Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
SOYBEAN GLUTATHIONE-S-TRANSFERASE ENZYMES
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More
specifically, this invention pertains to nucleic acid fragments encoding
soybean
glutathione-S-transferase (GST) enzymes involved in the detoxification of
xenobiotic compounds in plants and seeds.
BACKGROUND OF THE INVENTION
Glutathione-S-transferases (GST) are a family of enzymes which catalyze
the conjugation of glutathione, homoglutathione (hGSH) and other glutathione-
like analogs via a sulfhydiyl group, to a large range of hydrophobic,
electrophilic
compounds. The conjugation can result in detoxification of these compounds.
GST enzymes have been identified in a range of plants including maize (Wosnick
et al., Gene (Amst) 76 (1) (1989) 153-160; Rossini et al., Plant Physiology
(Rockville) 112 (4) (1996) 1595-1600; Holt et al., Planta (Heidelberg) 196 (2)
(1995) 295-302), wheat (Edwards et al., Pestic. Biochem. Physiol. (1996)
54(2),
96-104), sorghum (Hatzios et al., J. Environ. Sci. Health, Part B (1996),
B31(3),
545-553), arabidopsis (Van Der Kop et al., Plant Molecular Biology 30 (4)
(1996), sugarcane (Singhal et al., Phytochemistry (OXF) 30 (5) (1991)
1409-1414), soybean (Flury et al., Physiologia Plantarum 94 (1995) 594-604)
and
peas (Edwards R., Physiologia Plantarum 98 (3) (1996) 594-604). GST's can
comprise a significant portion of total plant protein, for example attaining
from 1
to 2% of the total soluble protein in etiolated maize seedlings (Timmermann,
Physiol. Plant. ( 1989) 77(3 ), 465-71 ).
Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the nucleophilic
attack of the thiol group of GSH to various electrophilic substrates. Their
functions and regulation in plants has been recently reviewed (Mans et al.,
Annu
Rev Plant Physiol Plant Mol Biol 47:127-58 (1996); Droog, F. JPlant Growth
Regul 16:95-107, (1997)). They are present at every stage of plant development
from early embryogenesis to senescence and in every tissue type examined. The
agents that have been shown to cause an increase in GST levels have the
potential to cause oxidative destruction in plants, suggesting a role for GSTs
in
the protection from oxidative damage. In addition to their role in the
protection
from oxidative damage, GSTs have the ability to nonenzymatically bind certain
small molecules, such as auxin (Zettl, et al., PNAS 91: 689-693, (1994)) and
perhaps regulate their bioavailabiiity. Furthermore the addition of GSH to a
molecule serves as an "address" to send that molecule to the plant vacuole
(Marts, et al., Nature 375: 397=400, (1995)).
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GSTs have also been implicated in the detoxification of certain
herbicides. Maize GSTs have been well characterized in relation to herbicide
metabolism. Three genes from maize have been cloned: GST 29 (Shah et al. ,
Plant Mol Biol 6, 203-211 (1986)), GST 27 (Jepson et al., Plant Mol Biol
26:1855-1866, (1994)), GST 26 (Moore et al., Nucleic AcidsRes 14:7227-7235
(1986)). These gene products form four GST isoforms: GST I (a homodimer of
GST 29), GST II (a heterodimer of GST 29 and GST 27), GST III (a homodimer
of GST 26), and GST IV (a homodimer of GST 27). GST 27 is highly induciblP
by safener compounds (Jepson (1994) supra; Holt et al., Planta 196:295-302,
(.1995)) and overexpression of GST 27 in tobacco confers alachlor resistance
to
transgenic tobacco (Jepson, personal communication). Additionally Bridges et
al. (U.S. 5589614) disclose the sequence of a maize derived GST isoform II
promoter useful for the expression of foreign genes in maize and wheat. In
soybean, herbicide compounds conjugated to hGSH have been detected and
correlated with herbicide selectivity (Frear et al., Physio120: 299-310
(I983);
Brown et al., Pest Biochem Physiol29:112-120, (1987)). This implies that
hGSH conjugation is an important determinant in soybean herbicide selectivity
although this hypothesis has not been characterized on a molecular level.
Glutathione (the tripeptide Y-glu-cys-gly, or GSH) is present in most
plants and animals. However, in some plants from the family Leguminaceae the
major free thioi is homoglutathione. For example, soybeans (Glycine max) have
nearly undetectable levels of glutathione with the tripeptide homoglutathione
(y-glu-cys-~3-ala) apparently substituting for the same functions. Some
herbicides are detoxified in soybeans by homoglutathione conjugation catalyzed
by glutathione S-transferase (GST) enzyme(s).
Homoglutathione (hGSH) was originally detected in Phaseolus vulgaris
and several other leguminous species (Price, C.A., Nature 180: 148-149,
(1957)). The structure of hGSH (Carnegie, P.R., Biochemical Journal
89:471-478 (1963)) was determined to be the tripeptide y-glu-cys-~3-ala.
Homoglutathione has not been found in non-leguminous species. In plants from
the family Leguminaceae, the ratio of hGSH to GSH varies according to both
species and tissue examined. In seeds and leaves of the tribe Vicieae, only
traces of hGSH were found in addition to the main thiol GSH, whereas in roots
the hGSH content exceeded the GSH content. - The tribe Trifolieae contained
both tripeptides and in the tribe Phaseoleae, hGSH predominated. In soybean
(Glycine max}, a member of the Phaseoleae, hGSH constitutes 99% of the free
thiol in leaves and seeds and greater than 95 % of the free thiol in soybean
roots
(Klapheck, S., Physiolgia Plantarum 74: 727-732 (1988)). As such, it is
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essential that soybean glutathione S-transferases be able to efficiently
utilize
hGSH.
Some efforts have been made to alter plant phenotypes by the expression
of either plant or mammalian foreign GST genes or their promoters in mature
plant tissue. For example, Helmer et al. (IJ.S. 5073677) teach the expression
of a
rat GST gene in tobacco under the control of a strong plant promoter.
Similarly,
Jepson et aI. (WO 97/11189) disclose a chemically inducible maize GST promoter
useful for the expression of foreign proteins in plants; Chilton et al. (EP
256223)
discuss the construction of herbicide tolerant plants expressing a foreign
plant
GST gene; and Bieseler et al. (WO 96/23072) teach DNA encoding GSTIIIc, its
recombinant production and transgenic plants containing the DNA having a
herbicide-tolerant phenotype.
Manipulation of nucleic acid fragments encoding soybean GST to use in
screening in assays, the creation of herbicide-tolerant transgenic plants, and
altered production of GST enzymes depend on the heretofore unrealized
isolation
of nucleic acid fragments that encode all or a substantial portion of a
soybean GST
enzyme.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid fragments isolated from
soybean encoding all or a substantial portion of a GST enzyme. The isolated
nucleic acid fragment is selected from the group consisting of (a) an isolated
nucleic acid fragment encoding all or a substantial portion of the amino acid
sequence selected from the group consisting of SEQ ID N0:2, SEQ ID N0:4,
SEQ ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID N0:14,
SEQ ID N0:16, SEQ ID N0:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID
N0:24, SEQ ID N0:26 and SEQ ID N0:28; (b) an isolated nucleic acid fragment
that is substantially similar to an isolated nucleic acid fragment encoding
all or a
substantial portion of the amino acid sequence sequence selected from the
group
consisting of SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8, SEQ
ID NO:10, SEQ ID N0:12, SEQ ID N0:14, SEQ ID N0:16, SEQ ID N0:18,
SEQ ID N0:20, SEQ ID N0:22, SEQ ID N0:24, SEQ ID N0:26, and SEQ ID
N0:28; and (c) an isolated nucleic acid fragment that is complementary to (a)
or
(b). The nucleic acid fragments and corresponding polypeptides are contained
in
the accompanying Sequence Listing and described in the Brief Description of
the
Invention.
In another embodiment, the instant invention relates to chimeric genes
encoding soybean GST enzymes or to chimeric genes that comprise nucleic acid
fragments as described above, the chimeric genes operably linked to suitable
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regulatory sequences, wherein expression of the chimeric genes results in
altered
levels of the encoded enzymes in transformed host cells.
The present invention further provides a transformed host cell comprising
the above described chimeric gene. The transformed host cells can be of
eukan~otic or prokaryotic origin. The invention also includes transformed
plants
that arise from transformed host cells of higher plants, and from seeds
derived
from such transformed plants, and subsequent progeny.
Additionally, the invention provides methods of altering the level of
expression of a soybean GST enzyme in a host cell comprising the steps of;
(i) transforming a host cell with the above described chimeric gene and;
(ii) growing the transformed host cell produced in step (i) under conditions
that
are suitable for expression of the chimeric gene wherein expression of the
chimeric gene results in production of altered levels of a plant GST enzyme in
the
transformed host cell relative to expression levels of an untransformed host
cell:
In an alternate embodiment, the present invention provides methods of
obtaining a nucleic acid fragment encoding all or substantially all of the
amino
acid sequence encoding a soybean GST enzyme comprising either hybridization
or primer-directed amplification methods known in the art and using the above
described nucleic acid fragment. A primer-amplification-based method uses SEQ
ID NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. The product of
these
methods is also part of the invention.
Another embodiment of the invention includes a method for identifying a
compound that inhibits the activity of a soybean GST enzyme encoded by the
nucleic acid fragment and substantially similar and complementary nucleic acid
fragments of SEQ ID NOS.: 1-28. The method has the steps: (a) transforming a
host cell with the above described chimeric gene; (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 GST
enzyme;
(c) optionally purifying the GST enzyme expressed by the transformed host
cell;
(d) contacting the GST enzyme with a chemical compound of interest; and
(e) identifying the chemical compound of interest that reduces the activity of
the
soybean GST enzyme relative to the activity of the soybean GST enzyme in the
absence of the chemical compound of interest.
This method may further include conducting step (d) in the presence of at
least one electrophilic substrate and at least one thiol donor. The isolated
nucleic
acid fragments of this method are chosen from the group represented by SEQ ID
NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27, and the soybean
GST
enzyme is selected from the group consisting of SEQ ID NOS.: 2, 4, 6, 8,10,
12,
14, 16, 18, 20, 22, 24, 26 and 28.
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The invention fiurther provides a method for identifying a chemical
compound that inhibits the activity of the soybean GST enzyme as described
herein, wherein the identification is based on a comparison of the phenotype
of a
plant transformed with the above described chimeric gene contacted with the
inhibitor candidate with the phenotype of a transformed plant that is not
contacted
with the inhibitor candidate. The isolated nucleic acid fragment of this
method is
selected from the group consisting of SEQ ID NOS.: l, 3, 5, 7, 9, 11, 13, 15,
17,
19, 21, 23, 25, 25, and 27 and the soybean GST enzyme is selected from the
group
consisting of SEQ ID NOS.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and
28.
In another embodiment, the invention provides a method for identifying a
substrate for the soybean GST enzyme. The method comprises the steps of:
(a) transforming a host cell with a chimeric gene comprising the nucleic acid
fragment as described herein, the chimeric gene encoding a soybean GST enzyme
operably linked to at least one suitable regulatory sequence; (b) growing the
transformed host cell of step (a) under conditions that are suitable for
expression
of the chimeric gene resulting in production of the GST enzyme; (c) optionally
purifying the GST enzyme expressed by the transformed host cell; (d)
contacting
the GST enzyme with a substrate candidate; and (e) comparing the activity of
soybean GST enzyme with the activity of soybean GST enzyme that has been
contacted with the substrate candidate and selecting substrate candidates that
increase the activity of the sobyean GST enzyme relative to the activity of
soybean GST enzyme in the absence of the substrate candidate. More preferably,
step (d) of this method is carried out in the presence of at least one thiol
donor.
The isolated nucleic acid fragment of this method is selected from the group
consisting of SEQ ID NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and
27
and the soybean GST enzyme is selected from the group consisting of SEQ ID
NOS.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.
Alternatively, methods are provided for identifying a soybean GST
substrate candidate wherein the identification of the substrate candidate is
based
on a comparison of the phenotype of a host cell transformed with a chimeric
gene
expressing a soybean GST enzyme and contacted with a substrate candidate with
the phenotype of a similarly transformed host cell grown without contact with
a
substrate candidate.
The isolated nucleic acid fragment of this method is selected from the
group consisting of SEQ ID NOS.: l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, and
27 and the soybean GST enzyme is selected from the group consisting of SEQ ID
NOS.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.
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BRIEF DESCRIPTION OF SEQUENCE DESCRIPTIONS
AND BIOLOGICAL DEPOSITS
The invention can be more fully understood from the following detailed
description and the accompanying sequence descriptions and biological deposits
which form a part of this application.
The following sequence descriptions and sequences listings 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. ~1.821-1.825. The
Sequence Descriptions contain the dne letter code~for nucleotide sequence
characters and the three letter codes for amino acids as defined in conformity
with
the IUPAC-IYUB standards described in Nucleic Acids Research 13:3021-3030
(1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are
herein incorporated by reference.
SEQ ID NO: I is the nucleotide sequence comprising the cDNA insert in
I5 clone seI.27b04 encoding a soybean type I GST.
SEQ ID N0:2 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone se1.27b04.
SEQ ID N0:3 is the nucleotide sequence comprising the cDNA insert in
clone ssm.pk0026.g11 encoding a soybean type II GST.
SEQ ID N0:4 is the deduced amino acid sequence of the r.~~cleotide
sequence comprising the cDNA insert in clone ssm.pk0026.g11.
SEQ ID NO:S is the nucleotide sequence comprising the cDNA insert in
clone GSTa encoding a soybean type III GST.
SEQ ID N0:6 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone GSTa.
SEQ ID N0:7 is the nucleotide sequence comprising the cDNA insert in
clone se3.03b09 encoding a soybean type III GST.
SEQ ID N0:8 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone se3.03b09.
SEQ ID N0:9 is the nucleotide sequence comprising the cDNA insert in
clone se6.pk0037.h4 encoding a soybean type III GST.
SEQ ID NO:10 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone se6.pk0037.h4.
SEQ ID NO:11 is the nucleotide sequence comprising the cDNA insert in
clone se6.pk0048.d7 encoding a soybean type III GST.
SEQ ID N0:12 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone se6.pk0048.d7.
SEQ ID N0:13 is the nucleotide sequence comprising the cDNA insert in
clone ses8w.pk0028.c6 encoding a soybean type III GST.
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SEQ ID N0:14 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ses8w.pk0028.c6.
SEQ ID NO:15 is the nucleotide sequence comprising the cDNA insert in
clone srl.pk0011.d6 encoding a soybean type III GST.
SEQ ID N0:16 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone srl.pk0011.d6.
SEQ ID N0:17 is the nucleotide sequence comprising the cDNA insert in
clone ssl.pk0002.f7 encoding a soybean type III GST.
