeds and suppression of DGAT may result in the diversion of
carbon into other metabolites.
SUMMARY OF THE INVENTION
[0005] The present invention relates to a composition comprising an
isolated polynucleotide or polypeptide of the present
invention.
[0006] The present invention relates to an isolated polynucleotide
of the present invention comprising the nucleotide sequence
comprising at least one of 30 contiguous nucleotides of a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:1,
3, 5, 7, 9, 11, 13, 15, 17, 19 and 21.
[0007] The present invention relates to an expression cassette
comprising an isolated polynucleotide of the present invention
operably linked to a promoter.
[0008] The present invention relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a first polypeptide of at
least 50 amino acids that has at least 60% identity based on the
Clustal method of alignment when compared to a polypeptide selected
from the group consisting of SEQ ID NOs:4, 6, 8, 10, 14, 20 and 22
or an isolated polynucleotide comprising the complement of the
nucleotide sequence.
[0009] The present invention relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a first polypeptide of at
least 50 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:18 and 20.
[0010] It is preferred that the isolated polynucleotide of the
claimed invention consists of a nucleic acid sequence selected from
the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,
19 and 21 that codes for the polypeptide selected from the group
consisting of SEQ ID NOs:2, 6, 8, 10, 14, 16, and 22. The present
invention also relates to an isolated polynucleotide comprising a
nucleotide sequences of at least one of 40 (preferably at least one
of 30) contiguous nucleotides derived from a nucleotide sequence
selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, and the complement of such nucleotide
sequences.
[0011] The present invention relates to a chimeric gene comprising
an isolated polynucleotide of the present invention operably linked
to suitable regulatory sequences.
[0012] The present invention relates to an isolated host cell
comprising a chimeric gene of the present invention or an isolated
polynucleotide of the present invention. The host cell may be
eukaryotic, such as a yeast or a plant cell, or prokaryotic, such
as a bacterial cell. The present invention also relates to a virus,
preferably a baculovirus, comprising an isolated polynucleotide of
the present invention or a chimeric gene of the present
invention.
[0013] The present invention relates to a process for producing an
isolated host cell comprising a chimeric gene of the present
invention or an isolated polynucleotide of the present invention,
the process comprising either transforming or transfecting an
isolated compatible host cell with a chimeric gene or isolated
polynucleotide of the present invention.
[0014] The present invention relates to a polypeptide of at least
50 amino acids that has at least 60% identity based on the Clustal
method of alignment when compared to a polypeptide selected from
the group consisting of a diacylglycerol acyltransferase
polypeptide of SEQ ID NOs:4, 6, 8, 10, 14, 20 and 22.
[0015] The present invention relates to a polypeptide of at least
50 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:18 and 20.
[0016] The present invention relates to a polypeptide of at least
50 amino acids that has at least 80% identity based on the Clustal
method of alignment when compared to a polypetide of SEQ ID
NO:2.
[0017] The present invention relates to a method of selecting an
isolated polynucleotide that affects the level of expression of a
diacylglycerol acyltransferase polypeptide in a host cell,
preferably a plant cell, the method comprising the steps of: [0018]
constructing an isolated polynucleotide of the present invention or
an isolated chimeric gene of the present invention; [0019]
introducing the isolated polynucleotide or the isolated chimeric
gene into a host cell; [0020] measuring the level a diacylglycerol
acyltransferase polypeptide in the host cell containing the
isolated polynucleotide; and [0021] comparing the level of a
diacylglycerol acyltransferase polypeptide in the host cell
containing the isolated polynucleotide with the level of a
diacylglycerol acyltransferase polypeptide in a host cell that does
not contain the isolated polynucleotide.
[0022] The present invention relates to a method of obtaining a
nucleic acid fragment encoding a substantial portion of a
diacylglycerol acyltransferase polypeptide gene, preferably a plant
diacylglycerol acyltransferase polypeptide gene, comprising the
steps of: synthesizing an oligonucleotide primer comprising a
nucleotide sequence of at least one of 40 (preferably at least one
of 30) contiguous nucleotides derived from a nucleotide sequence
selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, and the complement of such nucleotide
sequences; and amplifying a nucleic acid fragment (preferably a
cDNA inserted in a cloning vector) using the oligonucleotide
primer. The amplified nucleic acid fragment preferably will encode
a portion of a diacylglycerol acyltransferase amino acid
sequence.
[0023] The present invention also relates to a method of obtaining
a nucleic acid fragment encoding all or a substantial portion of
the amino acid sequence encoding a diacylglycerol acyltransferase
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.
[0024] A further embodiment of the instant invention is a method
for evaluating at least one compound for its ability to inhibit the
activity of a diacylglycerol acyltransferase, the method comprising
the steps of: (a) transforming a host cell with a chimeric gene
comprising a nucleic acid fragment encoding a diacylglycerol
acyltransferase, 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 diacylglycerol
acyltransferase in the transformed host cell; (c) optionally
purifying the diacylglycerol acyltransferase expressed by the
transformed host cell; (d) treating the diacylglycerol
acyltransferase with a compound to be tested; and (e) comparing the
activity of the diacylglycerol acyltransferase that has been
treated with a test compound to the activity of an untreated
diacylglycerol acyltransferase, thereby selecting compounds with
potential for inhibitory activity.
BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE DESCRIPTIONS
[0025] The invention can be more fully understood from the
following detailed description and the accompanying drawing and
Sequence Listing which form a part of this application.
[0026] FIGS. 1A, 1B, and 1C show an alignment of the amino acid
sequences from Mus musculus diacylglycerol acetyltransferase (SEQ
ID NO:25), the instant Arabidopsis thaliana diacylglycerol
acetyltransferase (araebcF; SEQ ID NO:2), the instant corn
diacylglycerol acetyltransferase (cpjlc.pk005.h23; SEQ ID NO:8),
the instant rice diacylglycerol acetyltransferase
(r1s24.pk0034.d8:fis; SEQ ID NO:14), the instant soybean
diacylglycerol acetyltransferase (srl.pk0098.a8; SEQ ID NO:16), and
the instant wheat diacylglycerol acetyltransferase
(wrl.pk0119.b6:fis; SEQ ID NO:22). Amino acids which are identical
among all sequences are indicated with an asterisk (*) above the
alignment while those conserved only among the plant sequences are
indicated by a plus sign (+). Dashes are used by the program to
maximize alignment of the sequences.
[0027] 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.
TABLE-US-00001 TABLE 1 Diacylglycerol Acyltransferases SEQ ID NO:
(Nucleo- (Amino Protein Clone Designation tide) Acid) Arabidopsis
Diacylgycerol araebcF 1 2 Acyltransferase Corn Diacylgycerol Contig
of: 3 4 Acyltransferase cpj1c.pk005.h23 cen3n.pk0010.c10
cco1.pk0029.b6 Corn Diacylgycerol cen3n.pk0113.e12 5 6
Acyltransferase Corn Diacylgycerol cpj1c.pk005.h23 7 8
Acyltransferase Corn Diacylgycerol Contig of: 9 10 Acyltransferase
p0042.cspaf49r p0122.ckamb57r p0125.czaau61rb Rice Diacylgycerol
rls24.pk0034.d8 11 12 Acyltransferase Rice Diacylgycerol
rls24.pk0034.d8:fis 13 14 Acyltransferase Soybean Diacylgycerol
sr1.pk0098.a8 15 16 Acyltransferase Soybean Diacylgycerol
src3c.pk013.h18 17 18 Acyltransferase Wheat Diacylgycerol
wr1.pk0119.b6 19 20 Acyltransferase Wheat Diacylgycerol
wr1.pk0119.b6:fis 21 22 Acyltransferase
[0028] The nucleotide sequences having SEQ ID NOs:3, 11, 17, and 19
and the amino acid sequences having SEQ ID NOs:4, 12, 18, and 20
were presented in the U.S. Provisional Application No. 60/110602,
filed Dec. 2, 1998. The nucleotide sequences having SEQ ID NOs:1
and 15 as well as the amino acid sequences having SEQ ID NOs:2 and
16 were added in the U.S. Provisional Application No. 60/127111,
filed Mar. 3, 1999. The nucleotide sequence presented in SEQ ID
NO:15 encodes an entire soybean diacylglycerol acyltransferase
whose amino acid sequence is presented in SEQ ID NO:16, the amino
acid sequence presented in SEQ ID NO:17 encodes only a portion of
the enzyme. The nucleotide sequence presented in SEQ ID NO:7
corresponds to the full insert sequence and encodes a protein
identical to that of SEQ ID NO:4. The nucleotide sequences
presented in SEQ ID NOs:11 and 19 correspond to a portion of those
presented in SEQ ID NOs:13 and 21.
[0029] 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
[0030] In the context of this disclosure, a number of terms shall
be utilized. As used herein, a "polynucleotide" is a nucleotide
sequence such as a nucleic acid fragment. A polynucleotide may be a
polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, or
synthetic DNA. An isolated polynucleotide of the present invention
may include at least one of 40 contiguous nucleotides, preferably
at least one of 30 contiguous nucleotides, most preferably one of
at least 15 contiguous nucleotides, of the nucleic acid sequence of
the SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and the
complement of such sequences.
[0031] 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.
[0032] 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-a-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.
[0033] Substantially similar nucleic acid fragments may be selected
by screening nucleic acid fragments representing subfragments or
modifications of the nucleic acid fragments of the instant
invention, wherein one or more nucleotides are substituted, deleted
and/or inserted, for their ability to affect the level of the
polypeptide encoded by the unmodified nucleic acid fragment in a
plant or plant cell. For example, a substantially similar nucleic
acid fragment representing at least one of 30 contiguous
nucleotides derived from the instant nucleic acid fragment can be
constructed and introduced into a plant or plant cell. The level of
the polypeptide encoded by the unmodified nucleic acid fragment
present in a plant or plant cell exposed to the substantially
similar nucleic fragment can then be compared to the level of the
polypeptide in a plant or plant cell that is not exposed to the
substantially similar nucleic acid fragment.
[0034] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by nucleic acid fragments
that do not share 100% sequence identity with the gene to be
suppressed. Moreover, alterations in a nucleic acid fragment which
result in the production of a chemically equivalent amino acid at a
given site, but do not effect the functional properties of the
encoded polypeptide, are well known in the art. Thus, a codon for
the amino acid alanine, a hydrophobic amino acid, may be
substituted by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue, such as valine,
leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine, can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the polypeptide molecule would also not be expected to alter the
activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
Consequently, an isolated polynucleotide comprising a nucleotide
sequence of at least one of 60 (preferably at least one of 40, most
preferably at least one of 30) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs:1, 5, 7, 9, 13, 15, 21, and the complement of such nucleotide
sequences may be used in methods of selecting an isolated
polynucleotide that affects the expression of a polypeptide in a
plant cell. A method of selecting an isolated polynucleotide that
affects the level of expression of a polypeptide such as
diacylglyercol acyltransferase, in a host cell (eukaryotic, such as
plant or yeast, prokaryotic such as bacterial, or viral) may
comprise the steps of: constructing an isolated polynucleotide of
the present invention or an isolated chimeric gene of the present
invention; introducing the isolated polynucleotide or the isolated
chimeric gene into a host cell; measuring the level a polypeptide
in the host cell containing the isolated polynucleotide; and
comparing the level of a polypeptide in the host cell containing
the isolated polynucleotide with the level of a polypeptide in a
host cell that does not contain the isolated polynucleotide.
[0035] 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.
[0036] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Suitable nucleic acid fragments
(isolated polynucleotides of the present invention) encode
polypeptides that are at least 70% identical, preferably at least
80% identical to the amino acid sequences reported herein.
