CA2339349A1 - Maize glutathione-s-transferase enzymes - Google Patents

Abstract

This invention relates to isolated nucleic acid fragments encoding all or a substantial portion of maize glutathione-S-transferase (GST) enzymes involve d in the detoxification of xenobiotic compounds in plants and seeds. The invention also relates to the construction of chimeric genes encoding all or a substantial portion of maize GST enzymes, host cells transformed with those genes and methods of the recombinant production of maize GST enzymes. Method s of constructing transgenic plants having altered levels of GST enzymes and screens for identifying maize GST enzyme substrates and maize GST enzyme inhibitor, are also provided.

Description

TITLE
MAIZE 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 maize glutathione-S-transfi~rase (GS'T) enzymes involved in the detoxification of xenobiotic compounds in plants and seeds.

Glutathione-S-transferases (GST) are a family of enzymes which catalyze the conjugation of glutathione, homoglutathione (hGSH) and other glutathione-like analogs via a sulfhydryi group, to a large range of hydrophobic, electrophilic compounds. The conjugation can result in detoxification of these compounds.
GST enzymes have lbeen identified in a range of plants including maize (Wosnick et al., Gene (Amst) 76 (1) (1989) 153-160; Rossini et al., Plant Physiology ZS (Rockville) I12 (4) 1,1996) 1595-1600; Holt et al., Planta (Heidelberg) 196 (2) (1995) 295-302), wheat (Edwards et al., Pestle. Biochem. Physiol. (1996) 54(2), 96-104), sorghum (Iiatzios et al., J. Environ. Sct. Healtlz, Part B (1996), B31 (3}, 545-553), arabidops:is (Van Der Kop et al., Plant Molecular Biology 30 (4) (1996), sugarcane (Singhal et al., Phytochemistry (OXF) 30 (5) (1991) 1409-I414), 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. (198'9) 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 (Marrs 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 class 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 bioavailability. Furthermore the addition of GSH to a molecule serves as an "address" to send that molecule to the plant vacuole (Mans et al., Nature 375:397-400, (1995)).

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, 2t)3-211 ( 1986)}, GST 27 (Jepson et al. , Plant Mo1 Biol 5 26:1855-1866, (1994)), GST 26 {Moore et al., Nucleic.4cids Res 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 hamodimer of GST 26), and GST IV {a homodimer of GST 27). GST 27 is highly inducible by safener compounds (Jepson (1994) supra; Holt et al., Planta 196:295-302;
IO (1995)) and overexpression of GST 27 in tobacco confers alachlor resistance to transgenic tobacco (Jepson, personal communication). Additianally, Bridges et al. (U.S. SS89614) disclose the sequence of a maize derived GST isoform a promoter useful for the expression of foreign genes in maize and wheat. In soybean, herbicide compounds conjugated to hGSH have been detected and 15 correlated with herbicide selectivity (Frear et al., Fhysiol 20: 299-310 (1983);
Brown et al., Pcst Biochem Physiol 29:112-120, (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.
Some effart;s have been made to alter plant phenotypes by the expression 20 of either plant or mammalian foreign GST genes or their promoters in mature plant tissue. For example, Helmer et al. (U.S. 5073677) teach the expression of a rat GST gene in tobacco under the control of a strong plant promoter.
Similarly, Jepson et al. (V~0 97/11189) disclose a chemically inducible maize GST
promoter useful for the expression of foreign proteins in plants; Chilton et al. (EP
256223}
25 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.p~henotype.
Manipulation of nucleic acid fragments encoding soybean GST to use in 30 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
35 The present invention provides nucleic acid fragments isolated from maize 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 NO:6, SEQ

3'7 PCT/US98I20502 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, and SEQ ID N0:24; (b) an isolated nucleic acid fi~agmenl that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence S sequence selected from the group consisting of SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ QED N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID N0:14, SEQ ID N0:16, SEQ! ID NO: I8, SEQ ID N0:20, SEQ ID N0:22, and SEQ ID
N0:24; 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 maize GST enzymes or to chimeric genes that comprise nucleic acid fragments as described above, the chimeric genes operably linked.to suitable 1S regulatory sequences, wherein expression of the chirneric genes results in altered levels of the encoded. enzymes in transformed host cells.
The pxesent invention further provides a transformed host cell comprising the above described c;himeric gene. The transformed host cells can be of eukaryotic or prokar;rotic origin. The~invenrion also includes transformed plants that arise from transfbrmed host cells of higher plants, and from seeds derived from such transformc;d plants, and subsequent progeny.
Additionally, the invention provides methods of altering the level of expression of a maize GST enzyme in a host cell comprising the steps of;
(i) transforming a host cell with the above described chimeric gene and;
2S (ii) growing the transformed host cell produced in step (i) under conditions that are suitable for expression of the chirneric gene wherein expression of the chimeric gene result;. 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 maize GST enzyme comprising either hybridization or primer-directed amp:Eification methods known in the art and using the above described nucleic acid fragment. A primer-amplification-based method uses SEQ
ID NOS.: 1, 3, S, 7, 9, 11, 13, 1S, 17, 19, 21, or 23. The product of these methods 3S is also part of the invention.
Another embodiment of the invention includes a method for identifying a compound that inhibits the activity of a maize GST enzyme encoded by the nucleic acid fragment and substantially similar and complementary nucleic acid fragments of SEQ II) NOS.:1-24. 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 G;ST enzyme with a chemical compound of interest; and (e) identifying the chemical compound of interest that reduces the activity of the maize GST enzyme relative to the activity of the maize 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.: i, 3, 5, 7, 9, I 1, 13, 15, I7, 19, 21, and 23, and the maize GST enzyme is selected from the group consisting of SEQ ID NOS.: 2, 4, 6, 8,10, I2, I4, I6, 18, 20, 22, and 24.
The invention further provides a method for identifying a chemical compound that inhibits the activitry of the maize 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 firansformed 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.: 1, 3, 5, 7, 9, 11, 13, i 5, 17, 19, 21, and 23 and the mai2;e GST enzyme is selected from the group consisting of SEQ
ID NOS.: 2, 4, 6, 8;, 10, 12, 14, 16, 18, 20, 22, and 24.
In another embodiment, the invention provides a method for identifying a substrate for the maize 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 maize GST enzyme operably linked to ax least one suitable regulatory sequence; (b} growing the transformed host cell of step (a) under conditions that are suitable for expression of the chimeric gens~ 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 maize GST enzyme with the activity of maize GST enzyme that has been contacted with the substrate candidate and selecting substrate candidates that increase the activity of the maize GST enzyme relative to the activity of maize 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.: :l, 3, 5, 7, 9, 11, 13, IS, 17, 19, 2I, and 23, 25 and the maize GST enzyme is selec;ted from the group consisting of SEQ ID NOS.: 2, 4, 6, 8, 10, I2, 14, 16, 18, 2(), 22, and 24.
Alternatively, methods are provided for identifying a maize GST substrate candidate wherein the identification of the substrate candidate is based on a 5 comparison of the phenotype of a host cell transformed with a chimeric gene expressing a maize C1ST 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 10 group consisting of SEQ ID NOS.: 1, 3, 5, 7, 9, I 1, 13, 15, 17, 19, 2I, and 23 and the maize GST enzyme is selected from the group consisting of SEQ ID NOS.: 2, 4, 6, 8, 10, 12, 14; 1 ti, 18, 20, 22, and 24.
BRIEF DESCRIPTION OF SEQUENCE DESCRIPTIONS
AND BIOLOGICAL DEPOSITS
15 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 tthe rules governing nucleotide andlor amino acid sequence 20 disclosures in patent applications as set forth in 37 C.F.R. ~I.821-1.825.
The Sequence Descriptions contain the one 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 Nucteic Acids Research 13:3021-3030 (1985) and in the Bi~~chemical.Yournal 219 (IVo. 2):345-373 (1984) which are 25 herein incorporated '.by reference.
SEQ ID NO:1 is the nucleotide sequence comprising the cDNA insert in clone bmsl.pk0023.g8 encoding a maize GST.
SEQ ID N0:2 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone bmsi..pk0023.g8.
30 SEQ ID N0:3 is the nucleotide sequence comprising the cDNA insert in clone cs.pk0010.c5 encoding a maize GST.
SEQ ID N0:4 is the deduced amino acid sequence of the nucleotide sequence comprism~; the cDNA insert in clone cs.pk0010.c5.
SEQ ID N0:5 is the nucleotide sequence comprising the cDNA insert in 35 clone cebl.pk0017.a5 encoding a maize GST.
SEQ ID N0:6 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone cebl.pk0017.a5.
SEQ ID N0:7 is the nucleotide sequence comprising the cDNA insert in clone cc7lse-a.pk000I.g2 encoding a maize class III GST.