SEQ ID N0:18 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ssl.pk0002.f7.
SEQ ID N0:19 is the nucleotide sequence comprising the cDNA insert in
clone ssl.pk0005.e6 encoding a soybean type III GST.
SEQ ID N0:20 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ssl.pk0005.e6.
SEQ ID N0:21 is the nucleotide sequence comprising the cDNA insert in
clone ssl.pk0014.a1 encoding a soybean type III GST.
SEQ ID N0:22 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ssl.pk0014.a1.
SEQ ID N0:23 is the nucleotide sequence comprising the cDNA insert in
clone ssl.pk0020.b10 encoding a soybean type III GST.
SEQ ID N0:24 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ssl.pk0020.b10.
SEQ ID N0:25 is the nucleotide sequence comprising the cDNA insert in
clone ssm.pk0067.g5 encoding a soybean type III GST.
SEQ ID N0:26 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone ssm.pk0067.g5.
SEQ ID N0:27 is the nucleotide sequence comprising the cDNA insert in
clone se 1.pk0017.f5 encoding a soybean type IV GST.
SEQ ID N0:28 is the deduced amino acid sequence of the nucleotide
sequence comprising the cDNA insert in clone sel.pk0017.f5.
The transformed E. coli srl.pk0011.d6/pET30(LIC)BL21(DE3)
comprising the E. toll host BL21(DE3), containing the gene srl.pk0011.d6 in a
pET30(LIC) vector encoding a soybean type III GST was deposited on
21 August 1997 with the American Type Culture Collection (ATCC),
12301 Parklawn Drive, Rockville, MD 20852 U.S.A. under the terms of the
Budapest Treaty on the International Recognition of the Deposit of Micro-
organisms for the Purpose of Patent Procedure. The deposit is designated as
ATCC 98512.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel GST nucleotide sequences and
encoded proteins isolated from soybean. GST enzymes are known to function in
the process of detoxification of a variety of xenobiotic compounds in plants,
most notably, herbicides. Nucleic acid fragments encoding at least a portion
of '
several soybean GST enzymes have been isolated and identified by comparison
of random plant cDNA sequences to public databases containing nucleotide and
protein sequences using the BLAST algorithms well known to those skilled in
the art. The sequences of the present invention are useful in the construction
of
herbicide-tolerant transgenic plants, in the recombinant production of GST
enzymes, in the development of screening assays to identify compounds
inhibitory
to the GST enzymes, and in screening assays to identify chemical substrates of
the
GSTs.
In the context of this disclosure, a number of terms shall be utilized.
"Glutathione S-Transferase" or "GST" refers to any plant-derived
glutathione S-transferase (GST) enzyme capable of catalyzing the conjugation
of
- glutathione, homoglutathione and other glutathione-like analogs via a
sulfhydryl
group to hydrophobic and electrophilic compounds. The term "GST" includes
amino acid sequences longer or shorter than the length of natural GSTs, such
as
functional hybrid or partial fragments of GSTs, or their analogues. "GST" is
not
intended to be limited in scope on the basis of enzyme activity and may
encompass amino acid sequences that possess no measurable enzyme activity but
are substantially similar to those sequences known in the art to possess the
above-
mentioned glutathione conjugating activity.
The term "class" or "GST class" refers to a grouping of the various GST
enzymes according to amino acid identity. Currently, four classes have been
identified and are referred to as "GST class I" "GST class II", "GST class
III"
and "GST class IV". The grouping of plant GSTs into three classes is described
by Droog et al. (Plant Physiology 107:1139-1146 (1995)). All available amino
acid sequences were aligned using the Wisconsin Genetics Computer Group
package (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),
Madison, WI), and graphically represented on a phylogenetic tree. Three groups
were identified: class one including the archetypical sequences from maize GST
I
(X06755) and GST III (X04375); class two including the archetypical sequence
from Dianthus caryophyllus (M64628); and class three including the
archetypical
sequence soybean GH2/4 (M20363). Recently, Applicants have established a
further subgroup of the plant GSTs known as class IV GSTs with its
archetypical
sequence being In2-1 (X58573):
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As used herein, an "isolated nucleic acid fragment" is a polymer. of RNA
or DNA that is single- or double-stranded, optionally containing synthetic,
non-
natural or altered nucleotide bases. An isolated nucleic acid fragment in the
forni
of a polymer of DNA may be comprised of one or more segments of cDNA,
genomic DNA or synthetic DNA.
As used herein, "substantially 'similar" refers to nucleic acid fragments
wherein changes in one or more nucleotide bases result in substitution of one
or
more amino acids, but do not affect the functional properties of the protein
encoded by the DNA sequence. "Substantially similar" also refers to nucleic
acid
fragments wherein changes in one or more nucleotide bases do not affect the
ability of the nucleic acid fragment to mediate alteration of gene expression
by
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 nucleotide bases that do not
substantially
affect the functional properties of the resulting transcript vis-a-vis the
ability to
mediate alteration of gene expression by antisense or co-suppression
technology
or alteration of the functional properties of the resulting protein molecule.
It is
therefore understood that the invention encompasses more than the specific
exemplary sequences.
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 that the entire coding region of a gene, and by nucleic acid
fragments that do not share 100% identity with the gene to be suppressed.
Moreover, alterations in a gene 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 protein, 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 protein molecule would also not be expected to alter the
activity of
the protein. 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. Moreover, the skilled artisan recognizes that substantially similar
sequences encompassed by this 'invention are also defined by their ability to
hybridize, under stringent conditions (O.1X SSC, 0.1% SDS, 65 °C), with
the
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sequences exemplified herein. Preferred substantially similar nucleic acid
fragments of the instant invention are those nucleic acid fragments whose
DN.~1
sequences are at least 80°~o identical to the DNA sequence of the
nucleic acid
fragments reported herein. More preferred nucleic acid fragments are at least
90%
identical to the identical to the DNA sequence of the nucleic acid fragments
reported herein. Most preferred are nucleic acid fragments that are at least
95%
identical to the DNA sequence of the nucleic acid fragments reported herein.
A "substantial portion" of an amino acid or nucleotide sequence
comprising enough of the amino acid sequence of a polypeptide or the
nucleotide
sequence of a gene to putatively identify that polypeptide or gene, either by
manual evaluation of the sequence by one skilled in the art, or by computer-
automated sequence comparison and identification using algorithms such as
BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J.
Mol.
Biol. 215:403-410; see also www.ncbi.nim.nih.gov/BLAST~. In general, a
sequence of ten or more contiguous amino acids or thirty or more 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 20-30 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
12-15 bases may be used as amplifcation primers in PCR in order to obtain a
particular nucleic acid fragment comprising the primers. Accordingly, a
"substantial portion" of a nucleotide sequence comprises enough of the
sequence
to specifically identify and/or isolate a nucleic acid fragment comprising the
sequence. The instant specification teaches partial or complete amino acid and
nucleotide sequences encoding one or more particular fungal 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.
The term "complementary" is used to describe the relationship between
nucleotide bases that are capable to hybridizing to one another. For example,
with
respect to DNA, adenosine is complementary to thymine and cytosine is
complementary to guanine. Accordingly, the instant invention also includes
isolated nucleic acid fragments that are complementary to the complete
sequences
as reported in the accompanying Sequence Listing as well as those
substantially
similar nucleic acid sequences.
CA 02340791 2001-02-22
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"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 that encodes all or a substantial portion of the amino acid
sequence
encoding the GST enzymes as set forth in SEQ ID Nos: SEQ ID N0:2, SEQ ID
N0:4, SEQ ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID
N0:14, SEQ ID N0:16, SEQ ID N0:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID
N0:24, SEQ ID N0:26, and SEQ ID N0:28. 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 gene for improved
expression in a host cell, it is desirable to design the gene such that its
frequency
of codon usage approaches the frequency of preferred codon usage of the host
cell.
"Synthetic genes".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 gene segments which are
then enzymatically assembled to construct the entire gene. "Chemically
synthesized", as related to a sequence of DNA, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of DNA 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 genes 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.
"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 to 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
11
CA 02340791 2001-02-22
WO 00118936 PCTIUS98120501
genes. A "transgene" is a gene that has been introduced into the genome by a
transformation procedure.
"Coding sequence" refers to a DNA sequence that codes for a specific
amino acid sequence. "Suitable 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.
IO "Promoter" refers to a DNA 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 DNA 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 DNA 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 gene 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, ( I 989) Biochemistry of Plants I5:1-82. It is
further
recognized that since in most cases the exact boundaries of regulatory
sequences
have not been completely defined, DNA fragments of different lengths may have
identical promoter activity.
The "translation leader sequence" refers to a DNA 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,
R. and Foster, G.D. (1995) Molecular Biotechnology 3:225).
The "3' non-coding sequences" refer to DNA sequences located
downstream of a coding sequence and include polyadenylation recognition
sequences and other sequences encoding regulatory signals capable of affecting
12
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mRNA processing or gene expression. The polyadenylation signal is usually
characterized by affecting the addition of polyadenyiic 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).
"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 protein by the cell. "cDNA" refers to a double-stranded DNA
that is complementary to and derived from mRNA. "Sense" RNA refers to RNA
transcript that includes the mRNA and so can be translated into protein by the
cell.
"Antisense RNA" refers to a 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 (LJ.S. Patent No. 5,107,065). The complementarity of an antisense RNA
may be with any part of the specific gene transcript, i.e., at the 5' non-
coding
sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional
RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not
translated yet has an effect on cellular processes.
The term "operably linked" refers to the association of nucleic acid
sequences 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.
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. Patent No. 5,231,020).
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WO 00/18936 PCT/US98/20501
''Altered levels'' refers to the production of gene products) in transgenic
organisms in amounts or proportions that differ from that of normal or non-
transformed organisms.
"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.
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, J.J., (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).
"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. Enrymol.
143:277) and particle-accelerated or "gene gun" transformation technology
(Klein
et al. (1987} Nature (London) 327:70-73; U.S. Patent No. 4,945,050).
The term "herbicide-tolerant plant" as used herein is defined as a plant that
survives and preferably grows normally at a usually effective dose of a
herbicide.
Herbicide tolerance in plants according to the present invention refers to
detoxification mechanisms in a plant, although the herbicide binding or target
site
is still sensitive.
"Thiol donor" refers to a compound that contains the structure RSH (where
R is not equal to H). Within the context of the present invention suitable
thiol
donors may include, but are not limited to, Glutathione and homoglutathione.
"Electrophilic substrate" refers to a compound that is amenable to
conjugation with glutathione or homoglutathione via a sulfhydryl group.
Electrophilic substrates include a wide variety of compounds including
pesticides,
anti-pathogenic compounds such as fungicides and profungicides, pheraxnones,
14
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WO 00/18936 PCT/US98/20501
and herbicides. Within the context of the present invention electrophiiic
substrates with herbicidal activity may include, but are not limited to,
chlorimuronethyl, alachlor, and atrazine, 1-chloro-2,4-dinitrobenzene (CDNB),
ethacrynic acid, t-stilbene oxide, and 1,2-epoxy-3-(p-nitrophenoxy)propane.
Standard recombinant DNA and molecular cloning techniques used herein
are well known in the art and are described more fully in Sambrook, J.,
Fritsch,
E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring
Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis").
The nucleic acid fragments of the instant invention may be used to isolate
cDNAs and genes encoding homologous enzymes 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).
For example, genes encoding other GST enzymes, either as cDNAs or
genomic DNAs, could be isolated directly by using all or a portion of the
instant
nucleic acid fragments as DNA hybridization probes to screen libraries from
any
desired plant using 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 primers 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 of or foil-length 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.
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 poiyadenylic 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} PNAS USA 85:8998) to
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
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) PNAS USA 86:5673; Loh et al., (1989)
Science 23:217). Products generated by the 3' and 5' RACE procedures can be
combined to generate full-length cDNAs (Frohman, M.A. and Martin, G.R.,
(1989) Techniques 1:165).
Availability of the instant nucleotide and deduced amino acid sequences
facilitates immunological screening 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, R.A. (1984) Adv. Immunol. 36:1; Maniatis).
The nucleic acid fragments of the instant invention may be used to create
transgenic plants in which the disclosed GST enzymes are present at higher or
lower levels than normal or in cell types or developmental stages in which
they
are not normally found. This would have the effect of altering the level of
GST
enzyme available as well as the herbicide-tolerant phenotype of the plant.
Overexpression of the GST enzymes of the instant .invention may be
accomplished by first constructing chimeric genes in which the coding region
are
operably linked to promoters capable of directing expression of a gene in the
desired tissues at the desired stage of development. For reasons of
convenience,
the chimeric genes may comprise promoter sequences and translation leader
sequences derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals must also be provided. The instant chimeric
genes may also comprise one or more introns in order to facilitate gene
expression.
Any combination of any promoter and any terminator capable of inducing
expression of a GST coding region may be used in the chimeric genetic
sequence.
Some suitable examples of promoters and terminators include those from
nopaline
synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV)
genes. One type of efficient plant promoter that may be used is a high level
plant
promoter. Such promoters, in operable linkage with the genetic sequence for
GST, should be capable of promoting expression of the GST such that the
transformed plant is tolerant to an herbicide due to the presence of, or
increased
levels of, GST enzymatic activity. High level plant promoters that may be used
in
16
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WO 00/18936 PCT/US98/20501
this invention include the promoter of the small subunit (ss) of the ribulose-
I,~-
bisphosphate carboxylase from example from soybean (Berry-Lowe et al.,
J. Molecular and App. Gen., I :483-498 1982)), and the promoter of the
chlorophyll a/b binding protein. These two promoters are known to be light-
s induced in plant cells (See, for example, Genetic En inky eering,,of Plants,
an
A~~ricuitural Perspective, A. Cashmore, Plenum, New York (1983), pages 29-38;
Coruzzi, G. et al., The Journal ofBiological Chemistry, 258:1399 (1983), and
Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:285 (1983)).
Plasmid vectors comprising the instant chimeric genes can then
constructed. The choice of plasmid vector depends 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 blots (Southern, J. Mol. Biol. 98, 503, (1975)).
Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl.,
618 (1-2) (1993) 133-145), Western analysis of protein expression, or
phenotypic
analysis.
For some applications it will be useful to direct the instant GST enzymes
to different cellular compartments or to facilitate enzyme secretion from a
recombinant host cell. It is thus envisioned that the chimeric genes described
above may be further supplemented by altering the coding sequences to encode
enzymes with appropriate intracellular targeting sequences such as transit
sequences (Keegstra, K., Cell 56:247-253 (1989)), signal sequences or
sequences
encoding endoplasmic reticulum localization (Chrispeels, J.J., Ann. Rev. Plant
Phys. Plant Mol. Biol. 42:21-53 (1991)), or nuclear localization signals
(Raikhel,
N. Plant Phys.100:1627-1632 (1992)) 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 that are useful in the invention.