Preferred nucleic acid fragments encode amino acid sequences that
are at least 85% identical to the amino acid sequences reported
herein. More preferred nucleic acid fragments encode amino acid
sequences that are at least 90% identical to the amino acid
sequences reported herein. Most preferred are nucleic acid
fragments that encode amino acid sequences that are at least 95%
identical to the amino acid sequences reported herein. Suitable
nucleic acid fragments not only have the above homologies but
typically encode a polypeptide having at least 50 amino acids,
preferably at least 100 amino acids, more preferably at least 150
amino acids, still more preferably at least 200 amino acids, and
most preferably at least 250 amino acids. Sequence alignments and
percent identity calculations were performed using the Megalign
program of the LASERGENE bioinformatics computing suite (DNASTAR
Inc., Madison, Wis.). Multiple alignment of the sequences was
performed using the Clustal method of alignment (Higgins and Sharp
(1989) CABIOS. 5:151-153) with the default parameters (GAP
PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise
alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,
WINDOW=5 and DIAGONALS SAVED=5.
[0037] 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). 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.
[0038] "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.
[0039] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized
using procedures known to those skilled in the art. These building
blocks are ligated and annealed to form larger nucleic acid
fragments which may then be enzymatically assembled to construct
the entire desired nucleic acid fragment. "Chemically synthesized",
as related to nucleic acid fragment, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
nucleic acid fragments may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the nucleic acid fragments can be tailored for optimal gene
expression based on optimization of nucleotide sequence to reflect
the codon bias of the host cell. The skilled artisan appreciates
the likelihood of successful gene expression if codon usage is
biased towards those codons favored by the host. Determination of
preferred codons can be based on a survey of genes derived from the
host cell where sequence information is available.
[0040] "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.
[0041] "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.
[0042] "Promoter" refers to a nucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a nucleotide sequence which can
stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic nucleotide segments. It is understood by those skilled in
the art that different promoters may direct the expression of a
gene in different tissues or cell types, or at different stages of
development, or in response to different environmental conditions.
Promoters which cause a nucleic acid fragment to be expressed in
most cell types at most times are commonly referred to as
"constitutive promoters". New promoters of various types useful in
plant cells are constantly being discovered; numerous examples may
be found in the compilation by Okamuro and Goldberg (1989)
Biochemistry of Plants 15:1-82. It is further recognized that since
in most cases the exact boundaries of regulatory sequences have not
been completely defined, nucleic acid fragments of different
lengths may have identical promoter activity.
[0043] The "translation leader sequence" refers to a nucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Mol. Biotechnol.
3:225-236).
[0044] The "3' non-coding sequences" refer to nucleotide sequences
located downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
[0045] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into polypeptide by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to an RNA transcript that includes the mRNA and
so can be translated into a polypeptide by the cell. "Antisense
RNA" refers to an RNA transcript that is complementary to all or
part of a target primary transcript or mRNA and that blocks the
expression of a target gene (see U.S. Pat. No. 5,107,065,
incorporated herein by reference). The complementarity of an
antisense RNA may be with any part of the specific nucleotide
sequence, i.e., at the 5' non-coding sequence, 3' non-coding
sequence, introns, or the coding sequence. "Functional RNA" refers
to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may
not be translated but yet has an effect on cellular processes.
[0046] The term "operably linked" refers to the association of two
or more nucleic acid fragments on a single nucleic acid fragment so
that the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0047] 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).
[0048] "Altered levels" refers to the production of gene product(s)
in transgenic organisms in amounts or proportions that differ from
that of normal or non-transformed organisms.
[0049] "Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present
in the primary translation product have been removed. "Precursor"
protein refers to the primary product of translation of mRNA; i.e.,
with pre- and propeptides still present. Pre- and propeptides may
be but are not limited to intracellular localization signals.
[0050] 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).
[0051] "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).
[0052] 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").
[0053] Nucleic acid fragments encoding at least a portion of
several diacylglycerol acyltransferases 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 diacylglycerol
acyltransferases, either as cDNAs or genomic DNAs, could be
isolated directly by using all or a portion of the instant nucleic
acid fragments as DNA hybridization probes to screen libraries from
any desired plant employing methodology well known to those skilled
in the art. Specific oligonucleotide probes based upon the instant
nucleic acid sequences can be designed and synthesized by methods
known in the art (Maniatis). Moreover, the entire sequences can be
used directly to synthesize DNA probes by methods known to the
skilled artisan such as random primer DNA labeling, nick
translation, or end-labeling techniques, or RNA probes using
available in vitro transcription systems. In addition, specific
primers can be designed and used to amplify a part or all of the
instant sequences. The resulting amplification products can be
labeled directly during amplification reactions or labeled after
amplification reactions, and used as probes to isolate full length
cDNA or genomic fragments under conditions of appropriate
stringency.
[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 60 (preferably one of at least 40, most
preferably one of at least 30) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and the complement of
such nucleotide sequences may be used in such methods to obtain a
nucleic acid fragment encoding a substantial portion of an amino
acid sequence of a polypeptide. The present invention relates to a
method of obtaining a nucleic acid fragment encoding a substantial
portion of a polypeptide of a gene (such as diacylglycerol
acyltransferases) preferably a substantial portion of a plant
polypeptide of a gene, comprising the steps of: synthesizing an
oligonucleotide primer comprising a nucleotide sequence of at least
one of 60 (preferably at least one of 40, most preferably at least
one of 30) contiguous nucleotides derived from a nucleotide
sequence selected from the group consisting of SEQ ID NOs:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, and the complement of such nucleotide
sequences; and amplifying a nucleic acid fragment (preferably a
cDNA inserted in a cloning vector) using the oligonucleotide
primer. The amplified nucleic acid fragment preferably will encode
a portion of a polypeptide.
[0056] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).
[0057] 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 oil
content in those cells.
[0058] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. For reasons of convenience, the chimeric gene may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals may also be provided. The instant
chimeric gene may also comprise one or more introns in order to
facilitate gene expression.
[0059] Plasmid vectors comprising the instant chimeric gene can
then be constructed. The choice of plasmid vector is dependent upon
the method that will be used to transform host plants. The skilled
artisan is well aware of the genetic elements that must be present
on the plasmid vector in order to successfully transform, select
and propagate host cells containing the chimeric gene. The skilled
artisan will also recognize that different independent
transformation events will result in different levels and patterns
of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida
et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple
events must be screened in order to obtain lines displaying the
desired expression level and pattern. Such screening may be
accomplished by Southern analysis of DNA, Northern analysis of mRNA
expression, Western analysis of protein expression, or phenotypic
analysis.
[0060] For some applications it may be useful to direct the instant
polypeptide to different cellular compartments, or to facilitate
its secretion from the cell. It is thus envisioned that the
chimeric gene described above may be further supplemented by
altering the coding sequence to encode the instant polypeptide with
appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys.100:1627-1632)
added and/or with targeting sequences that are already present
removed. While the references cited give examples of each of these,
the list is not exhaustive and more targeting signals of utility
may be discovered in the future.
[0061] 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.
[0062] Molecular genetic solutions to the generation of plants with
altered gene expression have a decided advantage over more
traditional plant breeding approaches. Changes in plant phenotypes
can be produced by specifically inhibiting expression of one or
more genes by antisense inhibition or cosuppression (U.S. Pat. Nos.
5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression
construct would act as a dominant negative regulator of gene
activity. While conventional mutations can yield negative
regulation of gene activity these effects are most likely
recessive. The dominant negative regulation available with a
transgenic approach may be advantageous from a breeding
perspective. In addition, the ability to restrict the expression of
specific phenotype to the reproductive tissues of the plant by the
use of tissue specific promoters may confer agronomic advantages
relative to conventional mutations which may have an effect in all
tissues in which a mutant gene is ordinarily expressed.
[0063] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppression technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense genes may require the use of different chimeric
genes utilizing different regulatory elements known to the skilled
artisan. Once transgenic plants are obtained by one of the methods
described above, it will be necessary to screen individual
transgenics for those that most effectively display the desired
phenotype. Accordingly, the skilled artisan will develop methods
for screening large numbers of transformants. The nature of these
screens will generally be chosen on practical grounds, and is not
an inherent part of the invention. For example, one can screen by
looking for changes in gene expression by using antibodies specific
for the protein encoded by the gene being suppressed, or one could
establish assays that specifically measure enzyme activity. A
preferred method will be one which allows large numbers of samples
to be processed rapidly, since it will be expected that a large
number of transformants will be negative for the desired
phenotype.
[0064] The instant polypeptide (or portions thereof) may be
produced in heterologous host cells, particularly in the cells of
microbial hosts, and can be used to prepare antibodies to the these
proteins by methods well known to those skilled in the art. The
antibodies are useful for detecting the polypeptide of the instant
invention in situ in cells or in vitro in cell extracts. Preferred
heterologous host cells for production of the instant polypeptide
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 polypeptide. This chimeric gene could
then be introduced into appropriate microorganisms via
transformation to provide high level expression of the encoded
diacylglycerol acyltransferase. An example of a vector for high
level expression of the instant polypeptide in a bacterial host is
provided (Example 7).
[0065] Additionally, the instant polypeptide 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 diacylglycerol acyltransferase described herein
catalyzes the committed step in triacylglycerol biosynthesis.
Accordingly, inhibition of the activity of the enzyme described
herein could lead to inhibition plant growth. Thus, the instant
diacylglycerol acyltransferase could be appropriate for new
herbicide discovery and design.
[0066] All or a substantial portion of the nucleic acid fragments
of the instant invention may also be used as probes for genetically
and physically mapping the genes that they are a part of, and as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid fragments may be
used as restriction fragment length polymorphism (RFLP) markers.
Southern blots (Maniatis) of restriction-digested plant genomic DNA
may be probed with the nucleic acid fragments of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al. (1987) Genomics 1:174-181) in order to construct a genetic
map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et
al. (1980) Am. J. Hum. Genet. 32:314-331).
[0067] 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.
[0068] 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).
[0069] 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.
[0070] 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.
[0071] 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 polypeptide. 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
polypeptide can be identified and obtained. This mutant plant can
then be used to determine or confirm the natural function of the
instant polypeptide disclosed herein.
EXAMPLES
[0072] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions.
Example 1
Composition of cDNA Libraries; Isolation and Sequencing of cDNA
Clones
[0073] cDNA libraries representing mRNAs from various Arabidopsis,
corn, rice, soybean, and wheat tissues were prepared. The
characteristics of the libraries are described below.
TABLE-US-00002 TABLE 2 cDNA Libraries from Arabidopsis, Corn, Rice,
Soybean, and Wheat Library Tissue Clone ara 3 day-old Arabidopsis
thaliana seedling hypocotyls araebcF cco1 Corn Cob of 67 Day Old
Plants Grown in Green House cco1.pk0029.b6 cen3n Corn Endosperm 20
Days After Pollination* cen3n.pk0010.c10 cen3n.pk0113.e12 cpj1c
Corn Pooled BMS Treated With Chemicals Related to cpj1c.pk005.h23
Membrane Ionic Force** p0042 Corn Seedling After 10 Day Drought
Stress Heat Shocked p0042.cspaf49r for 24 Hours at 45.degree. C.
p0122 Corn Pith Tissue from Internode Subtending Ear Node 5
p0122.ckamb57r Days After Pollination* p0125 Corn Anther Prophase
I* p0125.czaau61rb rls24 Rice Leaf 15 Days After Germination, 24
Hours After rls24.pk0034.d8 Infection of Strain Magaporthe grisea
4360-R-67 (AVR2-YAMO); Susceptible sr1 Soybean Root sr1.pk0098.a8
src3c Soybean 8 Day Old Root Infected With Cyst Nematode
src3c.pk013.h18 wr1 Wheat Root From 7 Day Old Seedling
wr1.pk0119.b6 *These libraries were normalized essentially as
described in U.S. Pat. No. 5,482,845,\ incorporated herein by
reference. **Chemicals used included valinomycin, bafilomycin A1,
oligomycin, and ionomycin.