SEQ ID NO:8 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone cc7lse-a.pk0001.g2.
SEQ ID NO:9 is the nucleotide sequence comprising the cDNA insert in clone cc71 se-b.pk00~ 14.b8 encoding a maize class III GST.
5 SEQ ID NO:1 ~0 is the deduced amino acid sequence of the nucleotide sequence comprisin~; the cDNA insert in clone cc7lse-b.pk0014.b8.
SEQ ID NO:11 is the nucleotide sequence comprising the cDNA insert in clone ceb5.pk0051.f8 encoding a maize class III GST.
SEQ ID N0:1:2 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ceb5.pk0051.f8.
SEQ ID N0:13 is the nucleotide sequence comprising the cDNA insert in clone crin.pk0003.b~1 encoding a maize class III GST.
SEQ ID N0:14 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone crin.pk0003.b1.
SEQ ID N0:15 is the nucleotide sequence comprising the cDNA insert in clone crln.pk0014.g;8 encoding'a maize class iII GST.
SEQ ID N0:16 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone crln.pk0014.g8.
SEQ ID N0:17 is the nucleotide sequence comprising the cDNA insert in clone m.15.5.d06.sk20 encoding a maize class II~GST.
SEQ ID N0:18 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone m.15.5.d06.sk20.
SEQ ID N0:19 is the nucleotide sequence comprising the cDNA. insert in clone crln.pk0040.e;12 encoding a maize class II GST.
SEQ ID N0:20 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone crln.pk0040.e12.
SEQ ID N0:21 is the nucleotide sequence comprising the cDNA insert in clone ceb5.pk0049.a1 l encoding a maize class III GST.
SEQ ID N0:22 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ceb5.pk0049.a11.
SEQ ID N0:23 is the nucleotide sequence comprising the cDNA insert in clone csl.pk0059.e2 encoding a maize class IIi GST.
SEQ iD N0:24 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone csl.pk0059.e2.
35 The transformed E. coli ceb5.pk0051.f8/pET30(LIC)BL21(DE3) containing the gene ceb5.pk005I .f8 in a pET30(LIC) vector encoding a maize class III GST was deposited on 21 August 1997 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MY 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 98511.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel GST nucleotide sequences and 5 encoded proteins isolated from maize. 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 maize GST e;nzymes~ have been isolated and identified by comparison of random plant cDNA. sequences to public databases containing nucleotide and 10 protein sequences using the BLAST algorithms well known to those skilled in the art. The sequences of the present invention are useful iwthe construction of herbicide-tolerant transgenic plants, in the recombinant production of GST
enzymes, in the deve:Iopment of screening assays to identify compounds inhibitory to the GST enzymes., and in screening assays to identify chemical substrates of the 15 GSTs.
In the context of this disclosure, a number of terms shall be utilized.
As use herein "Glutathione S-Transferase" or "GST" refers to any plant derived glutathione S-transferase (GST) enzyme capable of catalyzing the conjugation of glutai:hione, homogiutathione and. other glutathione-like analogs 20 via a sulfliydryl 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.
As used herein "GS7C" is not intended to be delimited on the basis of enzyme activity but may encompass amino acid sequences that possess no measurable 25 enzyme activity but are substantially similar in 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"
30 and "GST class IV" . The grouping of plant GSTs into three classes is 'described by Droog et al. (Plant Physiology 107:1139-1 i46 (1995)). AlI 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 35 were identified: class one including the archetypical sequences from maize GST I
(X06755) and GST IfII (X04375); class two including the archetypical sequence from Dianthus caryophyllus (M64628); and class three including the archetypical sequence soybean GH214 (M20363). Recently, Applicants have established a further subgroup of the plant GSTs known as class IV GSTs with its archetypical sequence being In2-:l (X58573).
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 5 natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, .
genomic DNA ar synthetic DNA.
As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one ar 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 does 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 15 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-~-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 20 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 25 fragments that do ncrt share 100% identity with the gene to be suppressed.
Moreover, alteratior,~s 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 30 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) o:r one positively charged residue for another (such as lysine for arginine) can also be expected to produce a functionally equivalent product.
35 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 oiP the proposed modifications is well within the routine skill in the art, as is determiination 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 sequences exemplifte:d herein. Preferred substantially similar nucleic acid fragments of the inst~~nt invention are those nucleic acid fragments whose DNA
5 sequences are at least 80% 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.
~0 A "substantia.l 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 oi.-'the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as 15 BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al:, (1993) J. Mol.
Biol. 215:403-410; see also www.ncbi.nlm.nih.govBLAST~. 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 20 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., ih situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a 25 particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion''' of a nucleotide sequence comprises enough of the sequence to specifically identify andlor 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 30 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.
35 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 WO 00!18937 PCTNS98/20502 as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.
"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
NO:4, SEQ ID NU:6" SEQ ID NO:B, 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, and SEQ ID N0:24. The skilled artisan is well aware of the "codon-bias" exlubited 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 th.e 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 oligonucieotide 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 far 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 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 to organism, but that is :introduced into the host organism by gene transfer.
Foreign genes can comprise native genes inserted into a non-native organism, ar chimeric 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 10 sequence. Regulatory sequences may include promoters, translation leader .
sequences, introns, and polyadenylation recognition sequences.
"Promoter" rc;fers to a DNA sequence capable of controlling the expression of a coding sequence ar functional RNA. Tn general, a coding sequence is located 3' to a promoter sequence. The promoter sequence consists ~of 15 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. Promoter:> may be derived in their entirety from a native gene, or be 20 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 developrnent, or in response to different environmental conditions. Promoters which cause a gene to be 25 expressed in mast 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 Gotdberg, (1989) Biochemistry ofPlahts IS:I-82. It is further recognized that since: in most cases the exact boundaries of regulatory sequences 30 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 promotc;r sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of 35 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) Mohcular Biotechnology 3:225).
lI

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 mRNA processing or gene expression. The polyadenyiation signal is usually characterized by affecting the addition of polyadenylic acid tracts to. the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. (;(1989) Plant Cell 1:671-680).
"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 b~e 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. "eDNA" 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" rei:ers 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 (U.S. Pat. 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 ope;rably 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" rel:ers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5;231,020).
"Altered levc;ls" 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 rc;moved: "Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and 10 propeptides may be but are not limited to intracellular localization signals.
A "chloropla.st 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 IS 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 20 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 (Railchel (1992) Plant Phys.100:1627-1632).
"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host or3ganism, resulting in genetically stable inheritance. Host 25 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. (I987) Meth. Enrymol.
143:277) and particle-accelerated or "gene gun" transformation technology (Klein et al.~(1987) Nature (London) 327:70-73; U.S. Pat. No.-4,945,050).
30 The term "hc;rbicide-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.
35 "Thial donor" refers to a compound that contains the stntcture 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.
"Electrophil:ic substrate"'refers to a compound that is amenable to conjugation with ghztathioze or homogiutathione via a sulfhydryl group:

Electrophilic substrates include a wide variety of compounds including pesticides, anti-pathogenic compounds such as fungicides and profungicides, pheromones, and herbicides. V~itlun the context of the present invention electrophiiic substrates with herbicidal activity may include, but are not. limited to, S 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 knov~rn 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").
"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.
IS "Electrophilic substrate" refers to a compound that is amenable to conjugation with glutathione or homoglutathione via a sulfhydryl group.
Eiectrophilic substraites include a wide variety of compounds including pesticides, anti-pathogenic compounds such as fungicides and profungicides, pheromones, and herbicides. Witrun the context of the present invention electrophilic 20 substrates with herbicidal activity may include, but are not limited to, chlorimuronethyl, alachior, and atrazine, 1-chloro-2,4-dinitrobenzene {CDNB), ethacrynic acid, t-stilbene oxide, and 1,2-epoxy-3-(p-nitrophenoxy)propane.
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 2S 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., polyrnerase chain reaction, ligase chain reaction).
30 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 3S designed and synthe:>ized 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 system:>. In addition, specific primers can be designed and used to amplify a part of or full-length of the instant sequences. The resulting , amplification products can be labeled directly during amplif cation reactions or labeled after amplificration reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
In addition, t<NO 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 ailso be performed on a library of cloned nucleic acid fragments wherein th.e sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts~to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE; protocol (Frohmarx et ai., (1988) PNAS USA 85:8998) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., (1989) PNAS USA 86:5673; Loh et al., {1989) Science 243:217). 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 amine acid sequences. These antibodies can be then be used to screen cDNA expres:cion libraries to isolate full-length cDNA clones of interest (Lerner, R.A. (1984) Adv. Immuhol. 36:1; Maniatis).
T'he nucleic acid fragments of the instant~invention may be used to create transgenic plants in vrhich 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 tlae 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 nnay 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 (Cad 10 genes. One type of efficient plant promoter that rriay be used is a high level plant promoter. Such promoters, in operable linY,age 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 IS this invention include the promoter of the small subunit (ss) of the ribulose-1,5 bisphosphate carboxylase from example from soybean (Berry-Lowe et al., J. Molecular and Abp. Gen., I :483-498 1982)), and the promoter of the chlorophyll alb binding protein. These two promoters are~known to be light-induced in plant cells (See, for example, Genetic Eneineerin~ of Plants, an 20 A 'ng cultural Perspective, A. Cashmore, Plenum, New York (1983), pages 29-38;
Conzzzi, G. et al., The Journal ofBiolvgical Chemistry, 258:1399 (1983), and Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:285 (1983)).
Plasmid veci:ors comprising the instant chimeric genes can then constructed. T'he choice of plasmid vector depends upon the method that will be 25 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) 30 EMBO J. 4:2411-2418; De Alrrieida 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 oiFDNA blots (Southern, J. Mol. Bivl. 98, 503, (1975)).
Northern analysis oiF mRNA expression (Kroczek, .I. Chromatogr. Biomed. Appl., 35 618 (1-2} (i993} 13:3-145), Western analysis of protein expression, or phenotypic analysis.
For some applications it will be useful to direct the instant GST enzymes to different cellular comparbmeiits or to facilitate enzyme secretion from a recombinant host ce;Il. 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 endopiasmic reticulum localization (Chrispeels, J.J., .~4nn. Rev.
Plant Phys. Plant Mol. Biol: 42:21-53 (i991)),~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 e:~haustive and more targeting signals of utility may be discovered in the futn~re that are useful in the invention.
It may also be desirable to reduce or eliminate expression of the genes encoding the instant tJST enzymes in plants for some applications. 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 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 plants 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 is known to result in sequestration and detoxification of a number of herbicides and other xenobiotics (Mans et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:127-58 (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 transgenic seedlings that overexpress gl_utathione S-transferase, Annual Meeting of the American Society of Plant Physiologists, (August 1997), abstract 1574, Final Program, Plant Biology and Supplement to Plant (Physiology, 301), exposure to ozone (Sharma et al., Plant Physiology, I05 (4) 11994) 1089-1096), and exposure to industrial pollutants such as sulfur dioxide (Navari-Izzo et al., Plant Science 96 (1-2) ((994) 31-40).
It is contemplated that t:ransgenic 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 direcit high level expression of foreign proteins are~well known to those skilled in the axt. Any of these could be used to construct chimeric genes for 5 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 c~~ssettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences 10 directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
Suitable vectors comprise a region 5' 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 15 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 expression of the genes encoding the GST enzymes in the desired host cell are 20 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, GAL1, GALIO, ADH1, PGK, PH05, GAPDH, ADG1, TRP1, UR.A3, LEU:z, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in ~'ichia); and lac, trp, ~,PL, ~,PR, T7, tac, and trc (useful 25 for expression in E. toll).
Termination control regions may also be derived from various genes native to the preferred pasta. Optionally, a termination site may be unnecessary, however, it is most (preferred if included.
An example of a vector far high level expression of the instant GST
30 enzymes in a bacterial host is provided (Example 5).
Additionally, the instant maize GST enzymes can be used as a targets to facilitate design andlor 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 sulfhydryl conjugation of glutathione to compounds 35 toxic to the plant. C:orijugation 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 maize GST enzymes could be appropriate for new herbicide oi'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 nnarkers 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 malyses using computer programs such as MapMaker (Lander et at., (1987) Genomics 1:174=181} in order to construct a genetic map.
In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclea.se-treated genomic DNAs of a set of individuals representing parent and progeny of a def ned genetic cross.
15 Segregation of the D:~1A polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously - obtained using this population (Botstein et al., (1980} Am. J. Hum. Genet.
32:314-331 ).
The production and use of plant gene-derived probes for use in genetic mapping are described by Bernatzky, R. and Tanlcsley, S.D. (Plant Mol. Biol.
Reporter 4(1): 37-41 (1986)). Nxunerous publications describe genetic mapping of specific eDNP. clones using the methodology outlined above or variations thereof.
For example, F2 intercross populations, backcross populations, randomly mated populatians, near iso;genic lines, and other sets of individuals may be used for mapping. Such metluodologies are well known to those skilled in the art.
Nucleic acid probes derived fram 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 199fi, pp. 319-346, and references cited therein).
In another err~bodiment, 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 FISI-I 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-specif c amplification, polymorphism of PCR-amplified fragments (CAPS}, allele-specif c iigation, nucleotide extension reactions, Radiation Hybrid Mapping and Happy Mapping. For these methods, the sequence wo oons93~ rcT~s9sizasoz 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 knov~m to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences 5 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 ahould 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 1S the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
GENERAL METHC
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 Cdoning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, {1989) (Maniatis) and by T. J.
Silhavy, M. L. Be~.an, and L. ~V. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F.
25 M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and wiley-Interscience (1987).
EXAMPLE I
Composition of cDNA Libraries' isolation and Sequencing of eDNA Clones cDNA libraries representing mRNAs from various maize tissues were prepared. The characteristics of the libraries are described in Table I .