It may also be desirable to reduce or eliminate expression of the genes
encoding the instant GST enzymes in plants. In order to accomplish this,
chimeric
genes designed for co-suppression of the instant GST enzymes can be
constructed
by linking the genes or gene fragments encoding the enzymes to plant promoter
sequences. Alternatively, chimeric genes designed to express antisense RNA for
17
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WO 00/18936 PCT/US98/20501
all or part of the instant nucleic acid fragments can be constructed by
linking the
genes or gene fragment in reverse orientation to plant promoter sequences.
Either
the co-suppression or antisense chimeric genes could be introduced into
piaTits via
transformation wherein expression of the corresponding endogenous genes are
reduced or eliminated.
Plants transformed with the present GST genes will have a variety of
phenotypes corresponding to the various properties conveyed by the GST class
of
proteins. Glutathione conjugation catalyzed by GSTs are known to result in
sequestration and detoxification of a number of herbicides and other
xenobiotics
(Marrs et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:17-58 (1996)) and
thus will be expected to produce transgenic plants with this phenotype. Other
GST proteins are known to be induced by various environmental stresses such as
salt stress (Roxas, et al., Stress tolerance in trans~enic seedlings that
overexpress
glutathione S-transferase, Annual Meeting of the American Society of Plant
IS Physiologists, (August 1997), abstract 1574, Final Program, Plant Biology
and
Supplement to Plant Physiology, 301), exposure to ozone (Sharma et al., Plant
Physiology, 105 (4) (1994) 1089-1096), and exposure to industrial pollutants
such
as sulfur dioxide (Navari-Izzo et al., Plant Science 96 (1-2) (1994) 31-40).
It is
contemplated that transgenic plants, tolerant to a wide variety of stresses,
may be
produced by the present method by expressing foreign GST genes in suitable
plant
hosts.
The instant GST enzymes produced in heterologous host cells, particularly
in the cells of microbial hosts, can be used to prepare antibodies to the
enzymes by
methods well known to those skilled in the art. The antibodies are useful for
detecting the enzymes in situ in cells or in vitro in cell extracts. Preferred
heterologous host cells for production of the instant GST enzymes 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 chimeric
genes for
production of the instant GST enzymes. These chimeric genes could then be
introduced into appropriate microorganisms via transformation to provide high
level expression of the enzymes.
Vectors or cassettes useful for the transformation of suitable host cells are
well known in the art. Typically, the vector or cassette contains sequences
directing transcription and translation of the relevant gene, a selectable
marker,
and sequences allowing autonomous replication or chromosomal integration.
Suitable vectors comprise a region S' of the gene which harbors
transcriptional
initiation controls and a region 3' of the DNA fragment which controls
transcriptional termination. It is most preferred when both control regions
are
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WO 00/18936 PCT/US98/ZO501
derived from genes homologous to the transformed host cell, although it is to
be
understood that such control regions need not be derived from the genes native
to
the specific species chosen as a production host.
Initiation control regions or.promoters, which are useful to drive
S expression of the genes encoding the GST enzymes in the desired host cell,
are
numerous and familiar to those skilled in the art. Virtually any promoter
capable
of driving these genes is suitable. for the present invention including but
not
limited to CYC1, HIS3, GALL, GAL10, ADH1, PGK, PHOS, GAPDH, ADC1,
TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1
(useful for expression in Pich:a); and lac, trp, ~.PL, ~,PR, T7, tac, and trc
(useful
for expression in E. coli).
Termination control regions may also be derived from various genes native
to the preferred hosts. Optionally, a termination site may be unnecessary,
however, it is most preferred if included.
An example of a vector for high level expression of the instant GST
enzymes in a bacterial host is provided (Example 5).
Additionally, the instant soybean GST enzymes can be used as a targets to
facilitate design and/or identification of inhibitors of the enzymes that may
be
useful as herbicides or herbicide synergists. This is desirable because the
enzymes
described herein catalyze the sulflrydryl conjugation of giutathione to
compounds
toxic to the plant. Conjugation can result in detoxification of these
compounds. It
is likely that inhibition of the detoxification process will result in
inhibition of
plant growth or plant death. Thus, the instant soybean GST enzymes could be
appropriate for new herbicide or herbicide synergist discovery and design.
All or a 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 expression of the instant
enzymes.
Such information may be useful in plant breeding in order to develop lines
with
desired phenotypes or in the identification of mutants.
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 at., Genomics 1:174-181 (1987)) 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
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WO 00/18936 PCT/US98/20SU1
position of the instant nucleic acid sequence in the genetic map previously
obtained using this population (Botstein et al., (1980) Am. J. Hum. Genet.
3:314-331).
The production and use of plant gene-derived probes for use in genetic
mapping are described by Bernatzky, R. and Tanksley, S.D. (Plant Mol. Biol.
Reporter 4(1):37-41 (1986)). 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 t~ those skilled in the art.
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, pp. 319-346 ( I 996), and references cited therein).
In another embodiment, nucleic acid probes derived from the instant
nucleic acid sequences may be used in direct fluorescence in situ
hybridization
(FISH) mapping. Although current methods of FISH mapping favor use of large
clones (several to several hundred KB), improvements in sensitivity may allow
performance of FISH mapping using shorter probes.
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, polymorphism of PCR-amplified
fragments (CAPS), allele-specific ligation, nucleotide extension reactions,
Radiation Hybrid Mapping and Happy Mapping: , 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, this is generally not necessary
for
mapping methods. Such information may be useful in plant breeding in order to
develop lines with desired starch phenotypes.
EXAMPLES
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
CA 02340791 2001-02-22
WO 00/18936 PCTNS98/20501
and scope thereof, can make various changes and modifications of the invention
to
adapt it to various usages and conditions.
GENERAL METHODS
Standard recombinant DNA and molecular cloning techniques used in the
Examples are well known in the art and are described by Sambrook, J., Fritsch,
E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring
Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J.
Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. ( 1984) and by Ausubel, F.
M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing
Assoc. and Wiley-Interscience (1987).
EXAMPLE 1
Composition of cDNA Libraries' Isolation and Sequencing of cDNA Clones
cDNA libraries representing mRNAs from various soybean tissues were
prepared. The characteristics of the libraries are described in Table 1.
TABLE 1
cDNA Libraries From Soybean Tissues
GST
LibraryClass Clone Tissue
sel I se1.27b04 Soybean embryo,
ssm II ssm.pk0026.g11soybean shoot meristem
NA III GSTa NA
sea III se3.03b09 Soybean embryo,
se6 III se6.pk0037.h4 Soybean embryo,
se6 III se6.pk0048.d7 Soybean embryo,
ses8w III ses8w.pk0028.c6mature embryo 8 weeks after
subculture
srl III srl.pk0011.d6 Soybean root library.
ssl III ssl.pk0002.f7 soybean seedling 5-10 day
ssl III ssl.pk0005.e6 soybean seedling 5-10 day
ssl III ssl.pk0014.a1 soybean seedling 5-10 day
ssl III ssLpk0020.b10 soybean seedling 5-IO day
ssm III ssm.pk0067.g5 soybean shoot meristem
sel IV sel.pk0017.f5 Soybean embryo,
cDNA Library Preparation
For clones other than GSTa, cDNA libraries were prepared in
Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, CA): The Uni-ZAPTM XR libraries were converted
into plasmid libraries according to the protocol provided by Stratagene. Upon
21
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WO OOJ18936 PCT/US98/20501
conversion, cDNA inserts were contained in the plasmid vector pBluescript.
cDIvTA inserts from randomly picked bacterial colonies containing recombinant
pBluescript plasmids were amplified via polymerase chain reaction using
primers
specific for vector sequences flanking the inserted cDNA sequences. Amplified
inserE DNAs were sequenced in dye-primer sequencing reactions to generate
partial cDNA sequences (expressed sequence tags or ''ESTs"; see Adams, M. D.
et
al., Science 25?:16~ 1 ( 1991 )). The resulting ESTs were analyzed using a
Perkin
Elmer Model 377 fluorescent sequencer.
Cloning of GSTa
The GSTa clone was isolated and cloned using primers derived from a
published GST sequence, GH2/4 (Flurry et al., Physiologia Plantarum 94 (1995)
594-604) according to the following protocol.
Soybeans (cv Williams 82) were germinated in vermiculite in a
controlled growth room at 23 °C with 14-h light110-h dark cycle at 330
~tE
m-2 s-1. One week old seedlings were treated with I mM 2,4-D for 24 h before
harvest. Seedlings were frozen in liquid nitrogen and ground with a mortar and
pestle and RNA was prepared using TriZol reagent (Life Technologies
Bethesda, MD). Approximately 1.5 ~tg of total RNA was reverse transcribed
using the GeneAmp Kit (Perkin Eliner, Branchburg, NJ) and oligo dT primer.
The resulting first strand cDNA was used as a template for PCR amplification
with AmpliTaq (Perkin Elmer) and the following primers: primer 1: (GAY
GAR GAN CTN CTN GAY TTY TGG) (SEQ ID N0:29) and primer 2: (GAC
TCG AGT CGA CAT GCT T16) (SEQ ID N0:30). Primer 1 and primer 3 (see
below) were designed based on N-terminal protein sequence previously
described (Flury et al., 1995, supra). A Perkin-Ehner Thermal Cycle was
allowed to cycle at 95 °C for 30 sec, 52 °C for 30 sec and 72
°C for 30 sec for
cycles. The resulting PCR product was cloned in pCR2.1 (Invitrogen, San
Diego, CA) according to the manufacturer's instructions, named pBDl6 and
sequenced using an ABI sequencer. Primer 1 was designed to take advantage of
30 the lack of degeneracy for encoding tryptophan. Because of this, the clone
did
not include the entire coding region and a second round of PCR was performed
using the following primers: Primer 3: CAT ATG AGT GAT GAG GTA GTG
TTA TTA GAT TTC TGG (SEQ ID N0:31) and Primer 4: TTA TTA CAC
AAA TAT TAC TTA TTT GAA AGG CTA A (SEQ ID N0:32) and using
.002 ~tg of Iinearized pBDl6 as a template. Again, the resulting PCR product
was cloned into pCR2.1 and named pBDl7 and sequenced using an ABI
sequencer. Additional gene specific primers were made and used to determine
the complete sequence. All regions were sequenced at least two times in both
22
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
directions. The nucleotide sequence and encoded protein sequence are shown in
SEQ ID NO:S and SEQ ID N0:6, respectively.
EXAMPLE 2
Identification and Characterization of cDNA Clones
cDNAs encoding soybean GST enzymes were identified by conducting
BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J.
Mol.
Biol. 215:403-410; see also www.ncbi.nlm.nih.govBLASTn 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 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, W. and States, D. J. (1993) Nature Genetics
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.
All comparisons were done using the BLASTNnr algorithm. The results
of the BLAST comparison is given in Table 2 and summarizes the clones and the
sequences to which they have the most similarity. Each cDNA identified encodes
at least a portion of either a GST Class I, II, III, or IV.
Example 5 describes the strategy for sequencing the above described
clones.
23
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
TABLE 2
BLAST Results For Clones
SEQ
ID
NO.
GST Similarity Blast pLog
Clone ClassIdentified Base PeptideA1 orithmScore
se1.27b04 I X06754jZMGST1 1 2 Nnr 41.35
Maize
mRNA for GSH
gluthathione
S-transferase
I
ssm.pk0026.gi1II ~X58390jDCCARSR83 4 Nnr 85.02
D.caryophyllus
CARSR8
mRNA for glutathione
s-transferase
GSTa III Y10820 j GMGLUTTR5 6 Nnr 257.95
G.max mRNA for
glutathione transferase
se3.03b09 III M20363 j SOYHSP 7 8 Nnr 28.72
Soybean heat-shock
protein (Gmhsp26-A)
gene
se6.pk0037.h4III M20363jSOYHSP 9 IO Nnr 247.44
Soybean heat-shock
protein (Gmhsp26-A)
gene, complete
cds
se6.pk0048.d7III Y10820jGMGLUTTR 11 12 Nnr 0.0
G.max mRNA for
glutathione uansferase
ses8w.pk0028.c6III M20363jSOYHSP 13 14 Nnr 269.17
Soybean heat-shock
protein (Gmhsp26-A)
gene, complete
cds.
srl .pk0011.d6III U20809 j VRU20809I 16 Nnr 229.82
S
Vigna radiata
clone MII-4
auxin-induced
protein
mRNA, partial
cds
ssl.pk0002.III X68819 j GMGLYO 17 18 Nnr 206.01
f7
G.max mRNA for
Glyoxalase I
ssl.pk0005.e6III Y10820jGMGLUTTR 19 20 Xnr 296.05
G.max tnRNA for
glutathione transferase
ssl.pk0014.III M20363 j SOYHSP 21 22 Nnr 166.96
a 1
Soybean heat-shock
protein (Gmhsp26-A)
gene, complete
cds
ssl.pk0020.b10III M20363jS0YHSP 23 24 Nnr 34.76
Soybean heat-shock
protein (Gmhsp26-A)
gene, complete
cds.
24
CA 02340791 2001-02-22
WO 00/18936 PCT/US98,'20501
SEQ ID NO.
GST Similarity Blast pLog
Clone Class Identified Base PeptideAlgorithm Score
ssm.pk0067.g5 III M20363 ~ SOYHSP25 26 Nnr 104.00
Soybean heat-shock
protein (Gmhsp26-A)
gene, complete cds
sel.pk0017.f5 IV ~ X58573 ~ ZMIN2127 28 Nnr 72.04
Maize
In2-1 mRNA
EXAMPLE 3
Expression of Chimeric Genes Encoding Soybean
GST Enzymes in Maize Cells (Monocotyledon)
A chimeric gene comprising a cDNA encoding a soybean GST enzyme in
sense orientation can be constructed by polymerase chain reaction (PCR) of the
cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or
SmaI) can be incorporated into the oligonucleotides to provide proper
orientation
of the DNA fragment when inserted into the digested vector pML 103 as
described
below. Amplification is then performed in a 100 uL volume in a standard PCR
mix consisting of 0.4 mM of each oligonucleotide and 0.3 pM of target DNA in
10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 200 mM dGTP, 200 mM
dATP, 200 mM dTTP, 200 mM dCTP and 0.025 unit DNA polymerase.