[0074] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651-1656). The resulting ESTs are analyzed using a
Perkin Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0075] cDNA clones encoding diacylglycerol acyltransferases were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches for
similarity to sequences contained in the BLAST "nr" database
(comprising all non-redundant GenBank CDS translations, sequences
derived from the 3-dimensional structure Brookhaven Protein Data
Bank, the last major release of the SWISS-PROT protein sequence
database, EMBL, and DDBJ databases). The cDNA sequences obtained in
Example 1 were analyzed for similarity to all publicly available
DNA sequences contained in the "nr" database using the BLASTN
algorithm provided by the National Center for Biotechnology
Information (NCBI). The DNA sequences were translated in all
reading frames and compared for similarity to all publicly
available protein sequences contained in the "nr" database using
the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272)
provided by the NCBI. For convenience, the P-value (probability) of
observing a match of a cDNA sequence to a sequence contained in the
searched databases merely by chance as calculated by BLAST are
reported herein as "pLog" values, which represent the negative of
the logarithm of the reported P-value. Accordingly, the greater the
pLog value, the greater the likelihood that the cDNA sequence and
the BLAST "hit" represent homologous proteins.
Example 3
Characterization of Corn, Rice, and Wheat cDNA Clones Encoding
Diacylglycerol Acyltransferase
[0076] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the proteins encoded by the cDNAs
to a putative Acyl CoA cholesterol acyltransferase related gene
product from Arabidopsis thaliana (NCBI General Identifier No.
3135276), and to diacylglycerol acyltransferases from Homo sapiens
and Mus musculus (NCBI General Identifier Nos. 3746533, and
3859934, respectively). Animal acyl CoA cholesterol
acyltransferases have recently been shown to be related to
diacylglycerol acyltransferases (Cases et al. (1998) Proc. Natl.
Acad. Sci. USA 95:13018-13023). The sequences included here are
therefore more likely to be diacylglycerol acyltransferases than
acyl CoA cholesterol acyltransferases since cholesterol is only a
very minor constituent of plant sterols. Shown in Table 3 are the
BLAST results for individual ESTs ("EST"), or contigs assembled
from two or more ESTs ("Contig"):
TABLE-US-00003 TABLE 3 BLAST Results for Clones Encoding
Polypeptides Homologous to Diacylglycerol Acyltransferase BLAST
pLog Score Clone Status 3746533 3859934 Contig of: Contig 59.70
59.52 cpj1c.pk005.h23 cen3n.pk0010.c10 cco1.pk0029.b6
cen3n.pk0113.e12 EST 38.00 39.00 rls24.pk0034.d8 EST 3.70 3.70
wr1.pk0119.b6 EST 4.52 4.40
[0077] The BLASTX search using the EST sequences from clones listed
in Table 4 revealed similarity of the proteins encoded by the cDNAs
to putative diacylglycerol acyltransferases from Arabidopsis
thaliana and Brassica napus (NCBI General Identifier Nos. 5050913
and 5579408, respectively). Shown in Table 4 are the BLAST results
for the sequences of the entire cDNA inserts comprising the
indicated cDNA clones ("FIS"), contigs assembled from two or more
ESTs ("Contig"), or sequences encoding the entire protein derived
from an FIS and PCR ("CGS"):
TABLE-US-00004 TABLE 4 BLAST Results for Clones Encoding
Polypeptides Homologous to Diacylelycerol Acyltransferase BLAST
pLog Score Clone Status 5050913 5579408 cpj1c.pk005.h23 FIS 113.00
116.00 Contig of: Contig 111.00 109.00 p0042.cspaf49r
p0122.ckamb57r p0125.czaau61rb rls24.pk0034.d8:fis CGS >250.00
173.00 wr1.pk0119.b6:fis CGS 177.00 173.00
[0078] Sequence alignments (see Example 4) and BLAST scores and
probabilities indicate that the nucleic acid fragments comprising
the instant cDNA clones encode a substantial portion of a corn
diacylglycerol acyltransferase and entire corn, rice, and wheat
diacylglycerol acyltransferases. These sequences represent the
first corn, rice, and wheat sequences encoding diacylglycerol
acyltransferases.
Example 4
Cloning and Sequencing of cDNAs Encoding Entire Soybean and
Arabidopsis thaliana Diacylglycerol Acyltransferases
[0079] The BLASTX search using the EST sequences from clones listed
in Table 5 revealed similarity of the proteins encoded by the cDNAs
to a hypothetical protein from Arabisopsis thaliana and the Mus
musculus DGAT (NCBI General Identifier Nos: 3135275 and 3859934,
respectively). The sequence of the entire cDNA insert in clone
src3c.pk013.h18 was determined, it was found to contain insertions
and deletions with respect to known diacylglycerol
acetyltransferases. Clone srl.pk0098.a8 was found by searching the
DuPont EST database for soybean sequences with similarities to the
entire cDNA sequence from clone src3c.pk013.h18.
[0080] Because it was suspected that the Arabidopsis thaliana
putative ACAT sequence encoded only the C-terminal half of a DGAT,
an Arabidopsis thaliana DGAT sequence was obtained by PCR from a
public library described by Kieber et al. (1993) Cell 72:427-441.
This library was prepared from polyA+RNA isolated from 3 day-old
Arabidopsis thaliana (Columbia) seedling hypocotyls and consisted
of 2 to 3 kb size-selected cDNA inserts cloned into the EcoRI site
of lambda-ZAPII (Stratagene). Prior to use in PCR reactions, the
library was converted into plasmid form by mass-excision following
Hay and Short (1992) Strategies 5:16-18) to yield pBluescript
SK(-)-containing cDNA inserts. Primers used for PCR were:
TABLE-US-00005 AtDGx5' 5' CTT AGC TTC TTC CTT CAA TC 3' AT-DGAT3'
5' TTT CTA GAC TCG AGT GAA CAG TTG TTT CAT GAC 3'
[0081] The PCR primers were designed based on EST and genomic
sequences in the public domain. An Arabidopsis thaliana EST
sequence (GenBank General Identifier No. 2414087) was used to
design the 3' primer (AT-DGAT3; SEQ ID NO:23). The 5' primer
(AtDGx5'; SEQ ID NO:24) was based on Arabidopsis genomic sequence
information found in NCBI General Identifier No. 3135250, but could
not have been readily predicted as the appropriate 5' end of the
cDNA, based on public sequences. The 5' primer was designed to be
located upstream of a stop codon located in the same reading frame
as the codon for the putative start methionine. The PCR product
from this primer is therefore likely to contain the entire
cDNA.
[0082] Shown in Table 5 are the BLAST results for individual ESTs
("EST"), or sequences encoding the entire protein derived from an
FIS and PCR ("CGS"):
TABLE-US-00006 TABLE 5 BLAST Results for Sequences Encoding
Polypeptides Homologous to Diacylglycerol Acyltransferase BLAST
pLog Score Clone Status 3135275 3859934 araebcF CGS 132.00 77.70
src3c.pk013.h18 EST 3.00 sr1.pk0098.a8 CGS 105.00 81.52
[0083] FIGS. 1A, 1B, and 1C present an alignment of the amino acid
sequences set forth in SEQ ID NOs:2, 8, 14, 16, and 22 and the Mus
musculus and Arabidopsis thaliana diacylglycerol acetyltransferase
sequences (SEQ ID NO:25 and SEQ ID NO:26). The data in Table 6
represents a calculation of the percent identity of the amino acid
sequences set forth in SEQ ID NOs:2, 8, 14, 16, and 22 and the Mus
musculus and Arabidopsis thaliana diacylglycerol acetyltransferase
sequences (SEQ ID NO:25 and SEQ ID NO:26).
TABLE-US-00007 TABLE 6 Percent Identity of Amino Acid Sequences
Deduced From the Nucleotide Sequences of cDNA Clones Encoding
Polypeptides Homologous to Diacylglycerol Acyltransferase Percent
Identity to SEQ ID NO. 3859934 5050913 2 31.9 99.6 8 31.5 56.0 14
29.3 57.4 16 30.9 65.9 22 29.9 58.7
[0084] 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 three corn, one entire
Arabidopsis, one entire rice, and one entire wheat diacylglycerol
acyltransferase. These sequences represent the first Arabidopsis,
corn, rice, soybean, and wheat sequences encoding diacylglycerol
acyltransferase.
Example 5
Expression of Chimeric Genes in Monocot Cells
[0085] A chimeric gene comprising a cDNA encoding the instant
polypeptide 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 Smal) 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 Smal 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 Smal-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 polypeptide,
and the 10 kD zein 3' region.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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 6
Expression of Chimeric Genes in Dicot Cells
[0092] 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
polypeptide 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.
[0093] 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.
[0094] Soybean embryos may then be transformed with the expression
vector comprising sequences encoding the instant polypeptide. 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.
[0095] Soybean embryogenic suspension cultures can maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0096] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic.TM. PDS 1000/HE instrument (helium retrofit) can
be used for these transformations.
[0097] A selectable marker gene which can be used to facilitate
soybean transformation is a chimeric gene composed of the 35S
promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The seed expression cassette
comprising the phaseolin 5' region, the fragment encoding the
instant polypeptide 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.
[0098] To 50 .mu.L of a 60 mg/mL 1 .sub.ilm 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.
[0099] 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.
[0100] 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.
[0101] 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 7
Expression of Chimeric Genes in Microbial Cells
[0102] The cDNAs encoding the instant polypeptide 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.
[0103] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% NuSieve GTGTM low melting
agarose gel (FMC). Buffer and agarose contain 10 .mu.g/m1 ethidium
bromide for visualization of the DNA fragment. The fragment can
then be purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20
.mu.L of water. Appropriate oligonucleotide adapters may be ligated
to the fragment using T4 DNA ligase (New England Biolabs, Beverly,
Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the instant polypeptide are then screened for the
correct orientation with respect to the T7 promoter by restriction
enzyme analysis.
[0104] 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 8
Evaluating Compounds for Their Ability to Inhibit the Activity of
Diacylglycerol Acyltransferases
[0105] The polypeptide 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 7, or expression in eukaryotic cell culture,
in planta, and using viral expression systems in suitably infected
organisms or cell lines. The instant polypeptide 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.
[0106] Purification of the instant polypeptide, 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 polypeptide 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 polypeptide 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.
[0107] 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 polypeptide disclosed herein. Assays may
be conducted under well known experimental conditions which permit
optimal enzymatic activity. For example, assays for diacylglycerol
acyltransferases are presented by M. Andersson et al. ((1994) J.
Lipid Res. 35:535-545).