WO 00/1$937 PCTIUS9$120502 cDNA Libraries From Corn Tissues GST

LibraryClass Clone Tissue .

bmsl I brnsl.pk0023.g8Maize BMS cell culture 1 day after subculture csl I cs,l.pk0010.c5Maize leaf, sheath 5 wk plant Stratogene #837201 cebl I ce;bl.pk0017.a5Maize embryo cc7lse III cc:7lse-a.pk000I.g2Maize class II callus tissue, somatic embryo formed, highly transformable cc7lse III cc:7lse-b.pk0014.b8Maize class II callus tissue, somatic embryo formed, highly transformable ceb5 III ce:b5.pk0051.f8Amplified maize embryo 30 day crln III crln.pk0003.b1Maize root from 7 day seedlings grown in light normalized crln III crln.pk0014.g8Maize root from 7 day seedlings grown in light normalized m II m.15.5.d06.sk20Maize 15 day embryo library crln II crln.pk0040.ei2Maize root from 7 day seedlings grown in light ' normalized ceb5 III ce:b5.pk0049.a11Amplified maize embryo 30 day csl III cs;l.pk0059.e2Maize leaf, sheath 5 wk plant Stratogene #837201 cDNA librariies were prepared in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). The Uni-ZAPTM XR librwies were converted into plasmid libraries according to the 5 protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasrnid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing :recombinant pBluescript plasmids were amplified via polymerase chain re;actir~n using primers specific for vector sequences flanking the inserted cDNA sequences. 'Amplified insert DNAs were sequenced in dye-primer 10 sequencing reactions to generate partial eDNA sequences (expressed sequence tags or "ESTs"; see .Adams, M. D. et al., (1991) Science 252:1651). The resulting ESTs were analyzed using. a Perkin Elmer Model 377 fluorescent sequencer.

Identification and Characterization of cDNA Clones 15 cDNAs encoding maize GST enzymes were identified by conducting BLAST (Basic Loca Alignment Search Tool; Altschul, S. F., et al., (1993) J.
Mol.
Biol. 215:403-410; ;;ee also www.ncbi.nlm.ni.h.gov/BLASTn searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 20 3-dimensional structure Brookhaven Protein Data Bank, 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 I;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 IO 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 acnd the BLAST "hit" represent homologous proteins.
All comparisons were done using the BLASTNnr algorithm with the exception of crln.pk0040.e12 where BLASTXnr was used. The results of the BLAST comparison is given in Table 2 and summarizes the clones and the sequences to which tl;~ey have the most similarity. Each cDNA identified encodes at least a portion of either a GST class I, II, or iII. All isolated clones contain a full length open reading frame (ORF) with the exception of cc71 se-a.pk0001.g2 which is only a partial clone. Example S describes the sequencing strategy for the above described clones.

BLAST Results For Clones SEQ
TD
NO.

GST Blast pLog Clone ClassSimilarity IdentifiedBasePeptideAlgorithmScore bmsl.pk0023.g8I )C79515jZMGST27 Z.mays1 2 Nnr 122.086 CiST-27 mRNA for glutathione-S-transferase csl.pk0010.c5I I)17673jATHERD13 3 4 Nnr 8.16 ~

Arabidopsis thaliana mRNA far g;lutathione S-transferase cebl.pk0017.a5I 7i;78203jHMGST H.muticusS 6 Nnr 21.51 rnRNA for glutathione S-transferase cc7lse-a.pk0001.g2III (AF004358) glutathione7 8 Nnr 16.48 S-transferase TSI-1 (Aegilops squarrosa) cc7lse-b.pk0014.b8III i)10861jRICORFC Rice9 10 Nnr 14.96 mRNA

for a protein related to chilling tolerance cebS.pk0051.f8III I)10861jRICOjitFC 11 12 Nnr 40.44 Rice mRNA

for a protein related to chilling tolerance SEQ ID NO.
GST Blast pLog Clone ClassSimilarity IdentifiedBase PeptideAlgorithmScore crln.pk0003.b1III U806154EGU80615 Eucalyptus13 14 Nnr 24.70 gifobulus auxin-induced protein (I:gPar) mRNA, complete cds crln.pk0014.g8III iv(16901~MZEGSTIB 15 16 Nm. 5.85 Maize ~

gfutathione S-transferase (GST-I) mRNA, camplete cds m.15.5.d06.sk20II ~M97702~DROGLUSTD 17 18 Nnr 3.63 D~rosophila meianogaster g'.lutathione S-transferase gene crln.pk0040.e12II 167970 (L05915) (GSTl)19 20 Xnr 42.03 gene product (Dianthus caryophyllus) ceb5.pk0049.a11III p.'12862~ ZYMYI2862 21 22 Nm. 0.0 Zea lviaize riiRNA for glutathione S-transferase csLpk0059.e2III D~108b1~RICORFC Rice24 25 Nnr 41.03 rr~RNA for a protein related to chilling tolerance Ex~~ression of Chimeric Genes Encoding Maize GS'C Enzymes in Maize Cells (Monocotyledon) A chimeric gene comprising a cDNA encoding a maize GST enzyme in sense orientation can be constructed by polymerase chain reaction (PCR) of the cDNA clone using alrpropriate 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 ~L 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, i.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 !a5 °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 Ncol and SmaI and fractionated on a 0.7% Iow melting point agarose gel in 40 mM Tris-acetate, pH 8.5, 1 mM EDTA. The appropriate band can be excised from ithe 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 at ATCC (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852), and bears wo oons~3~ ~cTms9snosox accession number A'rCC 97366. The DNA segment from pMLI03 contains a 1.05 kb SaII-NcoI promoter fragment of the maize 27 kD zero gene and a 0.96 kb Smal-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 5 DNA can be ligated .at 15 °C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E, coli XL1-Blue (Epicurian Coli XL-1; Stratagene). I3acterial 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). The 10 resulting piasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the mai~:e 27 kD zein promoter, a eDNA fragment encoding a plant GST enzyme, and the 10 kD zein 3' region. The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissc;cted from developing caryopses derived from crosses of the 15 inbred corn lines H9'9 and LH132 (Indiana Agric. Exp. Station, Indiana, USA).
The embryos are isollated 10 to 11 days after pollination when they are I .0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27 °C. Friable embryogenic 20 callus consisting of tmdifferentiated 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 25 Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectalble 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 30 of the 35S promoter from Cauliflower Mosaic Virus (Udell et al. (1985) Nature 313:810-812) and the 3M region of the nopaiine synthase gene from the T-DNA
of the Ti plasmid of Agrobacterium tumefaciens. The particle bombardment method (Klein et al., (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles ({1 ~cm in 35 diameter) are coated with DNA using the following technique. Ten ug of plasmid DNAs are added to '.>0 uL of a suspension of gold particles (60 mg per mL).
Calcium chloride (51) ~L of a 2.5 M solution) and spermidine free base (20 ~.L
of a 1.0 M solution) are; added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rprn) and the supernatant removed. The particles are resuspended in 200 wL of absoluite 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 ~tL) of the DNA-coated gold particles 5 can be placed in the center of a flying disc (Bio-Rad Labs, 861 Ridgeview Dr, Medina, OH). The particles are then accelerated into the corn tissue with a PDS-1000/He (Bio-Rad Labs, 86I 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. For bombardment, the embryogenic tissue is placed on filter paper over agarose-10 solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1 OOO/He approximately 8 cm from the stopping screen. The air in tha chamber is then evacuated to a vacuum of 28 inches of Hg.
The macrocarrier is accelerated with a helium shock wave using a rupture 15 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, 20 areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium. 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 25 can be transferred to regeneration medium (Fromm et al., (1990) BiolTechnology 8:833-839).