Reactions are carried out in a Perkin-Elmer Cetus ThermocyclerT"" for 30
cycles
comprising 1 min at 95 °C, 2 min at 55 °C and 3 min at 72
°C, with a final 7 min
extension at 72 °C after the last cycle. The amplified DNA is then
digested with
restriction enzymes NcoI and SmaI and fractionated on a 0.7% low melting point
agarose gel in 40 mM Tris-acetate, pH 8.5, 1 mM EDTA. The appropriate band
can be excised from the gel, melted at 68 °C 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 with the ATCC and bears
accession number ATCC 97366. The DNA segment from pML103 contains a
1.05 kb SaII-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb
SmaI-SaII fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega Corp., 7113 Benhart Dr., Raleigh, NC). Vector and insert
DNA can be ligated at 1 S °C overnight, essentially as described
(Maniatis). The
ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli
XL-1; Stratagene). Bacterial transformants can be screened by restriction
enzyme
digestion of plasmid DNA and limited nucleotide sequence analysis using the
dideoxy chain termination method (DNA Sequencing Kit, U. S. Biochemical).
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
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 a
plant gst enzyme, and the 10 kD zero 3' region.
The chimeric gene so constructed can then be introduced into corn cells b~~
the following procedure. Immature corn embryos can be dissected from
developing caryopses derived from crosses of the inbred corn lines H99 and
LH132 (Indiana Agric. Exp. Station, Indiana, USA). The embryos are isolated 1
~
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., Sci. Sin. Peking 18:659-668 (1975)). The embryos are
kept in the dark at 27 °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. The
plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, v 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. Nature 313:810-812
(1985)) and the 3M region of the nopaline synthase gene from the T-DNA of the
Ti plasmid of Agrobacterium tumefaciens. The particle bombardment method
(Klein et al., Nature 327:70-73 (1987)) may be used to transfer genes to the
callus
culture cells. According to this method, gold particles (i ~m in diameter) are
coated with DNA using the following technique. Ten ug of plasmid DNAs are
added to 50 ~L of a suspension of gold particles (60 mg per mL). Calcium
chloride (SO uL of a 2.5 M solution) and spermidine free base (20 pL 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 ~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 uL of ethanol. An aliquot (5 uL) of the DNA-coated gold particles
can be placed in the center of a flying disc (Bio-Rad Labs, 861 R.idgeview Dr,
Medina, OH). The particles are then accelerated into the corn tissue with a
PDS-1000/He (Bio-Rad Labs, 861 Ridgeview Dr., Medina, OH), using a helium
pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0
cm.
26
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
For bombardment, the embryogenic tissue is placed on filter paper over
agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covers
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.
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 grov~ring 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: 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., BiolTechnology
8:833-839 (1990)).
EXAMPLE 4
ExQression of Chimeric Genes in Tobacco Cells lDicotvledonl
Cloning sites (XbaI or SmaI) can be incorporated into the oligonucleotides
to provide proper orientation of the DNA fragment when inserted into the
digested
vector pBI121 (Clonetech Inc., 6500 Donlon Rd, Somis, CA) or other appropriate
transformation vector. Amplification could be performed as described above and
the amplified DNA would then be digested with restriction enzymes XbaI and
SmaI and fractionated on a 0.7% low melting point agarose gel in 40 mM Tris-
acetate, pH 8.5, 1 mM EDTA. The appropriate band can be excised from the gel,
melted at 68 °C and combined with a 13 kb XbaI-SmaI fragment of the
plasmid
pBI121 and handled as in Example 3. The resulting plasmid construct would
comprise a chimeric gene encoding, in the 5' to 3' direction, right border
region,
the nos promoter linked to the NPT II gene and a nos terminator region
followed
by a cauliflower mosaic virus 35S promoter linked to a cDNA fragment encoding
a plant GST enzyme and the nos terminator 3' region flanked by the left border
region. The resulting plasmid could be mobilized into the Agrobacterium strain
LBA4404/pAL4404 (Hoekema et al. Nature 303:179-180, (1983) using tri-
parental matings (Ruvkin and Ausubel, Nature 289:85-88, (1981)). The resulting
Agrobacterium strains could be then cocultivated with protoplasts (van den
Elzen
et al. Plant Mol. Biol, 5:149-154 (1985)) or leaf disks (Horsch et al. Science
227:1229-1231, (1985)) of Nicotiana tabacum cv Wisconsin 38 and kanamycin-
27
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
resistant transformants would be selected. Kanamycin-resistant transformed
tobacco plants would be regenerated.
EXAMPLE 5
Expression Of Chimeric Genes In Microbial Cells And
Purification Of Gene Product
Example 5 illustrates the expression of isolated full length genes encoding
class I, II, III or IV GST proteins in E. coli.
All clones listed in Table 2 were selected on the basis of homology to
known GSTs using the BLAST algorithm as described in Example 2. Plasmid
DNA was purified using QIAFilter cartridges (Qiagen. Inc., 9600 De Soto Ave,
Chatsworth, CA) according to the manufacturer's instructions. Sequence was
generated on an ABI Automatic sequencer using dye terminator technology (U.S.
5366860; EP 272007) using a combination of vector and insert-specific primers.
Sequence editing was performed in either DNAStar (DNA, Star Inc.) or the
Wisconsin GCG program (Wisconsin Package Version 9.0, Genetics Computer
Group (GCG), Madison, WI). All sequences represent coverage at least two times
in both directions.
cDNA from full length clones listed in Table 2 encoding the instant
soybean GST enzymes were inserted into the ligation independent cloning (LIC)
pET30 vector (Novagen, Inc., 597 Science Dr, Madison, WI) under the control of
the T7 promoter, according to the manufacturer's instructions (see Novagen
publications "LIC Vector Kits", publication number TBI63 and U.S. 4952496).
The vector was then used to transform BL21 (DE3) competent E. coli hosts.
Primers with a specific 3' extension designed for ligation independent cloning
were designed to amplify the GST gene (Maniatis). Amplification products were
gel-purified and annealed into the LIC vector after treatment with T4 DNA
polymerase (Novagen). Insert-containing vectors were then used to transform
NovaBlue competent E. coli cells and transformants were screened for the
presence of viable inserts. Clones in the correct orientation with respect to
the T7
promoter were transformed into BL2I (DE3) competent cells (Novagen) and
selected on LB agar plates containing 50 pg/mL kanamycin. Colonies arising
from this transformation were grown overnight at 37 °C in Lauria Broth
to OD
600 = 0.6 and induced with 1 mM IPTG and allowed to grow for an additional
two hours. The culture was harvested, resuspended in binding buffer, Iysed
with a
French press and cleared by centrifugation.
Expressed protein was purified using the HIS binding kit (Novagen)
according to the manufacturer's instructions. Purified protein was examined on
15-20% SDS Phast Gels (Bio-Rad Laboratories, 861 Ridgeview Dr, Medina, OH)
28
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
and quantitated spectrophotometrically using BSA as a standard. Protein data
is
tabulated below in Table 3.
TABLE 3
Protein Expression Data
CLONE OD. 280
se1.27b04 0.5
ssm.pk0026.g11 0.44
GSTa 53.6
se3.03b09 29.1
se6.pk0037.h4 0.6
se6.pk0048.d7 1.41
ses8w.pk0028.c6 0.56
srl .pk0011.d6 0.55
ssl.pk0002.f7 0.70
ssl.pk0005.e6 0.51
ssl.pk0014.a1 0.62
ssl.pk0020.b10 1.14
ssm.pk0067.g5 1.64
se l .pk0017.f5 0.37
EXAMPLE 6
Screening Of Expressed GST Enzymes For Substrate Metabolism
The GST enzymes, expressed and purified as described in Example S were
screened for their ability to metabolize a variety of substrates. Substrates
tested
included the three herbicide electrophilic substrates chlorimuron ethyl,
alachlor,
and Atrazine, and four model electrophilic substrates, 1-chloro-2, 4-dinitro-
benzene (CDNB), ethacrynic acid, t-stilbene oxide, and 1,2-epoxy-3-(p-nitro-
phenoxy) propane. The enzymes were purified as described in Example 5 and
used in the following assay.
For each enzyme, the conjugation reaction with each electrophilic
substrate was performed by incubating 0.3 to 30 ~g enzyme in 0.1 M MOPS
(pH 7.0) containing 0.4 mM of the electrophilic substrate. The reaction was
inititated by the addition of glutathione to a final concentration of 4 mM.
After 5
to 30 min, the reaction was terminated by the addition of 45 ~L acetonitrile,
microfuged for 10 min to :emove precipitated protein, and then the supernatent
was removed and added to 65 qL of water. This sample was chromatographed on
a Zorbax C8 reverse phase HPLC column (3 ~m particle size, 6.2 mm x 8 cm)
using a combination of linear gradients (flow = 1.5 mL/min) of 1% H3P04 in
29
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
water (solvent A) and I% H3P04 in acetonitrile. The gradient started with
5°,'°
solvent B, progressing from 5% to 75% solvent B between 1 and 10 min. and
from 75% to 95% solvent B between 10 and I2 min. Control reactions without
enzyme were performed to correct for uncatalyzed reaction. Quantitation of
metabolites were based on an assumption that the extinction coefficient of
th~~
conjugate was identical to that of the electrophilic substrate.
Table 4 shows the activity of each enzyme measured in nmol~min- ~ ~mg- ~
with the seven different substrates. Activities are related to the activity of
a
known and previously isolated and purified GST enzyme, GH2/4 (also called
GST 26) (Czarnecka et al., Plant Molecular Biolog~ 3:45-58 ( 1984); Ulmasoz et
al., Plant Physiol 108:919-927 ( 1995)).
CA 02340791 2001-02-22
WO 00/18936 PCT/US98120501
M
T
.-. M ~ O .-. p~ h N V' O~ O O O N O
M ~ N N
.r _
C
N
WG M it N O ~ N ~O ~D Iw0 I~ O ~ ~n
--a 'b O Oy O ~ O '~t O O O O ~ O O O ~
O I~ c!' O O C O O C O 0 0 0 0 0
H
U
C
~, 'C 00 ~ O~ CT ~T' 00 ~-, ~..~ ~ M
.-. O ~O ~'
w
W
'oT'~f'~ ~ N ~ ~ ~ ~ ~n V1O
V~ U N ~ N N ~"~'d'Ov~ M N ~ ~' r..
~ .r ""' "'_'
W
~ O~ M O ~DO M M M O ~DO ~ ~ p O
t?'O O O O O I'~O
0 0 - 0 0 0 0 0 0 0 0 0 0 0
4~ Q
O
N N
U O
~ O ~-~O V I'~W D 00O 00O O O
> U O .-.~. I~~D M ~O
., y .r .-r ~ ~r
Q Q
O
w.
~ N M .-. r. ..~ V1 ~t ~ O~ ~ ~ O O
~ O O C~ 0 0 0 0 0 ~-~ 0 0 ~' 0 0 0
_O W
U
c
U .... ~.. ~.., ~..r ~ ... .... _ ~.. ~ ~.. ... ..,
~n o
,.O N V "a ~ .~ ~ -~ ~ ~ 4~..
O M ~ ~ O ~ d' N Ov d' N .-.
E-' O ~~G O O O O r,.y O O 0 O O ~ O
C~ O R~ O. O .~G O i O O O ~D -O x O
G1. N 3 C,' C' ø' O, C/~ Q. p" O. ~ N ~ G~.