Sequence CWU 1
1
2611888DNAArabidopsis thaliana 1gcttcttcct tcaatccgct ctttccctct
ccattagatt ctgtttcctc tttcaatttc 60ttctgcatgc ttctcgattc tctctgacgc
ctcttttctc ccgacgctgt ttcgtcaaac 120gcttttcgaa atggcgattt
tggattctgc tggcgttact acggtgacgg agaacggtgg 180cggagagttc
gtcgatcttg ataggcttcg tcgacggaaa tcgagatcgg attcttctaa
240cggacttctt ctctctggtt ccgataataa ttctccttcg gatgatgttg
gagctcccgc 300cgacgttagg gatcggattg attccgttgt taacgatgac
gctcagggaa cagccaattt 360ggccggagat aataacggtg gtggcgataa
taacggtggt ggaagaggcg gcggagaagg 420aagaggaaac gccgatgcta
cgtttacgta tcgaccgtcg gttccagctc atcggagggc 480gagagagagt
ccacttagct ccgacgcaat cttcaaacag agccatgccg gattattcaa
540cctctgtgta gtagttctta ttgctgtaaa cagtagactc atcatcgaaa
atcttatgaa 600gtatggttgg ttgatcagaa cggatttctg gtttagttca
agatcgctgc gagattggcc 660gcttttcatg tgttggatat ccctttcgat
ctttcctttg gctgccttta cggttgagaa 720attggtactt cagaaataca
tatcagaacc tgttggcatc tttcttcata ttattatcac 780catgacagag
gttttgtatc cagtttacgt caccctaagg tgtgattctg cttttttatc
840aggtgtcact ttgatgctcc tcacttgcat tgtgtggcta aagttggttt
cttatgctca 900tactagctat gacataagat ccctagccaa tgcagctgat
aaggccaatc ctgaagtctc 960ctactacgtt agcttgaaga gcttggcata
tttcatggtc gctcccacat tgtgttatca 1020gccaagttat ccacgttctg
catgtatacg gaagggttgg gtggctcgtc aatttgcaaa 1080actggtcata
ttcaccggat tcatgggatt tataatagaa caatatataa atcctattgt
1140caggaactca aagcatcctt tgaaaggcga tcttctatat gctattgaaa
gagtgttgaa 1200gctttcagtt ccaaatttat atgtgtggct ctgcatgttc
tactgcttct tccacctttg 1260gttaaacata ttggcagagc ttctctgctt
cggggatcgt gaattctaca aagattggtg 1320gaatgcaaaa agtgtgggag
attactggag aatgtggaat atgcctgttc ataaatggat 1380ggttcgacat
atatacttcc cgtgcttgcg cagcaagata ccaaagacac tcgccattat
1440cattgctttc ctagtctctg cagtctttca tgagctatgc atcgcagttc
cttgtcgtct 1500cttcaagcta tgggcttttc ttgggattat gtttcaggtg
cctttggtct tcatcacaaa 1560ctatctacag gaaaggtttg gctcaacggt
ggggaacatg atcttctggt tcatcttctg 1620cattttcgga caaccgatgt
gtgtgcttct ttattaccac gacctgatga accgaaaagg 1680atcgatgtca
tgaaacaact gttcaaaaaa tgactttctt caaacatcta tggcctcgtt
1740ggatctccgt tgatgttgtg gtggttctga tgctaaaacg acaaatagtg
ttataaccat 1800tgaagaagaa aagaaaatta gagttgttgt atctgcaaaa
attttggtag agacacgcaa 1860acccgtttgg attttgttat ggagtaaa
18882520PRTArabidopsis thaliana 2Met Ala Ile Leu Asp Ser Ala Gly
Val Thr Thr Val Thr Glu Asn Gly1 5 10 15Gly Gly Glu Phe Val Asp Leu
Asp Arg Leu Arg Arg Arg Lys Ser Arg 20 25 30Ser Asp Ser Ser Asn Gly
Leu Leu Leu Ser Gly Ser Asp Asn Asn Ser 35 40 45Pro Ser Asp Asp Val
Gly Ala Pro Ala Asp Val Arg Asp Arg Ile Asp 50 55 60Ser Val Val Asn
Asp Asp Ala Gln Gly Thr Ala Asn Leu Ala Gly Asp65 70 75 80Asn Asn
Gly Gly Gly Asp Asn Asn Gly Gly Gly Arg Gly Gly Gly Glu 85 90 95Gly
Arg Gly Asn Ala Asp Ala Thr Phe Thr Tyr Arg Pro Ser Val Pro 100 105
110Ala His Arg Arg Ala Arg Glu Ser Pro Leu Ser Ser Asp Ala Ile Phe
115 120 125Lys Gln Ser His Ala Gly Leu Phe Asn Leu Cys Val Val Val
Leu Ile 130 135 140Ala Val Asn Ser Arg Leu Ile Ile Glu Asn Leu Met
Lys Tyr Gly Trp145 150 155 160Leu Ile Arg Thr Asp Phe Trp Phe Ser
Ser Arg Ser Leu Arg Asp Trp 165 170 175Pro Leu Phe Met Cys Trp Ile
Ser Leu Ser Ile Phe Pro Leu Ala Ala 180 185 190Phe Thr Val Glu Lys
Leu Val Leu Gln Lys Tyr Ile Ser Glu Pro Val 195 200 205Gly Ile Phe
Leu His Ile Ile Ile Thr Met Thr Glu Val Leu Tyr Pro 210 215 220Val
Tyr Val Thr Leu Arg Cys Asp Ser Ala Phe Leu Ser Gly Val Thr225 230
235 240Leu Met Leu Leu Thr Cys Ile Val Trp Leu Lys Leu Val Ser Tyr
Ala 245 250 255His Thr Ser Tyr Asp Ile Arg Ser Leu Ala Asn Ala Ala
Asp Lys Ala 260 265 270Asn Pro Glu Val Ser Tyr Tyr Val Ser Leu Lys
Ser Leu Ala Tyr Phe 275 280 285Met Val Ala Pro Thr Leu Cys Tyr Gln
Pro Ser Tyr Pro Arg Ser Ala 290 295 300Cys Ile Arg Lys Gly Trp Val
Ala Arg Gln Phe Ala Lys Leu Val Ile305 310 315 320Phe Thr Gly Phe
Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro Ile 325 330 335Val Arg
Asn Ser Lys His Pro Leu Lys Gly Asp Leu Leu Tyr Ala Ile 340 345
350Glu Arg Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu Cys
355 360 365Met Phe Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala
Glu Leu 370 375 380Leu Cys Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp
Trp Asn Ala Lys385 390 395 400Ser Val Gly Asp Tyr Trp Arg Met Trp
Asn Met Pro Val His Lys Trp 405 410 415Met Val Arg His Ile Tyr Phe
Pro Cys Leu Arg Ser Lys Ile Pro Lys 420 425 430Thr Leu Ala Ile Ile
Ile Ala Phe Leu Val Ser Ala Val Phe His Glu 435 440 445Leu Cys Ile
Ala Val Pro Cys Arg Leu Phe Lys Leu Trp Ala Phe Leu 450 455 460Gly
Ile Met Phe Gln Val Pro Leu Val Phe Ile Thr Asn Tyr Leu Gln465 470
475 480Glu Arg Phe Gly Ser Thr Val Gly Asn Met Ile Phe Trp Phe Ile
Phe 485 490 495Cys Ile Phe Gly Gln Pro Met Cys Val Leu Leu Tyr Tyr
His Asp Leu 500 505 510Met Asn Arg Lys Gly Ser Met Ser 515
52031281DNAZea maysunsure(356)n = a, c, g, or t 3tttaatgcta
catcattgcg agactggcca ctgctaatgt gttgccttag tctacccata 60tttccccttg
gtgcatttgc agtcgaaaag ttggcattca acaatctcgt tagtgatcct
120gctactacct gttttcacat cctttttaca acatttgaaa ttgtatatcc
agtgctcgtg 180attcttaagt gtgattctgc agttttatca ggctttgtgt
tgatgtttat tgcctgcatt 240gtttggctga agcttgtatc ttttgcacat
acaaaccatg atataaggaa aactgatcac 300aagcggcaag aaggttgata
atgaactgac cgcggctggc atagataatt tacaanctcc 360aactcttggg
agtctaacat acttcaagat ggctccgaca ctctgttatc aagccaaagt
420tatcctncga acaccttatg ttagaaaagg ttggctggtc cgtcaagtta
ttctatactt 480gatatttact ggtctccaag gattcattat tgagcaatac
ataaatccta ttgttgtgaa 540ctctcaacat ccattgatgg gaggattact
gaatgctgta gagactgttt tgaagctctc 600attaccaaat gtctacctgt
ggctttgcat gttttattgc cttttccatc tgtggttaaa 660catacttgct
gagattcttc gatttggtga ccgagaattc tacaaagact ggtggaatgc
720aaagacaatt gatgagtact ggagaaaatg gaacatgcct gtgcataaat
ggattgttcg 780tcatatatat ttcccttgca tgcgaaatgg tatatcaaag
gaagttgctg tttttatatc 840gttctttgtt tctgctgtac ttcatgagtt
atgtgttgct gttccctgcc acatactcaa 900gttctgggct ttcttaggaa
tcatgcttca gattcccctc atcatattga catcatacct 960caaaaataaa
ttcagtgaca caatggttgg caatatgatc ttttggtttt ttttctgcat
1020atacgggcag ccaatgtgtg ttctattgta ttaccatgat gtgatgaacc
ggactgagaa 1080ggcaaaataa ccatctgtag atctttttgg gtttcatttc
tccatcatgg aaactgaaac 1140ataactgtgc acacataaac agcatcgtgt
ctcaattttt taaaaaanaa aagaananca 1200caaaaaaccc aggggggccg
gtaccaatcc ccaaantatc gntnaccncc cacggcgtnt 1260taaacncgta
cggaaaaccn g 12814361PRTZea maysUNSURE(119)Xaa = ANY AMINO ACID
4Phe Asn Ala Thr Ser Leu Arg Asp Trp Pro Leu Leu Met Cys Cys Leu1 5
10 15Ser Leu Pro Ile Phe Pro Leu Gly Ala Phe Ala Val Glu Lys Leu
Ala 20 25 30Phe Asn Asn Leu Val Ser Asp Pro Ala Thr Thr Cys Phe His
Ile Leu 35 40 45Phe Thr Thr Phe Glu Ile Val Tyr Pro Val Leu Val Ile
Leu Lys Cys 50 55 60Asp Ser Ala Val Leu Ser Gly Phe Val Leu Met Phe
Ile Ala Cys Ile65 70 75 80Val Trp Leu Lys Leu Val Ser Phe Ala His
Thr Asn His Asp Ile Gly 85 90 95Lys Leu Ile Thr Ser Gly Lys Lys Val
Asp Asn Glu Leu Thr Ala Ala 100 105 110Gly Ile Asp Asn Leu Gln Xaa
Pro Thr Leu Gly Ser Leu Thr Tyr Phe 115 120 125Lys Met Ala Pro Thr
Leu Cys Tyr Gln Ala Lys Val Ile Leu Arg Thr 130 135 140Pro Tyr Val
Arg Lys Gly Trp Leu Val Arg Gln Val Ile Leu Tyr Leu145 150 155
160Ile Phe Thr Gly Leu Gln Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro
165 170 175Ile Val Val Asn Ser Gln His Pro Leu Met Gly Gly Leu Leu
Asn Ala 180 185 190Val Glu Thr Val Leu Lys Leu Ser Leu Pro Asn Val
Tyr Leu Trp Leu 195 200 205Cys Met Phe Tyr Cys Leu Phe His Leu Trp
Leu Asn Ile Leu Ala Glu 210 215 220Ile Leu Arg Phe Gly Asp Arg Glu