Expression of Chimeric Genes in Tobacco Cells (Dicotvledonl Cloning sites {XbaI or Smal) can be incorporated into the oligonucleotides 30 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 Smal and fractionated on a 0.7% low melting point agarose gel in 40 mM Tris-35 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 mus;aic virus 3~S promoter Linked to a cD~:~ tra~~ment encodin~' a plant GST enzyme .and the nos terminator 3' region flanked by the left border region. The resulting plasmid could be mobilized into the .~ s,~ruhucterirrm strain LBA44041pAL440.~ (Hoekema et al. :Vcrture 303:179-180, ( 1983) using tri-parental matings ( Ruvkin and .Ausubel, :Vaturc 289:8-88, ( 198I )). The resulting .~grobcrctcrizrm strains could be then cocultivated with protoplasts (van den Elzen et al. Plant .~lol. Bioh x:149-1 ~4 ( 1980) or leaf disks (Horsch et al.
Science 327:1229-1231. (198:5)) of ~Vicotiancr tabacum cv Wisconsin 38 and kanamycin-resistant transformamts would be selected. Kanamycin-resistant transformed IO tobacco plants would be regenerated.
EXAMPLE ~
Exnr~ession Of Chimeric Genes In Microbial Cells And Purification Of Gene Product Example S illustrates the expression of isolated full length genes encoding 15 either class I, II or III GST proteins in E. coli.
All clones listed in Table 2 were selected on the basis of homology to known GSTs using tl a BLAST algorithm as described in Example 2. Plasmid DNA was purified using QIAFilter cartridges (Qiagen. Ine., 9600 De Soto Ave, Chatsworth, CA) according to the manufacturer's instructions. Sequence was 20 generated on an ABI Automatic sequencer using dye terminator technology (U.S.
5366860; EP 2?2007;) 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 25 in both directions.
cDNA from the clones bmsl.pk0023.g8, csl.pk0010.c~, cebl.pk00i7.a5, m.15.5.d06.sk20, ceb5.pk0049.a.11, ceb5.pk0051.f8, and csl.pk0059.e2; encoding the instant maize GS'C enzymes were inserted into the ligation independent cloning (LIC) pET30 vector (Novagen, Inc., 597 Science Dr, Madison, WI) under 30 the control of the T7 promoter, according to the manufacturer's instructions (see Novagen publications "LIC Vector Kits", publication number TB 163 and U.S.
4952496). The vector was then used to transform BL21(DE3) competent E. toll hosts. Primers with a specific 3' extension designed for ligation independent cloning were designed to amplify the GST gene (Maniatis). Amplification 35 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. toll 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 BL21(DE3) competent cells (Novagen) and selected on LB agar plates containing ~0 l.tg/mL kanamycin. Colonies arising trom this transformation were grown overnight at 37 °C in Lauria Broth to OD
600 = 0.6 and induced with L 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, l~Iedina, OH) and quantitated spectrophotometrically using BSA as a standard. Protein data is tabulated below in Table 3.

Protein Expression Data CLONE OD. 280 ,_. ., bmsl.pk0023.g$ 0.57 cs l .pk00110.c5 0.53 ceb l .pk0017.a5 0.50 m.15.5.dt)6.sk20 0.39 ceb5.pk0049.a11 2.06 ceb5.pk01)51.f8 1:30 cs l.pk00:i9.e2 1.45 Screening Of Expressed GST Enzymes For Substrate Metabolism The GST enzymes, expressed and purified as described in Example 5 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 purif ed 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 p.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 remove precipitated protein, and then the supernatent was removed and added to 65 pl of water. This sample was chromatographed on a Zorbax C8 reverse phase HPLC column (3 pm particle size, 6.2 rnm x 8 cm) using a combination a~f linear gradients ttlow = l .s mL/minl of l°,~o H;PO.I in water (solvent A) and 1% H,P04 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 12 min. Control reactions without enzyme were performed to correct for uncataiyzed reaction. Quantitation of metabolites were based on an assumption that the extinction coefficient of the conjugate was identical to that of the electrophilic substrate:
Table 4 shows the activity of each enzyme measured in nmol~min-1~mg-~
with the seven different substrates. activities are related to the activities of the known and previously isolated and purified GST enzymes, BZ-II (Marrs et al..
Nature 375:397-400 (1995)), pIN2-I (Hershey et al., Plant Molecular Biology 17:679-690, (1991)), GST-I, GST-III, and GST-IV, collectively described in Shah et al., Plant Mol Biol 6, 203-211 (1986); Jepson et al., Plant Mol Biol 26:1855-1866, (1994); Moore et al., Nucleic Acids Res I4:7227-7235 (1986); and Hoit et al., Planta l9Ei:295-302, (1995).

1,2-epoxy-3-Chlor- (p-nitro-GST Imuron- Ethacrynict-Stilbenephenoxy) GST Name ClassEther AlachlorAtrazineCDNI3 Acid Oxide propane csl.pk0059.e2III O.I 8 0.02 1348 20 1.25 43 ceb5.pk0049.a1IIII 0.4 18 0.01 3939 102 0.01 30 ceb5.pk0051.f8III 1.9 27 0.08 2I36 117 0.02 14 BZ-II III 0.2 0 0.00 I S 23 0.05 0 cebl.pk0017.a5I 0.1 d 0.00 15 5 0.00 0 csl.pk0010.c5I 0.1 0 0.00 30 9 0.00 0 bmsLpk0023.g8I 0.2 0. 0.00 15 13 0.00 0 GST-IV I 0.3 1 U.00 15 13 0.00 0 GST-I F 0.4 77 0.60 46485 32 0.98 92 GST-III I 0.3 3 0.05 1803 1 0.31 28 m.I5.5.d06.sk20II 0.1 0 0.00 45 17 0.00 t p IN2-1 I 0 0 --- I S --- --- -V

MISSING AT THE TIME OF PUBLICATION

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) ADDRESSEE: ...I. DU PONT DE NEMOURS AND COMPANY
(Bi STREET: 1007 MARKET STREET
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(F) ZIP: 19898 (G) TELEPHONE: 302-892-7229 (H) TELEFAX: 302-773-0164 , (I) TELEX: 6717325 (ii) TITLE OF INVENTION: PLANT GLUTATHIONE-S-TRANSFERASE
ENZYMES
(iii) NUMBER OF SEQUENCES: 29 (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. KINB
(B) REGISTRATION NUMBER: 34,850 (C) REFERENCE/DOCKET NUMBER: CL-1128 (2) ~~IeORMATIC)N :t)R. SEQ ID .'~c):1:
( i ) SEp_UE:ICE CHARACT3RISTICS
(A) LENGTH: 844 base pairs (B) TYPE: nucleic acid (C) STRANDEDNES~: singly (D) TOPOLOGY: _inear (ii) MOLECULE TYPE: ~~NA
.(isi) HYE'OTHETICAL: NO
(iv) ANT'I-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
(B) CLONE: BMS1.PK0023.G8 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1;

GGTGTGGCGG CGACCACCGC: CGGCCGGAGC ACCTCGCCAA AAACGCGTTC GGTGAAATCC 180 ACGCGTGGAT GGAGGTGGAA, GCCCACCACA TGGAGCCGGC CCTGTGGCCC ATCATCCGCC 360 ACAGCATCAT CGGCCAGTAC' GTCGGCCGCG AGCGCGACCA CCAGGCCGTC ATCGACGAGA 920 GGTACGCCAG CGGGAACATA CCATAGGCTA GR.AGCGGTGG GCGTCCGTCA TTCTGCAGAT 720 CTGAGGTCTC TGAACCTCAG CGTTTCCGAT Ir.AACATGCAT GCTTTATGTA CTGTTTAr~.rlA 780 AACAAACCTG ATTGGTGCAG GGTATTTTAG T.CCTCTTAAA P,~~AAAAAAAA AAAAI~Ae'~1.~A 840 (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids (B) TYPE: amino acid ;C) STRRNDEDNESS; not relevar.'.
;Df TOPOLOGY: ::~t ~?lavan=
'_) MOLECULE T'fPE: ~rotein ri ) JRIt.;INAL SOURCE
(P) TISSUE TYPE: MAIZE
;o:.i) IMMEDIATE SOURCE:
(B) CLONE: BMS'_.PK0023.G3 ;ai) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Pro °ro IHet Lys Val Tyr G1y Trp Ala Val Ser Pro Trp Met i0 '5 Ala Arg Ala Leu 'Jal Cys Leu Glu Glu Ala Gly Ala Asp Tyr Glu Ile Val Pro Met Ser Arg Cys Gly Gly Asp His Arg Arg Pro Glu His Leu A1a Lys Asn Pro Phe Gly Glu Ile Pro Val Leu Glu Asp Giy Asp Leu 50 55 ~0 Thr Leu Tyr Gln ;Ser Arg Ala Ile Ala Arg Tyr Val Leu Arg Lys Leu Lys Pro Glu Leu :Leu Arg Glu Gly Asp Leu Glu Gly Ser Ala Met Val Asp Ala Trp Met Glu Val Glu Ala His His Met Glu Pro Ala Leu firp loo lay llo Pro Ile Ile Arg His Ser Ile Ile G1y Gln Tyr Val Gly Arg Glu Arg Asp His Gln Ala Val Ile Asp Glu Asn Leu Asp Arg Leu Arg Lys Val Leu Pro Ala Tyr Glu Ala Arg Leu Ser Val Cys Lys Tyr Leu Val Gly Asp Asp IIe Ser Ala Ala Asp Leu Cys His Phe Gly Phe Met Arg Tyr Phe Met Ala Thr Glu Tyr Ala Gly Leu Val Asp Ala Tyr Pro His Val Lys Ala Trp Trp Asp Ala Leu Leu Ala Arg Pro Ser VaI G1n Lys Va1 Met Ala Gly Met Pro Pro Asp Phe Gly Tyr Ala Ser Gly Asn Ile Pro ('?) ~~1FOR;~InTIOPt FOR SEQ iD iI0:3:
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 999 'case pairs (B) TIPE: nucleic dClG'~
(C. STRANDEDNESS: singly D) TOPOLOG'!: linaa~
;iij MOLi~CULE TYPE: CDNA
~'_i i) HYPOTHETICAL: NO
'i~) ANT.:L-SENSE: NO
:~i ) ORIc;IDIAL SOURCE
(F) TISSUE TYPE: MAIZE .
(vii) IMMEDIATE SOURCE:
(B) CLONE: CS.PKOOl0.C5 (xi) SEQCJENCE DESCRIPTION: SEQ ID N0:3:

GTGGCGAAAT GGTATGACAG GCTCTCGAAG CGCGAGACAT GGGTGCAGGT CGTC.~1AGATG 720 CAGAAGGAAC ATCCTGGTGC GTTCAAGTAA TGGCTTGTCT. TGGGGAGTTG TGAGTATGGC 780 TTCATCGTCC GTGTTGuTCT GGCTCATCAG TGTTAAAAGC CCATCAGTGT CGTCAACCAG 840 AATAATGTGA AGCCC.~.ACTG TGATGTATGG TCTTTTTTTT TTAAAAGCGC ATTTGTAA.~C 900 TATTGGCTAT TTCTTGCACG TGCCAATTCA TCGTCACATA TAAAATAP.AC TGTATCTTTG 960 ACCTTGTGTC ATGTACGCA.~1 F,FU~~AAAAAA AAAAAAAAA

t2) i~I=ORWTi~J~1 ~'OR SEQ ID N0:4:
.',i) 3EQ'UENCE CHARACTERISTICS:
(A) LENGTH: 226 amino acids (B) TYPE: amino acid (~C) STRANDEDNESS: not rele~ran=
Di T.OPOLOG'~: nc= relevant 'ii) MOLhCULE TYPE: g=otein !°~i) ORIGINAL SOURCE:
( c~) TISSUE TYPE: ~IA_TZ
(vii) IMMEDIATE SOURCE:
(A) LIHRARY: .CS.2KOOl0.C5 (xi) SEQ(JENCE DESCRIPTION: SEQ ID N0:4:
Met Ala Ala Gly Leu Gin Val Phe Gly Gln Pro Ala Ser Thr Asp Val Ala Arg Val Leu 'rhr Cys Leu Phe Glu Lys Lys Leu G1u Phe Glu Leu Val Arg Ile Asp 'Chr Phe Lys Thr His His Arg Leu Pro Glu Phe Ile Arg Leu Arg Asp 1?ro Asn Gly Gln Val Thr Phe Lys His Gly Asp Lys Thr Leu Val Asp :3er Arg A.sp Ile Cys Arg Tyr Val Cys Asn Gln Phe Pro Asn Tyr Gly i~sn Lys Ser Leu Tyr Gly Ser Gly Ala Leu Glu Arg Ala Ser Ile Glu c;ln Trp Leu Gln Ala Glu Ala Gln Asn Phe Gly Pro Pro Ser Ser Ala )Leu Val Phe Gln Leu Ala Phe Val Pro His Leu Ser His Leu Gly Val i~rg Gln Asp Pro Ala Val Ile Ala Glu Asn Glu Asp Lys Leu Lys Gln 'Jal Leu Asp Val Tyr Aso Glu Ile Leu Ser Lys Asn 195 150 155 i60 Glu Tyr Leu Ala c;ly Asp Glu Phe Thr Leu A1a Asp Leu Ser His Lau Pro Asn Ser His 'Pyr Ile Val Asn Thr Glu Arg Gly Arg Lys Leu Phe T.hr Asn Lys Lys ~Asn Val Ala Lys Trp Tyr Asp Arg Leu Ser Lys Arg J

~Jiu Thr ?',-._-, 'i~~ __.. 'J,~1 'J.~' ;5 Met :,ln 'y ___ :i?:, Pro «ly Aia 210 % 1 ~ = ? ':
Phe Lys (2) I~L-~ORMATTnN .OR SEQ iD ~i0:5:
(i; SEQCENCE CHARACT~RI~TICS:
(A) Lc.NGTH: 3CV base baits (B) T~~'~.~'"..: nl:Cl?iC aCl(j ,cJ; S'"RANDEDNESa: single (D) TOPOLOGY: _inear (ii; MOi,EC~JLE Tt°E: ~~NA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: , (E) TISSUE TYPE: MAIZE
{vii) IMMIEDIATE SOURCE:
(B) CLONE: CEBi.PK0017.A5 I
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
i TTACATATGC GACCAGTATC~ CGGACTCT'GG TARTCAGGCC CTCTTCGGCA AGAAAGAAGA 300 CGGCGCGGTT GGCCGCGCTGi CCATTGAACA GTGGATAGAG TCTGA.~GGCC AGAGCTTTAA 3C0 i TGACCTGGCT GTGGTTGAGC; AAATGAAGCG AAGCTTGCGA AGGTGCTTGA TGTGTATGAC 480 CACTGAGATG CAGAGCACGC: CGAGGCCCTC TTAGAGCTTT TTTTTGGGTT TCTTTGAGCA 720 GCTTCTGATG GCAATTAGT7.' GCATTCTCCT TGTTTTGTCA TCAAGTCCTT GTCTGTACCG 780 TTTCCTGTTC TCTTATTTA'l.' CGGTCTTAAT TCTTGATCTA TGTATGGTTT GGATCTGTTC 840 TTCTGGTCCT TTAGTTTATA TAAGTACCTA CPATTCTTCA AAA.~AAAAAA AAAAAAAAAA 900 (2) IN~ORMP.TIOf'I i:OR SEQ ID NO:o:
( i ) SEQi;JENCE CHAxZACTERISTICS:
(A) LENGTH: 199 amino acids (E3) TYPE: amino acid (C) STRANDEDNESS: not rele~ran=
(D) TOPOLOGY: got relevant (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
l,r) TISSUE TYPE: MAIZ~
;vii) IMMEDIATE SOfJRCE:
(B) CLONE: CE81.PK0017.A~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Ala Ala Pro Val Thr Val Tyr G1y Pro Met Leu Ser Pro Ala Val Ala Arg Val Ala Ala Cys Leu Leu Glu Lys Asp Val Pro Phe Gln I1e Glu Pro Val Asp Met Ser Lys Gly Glu His Lys Ser Pro Ser Phe Leu Lys Leu Gln Pro f?he Gly Gln Va1 Pro Ala Phe Lys Asp His Leu Thr Thr Val Phe Glu Ser Arg Ala Ile Cys Arg Tyr Ile Cys Asp Gln Tyr Ala Asp Ser Gly Asn Gln A1a Leu Phe Gly Lys Lys Glu Asp Gly A1a Et5 90 95 Val Gly Arg Ala Ala Ile Glu Gln Trp Ile Glu Ser Glu Gly Gln Ser .

Phe Asn Pro Pro Ser Leu Ala Ile Ile Phe Gln Leu Ala Phe Ala Pro Met Met Gly Arg Thr Thr Asp Leu Ala Va1 Val G1u Gln Asn Glu Ala Lys Leu Ala Lys Val Leu Asp Val Tyr Asp Gln Arg Leu Gly Glu Ser Gln Tyr Phe Ala Gly Asp Asp Phe Ser Pro Gly Arg Pro Cys Ala Leu 1.65 170 175 Ala Gln Cys Arg Phe Pro Cys Glu G1n Asn Gln Gln Gly Trp Leu Asp His Arg Glu Lys C:lu Ser Cys (2) E;IFORM.ATIOrI FOR SE;2 ID N0:?:
;ij SEQUENCE CHARACTWRISTT_CS:
(A) LENGTH: 458 bass pairs (BI TYPE: nuclaic acid (C:~ gTRANDL:DNEjs: Single (C)i TOPOLOGY: linsa=
(ii) MOhECULE TYPE: ~DPJi-s (iii) HY1?OTHETICAL: NO
( i v) AN':I-SENSE: NO
(vi) OR:CGINAL SOURCE:
(F;I TISSUE TYPE: MAIZE , (vii) IMMEDIATE SOURCE:
(B) CLONE: CC71SE-A.PK0001.G2 (xi} SE(2UENCE DESCRIPTION: SEQ ID N0:7:

GTGCAAGCAA TAACTGAAG,A GGGCATGGTG TATCCGTCAT GTGTTTCAGG TTTTCGTATA 240 CGTCTCGCCG TTAGTTCAG~D TTATGTGATG TGAGTGTTGC CGTGCATGTG TGTGTTACTT 360 AAAAAAAAAA 1~1,F~~A Afi.F~AAAHAAA AAAAAAAA 4 5 8 (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids (B} TYPE: amino acid (C} STRANDEDNESS: not relevant (DD TOPOLOGY: riot relevant (ii) MOLECULE TYPE: protein ( vi } OR:CGINAL SOURCE
(F) TISSUE TYPE: MAIZE
(vii} IMMEDIATE SOURCE:
(B) CLONE: CC71SE-A.PKOOOOl.G2 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ala Ser Glu Glu Leu His Gly Val Arg Pro Phe Asp Pro Glu Arg Thr g WO 0011$937 PCT/US9$/20502 Pro Leu Leu Ala Aia Trp Sar G;v Arg Phe Gly Ala Lan Asp Ala Val Gln Thr Val Met Pro Asp '.1a1 G'y Arg Leu Leu Glu ?he Gly Lys Ala 3 5 4 . -~ 5 Leu Met Ala Arg Leu Ala :'~la .__ Al.a Ala Pro Va 1 31r. Ala Ile Thr 50 ,., 60 Glu Glu Gly Met Val Tyr Pro Sar rys Val Ser Gly ?he arg Its Val 05 ~0 75 80 Asn Lys Lys Gly Lys Asn Asn A_a Ser Tyr Ala Ser Glu Arg Gly Phe Val Leu Cys Arg Leu Ala Val Se= Ser Ala Tyr Val Met (2) INFORMATION FOR SEQ ID N0:9:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 911 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) AN7.'I-SENSE: NO
(vi) OR7:GINAL SOURCE:
{F) TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
{B) CLONE: CC71SE-B.PK0014.B8 (xi) SE()UENCE DESCRIPTION: SEQ ID N0:9:
GCAAGGTCGA CATGTCGTC'C CCGCCGCCGG TGAAGCTGAT CGGCTTCTTC GGCAGCCCGT 60 AGGACCTGTT CGGCAGCAAG AGCGAGCTCC T~CTCCACCA CAACCCCGTG CACAAGAAGG 180 ACGTCGACGA GGCCTTCGAI~ GGGCCGCCGC TGCTCCCCGC CGACCCCTAC GCGCGCGCCG 300 CGCTGCTGGA GGCGCAGCTC GAGGGP.AAGA G,sTTCTTCGC CGGCGACAGG CCGGGGTACC 480 WO 00118937 PC1'/US98/20502 TGGCGCTGCT CAGCGAGGr"1T GACCACCCCA ACCTGTGCCG ~~TGGACCAGG GAC:'ACTGC.z 600 CCTTCGAGGC TCTCAAGCC;G TGCATGCCGG ATCGGGAGAA GCTCCTCGCC TACTTCACTA 66G
AGAACTTCGA CAGGTACAF,G GCGGC~_~.GTCA ATGCGACGCT ATCGCAGTC:G CAGCAGTAAT 72O
AACTGCCCAA CTGGGTACGC CTCTGCCCGG CCGTATGGCG GGCGTTTCTT TTTTTCTTT~ 780 TTCAGAATAA CGTAGCTGTG CCCAGTACTC ATGTTTTCAA TTCTGCAA~G TGCe'~P.ACCA~ 84G
CAAGTCGCTG TGTGGTTTA.C TCTTTTT.~r? At~.PG~AAAAAA .~AAr~AAR.Ar'1A
ArIAA.eAAAAA~. 900 AAAAAAAAAA a 91t (2) INFORMATItaN FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A:) LENGTH: 235 amino acids (B;I TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
(B) CLONE: CC71SE-B.PK0014.B8 (xi) SEC>.UENCE DESCRIPTION: SEQ ID N0:10:
Met Ser Ser Pro Pro Pro Val Lys Leu Ile Gly Phe Phe Gly Ser Pro Tyr Ala Phe Arg Ala Glu Ala Ala Leu Cys Leu Lys G1y Val Pro Tyr Glu Leu Ile Leu Glu Asp Leu Phe Gly Ser Lys Sex Glu Leu Leu Leu 35 ' 40 45 His His Asn Pro Val His Lys Lys Val Pro Val Leu Leu His Gly Asp Gly Arg Ala Ile Ser Glu Ser Leu Val Ile Ala Glu Tyr Val Asp Glu Ala Phe Asp Gly Pro Pro Leu Leu Pro Ala Asp Pro Tyr Ala Arg Ala Ale Aia Arg Phe Trp Ala Asp Phe I1e Glu Thr Arg Leu Thr Lys Pro Phe Phe Met Ala Ile Trp Val Glu Glu Arg Asp Ala Arg Leu Arg Phe Glu Glu Glu Ala Lys G1u heu Val Ala Lau Leu Gl:a Ala :~ln Lau Glu 130 13:, iaC
Gly Lys Arg Phe Phe Ala Gly Asp Arg Pro Gly Tyr Leu Asp ~Jal Ala Ala Ser Ala Leu Gly Pro 7.'rp Arg Ser Val Ile Giu ~~lu Leu Asn Gly Val Ala Leu Leu Ser Glu Asp Asp His Pro Asn Leu Cys Arg Trp Thr Arg Asp Tyr Cys Ala Phe Glu Ala Leu Lys Pro Cys Met Pro Asp Arg Glu Lys Leu Leu Ala Tyr Phe Thr Lys Asn Phe Asp Arg Tyr Lys Ala Ala Val Asn Ala Thr Leu Ser Gln Ser Gln Gln (2} INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 948 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii} MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi). ORIGINAL SOURCE:
(F) TISSUE TYPE: maize (vii) IMMEDIATE SOURCE:
(B) CLONE: ceb5.pk0051.f8 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:

CGTTCGGGTT CnT.GGACVT'G GCGCTCGTGC CCTTCGTGCC GTGGCTC'.',.'.C AGCTAC~~AGC 540 GGTACGGGGA C'r'TCAGCGTG GCGGAGATCG CGCCCAGGCT GGC;:~GCG"_'GG GCGCGCCGGT 600 GCGCGCAGCG GGAGAGCGTG GCCAGG?CCC TTCACCCGCC GGAAAAGGTG GACGAGTTCA. 6n0 TCAACCTGCT. CAAGAAGACC TACGGCATCG AGTAGTAGAG CGGACTACTA CTAGCAGAGG 720 AGATGGTACC GGCCGTACGT ACGTGGCTGC CATGCAGTTT TTGTTTCGG~T TTGTTTAAAC 780 AGGTGAACTA AAATCACGGT AAAAACTCGG AAATTAGTTT GTAAAGGGTC CAGCCCCCCT 90C .
CCTTTATAAA TAGAGAGGT.A TACGGCTGAT AAAAAAP.AAA A,P~F1AAAAA 948 (2) INFORMATION FOR SEQ ID N0:12:
(i) SE(ZUENCE CHARACTERISTICS:
(A) LENGTH: 225 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (vi) OR7:GINAL SOURCE:
(F) TISSUE TYPE: maize (vii) IMMEDIATE SOURCE:
(B) CLONE: ceb5.pk0051.f8 (xi) SEC>.UENCE DESCRIPTION: SEQ ID N0:12:
Met Ala Gly Glu Thr Lys Lys Gly Leu Val Leu Leu Asp Phe Trp Val Ser Pro Phe Gly Gln Arg Cys Arg Tle Ala Leu Ala G1u Lys Gly Ile Ala Tyr Glu Tyr Ser Glu Gln Glu.Leu Leu Gly Gly Ala Lys Ser Asp Ile Leu Leu Arg Sex Asn Pro Val His Lys Lys Ile Pro Val Lau Leu His Asp Gly Arg Pro Val Cys Glu Ser Leu Val Ile Leu Glu Tyr Leu Glu Glu Ala Phe Pro Glu A.la Ser Pro Arg Leu Leu Pro Asp Ala Ala Tyr Ala Arg Ala Gln Ala Arg Phe Trp Ala Ala Tyr Ser Asp Lys Val Tyr Lys Ala Gly Thr Arg Leu Trp Lys Leu Arg Gly Asp Ala Arg Ala Gln Ala Arg Ala G1~.~ Ila 'a1 Gln Val '!al Arg asr Leu Asp Gif Glu 130 i35 14 Leu Gly Asp Lys Ala Phe Phe Gly Gly Giu Ala Phe Gly Phe Vai Asp 145 150 I55 i60 Val Ala Leu Val Pro Phe Val ?ro Trp Leu aro Ser Tyr Glu Arg Tyr 1&5 170 175 Gly Asp Phe Ser Va1 Ala Glu Ile Ala ?ro Arg Leu Ala Ala Trp Ala Axg Arg Cys Ala Gln Arg Glu Ser Val Ala Arg Thr Leu His Pro Pro 195 zoo 205 Glu Lys Val Asp Glu Phe Ile Asn Leu Leu Lys Lys Thr Tyr Gly Ile Glu {2} INFORMATION FOR SEQ ID N0:13:
(i} SEC}UENCE CHARACTERISTICS:
(A) LENGTH: 840 base pairs (B} TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOhECULE TYPE: cDNA
(iii) HYE'OTHETICAL: NO
{iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: MAIZE
(vii) IMN.IEDIATE SOURCE:
(H) CLONE: CR1N.PK0003.B1 (xi) SE~!UENCE DESCRIPTION: SEQ ID N0:13:
GTTGGGGATG TGGGCGAGCC: CTATGGTGAT CAGGGTGGAG TGGGCGCTGC GGCTGAAGGG 60 CGACCCCTAC GAGCGTGCCC: AGGCAAGGTT CTGGGCCAGG TTCGCTGAAG ACAAGTGCA.~1 300 CGCTGCTCTG TACCCGATC9.' TCACCGCGAC CGGCGAGGCG CAGCGCAAGG CGGTGCACGA 360 GGACGCCGTG GGCTACCTC'G ACATC,_,TCGT CGGGTGGTTC GCGCaCT~:~: TCC=CGTCAT 43.;
CGAGGAGGTG ACCGGCGCCA GCGTCGTCAC CGACGAGGAG CTGCCGCTGA T~PAGGCCTG 590 CCTCGCCGCC P.ACAAGGCCC GCCGTGAGCA GCTCCTCTCC GCGTAGATGG CT.?~GTe'~ATTC 660 AATGGTAGTC CCATAATAAT GCATATACAT CATGCATAAA AAAAAAA.~1A AAPAAAAr'lArn 840 (2} I'1FORMATION FOR SEQ ID N0:14:
(i) SE~ZUENCE CHARACTERISTICS:
(A) LENGTH: 212 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii} MO?~ECULE TYPE: protein (vi) OR7~GINAL SOURCE:
(F) TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
(B) CLONE: CR1N.PK0003.B1 (xi} SECZUENCE DESCRIPTION: SEQ ID N0:14:
Met Trp Ala Ser Pro Met Vai Ile Arg Val Glu Trp Ala Leu Arg Leu Lys Gly Val Glu Tyr Glu Tyr Val Asp Glu Asp Leu Ala Asn Lys Sex Ala Asp Leu Leu Arg His Asn Pro Val Thr Lys Lys Val Pro Val Leu Val His Asp Gly Lys Pro VaI Ala Glu Ser Thr Ile Ile Val G1u Tyr Ile Asp Glu Val Trp Lys Gly Gly Tyr Pro Ile Met Pro Gly Asp Pro Tyr Glu Arg Ala Gln Ala Arg Phe Trp Ala Arg Phe Ala Glu Asp Lys g5 g0 95 Cys Asn Ala Ala Leu Tyr Pro Ile Phe Thr Ala Thr Gly Glu Ala Gln Arg Lys Ala Val His Giu Ala Gln Gln Cys Leu Lys Thr Leu G1u Thr Ala Leu Asp Gly Lys Lys Phe Phe Gly Gly Asp Ala Val Gly Tyr Leu Asp Ile Val Val Gly Trp Phe ala His Trp Leu Pro Val Ile Glu Glu Val Thr Gly Al~a Ser Val Val ~:ar Asp Glu Glu Leu Pro Leu Met Lys Ala Trp Phe Gl;y Arg Phe Leu a_a Val Asp Val Val Lys Ala Ala Leu Pro Asp Arg Asp Arg Leu Leu Ala Ala Asn Lys Ala Arg Arg Glu Gln 195 ''00 205 Leu Leu Ser Ala (2) INFORMAT:CON FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 861 base pairs (F3) TYPE: nucleic acid , (C) STRANDEDNESS: single , (t)) TOPOLOGY: linear (ii) MOLECULE TYPE: 'cDNA
(iii) H'tPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
{f?) TISSUE TYPE: MAIZE
{vii) ID~IMEDIATE SOURCE:
(f3) CLONE: CRIN. PK0019.G8 (xi) Sf:QUENCE DESCRIPTION: SEQ ID N0:15: ' CGGAGGCGCA GAGCTTCGi~.C GCGCCCAGCG CCGAGATGGT CTACAGCCTC GCCTTCCTGC 60 CGCCCACCCT GCCCAAGCi4G AACGAC~ACG GCAACGGCGG CGCGTTCAAC GCCAGGGACG i20 AGCAGAGGAA GAAGGACC'PG GAGAAGCTGC TGGACATCTA CGAGCAGCGC CTGGAGGAGG 300 CCACGTTCCT GGCCGGCGAC AACTTCACCA TCGCCGACCT GTCGCACCTG CCCTACGCGG 360 ;