vD ,'~ ~ .W O ~ ~ ~ M ~~
U s... U v~ vWn N in N 4J N U
31
CA 02340791 2001-02-22
WO 00/18936 PCT/U598/20501
SEQUENCE LISTING
(' ) GENERi~L INFOR~f~.TION:
(i) APPLICANT:
(A) ADDRESSEE: E.I. DU PONT DE NEMOURS AND COMPANY
(B! STREET: 1007 MARKET STREET
(C) CITY: WILMINGTON
(D) STATE: DELAWARE
(E) COUNTRY': UNITED STATES OF AMERICi-
(F) ZIP: 19898
(G) TELEPHONE: 302-892-7229
(H} TELEFAX: 302-773-0154
(I) TELEX: 6717325
(ii) TITLE OF INVENTION: SOYBEAN GLUTATHIONE-S-TRANSFERASE
ENZYMES
(iii) NUMBER OF SEQUENCES: 32
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: DISKETTE, 3.50 INCH
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MICROSOFT WINDOWS 95
(D) SOFTWARE: MICROSOFT WORD VERSION 7.OA
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vi) ATTORNEY/AGENT INFORMATION:
(A) NAME: KAREN K. KING
(B) REGISTRATION NUMBER: 34,850
(C) REFERENCE/DOCKET NUMBER: CL-1108
1
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(2) INFORMATION
FOR SEQ ID
N0:1:
(i) SEQU~IS~E CHARACTERT_STICS:
(A) LENGTH: 886 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(~' TOPOLOGY: linear
(iii MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE1.27B04
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
CAAACACTAC
ACGTGCCATG
ATCTGTCTCC
ATGAGAAAGA
GGTCGATTTT
GAACTTGTTC
60
CGGTCAATGT
GTTCGCTGCT
GAGCACAAGC
AGCCTCCTTT
TCTCTCCAAG
AATCCCTTTG
120
GTTTCATTCC AGTACTGGAA GATGGTGATC TCACTCTTTT TGAGTCCAGG GCCATTACCG 180
CATACGTGGC TGAAAAATTC AAGGAAACAG AACCCGATCT GATAAGGCAC AAGGATGCAA 290
AAGAAGCAGC ACTGGTGAAG GTATGGACAG AGGTAGAGTC TCATTACTAC GAGCCAGCAG 300
TGTCGCCCAT TATCTACGAG TACTTCGTGG CCCCTTTCCA AGGCAAAGAA CCCGACAAGT 360
CAGTGATTGA CACCAACGTT GAGAAGCTGA AGACGGTGCT TGATGTGTAC GAGGCCAAGC 420
TGAGCAGCAC CAAGTACCTT GCTGGGGACT TTTATAGCCT TGCTGATCTT AGCCATGTTT 980
CTGAAACTCA CTACTTGATG CAGACCCCTT GTGCTTCCAT GATCAATGAG CTTCCTCATG 590
TAAAGGCTTG GTGGGAGGAT ATCTCTTCTA GGCCTGCTTT CAATAAGGTT GTGGGAGGAA 600
TGAGTTTTGG TCAGAATCAT TGAGGAATGA GTGTGTTTTG TGAGGTTCAA TTACTACCTA 660
ATTTGTTGCA GTATCTAGTC AAGCAAATGT GGTGTTGGGT GTTCTTGAAA CTTGTTTCAT 720
TTCTTATAAC TAGAATTAAT TAGGAAAACG AATCAATTTT TAGAGGGGTC TTTAAGAAAA 780
AGGACTTTAA TAGTTCCTTT TGTCTTATTT GATTAATTTA AAATTTTATG TTGTAGTGTT 840
TTGATGATAT GTTTTAATAT CCTATTTCAA AAAAAAAAAA AAAAAA 886
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 201 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY.: not relevant
(ii) MOLECULE TYPE: protein
2
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(vi) ORIGINAL SOURCE:
~, ~rlcc~~F 'nvpE: SOYBEAis
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE1.27B09
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ile Cys Leu His Glu Lys Glu Val Asp Phe Glu Leu Val Pro Vai
1 5 10 15
Asn Val Phe Ala Ala Glu His Lys Gln Pro Pro Phe Leu Ser Lys Asn
20 25 30
Pro Phe Gly Phe Ile Pro Val Leu Glu Asp Gly Asp Leu Thr Leu Phe
35 90 45
Glu Ser Arg Ala ile Thr Ala Tyr Val Ala Glu Lys Phe Lys Glu Thr
50 55 60
Glu Pro Asp Leu Ile Arg His Lys Asp Ala Lys Glu Ala Ala Leu Val
65 70 75 80
Lys Val Trp Thr Glu Val Glu Ser His Tyr Tyr Glu Pro Ala Val Ser
85 90 95
Pro Ile Ile Tyr Glu Tyr Phe Val Ala Pro Phe Gln Gly Lys Glu Pro
100 105 110
Asp Lys Ser Val Ile Asp Thr Asn Val Glu Lys Leu Lys Thr Val Leu
115 120 125
Asp Val Tyr Glu Ala Lys Leu Ser Ser Thr Lys Tyr Leu Ala Gly Asp
130 135 140
Phe Tyr Ser Leu Ala Asp Leu Ser His Val Ser Glu Thr His Tyr Leu
145 ?.50 155 160
Met Gln Thr Pro Cys Ala Ser Met Ile Asn Glu Leu Pro His Val Lys
165 170 175
Ala Trp Trp Glu Asp Ile Ser Ser Arg Pro Ala Phe Asn Lys Val Val
180 185 190
Gly Gly Met Ser Phe Gly Gln Asn His
195 200
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1007 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
3
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(iii) HYPOTHETICAL: NO
(iv) AN'~'i-SENSE: uJ
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SSM.PK0026.G11
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
CACGACACTG AGCATCAGCA ATGGCAAGCG CAAGTGTTGG TAAAGAACTG ACGCTGTATT 60
CGTATTGGAG GAGCTCTTGT TCCCACCGAG TCCGAATCGC TCTCAACCTC AAAGGGCTTA 120
AATACGAATA CAAGCCCGTC AATCTGCTCA AGGGAGAACA ATCTCGCCCT GAGTTTCTCC 180
AGCTCAATCC TGTTGGTTGT GTCCCCGTTC TAGTGGATGA CCACGTTGTT CTCTATGACT 290
CTTTCGCCAT TATTATGTAT TTGGAAGATA AGTATCCTCA CAATCCTTTG CTCCCTCATG 300
ATATTTACAA GAGAGCAATC AATTTCCAGG CTGCTAGTGT TGTTTCCTCA ACAATACAAC 360
CTCTTCATAA CTTGAGTTTA CTGAACTACA TTGGGGAGAA AGTTGGCCCT GATGAAAAAC 420
TTCCTTGGGC CCAAAGTATA ATTAGAAGAG GCTTTAAAGC ACTGGAAAAG CTATTGAAAG 980
ACCACACAGG AAGATATGCA ACTGGAGATG AAGTTTTCCT GGCAGATATA TTTTTAGCAC 590
CTCAGTTACA TGCAGCATTT AAGAGATTCA ACATTCACAT GAACGAGTTC CCTATTCTAG 600
CAAGATTGCA TGAGACATAT AATGAGATCC CTGCATTCCA GGAGGCTCTG CCAGAGAACC 660
AGCCTGATGC AGTACACTAG TTGAACCAAT AATTTGGGAC AGAAATATGA GTTGATATTA 720
AGTTGGAGAA ATTGCAGCAG GAGCTACTTA TTCAGCATCC GGATGAATTC GTTGTTAAAG 780
TATTAAAATA TGATACTCAA TATAGCAATA AGGTTGCCAC ATGCAATATT TATTGCACAC 840
ATCATGTACA ATTGAAAAAA F.AAAATTGGT TTCGGGTGTA TGTCTATAAA GCCTTATGTT 900
TATTTTCCAT TTCATATTCT TCCCAGAATC CCAGTCAATG TAGCTTGATG GATGATTCTT 960
AATGGTGTTT ATGGTTGAAT TGGTGTTTCA AAAAAAAAAA AAAAAAA 1007
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
4
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(vii) IMMEDIATE SOURCE:
(B) CLONE: SSM.PIC0026.G11
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Ala Ser Ala Ser Val Gly Lys Glu Leu Thr Leu Tyr Ser Tyr Trp
1 5 10 15
Arg Ser Ser Cys Ser His Arg Val Arg Ile Ala Leu Asn Leu Lys Gly
20 25 30
Leu Lys Tyr Glu Tyr Lys Pro Val Asn Leu Leu Lys Gly Glu Gln Ser
35 90 95
Arg Pro Glu Phe Leu Gln Leu Asn Pro Val Gly Cys Val Pro Val Leu
50 55 60
Val Asp Asp His Val Val Leu Tyr Asp Ser Phe Ala Ile Ile Met Tyr
65 70 75 80
Leu Glu Asp Lys Tyr Pro His Asn Pro Leu Leu Pro His Asp Ile Tyr
85 90 95
Lys Arg Ala Ile Asn Phe Gln Ala Ala Ser Val Val Sex Ser Thr Ile
100 105 110
Gln Pro Leu His Asn Leu Ser Leu Leu Asn Tyr Ile Gly Glu Lys Val
115 120 125
Gly Pro Asp Glu Lys Leu Pro Trp Ala Gln Ser Ile Ile Arg Arg Gly
130 135 190
Phe Lys Ala Leu Glu Lys Leu Leu Lys Asp His Thr Gly Arg Tyr Ala
195 150 155 160
Thr Gly Asp Glu Val Phe Leu Ala Asp Ile Phe Leu Ala Pro Gln Leu
165 170 175
His Ala Ala Phe Lys Arg Phe Asn Ile His Met Asn Glu Phe Pro Ile
180 185 190
Leu Ala Arg Leu His Glu Thr Tyr Asn Glu Ile Pro Ala Phe Gln Glu
195 200 205
Ala Leu Pro Glu Asn Gln Pro Asp Ala Val His
210 215
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 902 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(iii) HYPOT~?ETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: GSTA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GGCTTGACGA GGAAGTGTTA TTAGAGTTCT GGCCAAGTCC ATTTGGGATG AGGGTCAGGA 60
TTGCACTTGC TGAAAAGGGT ATCP:AATATG AGTACAAAGA AGAGGACTTG AGGAACAAGA 120
GTCCTCTTCT CCTCCAAATG AACCCGGTTC ACAAGAAGAT TCCGGTTCTC ATCCACAATG 180
GCAAACCCAT TTGTGAATCC CTCATTGCTG TTCAGTACAT TGAGGAGGTT TGGAATGACA 240
GAAATCCCTT GTTGCCTTCT GACCCTTACC AGAGAGCTCA GACTAGATTC TGGGCTGATT 300
ATGTTGATAA GAAGATATAT GATCTTGGAA GGAAGATTTG GACATCAAAA GGAGAAGAAA 360
AAGAAGCTGC CAAGAAGGAG TTCATAGAAG CCCTTAAATT GTTGGAGGAA CAGCTGGGAG 920
ACAAGACTTA TTTTGGAGGA GACAATCTAG GTTTTGTGGA TATAGCGCTT GTTCCATTCT 480
ACACTTGGTT CAAAGCCTAT GAGACTTTTG GCACCCTCAA CATAGAGAGT GAGTGCCCCA 540
AGTTTATTGC TTGGGCCAAG AGGTGCCTTC AGAAAGAAAG CGTTGCCAAG TCTCTTCCTG 600
ATCAGCAAAA GGTTTATGAG TTCATTATGG ATCTAAGAAA GAAGTTAGGC ATTGAGTAGG 660
TTGGAGCTTA ATGGCCATTG TGAAGTAGTG GTTTTCCATT GGTCGTTCTT AGCCTTTCAA 720
ATAAGTAATA TTTGTGTAAT AAAAGGCACT TAGATGTGCC AAACTTCGTG CTTTCTGTAG 780
GAATGTGTGG GTTTTGGAAA ATCTCTGATG TATCTTTCAT GTGTTTGTTG GTTTTGTAAT 890
TTTTTTTTGG TATTGTCTTA TACTTGAATA ATTTGAGACT AAAAAAAAAA AAAAAAAAAA 900
902
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE T-YPE: SOYBEAN
6
CA 02340791 2001-02-22
WO 00/18936 PCT/US98I20501
(vii) IMMEDIATE SOURCE:
(B) CLONE: GSTA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Ser Asp Glu Val Val Leu Leu Asp Phe Trp Pro Ser Pro Phe Gly
1 5 10 15
Mec Arg Val Arg Ile Ala Leu Ala Glu Lys Gly Ile Lys Tyr Glu Tyr
20 25 30
Lys Glu Glu Asp Leu Arg Asn Lys Ser Pro Leu Leu Leu Gln Met Asn
35 40 45
Pro Val His Lys Lys Ile Pro Val Leu Ile His Asn Gly Lys Pro Ile
50 55 60
Cys Glu Ser Leu Ile Ala Val Gln Tyr Ile Glu Glu Val Trp Asn Asp
65 70 75 BO
Arg Asn Pro Leu Leu Pro Ser Asp Pro Tyr Gln Arg Ala Gln Thr Arg
85 90 95
Phe Trp Ala Asp Tyr Val Asp Lys Lys Ile Tyr Asp Leu Gly Arg Lys
100 105 110
Ile Trp Thr Ser Lys Gly Glu Glu Lys Glu Ala Ala Lys Lys Glu Phe
115 120 125
Ile Glu Ala Leu Lys Leu Leu Glu Glu Gln Leu Gly Asp Lys Thr Tyr
130 135 140
Phe Gly Gly Asp Asn Leu Gly Phe Val Asp Ile Ala Leu Val Pro Phe
145 150 155 160
Tyr Thr Trp Phe Lys Ala Tyr Glu Thr Phe Gly Thr Leu Asn Ile Glu
165 170 175
Xaa Glu Cys Pro Lys Phe Ile Ala Trp Ala Lys Arg Cys Leu Gln Lys
180 185 190
Glu Ser Val Ala Lys Ser Leu Pro Asp Gln Gln Lys Val Tyr Glu Phe
195 200 205
Ile Met Asp Leu Arg Lys Lys Leu Gly Ile Glu
210 215
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 895 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
CA 02340791 2001-02-22
WO 00118936 PCT/US98/20501
(iv) ANTI-SENSE: NO
(vi) ORIGIN~.L SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE3.03B09
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
CACAACTTTG CCCCCTTGTA AAACTTCTTA TTGTGATGTC TAAAAGCGAA GACTTGAAGC 60
TTTTGGGAGG CTGGTTCAGC CCATTTGCCC TGAGGGTGCA GATTGCCCTT AACCTCAAGG 120
GTCTAGAATA TGAGGTTGTT GAAGAGACCT TGAATCCCAA AAGTGACCTG CTTCTTAAGT 180
CCAACCCTGT GCACAAGAAA ATCCCAGTTT TCTTCCATGG AGATAAAGTC ATTTGTGAAT 240
CTGCAATCAT AGTTGAGTAC ATTGATGAGG CTTGGACTAA TGTTCCCTCC ATCCTTCCAC 300
AAAATGCTTA TGATCGTGCT AATGCTCGAT TTTGGTTTGC CTACATTGAT GAGAAGTGGT 360
TTACGTCCTT GAGAAGTGTT CTAGTGGCTG AAGATGATGA GGCAAAGAAG CCACACTTTG 420
AGCAAGCAGA AGAAGGGCTT GAGAGGTTGG AAGAAGTGTT CAACAAGTAC AGTGAAGGGA 980