Phe Tyr Lys Asp Trp Trp Asn Ala225 230 235 240Lys Thr Ile Asp Glu
Tyr Trp Arg Lys Trp Asn Met Pro Val His Lys 245 250 255Trp Ile Val
Arg His Ile Tyr Phe Pro Cys Met Arg Asn Gly Ile Ser 260 265 270Lys
Glu Val Ala Val Phe Ile Ser Phe Phe Val Ser Ala Val Leu His 275 280
285Glu Tyr Val Leu Leu Phe Leu His Ile Leu Lys Phe Trp Ala Phe Leu
290 295 300Gly Ile Met Leu Gln Ile Pro Leu Ile Ile Leu Thr Ser Tyr
Leu Lys305 310 315 320Asn Lys Phe Ser Asp Thr Met Val Gly Asn Met
Ile Phe Trp Phe Phe 325 330 335Phe Cys Ile Tyr Gly Gln Pro Met Cys
Val Leu Leu Tyr Tyr His Asp 340 345 350Val Met Asn Arg Thr Glu Lys
Ala Lys 355 3605978DNAZea mays 5ggcacgaggt tagaaaaggt tggctggtcc
gtcaagttat tctatacttg atatttactg 60gtctccaagg attcattatt gagcaataca
taaatcctat tgttgtgaac tctcaacatc 120cattgatggg aggattactg
aatgctgtag agactgtttt gaagctctca ttaccaaatg 180tctacctgtg
gctttgcatg ttttattgcc ttttccatct gtggttaaac atacttgctg
240agattcttcg atttggtgac cgagaattct acaaagactg gtggaatgca
aagacaattg 300atgagtactg gagaaaatgg aacatgcctg tgcataaatg
gattgttcgt catatatatt 360tcccttgcat gcgaaatggt atatcaaagg
aagttgctgt ttttatatcg ttctttgttt 420ctgctgtact tcatgagctg
cagattactt ggatgaagtg ctctatataa aattaaatat 480ttcataatcc
agtccctttc gagaaaatta tgatacattt tgtttgcaat tgtacaccag
540ttatgtgttg ctgttccctg ccacatactc aagttctggg ctttcttagg
aatcatgctt 600cagattcccc tcatcatatt gacatcatac ctcaaaaata
aattcagtga cacaatgcca 660atgtgtgttc tattgtatta ccatgatgtg
atgaaccgga ctgagaaggc aaaataacca 720tctgtagatc ttttttggtg
tttcatttct tccatcatgg aaactgaaag caataatctg 780tgcacacagt
aaaccagcat cgtgtcttcc agtttttttt gttgttgttg gaatctatcc
840tagatcttta tcatgtgtat ggtggataac ctcatgtcac catcgtatct
gtatacaata 900agcctaaatc agctgacgtt ctatatgtaa attagtaaat
gtaatgacta attagtgcca 960aaaaaaaaaa aaaaaaaa 9786155PRTZea mays
6His Glu Val Arg Lys Gly Trp Leu Val Arg Gln Val Ile Leu Tyr Leu1 5
10 15Ile Phe Thr Gly Leu Gln Gly Phe Ile Ile Glu Gln Tyr Ile Asn
Pro 20 25 30Ile Val Val Asn Ser Gln His Pro Leu Met Gly Gly Leu Leu
Asn Ala 35 40 45Val Glu Thr Val Leu Lys Leu Ser Leu Pro Asn Val Tyr
Leu Trp Leu 50 55 60Cys Met Phe Tyr Cys Leu Phe His Leu Trp Leu Asn
Ile Leu Ala Glu65 70 75 80Ile Leu Arg Phe Gly Asp Arg Glu Phe Tyr
Lys Asp Trp Trp Asn Ala 85 90 95Lys Thr Ile Asp Glu Tyr Trp Arg Lys
Trp Asn Met Pro Val His Lys 100 105 110Trp Ile Val Arg His Ile Tyr
Phe Pro Cys Met Arg Asn Gly Ile Ser 115 120 125Lys Glu Val Ala Val
Phe Ile Ser Phe Phe Val Ser Ala Val Leu His 130 135 140Glu Leu Gln
Ile Thr Trp Met Lys Cys Ser Ile145 150 15571559DNAZea
maysunsure(1542)..(1543)n = a, c, g, or t 7ttttggttta atgctacatc
attgcgagac tggccactgc taatgtgttg ccttagtcta 60cccatatttc cccttggtgc
atttgcagtc gaaaagttgg cattcaacaa tctcgttagt 120gatcctgcta
ctacctgttt tcacatcctt tttacaacat ttgaaattgt atatccagtg
180ctcgtgattc ttaagtgtga ttctgcagtt ttatcaggct ttgtgttgat
gtttattgcc 240tgcattgttt ggctgaagct tgtatctttt gcacatacaa
accatgatat aagaaaactg 300atcacaagcg gcaagaaggt tgataatgaa
ctgaccgcgg ctggcataga taatttacaa 360gctccaactc ttgggagtct
aacatacttc atgatggctc cgacactctg ttatcagcca 420agttatcctc
gaacacctta tgttagaaaa ggttggctgg tccgtcaagt tattctatac
480ttgatattta ctggtctcca aggattcatt attgagcaat acataaatcc
tattgttgtg 540aactctcaac atccattgat gggaggatta ctgaatgctg
tagagactgt tttgaagctc 600tcattaccaa atgtctacct gtggctttgc
atgttttatt gccttttcca tctgtggtta 660aacatacttg ctgagattct
tcgatttggt gaccgagaat tctacaaaga ctggtggaat 720gcaaagacaa
ttgatgagta ctggagaaaa tggaacatgc ctgtgcataa atggattgtt
780cgtcatatat attttccttg catgcgaaat ggtatatcaa aggaagttgc
tgtttttata 840tcgttctttg tttctgctgt acttcatgag gtaacttatt
tactttttca ctcttcatct 900gcatatatta attatatagt tctctatttt
caaatgtgtc ctttcgagtt tcgacatgct 960tttgttcaaa cttaccagct
gtagattact tggatgaagt gctctatata aaattcaata 1020tttcacaatc
cagtcccttt cgagaaaatt atgatacatt ttgtttgcat ttgtacacca
1080gttatgcgtt gcagttccct gccacatact caagttctgg gctttcttag
gaatcatgct 1140tcagattccc ctcatcatat tgacatcata cctcaaaaat
aaattcagtg acacaatggt 1200tggcaatatg atcttttggt tttttttctg
catatacggg cagccaatgt gtgttctatt 1260gtattaccat gatgtgatga
accggactga gaaggcaaaa taaccatctg tagatctttt 1320ttggtgtttc
atttctgcca tcatggaaac tgaaagcaat aatctgtgca cacagtaaac
1380cagcatcgtg tcttccagtt ttctttttgt tgttggaatc tatcctagat
ctttatcatg 1440tgtatggtgg ataacctcat gtcaccatcg tatctgtata
caataagcct aaatcagctg 1500acgttatata tgtataatta gtaaatgtag
cgataaatgt cnnccccctg agagnnacg 15598327PRTZea mays 8Phe Trp Phe
Asn Ala Thr Ser Leu Arg Asp Trp Pro Leu Leu Met Cys1 5 10 15Cys Leu
Ser Leu Pro Ile Phe Pro Leu Gly Ala Phe Ala Val Glu Lys 20 25 30Leu
Ala Phe Asn Asn Leu Val Ser Asp Pro Ala Thr Thr Cys Phe His 35 40
45Ile Leu Phe Thr Thr Phe Glu Ile Val Tyr Pro Val Leu Val Ile Leu
50 55 60Lys Cys Asp Ser Ala Val Leu Ser Gly Phe Val Leu Met Phe Ile
Ala65 70 75 80Cys Ile Val Trp Leu Lys Leu Val Ser Phe Ala His Thr
Asn His Asp 85 90 95Ile Arg Lys Leu Ile Thr Ser Gly Lys Lys Val Asp
Asn Glu Leu Thr 100 105 110Ala Ala Gly Ile Asp Asn Leu Gln Ala Pro
Thr Leu Gly Ser Leu Thr 115 120 125Tyr Phe Met Met Ala Pro Thr Leu
Cys Tyr Gln Pro Ser Tyr Pro Arg 130 135 140Thr Pro Tyr Val Arg Lys
Gly Trp Leu Val Arg Gln Val Ile Leu Tyr145 150 155 160Leu Ile Phe
Thr Gly Leu Gln Gly Phe Ile Ile Glu Gln Tyr Ile Asn 165 170 175Pro
Ile Val Val Asn Ser Gln His Pro Leu Met Gly Gly Leu Leu Asn 180 185
190Ala Val Glu Thr Val Leu Lys Leu Ser Leu Pro Asn Val Tyr Leu Trp
195 200 205Leu Cys Met Phe Tyr Cys Leu Phe His Leu Trp Leu Asn Ile
Leu Ala 210 215 220Glu Ile Leu Arg Phe Gly Asp Arg Glu Phe Tyr Lys
Asp Trp Trp Asn225 230 235 240Ala Lys Thr Ile Asp Glu Tyr Trp Arg
Lys Trp Asn Met Pro Val His 245 250 255Lys Trp Ile Val Arg His Ile
Tyr Phe Pro Cys Met Arg Asn Gly Ile 260 265 270Ser Lys Glu Val Ala
Val Phe Ile Ser Phe Phe Val Ser Ala Val Leu 275 280 285His Glu Val
Thr Tyr Leu Leu Phe His Ser Ser Ser Ala Tyr Ile Asn 290 295 300Tyr
Ile Val Leu Tyr Phe Gln Met Cys Pro Phe Glu Phe Arg His Ala305 310
315 320Phe Val Gln Thr Tyr Gln Leu 3259901DNAZea maysunsure(491)n =
a, c, g, or t 9ccggaattcc cgggtcgacc cacgcgtccg gtctcttatg
cacatacaaa ttatgatata 60agggtattgt ccaaaagtac tgagaagggt gctgcatatg
gaaattatgt cgatcctgag 120aatatgaaag atccaacctt taaaagtcta
gtgtacttca tgttggcccc aacactttgt 180taccagccaa cttatcctca
aactacatgt attagaaagg gttgggtgac ccagcaactc 240ataaagtgcg
tggtttttac aggcttgatg ggcttcataa ttgagcaata tataaaccca
300attgtgaaga attccaaaca tccactgaaa gggaattttt tgaatgctat
agaaagagtc 360ttaaaactct cagtgccaac attatatgta tggctttgca
tgttctattg cttttttcat 420ttatggctga acattgtagc ttaactcctc
tgtttcggtg accgtgaatt ctataaggac 480tggtggaatg ncaaaactgt
tgaagagtac tggaggatgt ggaacatgcc tgttcataag 540tggatcatca
gacacatata ttttccatgt ataaggnaag gcttttccag gggtgtagct
600attctaatct cgtttctggt ttcagctgta ttccatgaga tatgtattgc
ggtgccgtgc 660cacattttca aattctgggc attttctggg atcatgtttc
agataccgtt ggtattcttg 720acaagatatc
tccatgctac gttcaagcat gtaatggtgg gcaacatgat attttggttc
780ttcagtatag tccgacagcc gatgtngtgt ctctataact aacatgacgt
catgaaacaa 840gcaaggccaa gcaagtagat agttcggcag agacatgtaa
cttcaacatc gancatcaga 900a 90110285PRTZea maysUNSURE(148)Xaa = ANY
AMINO ACID 10Pro Glu Phe Pro Gly Arg Pro Thr Arg Pro Val Ser Tyr
Ala His Thr1 5 10 15Asn Tyr Asp Ile Arg Val Leu Ser Lys Ser Thr Glu
Lys Gly Ala Ala 20 25 30Tyr Gly Asn Tyr Val Asp Pro Glu Asn Met Lys
Asp Pro Thr Phe Lys 35 40 45Ser Leu Val Tyr Phe Met Leu Ala Pro Thr
Leu Cys Tyr Gln Pro Thr 50 55 60Tyr Pro Gln Thr Thr Cys Ile Arg Lys
Gly Trp Val Thr Gln Gln Leu65 70 75 80Ile Lys Cys Val Val Phe Thr