AGGCCTATTT.CGGAGGAGAT AGCATTGGAT TCATTGACAT TGGTTTTGGG AGCTTCTTGA 590
GTTGGATGAG AGTCATAGAG GAGATGAGTG GAAGAAAATT GCTTGATGAA AAGAAGCACC 600
CTGGTTTGAC CCAATGGGCT GAAACGTTTG CTGCTGATCC TGCTGTGAAG GGCATTCTTC 660
CAGAGACTGA TAAGCTTGTT GAGTTTGCCA AGATTCTTCA GCTAAAATGG ACTGCTGCAG 720
CAGCTGCAGC TGCAAAGTAA ATGGAATCAA ATTAATTGCG AGAGTATTTT CAAAATTGTT 780
GTCCAAGTTG TTTTTATCTC AGGCTATGTT GTTGCAACTT TATTTATTTA AAAGTTATTT 840
TAAATTTAAA ATGTAAAATA
TTAAGAAAGT TTAAGTAAGT
TAGTTGAAAA ATTTT 895
(2) INFORMATION FOR SEQ
ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE3.03B09
g
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Ser Lys Ser Glu Asp Leu Lys Leu Leu Gly Gly Trp Phe Ser Pro
1 5 10 15
Phe Ala Leu Arg Val Gln Ile Ala Leu Asn Leu Lys Gly Leu Glu Tyr
20 25 30
Glu Val Val Glu Glu Thr Leu Asn Pro Lys Ser Asp Leu Leu Leu Lys
35 90 45
Ser Asn Pro Val His Lys Lys Ile Pro Val Phe Phe His Gly Asp Lys
50 55 60
Val Ile Cys Glu Ser Ala Ile Ile Val Glu Tyr Ile Asp Glu Ala Trp
65 70 75 80
Thr Asn Val Pro Ser Ile Leu Pro Gln Asn Ala Tyr Asp Arg Ala Asn
B5 90 95
Ala Arg Phe Trp Phe Ala Tyr Ile Asp Glu Lys Trp Phe Thr Ser Leu
100 105 110
Arg Ser Val Leu Val Ala Glu Asp Asp Glu Ala Lys Lys Pro His Phe
115 120 125
Glu Gln Ala Glu Glu Gly Leu Glu Arg Leu Glu Glu Val Phe Asn Lys
130 135 140
Tyr Ser Glu Gly Lys Ala Tyr Phe Gly Gly Asp Ser Ile Gly Phe Ile
195 150 155 160
Asp Ile Gly Phe Gly Ser Phe Leu Ser Trp Met Arg Val Ile Glu Glu
165 170 175
Met Ser Gly Arg Lys Leu Leu Asp Glu Lys Lys His Pro Gly Leu Thr
180 185 190
Gln Trp Ala Glu Thr Phe Ala Ala Asp Pro Ala Val Lys Gly Ile Leu
195 200 205
Pro Glu Thr Asp Lys Leu Val Glu Phe Ala Lys Ile Leu Gln Leu Lys
210 215 220
Trp Thr Ala Ala Ala Ala Ala Ala Ala Lys
225 230
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 931 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
9
' CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE6.PK0037.H9
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CTGCAGGTAG TTTTTCTGTT TGAAGTGCTA CAAACAATGG CAGCTACTCA GGAAGATGTG 60
ACGCTTTTGG GAGTTGTTGG AAGCCCGTTT GTGTGCAGGG TCCAGATTGC CCTCAAATTG 120
AAGGGAATTG AATGCAAATT TTTGGAAGAA AATTTGGCAA ACAAGAGTGA TCTACTTCTC i80
AAATCCAACC CCGTTTACAA GAAGGTTCCA GTGTTTATTC ATAATGAGAA GCCCATAGCA 240
GAGTCTCTTG TGATTGTTGA GTACATTGAT GAGACATGGA AGAACAACCC CATCTTGCCT 300
TCTGATCCTT ACCAAAGATC CTTTGCTCGG TTTTGGTCCA AGTTCATAGA TGACAAGATT 360
GTGGGTGCTT CATGGAAATC TGTTTTCACG GTTGATGAGA AAGAGCGTGA GAAGAATGTT 420
GAAGAATCGT TGGAGGCTCT GCAGTTTCTT GAGAATGAAC TACAGGACAA AAGGTTCTTT 480
GGAGGAGATG AATTTGGATT TGTAGATATT GCTGGTGTCT TCATTGCATT TTCAATCCCA 540
ATTTTCCAAG AAGTAGCAGG GTTGCAATTA TTCACCAGTG AGAAATTTCC TAAGCTCTTC 600
AAATGGAGCC AAGAGTTGAT CAACCACCCT GTTGTCAAAG ATGTCCTTCC TCCTAGAGAA 660
CCACTTTTTG CCTTCTTCAA ATCCCTCTAT GAAAGCCTTT CTGCTTCAAA ATAGATTGTT 720
TAAGAATGAT TGTGTGAACT ACTTGTCGCT CATTGAATTA TTGTTGTTTG AATTTCATGT 780
CAATTTGATA CTATATGTAA TTTAGTAACC TGGGATATTA GGATATCCCC AAGGAACAAA 840
GAATCCTAGG ATTTTGTTTC CATTTTGGCC ATTTCAGTTA ATAATTAAAG AAACTCTATT 900
TTTTCTTGTT AC.~AAAAAAA AAAAAAAAAA A 931
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE6.PK0037.H9
1~
' CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(xi? SEQUENCE DESCRIPTION: SEQ ID N0:10:
Met Ala Ala Thr Gln Glu Asp Val Thr Leu Leu Gly Val Val Gly Ser
1 5 10 15
Pro Phe Val Cys Arg Val Gln Ile Ala Leu Lys Leu Lys Gly Ile Glu
20 25 30
Cys Lys Phe Leu Glu Glu Asn Leu Ala Asn Lys Ser Asp Leu Leu Leu
35 90 95
Lys Ser Asn Pro Val Tyr Lys Lys Val Pro Val Phe Ile His Asn Glu
50 55 60
Lys Pro Ile Ala Glu Ser Leu Val Ile Val Glu Tyr Ile Asp Glu Thr
65 70 ~ 75 80
Trp Lys Asn Asn Pro Ile Leu Pro Ser Asp Pro Tyr Gln Arg Ser Phe
85 90 95
Ala Arg Phe Trp Ser Lys Phe Ile Asp Asp Lys Ile Val Gly Ala Ser
100 105 110
Trp Lys Ser Val Phe Thr Val Asp Glu Lys Glu Arg Glu Lys Asn Val
115 120 125
Glu Glu Ser Leu Glu A1a Leu Gln Phe Leu Glu Asn Glu Leu Gln Asp
130 135 140
Lys Arg Phe Phe Gly Gly Asp Glu Phe Gly Phe Val Asp Ile Ala Gly
145 150 155 160
Val Phe Ile Ala Phe Ser Ile Pro Ile Phe Gln Glu Val Ala Gly Leu
165 170 175
Gln Leu Phe Thr Ser Glu Lys Phe Pro Lys Leu Phe Lys Trp Ser Gln
180 185 190
Glu Leu Ile Asn His Pro Val Val Lys Asp Val Leu Pro Pro Arg Glu
195 200 205
Pro Leu Phe Ala Phe Phe Lys Ser Leu Tyr Glu Ser Leu Ser Ala Ser
210 215 220
Lys
225
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 946 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE'TYPE: cDNA
11
~ CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(R) CLONE: SE6.PK0098.D7
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
TTGCACTACA AATCAGTTTT CTACTTGAAT CTTCGTTATC CTTCTTTTTT TCTCCTTGAA 60
CTCGAATATT CACTATGGCA GATGAGGTGG TTCTGCTAGA TTTCTGGCCA AGTCCATTTG 120
GGATGAGGGT CAGGATTGCA CTTGCTGAAA AGGGTATCAA ATATGAGTCC AAAGAAGAGG 180
ACTTGCAGAA CAAGAGCCCT TTGCTCCTCA AAATGAACCC GGTTCACAAG AAAATCCCGG 240
TTCTCATCCA CAATGGCAAA CCCATTTGTG AATCTCTCGT TGCTGTTCAG TACATTGAGG 300
AGGTCTGGAA TGACAGAAAT CCCTTGTTGC CTTCTGACCC TTACCAGAGA GCTCAGGCTA 360
GATT'CTGGGC TGACTTTGTT GACAATAAGA TATTTGATCT TGGAAGAAAG ATTTGGACAT 920
CAAAGGGAGA AGAAAAAGAA GCTGCCAAAA AGGAGTTCAT AGAGGCCCTT AAATTATTGG 480
AGGAACAGCT GGGAGACAAG ACTTATTTTG GAGGAGACGA TCTAGGTTTT GTGGATATAG 540
CACTTATTCC ATTCGACACT TGGTTCAAGA CTTTTGGCAG CCTCAACATA GAGAGTGAGT 600
GCCCCAAGTT TGTTGCTTGG GCCAAGAGGT GCCTGCAGAA AGACAGTGTT GCCAAGTCTC 660
TTCCTGATCA ACACAAGGTC TATGAGTTCA TTATGGACAT AAGAAAGAAG TTCGACATTG 720
AGTAGGTTCA TGTTGGATTT TAATAGCCAT AGTGACGTAT TGATCATTCT TGGCCTTTCA 780
ACTAAATAGT ATTTGTGTAG TAAATTAAAG GCACTTGGAT GTACCAAACT TCATGCTTTT 840
TGTAGGAGTG CGTAGGTTTT AAAAATTTTC TGATGTATCT TTCATGTGTT TGTTGGTTTT 900
GTAACAGAAT ATTTCCTATA TTATACATAA AAAAAAAAAA AAAAAA 946
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 216 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
12
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE6.PK0048.D7
(xi) SEQUENCE DESCRIPTION: SEQ ID 140:12:
Met Ala Asp Glu Val Val Leu Leu Asp Phe Trp Pro Ser Pro Phe Gly
10 15
Met Arg Val Arg Ile Ala Leu Ala Glu Lys Gly Ile Lys Tyr Glu Ser
20 25 30
Lys Glu Glu Asp Leu Gln Asn Lys Ser Pro Leu Leu Leu Lys Met Asn
35 40 95
Pro Val His Lys Lys Ile Pro Val Leu Ile His Asn Gly Lys Pro Ile
50 55 60
Cys Glu Ser Leu Val Ala Val Gln Tyr Ile Glu Glu Val Trp Asn Asp
65 70 75 80
Arg Asn Pro Leu Leu Pro Ser Asp Pro Tyr Gln Arg Ala Gln Ala Arg
85 90 95
Phe Trp Ala Asp Phe Val Asp Asn Lys Ile Phe Asp Leu Gly Arg Lys
100 105 110
Ile Trp Thr Ser Lys Gly Glu Glu Lys Glu Ala Ala Lys Lys Glu Phe
115 120 125
Ile Glu Ala Leu Lys Leu Leu Glu Glu Gln Leu Gly Asp Lys Thr Tyr
130 135 140
Phe Gly Gly Asp Asp Leu Gly Phe Val Asp Ile Ala Leu Ile Pro Phe
145 150 155 160
Asp Thr Trp Phe Lys Thr Phe Gly Ser Leu Asn Ile Glu Ser Glu Cys
165 170 175
Pro Lys Phe Val Ala Trp Ala Lys Arg Cys Leu Gln Lys Asp Ser Val
180 185 190
Ala Lys Ser Leu Pro Asp Gln His Lys Val Tyr Glu Phe Ile Met Asp
195 200 205
Ile Arg Lys Lys Phe Asp Ile Glu
210 215
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 977 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
13
' CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SESBW.PK0028.C6
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
CTGATTCCCG GCTCAATAAG AGGAGAATAC CTTAGGAATC CATAAGAAAC ATTAATTCAC 60
CACTATAGTT GTTCTGTTAG AAGTGCTACA AACAACAATG GCTGCTAATC AGGAAGATGT 120
GAAGCTTTTG GGAGCTACTG GAAGCCCATT TGTGTGCAGG GTTCAGATTG CCCTCAAGTT 180
GAAGGGAGTT CAATACAAAT TTTTGGAAGA AAATTTGAGG AACAAGAGTG AACTGCTTCT 240
CAAATCCAAC CCAGTTCACA AGAAGGTTCC AGTGTTTATT CACAATGAGA AGCCCATAGC 300
AGAGTCTCTT GTGATTGTTG AATACATTGA TGAGACATGG AAGAACAACC CCATCTTGCC 360
TTCTGATCCT TACCAAAGAG CCTTGGCTCG TTTCTGGTCC AAATTCATTG ATGACAAGGT 920
TGTGGGTGCT GCATGGAAAT ATATTTATAC TGTTGATGAG AAAGAGCGTG AGAAGAATGT 480
TGAAGAGTCA TATGAGGCTC TGCAGTTTCT TGAGAATGAG CTGAAGGACA AGAAGTTTTT 590
TGGAGGAGAG GAAATTGGGT TGGTAGATAT TGCTGCTGTC TTCATAGCAT TTTGGATCCC 600
TATAATTCAA GAAGTATTGG GTTTGAAGTT ATTCACAAGT GAGAAATTTC CTAAGCTCTA 660
CAAATGGAGC CAAGAGTTCA TCAACCACCC TGTTGTCAAA CAAGTCCTTC CTCCTAGAGA 720
TCAACTTTTT GCCTTCTACA AAGCCTGCCA TGAAAGTCTT TCTGCTTCAA AATAGACTTA 780
TTTAAGGATA GTTGTGTGAA CTACTGGTCT CTCATTTGTG AGTTATTGCA GTTTGAATTT 890
CATGTCAATT TGGTTTTATA TGTAATTTAG TAACCTGGGA TATCTCCCAT GGAGAAAATA 900
ATCCTTGGAT CTTGTTTCCA TTTTGGCCAT TTCAGTTAAT AAAGAAATTC ATTTTTTCCA 960
977
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
14
' CA 02340791 2001-02-22
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(vii) IMMEDIATE SOURCE:
(B) CLONE: SESBW.PK0028.C6
(xi) SEQUENCE DESCRIFTION: SEQ ID NG:14:
Met Ala Ala Asn Gln Glu Asp Val Lys Leu Leu Gly Ala Thr Gly Ser
1 5 10 15
Pro Phe Val Cys Arg Val Gln Ile Ala Leu Lys Leu Lys Gly Val Gln
20 25 30
Tyr Lys Phe Leu Glu Glu Asn Leu Arg Asn Lys Ser Glu Leu Leu Leu
35 90 45
Lys Ser Asn Pro Val His Lys Lys Val Pro Val Phe Ile His Asn Glu
50 55 60
Lys Pro Ile Ala Glu Ser Leu Val Ile Val Glu Tyr Ile Asp Glu Thr
65 70 75 80
Trp Lys Asn Asn Pro Ile Leu Pro Ser Asp Pro Tyr Gln Arg Ala Leu
85 90 95
Ala Arg Phe Trp Ser Lys Phe Ile Asp Asp Lys Val Val Gly Ala Ala
100 105 110
Trp Lys Tyr Ile Tyr Thr Val Asp Glu Lys Glu Arg Glu Lys Asn Val
115 120 125
Glu Glu Ser Tyr Glu Ala Leu Gln Phe Leu Glu Asn Glu Leu Lys Asp
130 135 140
Lys Lys Phe Phe Gly Gly Glu Glu Ile Gly Leu Val Asp Ile Ala Ala
145 150 155 160
Val Phe Ile Ala Phe Trp Ile Pro Ile Ile Gln Glu Val Leu Gly Leu
165 170 175
Lys Leu Phe Thr Ser Glu Lys Phe Pro Lys Leu Tyr Lys Trp Ser Gln
180 185 190
Glu Phe Ile Asn His Pro Val Val Lys Gln Val Leu Pro Pro Arg Asp
195 200 205
Gln Leu Phe Ala Phe Tyr Lys Ala Cys His Glu Ser Leu Ser Ala Ser
210 215 220
Lys
225
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:'
(A) LENGTH: 1006 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SR1.PKOO11.D6
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
ATAGTGCTGC AATGGCTTCA AGTCAGGAGG AGGTGACCCT TTTGGGAGCT ACTGGAAGCC 60
CATTTGTGTG CAGGGTTCAT ATTGCCCTCA AGTTGAAGGG AGTTCAATAC AAATATGTCG 120
AAGAAAATTT GAGGAACAAG AGTGAACTGC TTCTCAAATC CAACCCAGTT CACAAGAAGG 180
TTCCAGTGTT TATTCACAAT GAGAAGCCCA TAGCAGAGTC TCTTGTGATT GTTGAATACA 290
TTGATGAGAC ATGGAAGAAC AACCCCATCT TGCCTTCTGA TCCTTACCAA AGAGCCTTGG 300
CTCGTTTCTG GTCCAAATTC ATTGATGATA AGGTTTTTGG TGCTGCATGG AAATCCGTTT 360
TCACAGCTGA TGAGAAAGAG CGTGAGAAGA ATGTTGAGGA AGCAATTGAG CTCTGCAGTT 920
TCTTGAGAAT GAGATAAAGG ACAAGAAGTT CTTTGGAGGA GAGGAGATTG GGTTGGTAGA 480
TATTGCTGCT GTCTACATAG CATTTTGGGT CCCTATGGTT CAAGAAATTG CAGGGTTGGA 540
GTTATTCACA AGTGAGAAAT TTCCTAAGCT CCACAATTGG AGCCAAGAAT TTTTGAACCA 600
TCCAATTGTC AAAGAAAGTC TGCCCCCTAG AGATCCTGTT TTCTCCTTTT TCAAGGGTCT 660
CTATGAAAGC CTTTTTGGTT CAAAATAGAT TTGATGATGT GGTGTGAGAC TTAGTATTTC 720
TAAGAATTAT GTGTTTGTTA AAGGCTTCTA TGAAAGCCTC ACTGCTTCAA AATAGATTCA 780
TGTATGTGAG ACTCAGAATC TCTGGGGAAA ATTGTGTGTG GTGTGGACTA CTTGTTTTGT 840
TTGTCATTGA GCTATATCGC TGTTAATTAG GATTTTGTTT CAAAATGATG CTTATAAGTT 900
GTAATCTAGG ATTTCTCCCT TTGAAATCCT AGGTTGTTCT TGACATTTGC TATTTCAAAG 960
AATAAATATA TAGCATCTTT CTATTTCTCA F,F~~AAP~AAAA AAAAAA 1006
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
16
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(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SR1.PKOO11.D6
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Ala Ser Ser Gln Glu Glu Val Thr Leu Leu Gly Ala Thr Gly Ser
1 5 10 15
Pro Phe Val Cys Arg Val His Ile Ala Leu Lys Leu Lys Gly Val Gln
20 25 30
Tyr Lys Tyr Val Glu Glu Asn Leu Arg Asn Lys Ser Glu Leu Leu Leu
35 40 45
Lys Ser Asn Pro Val His Lys Lys Val Pro Val Phe Ile His Asn Glu
50 55 60
Lys Pro Ile Ala Glu Ser Leu Val Ile Val Glu Tyr Ile Asp Glu Thr
65 70 75 80
Trp Lys Asn Asn Pro Ile Leu Pro Ser Asp Pro Tyr Gln Arg Ala Leu
85 90 95
Ala Arg Phe Trp Ser Lys Phe Ile Asp Asp Lys Val Phe Gly Ala Ala
. 100 105 110
Trp Lys Ser Val Phe Thr Ala Asp Glu Lys Glu Arg Glu Lys Asn Val
115 120 125
Glu Glu Ala Ile Glu Ala Leu Gln Phe Leu Glu Asn Glu Ile Lys Asp
130 135 140
Lys Lys Phe Phe Gly Gly Glu Glu Ile Gly Leu Val Asp Ile Ala Ala
145 150 155 160
Val Tyr Ile Ala Phe Trp Val Pro Met Val Gln Glu Ile Ala Gly Leu
165 170 175
Glu Leu Phe Thr Ser Glu Lys Phe Pro Lys Leu His Asn Trp Ser Gln
180 185 190
Glu Phe Leu Asn His Pro Ile Val Lys Glu Ser Leu Pro Pro Arg Asp
195 200 205
Pro Val Phe Ser Phe Phe Lys Gly Leu Tyr Glu Ser Leu Phe Gly Ser
210 215 220
Lys
225
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 993 base pairs
(B) TYPE: nucleic acid
1~
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SS1.PK0002.F7
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
AGCTAGTTCA CAGCTTCAGT TCGTTTTTGT TGATCCTGTG AACTTATGGC TGACGGGGTG 60
GTTCTGTTGG ATACATGGGC CAGCATGTTT GGGATGAGGG TTAGGATTGC ATTAGCTGAA 120
AAGGGTGTTG AGTATGAATA CAAGGAAGAA AATCTCAGGA ACAAGAGTCC TTTGCTTTTG 180
CAAATGAACC CAATTCACAA GAAAATTCCA GTTCTGATCC ATAATGGCAA ACCAATTTGT 240
GAATCTGCAA TTATAGTGCA GTACATTGAT GAGGTCTGGA ATGATAAAGC TCCAATCTTG 300
CCCTCTGACC CTTATGAGAG AGCTCAAGCC AGATTCTGGG TAGATTACAT TGACAAAAAG 360
GTGTATGACA CTTGGAGGAA AATGTGGCTT TCTAAAGGAG AGGAGCATGA GGCAGGGAAG 420
AAGGAGTTTA TCTCTATCTT TAAGCAGCTA GAAGAGACAC TGAGTGACAA AGCTTATTAT 480
GGAAGTGACA CCTTTGGGTT CCTTGATATT GGTTTGATCC CTTTCTACAG TTGGTTTTAT 540
ACCTTTGAGA CATATGGTAA CTTCAAAATG GAAGAAGAGT GTCCTAAACT CGTTGCTTGG 600
GCTAAGAGAT GCATGCAAAG AGAGGCTGTG TCCAAATCTC TTTCCTGATG AGAAGAAGGT 660
GTATGACTAT GTTGTGGCCG TAACAAAATT ACTTGAGTCA AACTAGAGAG ACTTCTTGAA 720
TAAATTCACG TAAGGTCTTG TGTAATTTTT ATCTTATGTT TGCTTGGGAG TTACTTATAG 780
CTTCCTAGAC ACTTGAGTGT GTCTAGTGTC TGCAGGATTT GTAACTTTAT CTTATGTTTG 840
CTAGCCTTCA GTTACTTATG ATTGCTAGAC CCTTGAGTGT GTCTACAGGA TTTGGAGCTG 900
AGGAAGGATG GATGTTGTAA TGTTTGTTTT AAGTTGTGTG TTTATGATCA ATAAATCACT 960
CATTTTATAA GGACAAAAAA F,F~~;AAAAAAA AAA 993
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
18
CA 02340791 2001-02-22
WO 00118936 PCT/US98I20501
(ii) MOLECULE TYPE: protein
(ui) ORIGID7AL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDT_ATE SOURCE:
(B) CLONE: SSi.PK0002.F7
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Ala Asp Gly Val Val Leu Leu Asp Thr Trp Ala Ser Met Phe Gly
1 5 10 15
Met Arg Val Arg Ile Ala Leu Ala Glu Lys Gly Val Glu Tyr Glu Tyr
20 25 30
Lys Glu Glu Asn Leu Arg Asn Lys Ser Pro Leu Leu Leu Gln Met Asn
35 40 45
Pro Ile His Lys Lys Ile Pro Val Leu Ile His Asn Gly Lys Pro Ile
50 55 60
Cys Glu Ser Ala Ile Ile Val Gln Tyr Ile Asp Glu Val Trp Asn Asp
65 70 75 80
Lys Ala Pro Ile Leu Pro Ser Asp Pro Tyr Glu Arg Ala Gln Ala Arg
85 90 95
Phe Trp Val Asp Tyr Ile Asp Lys Lys Val Tyr Asp Thr Trp Arg Lys
100 105 110
Met Trp Leu Ser Lys Gly Glu Glu His Glu Ala Gly Lys Lys Glu Phe
115 120 125
Ile Ser Ile Phe Lys Gln Leu Glu Glu Thr Leu Ser Asp Lys Ala Tyr
130 1,35 140
Tyr Gly Ser Asp Thr Phe Gly Phe Leu Asp Ile Gly Leu Ile Pro Phe
145 150 155 160
Tyr Ser Trp Phe Tyr Thr Phe Glu Thr Tyr Gly Asn Phe Lys Met Glu
165 - 170 175
Glu Glu Cys Pro Lys Leu Val Ala Trp Ala Lys Arg Cys Met Gln Arg
180 185 190
Glu Ala Val Ser Lys Ser Leu Ser
195 200
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 935 base pairs
(B) TYPE: nucleic acid
(C) STR~~NDEDNESS: single
(D) TOPOLOGY: linear
19
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICF:L: NO
(iv) ANTI-SENSE: NO
(vi) ORIGT_NAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SS1.PK0005.E6
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
ATTTTCTTCA TCCTTCTCTG TTCTCCTAGA ACTTGATTAC TTGAACATTC CCTATGACAG 60
ATGAGGTGGT TCTTCTGGAT TTCTGGCCAA GTCCATTTGG GATGAGGGTC AGGATTGCAC 120
TTGCTGAAAA GGGTATCGAA TATGAGTACA AAGAAGAGGA CTTGAGGAAC AAGAGTCCTC 180
TTCTCTTACA AATGAACCCG GTTCACAAGA AGATTCCGGT TCTCATCCAC AATGGCAAAC 240
CCATTTCCGA ATCCCTCATT GCTGTTCAGT ACATTGAGGA GGTTTGGAAT GACAGAAATC 300
CCTTGTTGCC TTCAGACCCT TACCAGAGAG CTCAGGCTAG ATTCTGGGCT GATTATGTTG 360
ACATTAAGAT ACATGATCTT GGAAAGAAAT TTGGACATCA AAGGGAGAAG AAAAAGAAGC 420
TGCCAAGAAG GAGTTCATAG AGGCCCTTAA ATTGTTGGAG GAACAGCTGG GAGATAAGAC 480
TTATTTTGGA GGAGACAATA TTGGTTTTGT GGATATAGCA CTTGTTCCAT TCTACACTTG 540
GTTCAAAGTC TATGAGACTT TTGGCAGCCT CAACATTGAG AATGAGTGCC CCAGGTTTGT 600
TGCTTGGGCC AAGAGGTGCC TACAGAAAGA GAGTGTTGCA AAGTCTCTTC CTGATCAGCA 660
CAAGGTCTAT GAGTTCGTTG TGGAGATAAG AAAGAAGTTA GTCATCGAGT AGGTTTCATG 720
TTGGATCTTA ATAGCCATAG TGAAGTATTG GTCGTTCTTG ACCTTTCAAC TAAATAATAT 780
TTGTGTAATA AAAAGGCATT TGGATGTGCC AAACTTCATG CTTTCTGTTG GATTGTGTAG 890
GTTTTAAAAT TTTTCTGATG TATCTTTCAT GTGTTTGTTG GTTTTGCAAT AGAGTATTTT 900
CCGTATTATC ATATAAAAAA A,F~1AAAAAAA AAAAA 935
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(vii) IMMEDIATE SOURCE:
(B) CLONE: SS1.PK0005.E6
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Met m;-,r Asp G1u Val Val Leu Leu Asp Phe Trp Pro Ser Pro Phe Gly
1 5 10 15
Met Arg Val Arg Ile Ala Leu Ala Glu Lys Gly Ile Glu Tyr Glu Tyr
20 25 30
Lys Glu Glu Asp Leu Arg Asn Lys Ser Pro Leu Leu Leu Gln Met Asn
35 40 45
Pro Val His Lys Lys Ile Pro Val Leu Ile His Asn Gly Lys Pro Ile
50 55 60
Ser Glu Ser Leu Ile Ala Val Gln Tyr Ile Glu Glu Val Trp Asn Asp
65 70 75 80
Arg Asn Pro Leu Leu Pro Ser Asp Pro Tyr Gln Arg Ala Gln Ala Arg
85 90 95
Phe Trp Ala Asp Tyr Val Asp Ile Lys Ile His Asp Leu Gly Lys Lys
100 105 110
Ile Trp Thr Ser Lys Gly Glu Glu Lys Glu Ala Ala Lys Lys Glu Phe
115 120 125
Ile Glu Ala Leu Lys Leu Leu Glu Glu Gln Leu Gly Asp Lys Thr Tyr
130 135 140
Phe Gly Gly Asp Asn Ile Gly Phe Val Asp Ile Ala Leu Val Pro Phe
145 150 155 160
Tyr Thr Trp Phe Lys V~al Tyr Glu Thr Phe Gly Ser Leu Asn Ile Glu
165 170 175
Asn Glu Cys Pro Arg Phe Val Ala Trp Ala Lys Arg Cys Leu Gln Lys
180 185 190
Glu Ser Val Ala Lys Ser Leu Pro Asp Gln His Lys Val Tyr Glu Phe
195 200 205
Val Val Glu Ile Arg Lys Lys Leu Val Ile Glu
210 215
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 895 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
21
CA 02340791 2001-02-22
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NC;
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(H) CLONE: SS1.PK0014.A1
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
AAATAAGTAT CTTCGTAGTT GCATAAGTCA AGAGAAGAAG TGAAGTGGCT GCAATGGCTT 60
CAAGTCAGGA AGAGGTGACC CTTTTGGGAG TTGTGGGAAG CCCATTTCTA CACAGGGTTC 120
AGATTGCTCT CAAGTTGAAG GGAGTTGAAT ACAAATATTT GGAAGACGAT TTGAACAACA 180
AGAGTGATTT GCTCCTCAAG TATAACCCAG TTTACAAAAT GATTCCAGTG CTTGTTCACA 240
ATGAGAAGCC CATTTCAGAG TCCCTTGTGA TTGTTGAGTA CATTGATGAC ACATGGAAAA 300
ACAATCCCAT CTTGCCTTCT GATCCCTACC AAAGAGCCTT GGCTCGTTTC TGGGCTAAGT 360
TCATTGATGA CAAGTGTGTG GTTCCAGCAT GGAAATCTGC TTTTATGACT GATGAGAAAG 420
AGAAAGAGAA GGCTAAAGAA GAGTTATTTG AGGCTCTGAG TTTTCTTGAG AATGAGTTGA 480
AGGGCAAGTT TTTTGGTGGA GAGGAGTTTG GCTTTGTGGA TATTGCTGCT GTGTTAATAC 540
CTATAATTCA AGAGATAGCA GGGTTGCAAT TGTTCACAAG TGAGAAATTC CCAAAGCTCT 600
CTAAATGGAG CCAAGACTTT CACAACCATC CAGTTGTCAA CGAAGTTATG CCTCCTAAGG 660
ATCAACTTTT TGCCTATTTC AAGGCTCGGG CTCAAAGCTT CGTTGCTAAA AGAAAGAATT 720
AATATAGTGA GACTCAGAAT TTCCATCGAG GTTTCAGTAT TGTATGAAAT GAAAGCTACT 780
TGTCTATGTT TCGTTATTGC GGTTGTATTT TCATTTTTCA ATGAATTATG TGATATAGGA 890
TTTCTCCATG TCAAAAGATA GTTCAATTCA ATCAATAAAA TAAACGAATG AGCGG 895
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 222 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SS1.PK0019.A1
22
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Met Ala Ser Ser G1:~ Glu Glu Val Thr Leu Leu Gly Val Val Gly Ser
1 5 10 15
Pro Phe Leu His Arg Val Gln Ile Ala Leu Lys Leu Lys Gly Val Glu
20 25 30
Tyr Lys Tyr Leu Glu Asp Asp Leu Asn Asn Lys Ser Asp Leu Leu Leu
35 40 45
Lys Tyr Asn Pro Val Tyr Lys Met ile Pro Val Leu Val His Asn Glu
50 55 60
Lys Pro Ile Ser Glu S~__°r Leu Val Ile Val Glu Tyr Ile Asp Asp Thr
65 70 75 80
Trp Lys Asn Asn Pro Ile Leu Pro Ser Asp Pro Tyr Gln Arg Ala Leu
85 90 95