Gly Leu Met Gly Phe Ile Ile Glu Gln 85 90 95Tyr Ile Asn Pro Ile Val
Lys Asn Ser Lys His Pro Leu Lys Gly Asn 100 105 110Phe Leu Asn Ala
Ile Glu Arg Val Leu Lys Leu Ser Val Pro Thr Leu 115 120 125Tyr Val
Trp Leu Cys Met Phe Tyr Cys Phe Phe His Leu Trp Leu Asn 130 135
140Ile Val Ala Xaa Leu Leu Cys Phe Gly Asp Arg Glu Phe Tyr Lys
Asp145 150 155 160Trp Trp Asn Xaa Lys Thr Val Glu Glu Tyr Trp Arg
Met Trp Asn Met 165 170 175Pro Val His Lys Trp Ile Ile Arg His Ile
Tyr Phe Pro Cys Ile Arg 180 185 190Xaa Gly Phe Ser Arg Gly Val Ala
Ile Leu Ile Ser Phe Leu Val Ser 195 200 205Ala Val Phe His Glu Ile
Cys Ile Ala Val Pro Cys His Ile Phe Lys 210 215 220Phe Trp Ala Phe
Ser Gly Ile Met Phe Gln Ile Pro Leu Val Phe Leu225 230 235 240Thr
Arg Tyr Leu His Ala Thr Phe Lys His Val Met Val Gly Asn Met 245 250
255Ile Phe Trp Phe Phe Ser Ile Val Arg Gln Pro Met Xaa Cys Leu Tyr
260 265 270Asn Xaa His Asp Val Met Lys Gln Ala Arg Pro Ser Lys 275
280 28511254DNAOryza sativa 11ggcatacggc ggtggggact tctccgcgtt
cacgttccgc gcggcggcgc cggtgcaccg 60caaggccaag gagagccccc tcagctccga
cgccatcttc aagcagagtc atgcaggcct 120tttcaaccta tgcattgttg
ttctagttgc agtgaacagc aggcttatta tcgagaactt 180aatgaagtat
ggcttattaa taagagctgg gttttggttt aatgataaat cattgcggga
240ctggccactt ctaa 2541280PRTOryza sativa 12Ala Tyr Gly Gly Gly Asp
Phe Ser Ala Phe Thr Phe Arg Ala Ala Ala1 5 10 15Pro Val His Arg Lys
Ala Lys Glu Ser Pro Leu Ser Ser Asp Ala Ile 20 25 30Phe Lys Gln Ser
His Ala Gly Leu Phe Asn Leu Cys Ile Val Val Leu 35 40 45Val Ala Val
Asn Ser Arg Leu Ile Ile Glu Asn Leu Met Lys Tyr Gly 50 55 60Leu Leu
Ile Arg Ala Gly Phe Trp Phe Asn Asp Lys Ser Leu Arg Asp65 70 75
80131587DNAOryza sativa 13gcacgagggc atacggcggt ggggacttct
ccgcgttcac gttccgcgcg gcggcgccgg 60tgcaccgcaa ggccaaggag agccccctca
gctccgacgc catcttcaag cagagtcatg 120caggcctttt caacctatgc
attgttgttc tagttgcagt gaacagcagg cttattatcg 180agaacttaat
gaagtatggc ttattaataa gagctgggtt ttggtttaat gataaatcat
240tgcgggactg gccacttcta atgtgttgtc ttagtctgcc tgctttcccc
ctgggtgcat 300ttgcagttga aaagttggca tttaacaatg ttattactga
tgctgttgct acctgcctcc 360atatcttcct ttcaacaacc gaaattgtat
atccagtgct tgtgattctt aagtgtgatt 420ctgcagtttt gtctggcttt
ttgttgatat ttattgcctg tattgtttgg ctgaagcttg 480tatcttttgc
acatacaaac catgatataa ggcaactgac catgggcggc aagaaggttg
540ataatgaact aagcacagtt gacatggata atttacaacc tccaacttta
gggaatctaa 600tatacttcat gatggctcct acactctgtt atcagccaag
ctatccccga acttcatgtg 660ttagaaaagg ttggctgatt cgtcaaatta
ttctgtactt gatctttact ggtcttcaag 720gcttcattat tgagcaatac
ataaatccaa ttgttgtgaa ttctcagcat ccattgaaag 780gaggactcct
aaatgctgta gagactgttt tgaaactctc attaccaaat gtttacctgt
840ggctttgcat gttctatgct tttttccatc tctggttaag tatacttgct
gagattcttc 900gatttggtga ccgtgaattc tacaaagatt ggtggaatgc
aaaaacaatt gatgagtatt 960ggagaaaatg gaatatgcct gtacataaat
gggttgttcg ccatatttac tttccttgca 1020tgcgaaatgg tatatcaaag
gaagttgctg tcttgatatc attccttgtt tctgccgtac 1080tccatgagat
atgtgtcgct gttccctgcc gcattctcaa gttctgggca ttcttaggaa
1140taatgctaca gatccccctt atcgtattga cagcatacct caaaagtaaa
ttcagagata 1200caatggttgg caacatgata ttttggttct ttttctgcat
ctatgggcag ccaatgtgcc 1260ttctcctgta ctatcatgat gtgatgaaca
ggattgagaa ggcaagataa atgcgtgttg 1320ccatcttttt cctctgtttc
attttgtacc agcagaagca caagcaataa tccacatgct 1380agccataaaa
cagcatgatt cccaacggtg tggtacagcc aaccttcctg ttattctatt
1440ttcttggctg tggtgtagat ttagttttta acttgtggct aaccgcagga
atgcctgtag 1500ataagcatct gtcattctgt ctggcgacgt tctccttatt
aatgtgtaga tgtagaactg 1560tttccgaaaa aaaaaaaaaa aaaaaaa
158714500PRTOryza sativa 14Met Val Gly Ser Asp Gly Asp Gly Asp Gly
Gly Gly Gly Glu Ala His1 5 10 15Ala Gly Gly Pro Arg Arg Arg Ala Gly
Gln Leu Arg Gly Arg Leu Arg 20 25 30Asp Glu Ala Ala Pro Gly Ser Pro
Pro Arg Pro Arg Pro Arg Pro Arg 35 40 45Pro Arg Gly Gly Asp Ser Asn
Gly Arg Ser Val Leu Arg Pro Gly Gly 50 55 60Gly Gly Gly Arg Gly Gly
Gly Gly Asp Phe Ser Ala Phe Thr Phe Arg65 70 75 80Ala Ala Ala Pro
Val His Arg Lys Ala Lys Glu Ser Pro Leu Ser Ser 85 90 95Asp Ala Ile
Phe Lys Gln Ser His Ala Gly Leu Phe Asn Leu Cys Ile 100 105 110Val
Val Leu Val Ala Val Asn Ser Arg Leu Ile Ile Glu Asn Leu Met 115 120
125Lys Tyr Gly Leu Leu Ile Arg Ala Gly Phe Trp Phe Asn Asp Lys Ser
130 135 140Leu Arg Asp Trp Pro Leu Leu Met Cys Cys Leu Ser Leu Pro
Ala Phe145 150 155 160Pro Leu Gly Ala Phe Ala Val Glu Lys Leu Ala
Phe Asn Asn Val Ile 165 170 175Thr Asp Ala Val Ala Thr Cys Leu His
Ile Phe Leu Ser Thr Thr Glu 180 185 190Ile Val Tyr Pro Val Leu Val
Ile Leu Lys Cys Asp Ser Ala Val Leu 195 200 205Ser Gly Phe Leu Leu
Ile Phe Ile Ala Cys Ile Val Trp Leu Lys Leu 210 215 220Val Ser Phe
Ala His Thr Asn His Asp Ile Arg Gln Leu Thr Met Gly225 230 235
240Gly Lys Lys Val Asp Asn Glu Leu Ser Thr Val Asp Met Asp Asn Leu
245 250 255Gln Pro Pro Thr Leu Gly Asn Leu Ile Tyr Phe Met Met Ala
Pro Thr 260 265 270Leu Cys Tyr Gln Pro Ser Tyr Pro Arg Thr Ser Cys
Val Arg Lys Gly 275 280 285Trp Leu Ile Arg Gln Ile Ile Leu Tyr Leu
Ile Phe Thr Gly Leu Gln 290 295 300Gly Phe Ile Ile Glu Gln Tyr Ile
Asn Pro Ile Val Val Asn Ser Gln305 310 315 320His Pro Leu Lys Gly
Gly Leu Leu Asn Ala Val Glu Thr Val Leu Lys 325 330 335Leu Ser Leu
Pro Asn Val Tyr Leu Trp Leu Cys Met Phe Tyr Ala Phe 340 345 350Phe
His Leu Trp Leu Ser Ile Leu Ala Glu Ile Leu Arg Phe Gly Asp 355 360
365Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala Lys Thr Ile Asp Glu Tyr
370 375 380Trp Arg Lys Trp Asn Met Pro Val His Lys Trp Val Val Arg
His Ile385 390 395 400Tyr Phe Pro Cys Met Arg Asn Gly Ile Ser Lys
Glu Val Ala Val Leu 405 410 415Ile Ser Phe Leu Val Ser Ala Val Leu
His Glu Ile Cys Val Ala Val 420 425 430Pro Cys Arg Ile Leu Lys Phe
Trp Ala Phe Leu Gly Ile Met Leu Gln 435 440 445Ile Pro Leu Ile Val
Leu Thr Ala Tyr Leu Lys Ser Lys Phe Arg Asp 450 455 460Thr Met Val
Gly Asn Met Ile Phe Trp Phe Phe Phe Cys Ile Tyr Gly465 470 475
480Gln Pro Met Cys Leu Leu Leu Tyr Tyr His Asp Val Met Asn Arg Ile
485 490 495Glu Lys Ala Arg 500151942DNAGlycine max 15tagaaaacac
gctcggtctt cttctccaat ggcgatttcc gatgagcctg aaagtgtagc 60cactgctctc
aaccactctt ccctgcgccg ccgtccctcc gccacctcca ccgccggcct
120cttcaattcg cctgagacaa ccaccgacag ttccggtgat gacttggcca
aggattctgg 180ttccgacgac tccatcaaca gcgacgacgc cgccgtcaat
tcccaacagc aaaacgaaaa 240acaagacact gatttctccg tcctcaaatt
cgcctaccgt ccttccgtcc ccgctcaccg 300caaagtgaag gaaagtccgc
tcagctccga cactattttc cgtcagagtc acgcgggcct 360cttcaacctt
tgtatagtag tccttgttgc tgtgaatagc cgactcatca ttgagaattt
420aatgaagtat ggttggttga tcaaatctgg cttttggttt agttcaaagt
cattgagaga 480ctggcccctt ttcatgtgtt gtctttctct tgtggtattt
cctttcgctg cctttatagt 540ggagaagttg gcacaacgga agtgtatacc
cgaaccagtt gttgttgtac ttcatataat 600cattacctca acttcgcttt
tctatccagt tttagttatt ctcaggtgtg attctgcttt 660tgtatcaggt
gtcacgttaa tgctgttttc ttgtgttgta tggttaaaat tggtgtctta
720tgcacataca aactatgata tgagagcact taccaaatta gttgaaaagg
gagaagcact 780gctcgatact ctgaacatgg actatcctta caacgtaagc
ttcaagagct tggcatattt 840cctggttgcc cctacattat gttaccagcc
aagctatcct cgcacacctt atattcgaaa 900gggttggttg tttcgccaac
ttgtcaagct gataatattt acaggagtta tgggatttat 960aatagaccaa
tatattaatc ccatagtaca aaattcacag catcctctca agggaaacct
1020tctttacgcc accgagagag ttctgaagct ttctgttcca aatttatatg
tgtggctctg 1080catgttctat tgctttttcc acctttggtt aaatatcctg
gcagagcttc ttcgatttgg 1140tgatcgtgaa ttctacaagg attggtggaa
tgccaaaact gtcgaagatt attggaggat 1200gtggaatatg cctgttcaca
aatggatgat ccgccaccta tattttccat gtttaaggca 1260cggtctacca
aaggctgctg ctcttttaat tgccttcctg gtttctgctt tattccatga
1320gctgtgcatt gctgttcctt gccacatatt caagttgtgg gctttcggtg
gaattatgtt 1380tcaggttcct ttggtcttga tcactaatta tctgcaaaat
aaattcagaa actcaatggt 1440tggaaatatg attttttggt tcatattcag
tatccttggt caacctatgt gtgtactgct 1500atactaccat gacttgatga
ataggaaagg caaacttgac tgaagctacg gccattacat 1560tttaaaggtg
cacatggatg agcttttcag ttttcagatt gtaaaattga tgtggatatg
1620ttggtcaata tttgttttct acgaatgctt tcatctacca tggcattggc
tgctctgaag 1680gaattccacg ggatatgcca gttcacgagg ctaattcatt
atcttgatct atgtacttac 1740caactctcct ctggcaattg tatcaaaata
tgcaattttg agagccatac actggcattg 1800ataactgcca aggaacactc
taactgtttt ctgttaactg ttaattagta gagggctaga 1860tgtaaatggt
ttatgctcaa tatatttatt tcctcctaaa aaaaaaaaaa aaaaaaaaaa
1920aaaaaaaaaa aaaaaaaaaa aa 194216504PRTGlycine max 16Met Ala Ile
Ser Asp Glu Pro Glu Ser Val Ala Thr Ala Leu Asn His1 5 10 15Ser Ser
Leu Arg Arg Arg Pro Ser Ala Thr Ser Thr Ala Gly Leu Phe 20 25 30Asn
Ser Pro Glu Thr Thr Thr Asp Ser Ser Gly Asp Asp Leu Ala Lys 35 40
45Asp Ser Gly Ser Asp Asp Ser Ile Asn Ser Asp Asp Ala Ala Val Asn
50 55 60Ser Gln Gln Gln Asn Glu Lys Gln Asp Thr Asp Phe Ser Val Leu
Lys65 70 75 80Phe Ala Tyr Arg Pro Ser Val Pro Ala His Arg Lys Val
Lys Glu Ser 85 90 95Pro Leu Ser Ser Asp Thr Ile Phe Arg Gln Ser His
Ala Gly Leu Phe 100 105 110Asn Leu Cys Ile Val Val Leu Val Ala Val
Asn Ser Arg Leu Ile Ile 115 120 125Glu Asn Leu Met Lys Tyr Gly Trp
Leu Ile Lys Ser Gly Phe Trp Phe 130 135 140Ser Ser Lys Ser Leu Arg
Asp Trp Pro Leu Phe Met Cys Cys Leu Ser145 150 155 160Leu Val Val
Phe Pro Phe Ala Ala Phe Ile Val Glu Lys Leu Ala Gln 165 170 175Arg
Lys Cys Ile Pro Glu Pro Val Val Val Val Leu His Ile Ile Ile 180 185
190Thr Ser Thr Ser Leu Phe Tyr Pro Val Leu Val Ile Leu Arg Cys Asp
195 200 205Ser Ala Phe Val Ser Gly Val Thr Leu Met Leu Phe Ser Cys
Val Val 210 215 220Trp Leu Lys Leu Val Ser Tyr Ala His Thr Asn Tyr
Asp Met Arg Ala225 230 235 240Leu Thr Lys Leu Val Glu Lys Gly Glu
Ala Leu Leu Asp Thr Leu Asn 245 250 255Met Asp Tyr Pro Tyr Asn Val
Ser Phe Lys Ser Leu Ala Tyr Phe Leu 260 265 270Val Ala Pro Thr Leu
Cys Tyr Gln Pro Ser Tyr Pro Arg Thr Pro Tyr 275 280 285Ile Arg Lys
Gly Trp Leu Phe Arg Gln Leu Val Lys Leu Ile Ile Phe 290 295 300Thr
Gly Val Met Gly Phe Ile Ile Asp Gln Tyr Ile Asn Pro Ile Val305 310
315 320Gln Asn Ser Gln His Pro Leu Lys Gly Asn Leu Leu Tyr Ala Thr
Glu 325 330 335Arg Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp
Leu Cys Met 340 345 350Phe Tyr Cys Phe Phe His Leu Trp Leu Asn Ile
Leu Ala Glu Leu Leu 355 360 365Arg Phe Gly Asp Arg Glu Phe Tyr Lys
Asp Trp Trp Asn Ala Lys Thr 370 375 380Val Glu Asp Tyr Trp Arg Met
Trp Asn Met Pro Val His Lys Trp Met385 390 395 400Ile Arg His Leu
Tyr Phe Pro Cys Leu Arg His Gly Leu Pro Lys Ala 405 410 415Ala Ala
Leu Leu Ile Ala Phe Leu Val Ser Ala Leu Phe His Glu Leu 420 425
430Cys Ile Ala Val Pro Cys His Ile Phe Lys Leu Trp Ala Phe Gly Gly
435 440 445Ile Met Phe Gln Val Pro Leu Val Leu Ile Thr Asn Tyr Leu
Gln Asn 450 455 460Lys Phe Arg Asn Ser Met Val Gly Asn Met Ile Phe
Trp Phe Ile Phe465 470 475 480Ser Ile Leu Gly Gln Pro Met Cys Val
Leu Leu Tyr Tyr His Asp Leu 485 490 495Met Asn Arg Lys Gly Lys Leu
Asp 50017470DNAGlycine maxunsure(372)n = a, c, g, or t 17taaacacgct
cgctcggtct tcttttccaa tggcgatttc cgatgagcct gaaactgtag 60ccactgctct
caaccactct tccctgcgcc gccgtcccac cgccgctggc ctcttcaatt
120cgcccgagac gaccaccgac agttccggtg atgacttggc caaggattcc
ggttccgacg 180actccatcag cagcgacgcc gccaattcgc aaccgcaaca
aaaacaagac actgatttct 240ccgtcctcaa attcgcctac cgtccttccg
tccccgctca tcgcaaagtg aaggaaagtc 300cgctcagctc ccgacaccat
tttccgtcag aagtcacgcg gggcctcttc aacctcctgt 360atagtaagtc
cntgttgctg tgaataagcc gactcatcat tgagaatttt aaatgaaata
420tggnttgggt tgatcaaatc cnggcntttt gggttaagct caaagtcant
4701838PRTGlycine max 18Asp Phe Ser Val Leu Lys Phe Ala Tyr Arg Pro
Ser Val Pro Ala His1 5 10 15Arg Lys Val Lys Glu Ser Pro Leu Ser Ser
Asp Thr Ile Phe Val Arg 20 25 30Ser His Ala Gly Pro Leu
3519646DNATriticum aestivumunsure(240)n = a, c, g, or t
19ctccgacgcc atcttccgac agagccatgc aggtcttctg aatctatgca ttgttgtgct
60gattgcagtg aacagcaggc tcattattga gaacttaatg aagtatggcc tattaataag
120agctgggttt tggtttaagt gcaagatcgc tgggagattg gccacttctg
atgtgctgcc 180tcactttacc cattttccca cttgctgctc tcatgaccgg
agaattgggt caaaagaaan 240tcatccgtgg atcatgtgtc tatcctcccc
catataatta ttacaaccac tgtccttatc 300ctatccggtg ntgtgatcct
taaagtgtga accacantat atcctggttt gtgnttatgt 360ccattgcaan
atacttgggt gancttgncc cttttgctcc atacaattag atataagtat
420tgnccccaaa ntatngaaag ggtgctacac agggattcta ccnagaagaa
aattaaagcc 480caactncaac aagtgtgtat cangttggcc caacactggt
acaaccaatt tacccggcan 540attatanaaa ggtggtcacc ggaactataa
agtgtatttt aagcttatgg ctcaaatggc 600ataataacca ttgganatca
acacatgacg aanttttgnc atgaaa 6462039PRTTriticum aestivum 20Ser Asp
Ala Ile Phe Arg Gln Ser His Ala Gly Leu Leu Asn Leu Cys1 5 10 15Ile
Val Val Leu Ile Ala Val Asn Ser Arg Leu Ile Ile Glu Asn Leu 20 25
30Met Lys Tyr Gly Leu Leu Ile 35211975DNATriticum
aestivumunsure(93)n = a, c, g, or t 21acgagggcct aggtcgcctc
cgcsactgtg tcagcgcgca agtcggccgc ctccctccgc 60tttmcgcttt tgcgcgtcmg
tgctggcgcg ggnccaccac catcgcatgt caaaagggaa 120cccagacccg
cacctccccg gcagcttcct cccttcccac ggcgggccgc caccgaaacc
180caaaaccccg ccccgaacct tccggaacct cccctccagt tccacccatg
gccccgcccc 240gtccgtggcc gctgccacga tcgcgacgac ccctccctcc
gcctccgccg cgcccctgcc 300gccgacggtc cacggagagg cggcgcatgg
agcagccgca gcggcacgac gagatgccct 360gctaccgggc gtcggcgccg
cccaccgccg ggtcaaggag agcccgctta gctccgacgc 420catcttccga
cagagccatg caggtcttct gaatctatgc attgttgtgc tgattgcagt
480gaacagcagg ctcattatcg agaacttaat gaagtatggc ctattaataa
gagctgggtt 540ttggtttagt gcaagatcgc tgggagattg gccacttctg
atgtgctgcc tcactttacc 600cattttccca cttgctgctc tcatgaccga
gaagtgggct caaagaaagc tcatccgtga
660tcatgtgtct attcttctcc atataattat tacaaccact gtccttatct
atccggttgt 720tgtgattctt aagtgtgaat cagcagtatt atctggattt
gtgttaatgt tcattgcaag 780cattacttgg ttgaagcttg tctcttttgc
tcatacaaat tatgatataa ggatattgtc 840ccaaagtatt gaaaagggtg
ctacacatgg cagttctatc gatgaggaaa acattaaagg 900cccaactatc
aacagtgttg tgtatttcat gttggcccca acactttgtt accagccaag
960ttatccccgg acagcattta ttagaaaagg ctgggtcacc cggcagctta
taaaatgtgt 1020agtttttaca ggcttgatgg gcttcataat tgagcaatac
attaatccaa ttgtgcagaa 1080ttcgaagcat ccattgaacg gaaatttctt
ggatgctatt gagagagtct tgaaactctc 1140agtgccaaca ttatatgtat
ggctttgtat gttctattcc tttttccatc tgtggttgaa 1200tattctagcc
gaactcctcc gttttggtga tcgtgaattc tacaaggact ggtggaacgc
1260caaaacagtt gaagagtact ggagaatgtg gaatatgcct gttcataagt
ggatcgttcg 1320acatatatat tttccatgca taaggaatgg cttatcaaag
ggttgtgcca ttctcatcgc 1380atttctggtt tcagctgtat ttcatgagct
atgtattgct gttccgtgcc acattttcaa 1440attatgggca ttttctggaa
tcatgtttca gattcccctg ctattcttga cgaaatatct 1500tcaagataag
ttcaagaata caatggtggg caacatgata ttttggttct tcttcagcat
1560agttgggcaa ccaatgtgtg ttctcttgta ctaccatgat gtcatgaaca
gacaggctca 1620gacaaatggc tagttctgtt ttagaagtgc actataacac
agatcgtccg aagcaaattg 1680gcccgaggca atggaggggc ggcctcctta
atgtttcgcc atgggctgtt agagcttgct 1740atgctacgaa tccaagtttg
tcagcatgat atgttccaat ccgttccagt tagctcgctg 1800cgttccaaat
gtatgatatg ccggccgggg tgtgtaccga agatacccca gtgatgaagc
1860cgaagataac acgacctgcc acatgtgttt tgtgtatacg tttcggttca
tgtgccagca 1920gagttacgta cgtgatgccc tgttggatat aaagtgtacg
tgccgtatga aaaaa 197522508PRTTriticum aestivum 22Met Ser Lys Gly
Asn Pro Asp Pro His Leu Pro Gly Ser Phe Leu Pro1 5 10 15Ser His Gly