Ala Arg Phe Trp Ala Lys Phe Ile Asp Asp Lys Cys Val Val Pro Ala
100 105 110
Trp Lys Ser Ala Phe Met Thr Asp Glu Lys Glu Lys Glu Lys Ala Lys
115 120 125
Glu Glu Leu Phe Glu Ala Leu Ser Phe Leu Glu Asn Glu Leu Lys Gly
130 - 135 140
Lys Phe Phe Gly Gly Glu Glu Phe Gly Phe Val Asp Ile Ala Ala Val
195 150 155 160
Leu Ile Pro Ile Ile Gln Glu Ile Ala Gly Leu Gln Leu Phe Thr Ser
165 170 175
Glu Lys Phe Pro Lys Leu Sex Lys Trp Ser Gln Asp Phe His Asn His
180 185 190
Pro Val Val Asn Glu Val Met Pro Pro Lys Asp Gln Leu Phe Ala Tyr
195 200 205
Phe Lys Ala Arg Ala Gln Ser Phe Val Ala Lys Arg Lys Asn
210 215 220
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 885 base pairs
(B) TYPE: nucleic 'acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
23
CA 02340791 2001-02-22
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(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SS1.PK0020.B10
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
CCATAGCAAT GGCAGAGCAA GACAAGGTGA TCCTACACGG GATGTGGGCC AGCCCTTATG 60
CCAAGAGGGT GGAATTGGCC CTTAATTTTA AGGGCATACC CTATGAGTAT GTTGAAGAAG 120
ACTTGAGAAA TAAGAGTGAT TTGCTTCTAA AGTACAACCC TGTTCACAAG AAGGTTCCTG 180
TACTTGTTCA TAATGGAAAG GCCATTGCTG AATCCATGGT GATCCTTGAG TATATTGATG 290
AAACATGGAA AGATGGTCCT AAACTGCTTC CAAGTGATTC TTACAAACGA GCCCAAGCTC 300
GATTCTGGTG TCATTTCATC CAGGATCAGT TAATGGAGAG CACTTTTCTA GTAGTCAAAA 360
CTGATGGAGA AGCACAACAA AAGGCCATTG ACCACGTGTA TGAGAAACTG AAAGTGCTAG 420
AAGATGGAAT GAAGACCTAT CTGGGAGAAG GCAATGCTAT TATCTCTGGT GTTGAAAACA 980
ACTTTGGAAT CCTTGACATT GTGTTTTGTG CTTTATATGG TGCCTACAAG GCTCATGAAG 590
AAGTTATTGG CCTCAAGTTC ATAGTGCCAG AAAAGTTTCC TGTGTTGTTT TCTTGGTTGA 600
TGGCTATTGC TGAGGTTGAA GCTGTGAAAA TTGCAACTCC TCCACATGAA AAAACAGTGG 660
GAATTCTTCA GTTGTTCAGG CTGTCTGCAC TGAAATCTTC TTCTGCCACA GAATGATATA 720
TACTTCAACA CTTTAATAGA CTGTCCATCG TTTGCTTCTT CTGCGAGTCT TTAGTGTATG 780
TATCTTTCAA TAACAGGATG AGTAACACCT GAGTATGTAA AGCGTGATGA TATAGAGATA 890
TACCTCTATA TATCAAATAC TCTTCTATAA AAAAAAAAAA AAAAA 885
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 235 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SS1.PK0020.810
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Met Ala Glu Gln Asp Lys Val Ile Leu His Gly Met Trp Ala Ser Pro
1 5 10 15
24
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
Tyr Ala Lys Arg Val Glu Leu Ala Leu Asn Phe Lys Gly Ile Pro Tyr
20 25 30
Glu Tyr Val Glu Glu Asp Leu Arg Asn Lys Ser Asp Leu Leu Leu Lys
35 40 45
Tyr Asn Pro Val His Lys Lys Val Pro Val Leu Val His Asn Gly Lys
50 55 60
Ala Ile Ala Glu Ser Met Val Ile Leu Glu Tyr Ile Asp Glu Thr Trp
65 70 75 80
Lys Asp Gly Pro Lys Leu Leu Pro Ser Asp Ser Tyr Lys Arg Ala Gln
85 90 95
Ala Arg Phe Trp Cys His Phe Ile Gln Asp Gln Leu Met Glu Ser Thr
100 105 110
Phe Leu Val Val Lys Thr Asp Gly Glu Ala Gln Gln Lys Ala Ile Asp
115 120 125
His Val Tyr Glu Lys Leu Lys Val Leu Glu Asp Gly Met Lys Thr Tyr
130 135 190
Leu Gly Glu Gly Asn Ala Ile Ile Ser Gly Val Glu Asn Asn Phe Gly
145 150 155 160
Ile Leu Asp Ile Val Phe Cys Ala Leu Tyr Gly Ala Tyr Lys Ala His
165 170 175
Glu Glu Val Ile Gly Leu Lys Phe Ile Val Pro Glu Lys Phe Pro Val
180 185 190
Leu Phe Ser Trp Leu Met Ala Ile Ala Glu Val Glu Ala Val Lys Ile
195 200 205
Ala Thr Pro Pro His Glu Lys Thr Val Gly Ile Leu Gln Leu Phe Arg
210 215 220
Leu Ser Ala Leu Lys Ser Ser Ser Ala Thr Glu
225 230 235
(2) INFORMATION FOR SEQ
ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 991 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: .NO
- . CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SSM.PK0067.G5
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
CTCGTGCCGT TTCTATAAAG GCCAAACTCA CAAACCACAC CCTAACAAAT TCATCTTATT 60
TTGCAACACA ATTCAATTTT GAGCACTTAC CAACACCACT TCCAATGGCT TCATATCATG 120
AAGAAGAAGT GAGGCTATTG GGCAAGTGGG CCAGCCCATT TAGCAACAGA GTAGACCTTG 180
CTCTCAAGCT CAAGGGTGTT CCCTACAAAT ACTCCGAGGA AGATCTTGCT AACAAGAGTG 29fl
CTGATCTTCT CAAGTACAAC CCCGTTCACA AGAAGGTTCC GGTTTTGGTC CACAATGGGA 300
ACCCATTGCC CGAGTCACTC ATCATTGTTG AATACATAGA TGAGACGTGG AAAAATAACC 360
CACTATTGCC TCAAGACCCA TATGAAAGAG CCTTGGCTCG TTTTTGGTCT AAGACCTTAG 420
ATGACAAGAT CTTGCCAGCT ATATGGAATG CTTGCTGGAG TGACGAGAAT GGGCGTGAGA 980
AAGCAGTGGA GGAAGCCTTG GAAGCATTGA AAATCCTACA GGAAACACTG AAAGACAAGA 540
AATTCTTTGG AGGAGAGAGC ATAGGATTGG TAGATATTGC TGCCAATTTC ATTGGGTATT 600
GGGTTGCCAT ATTGCAAGAG ATTGCAGGGT TGGAGTTGCT CACCATTGAG AAATTTCCCA 660
AGTTATATAA TTGGAGTCAA GACTTTATCA ACCACCCTGT GATCAAGGAG GGTCTGCCTC 720
CTAGAGATGA ATTGTTTGCT TTCTTCAAAG CTTCTGCTAA AAAGTAGAAC CATTTTAGAG 780
GTAGGATTCA TAATAAGTTA GTATGATTTT GTTGGGAAAC AATTATCTTG TTGTGAGCAA 840
AGGATTGTTC TGTTTTAAAT TTAATTGACT GTGATTTGGT TGGGTATTGG CTATTTTAAT 900
TTTAACTAAA AAAAGTGTTC AGTTTTAAAA A,P~AAAAAAAA F,F~~iAAAAAAA
p~~i°~AAAAAAA 960
p,~~~Ap AAAAA AAAAAAAAAA A 9 91
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 220 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE. TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SSM.PK0067.G5
26
CA 02340791 2001-02-22
VVO 00/18936 PCT/US98120501
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Met Ala Ser Tyr His Glu Glu Glu Val Arg Leu Leu Gly Lys Trp Ala
1 5 10 15
Ser Pro Phe Ser Asn Arg Val Asp Leu Ala Leu Lys Leu Lys Gly Val
20 25 30
Pro Tyr Lys Tyr Ser Glu Glu Asp Leu Ala Asn Lys Ser Ala Asp Leu
35 40 95
Leu Lys Tyr Asn Pro Val His Lys Lys Val Pro Val Leu Val His Asn
50 55 60
Gly Asn Pro Leu Pro Glu Ser Leu Ile Ile Val Glu Tyr Ile Asp Glu
65 70 75 80
Thr Trp Lys Asn Asn Pro Leu Leu Pro Gln Asp Pro Tyr Glu Arg Ala
85 90 95
Leu Ala Arg Phe Trp Ser Lys Thr Leu Asp Asp Lys Ile Leu Pro Ala
100 105 110
Ile Trp Asn Ala Cys Trp Ser Asp Glu Asn Gly Arg Glu Lys Ala Val
115 120 125
Glu Glu Ala Leu Glu Ala Leu Lys Ile Leu Gln Glu Thr Leu Lys Asp
130 135 140
Lys Lys Phe Phe Gly Gly Glu Ser Ile Gly Leu Val Asp Ile Ala Ala
145 150 155 160
Asn Phe Ile Gly Tyr Trp Val Ala Ile Leu Gln Glu Ile Ala Gly Leu
165 170 175
Glu Leu Leu Thr Ile Glu Lys Phe Pro Lys Leu Tyr Asn Trp Ser Gln
180 185 190
Asp Phe Ile Asn His Pro Val Ile Lys Glu Gly Leu Pro Pro Arg Asp
195 200 205
Glu Leu Phe Ala Phe Phe Lys Ala Ser Ala Lys Lys
210 215 220
(2) INFORMATION FOR SEQ ID N0:27:
(i)- SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1024 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
27
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WO 00/18936 PCT/US98/20501
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE1.PK0017.F5
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
CCAAATCTTA AAAATATTCA GTGAAGATCA ACCTCAATGG CATCTCTTGG CGTGCGACCA 60
GTTCTTCCCC CTCCATTAAC TTCCATCTCC GACCCACCTC CTCTTTTCGA TGGCACCACC 120
AGGTTGTACA TCAGTTATTC TTGCCCCTAT GCACAACGTG TGTGGATCGC TAGGAACTAC 180
AAGGGGCTAC AAGATAAGAT CAATTTGGTC CCTATTAACC TTCAAGACAG GCCAGCTTGG 240
TATAAGGAGA AAGTCTACCC TGAAAATAAG GTGCCATCCT TGGAGCACAA TGGCAAGGTG 300
TTGGGAGAAA GTCTTGATTT GATCAAATAT GTAGATGCAA ACTTTGAAGG GACACCTTTG 360
TTTCCCAGTG ATCCTGCCAA GAAAGAGTTC GGTGAGCAAT TGATATCCCA TGTTGATACA 420
TTCAGCAAAG ACCTGTTCGT TTCATTGAAA GGGGATGCTG TACAGCAAGC CAGTCCCGCT 980
TTTGAATACT TGGAGAATGC TCTTGGTP.AA TTTGATGATG GGCCATTCTT GCTTGGCCAA 590
TTCAGTTTGG TGGATATTGC TTATATTCCA TTTGTTGAAA GATTCCAAAT TGTCTTTGCT 600
GAGGTGTTCA AACATGACAT CACAGAAGGA AGGCCTAAAC TTGCAACATG GTTTGAGGAG 660
TTGAATAAGC TAAATGCTTA TACCGAGACT AGAGTCGATC CTCAGGAGAT CGTTGATCTT 720
TTCAAGAAAC GCTTCCTGCC TCAACAGTGA ACGTTGTATT GCTGCAGGCT TCCTCTAAAA 780
TGTAGACTCT GCCCATATAG CGTCCTTTCA TTCACGGGAT GGGATGCATC TGCAGTCAAA B40
TGTCGGTTGT GTTTATCTGC CAGAGTTGCA GGATAGTTTG AAGTCATAAT CACGTTCATT 900
TTTCAGCTTG TTTGTTTGAT GTCATAATAA TGTTTATGTA CCAGTTTGTG ATCACTGATC 960
AATATGATAT AATGACCAAT ATGGTATTAT TATCCTATTT GAACTAAAAA F~F~~,HAAAAAA 1020
pip 1029
(2) INFORMATION FOR SEQ
ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 237 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: SOYBEAN
(vii) IMMEDIATE SOURCE:
(B) CLONE: SE1.PK0017.F5
28
w CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Met Ala Ser Leu Gly Val Arg Pro Val Leu Pro Pro Pro Leu Thr Ser
1 5 10 15
Ile Ser Asp Pro Pro Pro Leu Phe Asp Gly Thr Thr Arg Leu Tyr Ile
20 25 30
Ser Tyr Ser Cys Pro Tyr Ala Gln Arg Val Trp Ile Ala Arg Asn Tyr
35 40 45
Lys Gly Leu Gln Asp Lys Ile Asn Leu Val Pro Ile Asn Leu Gln Asp
50 55 60
Arg Pro Ala Trp Tyr Lys Glu Lys Val Tyr Pro Glu Asn Lys Val Pro
65 70 75 80
Ser Leu Glu His Asn Gly Lys Val Leu Gly Glu Ser Leu Asp Leu Ile
85 90 95
Lys Tyr Val Asp Ala Asn Phe Glu Gly Thr Pro Leu Phe Pro Ser Asp
100 105 ~ 110
Pro Ala Lys Lys Glu Phe Gly Glu Gln Leu Ile Ser His Val Asp Thr
115 120 125
Phe Ser Lys Asp Leu Phe Val Ser Leu Lys Gly Asp Ala Val Gln Gln
130 135 190
Ala Ser Pro Ala Phe Glu Tyr Leu Giu Asn Ala Leu Gly Lys Phe Asp
145 150 155 160
Asp Gly Pro Phe Leu Leu Gly Gln Phe Ser Leu Val Asp Ile Ala Tyr
165 170 175
Ile Pro Phe Val Glu Arg Phe Gln Ile Val Phe Ala Glu Val Phe Lys
180 185 190
His Asp Ile Thr Glu Gly Arg Pro Lys Leu Ala Thr Trp Phe Glu Glu
195 200 205
Leu Asn Lys Leu Asn Ala Tyr Thr Glu Thr Arg Val Asp Pro Gln Glu
210 215 220
Ile Val Asp Leu Phe Lys Lys Arg Phe Leu Pro Gln Gln
225 230 235
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE.: other nucleic acid
(A) DESCRIPTION: /desc = "PRIMER"
29
CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GAYGARGANC TNCTNGAYTT YTGG 24
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(Ay LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PRIMER"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GACTCGAGTC GACATGCTT 19
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(By TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
CATATGAGTG ATGAGGTAGT GTTATTAGAT TTCTGG 36
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
~
~ CA 02340791 2001-02-22
WO 00/18936 PCT/US98/20501
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
TTATTACACA AATATTACTT ATTTGAAAGG CTAA 39
31