Gly Pro Pro Pro Lys Pro Lys Thr Pro Pro Arg Thr Phe 20 25 30Arg Asn
Leu Pro Ser Ser Ser Thr His Gly Pro Ala Pro Ser Val Ala 35 40 45Ala
Ala Thr Ile Ala Thr Thr Pro Pro Ser Ala Ser Ala Ala Pro Leu 50 55
60Pro Pro Thr Val His Gly Glu Ala Ala His Gly Ala Ala Ala Ala Ala65
70 75 80Arg Arg Asp Ala Leu Leu Pro Gly Val Gly Ala Ala His Arg Arg
Val 85 90 95Lys Glu Ser Pro Leu Ser Ser Asp Ala Ile Phe Arg Gln Ser
His Ala 100 105 110Gly Leu Leu Asn Leu Cys Ile Val Val Leu Ile Ala
Val Asn Ser Arg 115 120 125Leu Ile Ile Glu Asn Leu Met Lys Tyr Gly
Leu Leu Ile Arg Ala Gly 130 135 140Phe Trp Phe Ser Ala Arg Ser Leu
Gly Asp Trp Pro Leu Leu Met Cys145 150 155 160Cys Leu Thr Leu Pro
Ile Phe Pro Leu Ala Ala Leu Met Thr Glu Lys 165 170 175Trp Ala Gln
Arg Lys Leu Ile Arg Asp His Val Ser Ile Leu Leu His 180 185 190Ile
Ile Ile Thr Thr Thr Val Leu Ile Tyr Pro Val Val Val Ile Leu 195 200
205Lys Cys Glu Ser Ala Val Leu Ser Gly Phe Val Leu Met Phe Ile Ala
210 215 220Ser Ile Thr Trp Leu Lys Leu Val Ser Phe Ala His Thr Asn
Tyr Asp225 230 235 240Ile Arg Ile Leu Ser Gln Ser Ile Glu Lys Gly
Ala Thr His Gly Ser 245 250 255Ser Ile Asp Glu Glu Asn Ile Lys Gly
Pro Thr Ile Asn Ser Val Val 260 265 270Tyr Phe Met Leu Ala Pro Thr
Leu Cys Tyr Gln Pro Ser Tyr Pro Arg 275 280 285Thr Ala Phe Ile Arg
Lys Gly Trp Val Thr Arg Gln Leu Ile Lys Cys 290 295 300Val Val Phe
Thr Gly Leu Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn305 310 315
320Pro Ile Val Gln Asn Ser Lys His Pro Leu Asn Gly Asn Phe Leu Asp
325 330 335Ala Ile Glu Arg Val Leu Lys Leu Ser Val Pro Thr Leu Tyr
Val Trp 340 345 350Leu Cys Met Phe Tyr Ser Phe Phe His Leu Trp Leu
Asn Ile Leu Ala 355 360 365Glu Leu Leu Arg Phe Gly Asp Arg Glu Phe
Tyr Lys Asp Trp Trp Asn 370 375 380Ala Lys Thr Val Glu Glu Tyr Trp
Arg Met Trp Asn Met Pro Val His385 390 395 400Lys Trp Ile Val Arg
His Ile Tyr Phe Pro Cys Ile Arg Asn Gly Leu 405 410 415Ser Lys Gly
Cys Ala Ile Leu Ile Ala Phe Leu Val Ser Ala Val Phe 420 425 430His
Glu Leu Cys Ile Ala Val Pro Cys His Ile Phe Lys Leu Trp Ala 435 440
445Phe Ser Gly Ile Met Phe Gln Ile Pro Leu Leu Phe Leu Thr Lys Tyr
450 455 460Leu Gln Asp Lys Phe Lys Asn Thr Met Val Gly Asn Met Ile
Phe Trp465 470 475 480Phe Phe Phe Ser Ile Val Gly Gln Pro Met Cys
Val Leu Leu Tyr Tyr 485 490 495His Asp Val Met Asn Arg Gln Ala Gln
Thr Asn Gly 500 5052320DNAArtificial SequenceDescription of
Artificial SequencePCR primer 23cttagcttct tccttcaatc
202433DNAArtificial SequenceDescription of Artificial SequencePCR
primer 24tttctagact cgagtgaaca gttgtttcat gac 3325497PRTMus
musculus 25Met Gly Asp Arg Gly Gly Ala Gly Ser Ser Arg Arg Arg Thr
Gly Ser1 5 10 15Arg Val Ser Val Gln Gly Gly Ser Gly Pro Lys Val Glu
Glu Asp Glu 20 25 30Val Arg Asp Ala Ala Val Ser Pro Asp Leu Gly Ala
Gly Gly Asp Ala 35 40 45Pro Ala Pro Ala Pro Ala Pro Ala His Thr Arg
Asp Lys Asp Gly Arg 50 55 60Thr Ser Val Gly Asp Gly Tyr Trp Asp Leu
Arg Cys His Arg Leu Gln65 70 75 80Asp Ser Leu Phe Ser Ser Asp Ser
Gly Phe Ser Asn Tyr Arg Gly Ile 85 90 95Leu Asn Trp Cys Val Val Met
Leu Ile Leu Ser Asn Ala Arg Leu Phe 100 105 110Leu Glu Asn Leu Ile
Lys Tyr Gly Ile Leu Val Asp Pro Ile Gln Val 115 120 125Val Ser Leu
Phe Leu Lys Asp Pro Tyr Ser Trp Pro Ala Pro Cys Val 130 135 140Ile
Ile Ala Ser Asn Ile Phe Val Val Ala Ala Phe Gln Ile Glu Lys145 150
155 160Arg Leu Ala Val Gly Ala Leu Thr Glu Gln Met Gly Leu Leu Leu
His 165 170 175Val Val Asn Leu Ala Thr Ile Ile Cys Phe Pro Ala Ala
Val Ala Leu 180 185 190Leu Val Glu Ser Ile Thr Pro Val Gly Ser Val
Phe Ala Leu Ala Ser 195 200 205Tyr Ser Ile Met Phe Leu Lys Leu Tyr
Ser Tyr Arg Asp Val Asn Leu 210 215 220Trp Cys Arg Gln Arg Arg Val
Lys Ala Lys Ala Val Ser Thr Gly Lys225 230 235 240Lys Val Ser Gly
Ala Ala Ala Gln Gln Ala Val Ser Tyr Pro Asp Asn 245 250 255Leu Thr
Tyr Arg Asp Leu Tyr Tyr Phe Ile Phe Ala Pro Thr Leu Cys 260 265
270Tyr Glu Leu Asn Phe Pro Arg Ser Pro Arg Ile Arg Lys Arg Phe Leu
275 280 285Leu Arg Arg Val Leu Glu Met Leu Phe Phe Thr Gln Leu Gln
Val Gly 290 295 300Leu Ile Gln Gln Trp Met Val Pro Thr Ile His Asn
Ser Met Lys Pro305 310 315 320Phe Lys Asp Met Asp Tyr Ser Arg Ile
Ile Glu Arg Leu Leu Lys Leu 325 330 335Ala Val Pro Asn His Leu Ile
Trp Leu Ile Phe Phe Tyr Trp Phe Phe 340 345 350His Ser Cys Leu Asn
Ala Val Ala Glu Leu Leu Gln Phe Gly Asp Arg 355 360 365Glu Phe Tyr
Arg Asp Trp Trp Asn Ala Glu Ser Val Thr Tyr Phe Trp 370 375 380Gln
Asn Trp Asn Ile Pro Val His Lys Trp Cys Ile Arg His Phe Tyr385 390
395 400Lys Pro Met Leu Arg His Gly Ser Ser Lys Trp Val Ala Arg Thr
Gly 405 410 415Val Phe Leu Thr Ser Ala Phe Phe His Glu Tyr Leu Val
Ser Val Pro 420 425 430Leu Arg Met Phe Arg Leu Trp Ala Phe Thr Ala
Met Met Ala Gln Val 435 440 445Pro Leu Ala Trp Ile Val Gly Arg Phe
Phe Gln Gly Asn Tyr Gly Asn 450 455 460Ala Ala Val Trp Val Thr Leu
Ile Ile Gly Gln Pro Val Ala Val Leu465 470 475 480Met Tyr Val His
Asp Tyr Tyr Val Leu Asn Tyr Asp Ala Pro Val Gly 485 490
495Val26520PRTArabidopsis thaliana 26Met Ala Ile Leu Asp Ser Ala
Gly Val Thr Thr Val Thr Glu Asn Gly1 5 10 15Gly Gly Glu Phe Val Asp
Leu Asp Arg Leu Arg Arg Arg Lys Ser Arg 20 25 30Ser Asp Ser Ser Asn
Gly Leu Leu Leu Ser Gly Ser Asp Asn Asn Ser 35 40 45Pro Ser Asp Asp
Val Gly Ala Pro Ala Asp Val Arg Asp Arg Ile Asp 50 55 60Ser Val Val
Asn Asp Asp Ala Gln Gly Thr Ala Asn Leu Ala Gly Asp65 70 75 80Asn
Asn Gly Gly Gly Asp Asn Asn Gly Gly Gly Arg Gly Gly Gly Glu 85 90
95Gly Arg Gly Asn Ala Asp Ala Thr Phe Thr Tyr Arg Pro Ser Val Pro
100 105 110Ala His Arg Arg Ala Arg Glu Ser Pro Leu Ser Ser Asp Ala
Ile Phe 115 120 125Lys Gln Ser His Ala Gly Leu Phe Asn Leu Cys Val
Val Val Leu Ile 130 135 140Ala Val Asn Ser Arg Leu Ile Ile Glu Asn
Leu Met Lys Tyr Gly Trp145 150 155 160Leu Ile Arg Thr Asp Phe Trp
Phe Ser Ser Arg Ser Leu Arg Asp Trp 165 170 175Pro Leu Phe Met Cys
Cys Ile Ser Leu Ser Ile Phe Pro Leu Ala Ala 180 185 190Phe Thr Val
Glu Lys Leu Val Leu Gln Lys Tyr Ile Ser Glu Pro Val 195 200 205Val
Ile Phe Leu His Ile Ile Ile Thr Met Thr Glu Val Leu Tyr Pro 210 215
220Val Tyr Val Thr Leu Arg Cys Asp Ser Ala Phe Leu Ser Gly Val
Thr225 230 235 240Leu Met Leu Leu Thr Cys Ile Val Trp Leu Lys Leu
Val Ser Tyr Ala 245 250 255His Thr Ser Tyr Asp Ile Arg Ser Leu Ala
Asn Ala Ala Asp Lys Ala 260 265 270Asn Pro Glu Val Ser Tyr Tyr Val
Ser Leu Lys Ser Leu Ala Tyr Phe 275 280 285Met Val Ala Pro Thr Leu
Cys Tyr Gln Pro Ser Tyr Pro Arg Ser Ala 290 295 300Cys Ile Arg Lys
Gly Trp Val Ala Arg Gln Phe Ala Lys Leu Val Ile305 310 315 320Phe
Thr Gly Phe Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro Ile 325 330
335Val Arg Asn Ser Lys His Pro Leu Lys Gly Asp Leu Leu Tyr Ala Ile
340 345 350Glu Arg Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp
Leu Cys 355 360 365Met Phe Tyr Cys Phe Phe His Leu Trp Leu Asn Ile
Leu Ala Glu Leu 370 375 380Leu Cys Phe Gly Asp Arg Glu Phe Tyr Lys
Asp Trp Trp Asn Ala Lys385 390 395 400Ser Val Gly Asp Tyr Trp Arg
Met Trp Asn Met Pro Val His Lys Trp 405 410 415Met Val Arg His Ile
Tyr Phe Pro Cys Leu Arg Ser Lys Ile Pro Lys 420 425 430Thr Leu Ala
Ile Ile Ile Ala Phe Leu Val Ser Ala Val Phe His Glu 435 440 445Leu
Cys Ile Ala Val Pro Cys Arg Leu Phe Lys Leu Trp Ala Phe Leu 450 455
460Gly Ile Met Phe Gln Val Pro Leu Val Phe Ile Thr Asn Tyr Leu
Gln465 470 475 480Glu Arg Phe Gly Ser Thr Val Gly Asn Met Ile Phe
Trp Phe Ile Phe 485 490 495Cys Ile Phe Gly Gln Pro Met Cys Val Leu
Leu Tyr Tyr His Asp Leu 500 505 510Met Asn Arg Lys Gly Ser Met Ser
515 520
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