ACTTCTAGCT GTTGCCGTC;C CTTCCCGCCG ACGAATAAAC TACCTGC~.:~ GCCGC~ACCG EinO
CCGCCATCCA TCAACATGCiT TCCTTGTGCT GTTCGTGTCG TTTTCATA~,G 'TCATACGTGT 7~~
CTTGCTGCTT TTGAAGCTC;C GTTCCCGGGT GCAGGGACCT ACGAGTC~aT TCCGTCGTTT 780 GCTGATTCTG T.TCGTCGTCiT AATAAAATGA ?.AACCCCACC CCGTTT'" ~.a TGAAP.AAAAA 840 AAAAAAA~1AA AAAARAAAP~A A 8 61 (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 190 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: maize (vii) IMMEDIATE SOURCE:
(B) CLONE: crln.pk0014.g8 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Val Tyr Ser Leu Ala Phe Leu Pro Pro Thr Leu Pro Lys Gln Asn Asp Asn Gly Asn G1y Gly Ala Phe Asn Ala Arg Asp Ala Thr Val Gly Ser Asn Ala Asp Ala Ser Ser Gly Lys Arg Gly Val Ala Gly Ser Gln Pro Ala Ala Ser Gln Thr Lys Val Ser Ala Gln Lys Glu Glu Glu Met Leu Lys Leu Phe Glu Gln Arg Lys Lys Asp Leu Glu Lys Leu Leu Asp Ile Tyr Glu Gln Arg Leu Glu Glu Ala Thr Phe Leu Ala Gly Asp Asn Phe Thr Ile Ala Asp Leu Ser His Leu Pro Tyr Ala Asp His Leu Val Ser Asp Pro Arg Ser Arg Arg Met Phe G1u Ser Arg Lys Asn Val Ser Arg Trp Trp His Asp Val Ser Gly Arg Asp Thr Trp Lys Tyr Val Lys Thr Leu Gln Arg Pro Pro Ser Thr Ser Thr Asp Ala Ser Ala Lys Asn Ifi Gly Gln Leu .Giy~~Gln Gln Gln H_s Leu Pro Ser Ser Thr Asp GIy His Gly Vai Lys Thr Gln Arg Leu 'Jal Gln Asn Glu Arg His Phe .80 185 190 (2) INFORMATION FOR aSEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 917 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D} TOPOLOGY: linear (ii) MOLECULE T'1PE: cDNA
(iii} HYPOTHETICAL: NO
(iv) AN'CI-SENSE: NO
(vi) OR:CGINAL SOURCE:
(F) TISSUE TYPE: MAIZE
(vii) IMD4EDIATE SOURCE:
(B) CLONE: M.15.5.D06.SK20 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
ATGGCGGAGG TGGAGGCGAC' GGTGGGGCGA CTGATGCTGT ACTCGTACTG GCGCAGCTCG 60 TGCTCCCACC GTGCCCGCA'.C CGCTCTCAAT CTCAAAGGTG TGGATTACGA GTACAAGGCG 120 TATTTGGAGG ACAAGTACCC: AGAGCCTCCT CTTCTACCTC AAGACCTTCA AAAGAAAGCT 300 CAAATCGAGA GAGGTTTCAC: AGCTATTGAG AACCTGATAC AACTAAAAGG ATGCGCCGGG 480 AAGTATGCAA CAGGAGATGP, AGTCCAACTG GCAGATGTAT TCCTTGCACC CCAGATCTAT 540 GCAGCCATTG AACGCACTAA, AATTGACATG TCAAACTACC TCACTCTTGC TAGGCTCCAC 600 TCGGAGTACA TGTCACACCC' TGCGTTTGAA GCAGCGCTCC CTGGCAAGCA ACCGGACGCC 660 1~

AAAAr~.t'u~AAA AAP.AAAA 9 t (2) INFORMATION FOR SEQ ID N0:18:
(i) SE;QUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids (8~) TYPE: amino acid (C') STRADiDEDPIESS: not r=levan=
(D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
(B) CLONE: M.15.5.D06.SK20 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Ala Glu Val Giu Ala Thr Val Gly Arg Leu Met Leu Tyr Ser Tyr Trp Arg Ser Ser Cys Ser His Arg Ala Arg Ile Ala Leu Asn Leu Lys Gly Val Asp Tyr Glu Tyr Lys Ala Val Asn Leu Leu Lys Gly Glu Gln Ser Asp Pro Glu Phe Val Lys Leu Asn Pro Met Lys Phe Val Pro AIa LeuValAspGly SerSerValIleGly SerTyr AlaIleThr Leu Asp TyrLeuGluAsp LysTyrProGluPro ProLeuLeu ProGlnAsp Leu GlnLysLysAla LeuAsnHisGlnIle AlaSerIle ValAlaSer Gly IleGlnProLeu HisAsnLeuThrVal LeuArgPhe IleAspGln Lys Val Gly Ala Gly Glu Ser Val Leu Trp Thr Gln Gln Gln Ile Glu Arg Gly Phe Thr Ala Ile Glu Asn Leu Ile Gln Leu Lys Gly Cys Aia Gly Lys Tyr Ala Thr Gly Asp Glu Val Gln Leu Ala Asp Val Phe Leu Ala Pro Gln Ile Tyr Ala Ala Ile Glu Arg Thr Lys Ile Asp Met Ser Asn 18.0 185 190 Tyr Leu Thr Leu Ala Arg Leu His Ser G1u Tyr Met Ser His Pro Ala Phe Glu Ala Ala Leu Pro nly Lls Gln Pro Asp Aia °ro Sir Sec Ser (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(.A) LENGTH: 9I9 base pairs (13) TYPE: nucleic acid (C) STRANDEDNESS: singly (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) H'.CPOTHETTCAL: NO
(iv) ANTI-SENSE: NO
(vi) OFtIGINAL SOURCE:
{ ):') TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
{E~) CLONE: CR1N.PK0090.EI2 (xi) SE;QUENCE DESCRIPTION: SEQ ID N0:19:

GAGGGCCATG GCGACCGAG~A AGCCCATCCT GTACAACGCC TGGATCAGCT CCTGCTCCCA 120 CCGTGTTCGC ATCGCACTC;A ACCTCAAAGG TGTGGATTAC GAGTACAAGT CGGTAAACCC 180 TGGGGACATA GTCGTTTCT'G ATTCTCTTGC CATCTCATTG TATTTGGAAG ATAAGTATCC 300 i ATTGGCAGAT GTGTTCCTTG AACCACAGAT ACATGCCGGC ATAP.ATCGCT TCCAAATCGA 600 TCAATAATTT GCATGTCAT'T TTGTAATAAT TTGGATAGGG AGCCACTGCT TCCTCCATCC 780 TTTCTTAAAC AGATACTAT'P TACGGCTATT GTAATTTAAG CCCAAAAAAA AAAAAAAAAA 900 (2) iNE'ORMATION FOR SEQ ID N0:20:
(i) SEQUENCE ~HARACT~RISTICS:
(~a) LENGTH: 212 amino acids (;3) TYPE: amino acid (C) STRAC1DEGNESS: ~ot relevant (!)) TO POLr)GY: not relevant (ii) Mt)LECULE TYPE: protein (vi) ORIGINAL SOURCE:
(.') TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
(Ef) CLONE: CR1N.PK0040.E12 (xi) SE:QUENCE DESCRIPTION: SEQ ID N0:20:
Met Ala Thr Giu Lys Pro Ile Leu Tyr Asn Ala Trp Ile Ser Ser Cys 1 5 i0 15 Ser His Arg Val Arg Ile Ala Leu Asn Leu Lys Gly Val Asp Tyr Glu Tyr Lys Ser Val Asn Pro Arg Thr Asp Pro Asp Tyr Glu Lys Ile Asn Pro Ile Lys Tyr Ile Pro Ala Leu Val Asp Gly Asp Ile Val Val Ser Asp Ser Leu Ala Ile Ser Leu Tyr Leu Glu Asp Lys Tyr Pro Glu His Pro Leu Leu Pro Lys Asp Leu Lys Arg Lys Ala Leu Asn Leu Gln Ile Ala Asn Ile Val Cys Ser Ser Ile Gln Pro Leu Gln Gly Tyr Ala Val Ile Gly Leu His Glu Gly Arg Met Ser Pro Asp Glu Gly Leu His Ile VaI Gln Ser Tyr Ile Asp Lys Gly Phe Arg Ala Ile Glu Lys Leu Leu Glu Gly Cys Glu Ser Lys Tyr Ala Thr Gly Asp Asp Va1 Gln Leu Ala Asp Val Phe Leu Glu Pro Gln Ile His Ala Gly Ile Asn Arg Phe Gln Ile Asp Met Ser Met Tyr Pro Ile Leu Glu Arg Leu His Asp Ala Tyr Met Gln Ile Pro Ala Phe Gln Ala Ala Leu Pro Lys Asn Gln Pro Asp WO 00/18937 PCT/US9$/20502 Ala Pro Sar Ser (2) INFORMATION FOR SEQ ID V0:2i:
ti) SEQUEDICE CHARAC-'=RIST.ICS:
tA) LENGTH:' 99o base pairs (B) TYPE: nuc_eic acid (C) STRANDEDNES~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: :v0 (iv) AF~TI-SENSE: NO
(vi) ORIGINAL SOURCE:
(f) TISSUE TYPE: MAIZE
(vii) ID9MEDIATE SOURCE:
(B) CLONE: CEB5.PFC0099.A11 (xi) SE:QUENCE DESCRIPTION: SEQ ID N0:2I:
CATCGATCCG CCATTGCTC:A CCGCACAAGT GCACGCTCAC CTGACACACG CAGCTAAGTA 60 GCTAAGGCCG TAGGGGAGAA CAAGAAAAGG CTCGACATGG CCGAGGAGAA GAAGCAGGGC 120 ' CTGCAGCTGC TGGACTTG7~G GGTGAGCCCA TTCGGGCAGG GCTGCCGCAT CGCGCTGGAC 180 CTCCGCGCCA ACCCGGTGC;A CAAGAAGATC CCCGTGCTGC TGGACGACGG CCGCCCCGTC 300 CTGCTCCCCG CCGACCCCT'A CGCGCGCGCG CAGGCCCGCT TCTGGGCGGA CTAGGTCGAC 420 AAGAAGCTGT ACGACTGCGG CACCCGGCTG TGGAAGCTCA AGGGGGACGG CCAGGCGCAG 4.80 AGTGTGCTCA GCAGAGCAG'T TCGTGTTCAT GAGTTCGTCG TCGTTGTATT TTCTATTGTC 900 TTCTTCTTGT TGTC;TTAAA~4 AAAAAAAAAA P.AAAAA 996 WO 00/1$937 PCT/IJS9$/20502 (2) INFORMATIODI FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids (B) TYPE: amino acid (~C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) McJLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: MAIZE
(vii) IMMEDIATE SOURCE:
(B) CLONE: CEB5.PK0049.A11 (xi) SE:QUENCE DESCRIPTION: SEQ ID N0:22:
Met Ala Glu Glu Lys Lys Gln Gly Leu Gln Leu Leu Asp Phe Trp Val Ser Pro Phe Gly Gln Arg Cys Arg Ile Ala Leu Asp Glu Lys Gly Leu Ala Tyr Glu Tyr Leu Glu Gln Asp Leu Arg Asn Lys Ser Glu Leu Leu Leu Arg Ala Asn Pro Val His Lys Lys Ile Pro Val Leu Leu His Asp Gly Arg Pro Val Cys Glu Ser Leu Val Ile Val Gln Tyr Leu Asp Glu Ala Phe Pro Glu Ala Ala Pro Ala Leu Leu Pro Ala Asp Pro Tyr Ala Arg Ala Gln Ala Arg Phe Trp Ala Asp Tyr Val Asp Lys Lys Leu Tyr Asp Cys Gly Thr Arg Leu Trp Lys Leu Lys Gly Asp Gly Gln Ala Gln Ala Arg Ala Glu Met Val Glu Ile Leu Arg Thr Leu Glu Gly A1a Leu Gly Asp Gly Pro Phe Phe Gly Gly Asp Ala Leu Gly Phe Val Asp Val Ala Leu Val Pro Phe Thr Ser Trp Phe Leu Ala Tyr Asp Arg Phe Gly Gly Val Ser Val Glu Lys Glu Cys Pro Arg Leu Ala Ala Trp Ala Lys Arg Cys Ala Glu Arg Pro Ser Val Ala Lys Asn Leu Tyr Pro Pro Glu Lys Val Tyr Asp Phe V31 Cys Gly Met Lys Lys Arw Leu Gly Ile Glu 210 215 22~
(2) INFORMATION FOR SEQ ID N0:23:
(i) SIaQUENCE CHARACTERISTICS:
(A) LENGTH: 895 base pairs (~3) TYPE: nucleic acid (C:) STRANDEDNESS: single (i)) TOPOLOGY: linear { i i ) MC>LECULE TYPE : cDNA
(iii) H~'POTaETICAL: NO
(iv) ANTI-SENSE: NO
(vi} ORIGINAL SOURCE:
(F') TISSUE TYPE: MAIZE
(viiy IMMEDIATE SOURCE:
(B) CLONE: CSl.PK0059.E2 (xi} SEQUENCE DESCRIPTION: SEQ ID N0:23:
GGCACGAGAC GACATCGAA.G GAGCCTGCGA AGCGAGCGAG AGTCTATAAT GGCGGACGGA 60 CCCGTGTGCG AGTCGCTCG'T CATCCTGCAG TACGTCGACG AGACCTGGGG AGGCACCGGG 300 ACCCCTCTCC TCCCCGCCGiA CGCCTACGAC CGCGCCATGG CTCGCTTCTG GGCAGCCTAC 360 GCGGCGGAGG CGCTCGGCG'.C CGTCGTCCCC GTGGTGGAGA CGCTGGAGCA GGCGTTCAGG 480 GAGTGCTCCA AAGGGAAACC: TTCTTCGGCG GCGACGCCGT CGGGCTCGTG GACATCGCGC 540 ACGAGGCCAA GTTCCCGGCC: TTGACGGCGT GGGCGGAGCG CTTCTTGGCG GTGGACGCCG 660 TGAAGGAGGT GATGCCGGAC: GCCGGAAGGC TGTTGGAGCA CTACAAGGGG TTTCTGGCTA 720 AACGGTCTCC ACCTGCTGG'F' TACTGAACGC TGTAACTGTA AGCCTGTAAC AGCAAGCTCA 780 (2) I,iFORMATI:ON FOR SEQ iD N0:24:
(i) SE;QUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids (B.) TYPE: amino acid (C') STRANDEDNESS: not relevant (D) TOPOLOGY: not releva~=
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(F) TISSUE TYPE: MAIZE
( vii ) IM1KEDIATE SOURCE
(A) LIBRARY: CS1.PK0059.E2 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Met Ala Asp Gly Gly Glu Leu Gln Leu Leu Gly Ser Trp Tyr Ser Pro Tyr Val Ile Arg Ala Lys Val Ala Leu Gly Leu Lys Gly Leu Ser Tyr Glu Phe Val Glu Glu Asp Leu Ser Arg Lys Ser Asp Leu Leu Leu Lys Leu Asn Pro Val His Arg Lys Val Pro Val Leu Val His Gly Gly Arg Pro Val Cys Glu Ser Leu Va1 Ile Leu Gln Tyr Val Asp Glu Thr Trp Aia Gly Thr Gly Thr Pro Leu Leu Pro Ala Asp Ala Tyr Asp Arg Ala Met Ala Arg Phe Trp Ala Ala Tyr Val Asp Asp Lys Phe Tyr Lys Glu Trp Asn Arg Leu Phe Trp Ser Thr Thr Ala Glu Lys Ala Ala Glu Ala Leu Gly Val Val Val Pro Val Val Glu Thr Leu Glu G1n Ala Phe Arg Glu Cys Ser Lys CJly Lys Pro Ser Ser Ala Ala Thr Pro Ser Gly Ser Trp Thr Ser Arg S_er Gly Ala Ser Trp Cys Gly Ser Gly Trp Trp Thr x.65 170 175 Arg Arg Pro Ala

Claims (22)

What is claimed is:

1. An isolated nucleic acid fragment encoding a Glutathione S-Transferase enzyme selected from the group consisting of (a) an isolated nucleic acid fragment encoding the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO: 16, SEQ ID NO:18, and SEQ ID NO:20;
(b) an isolated nucleic acid fragment that hybridizes with (a) under the selectee hybridization conditions: 0.1X SSC, 0.1% SDS at 65 degrees C; and (c) an isolated nucleic acid fragment that is completely complementary to (a) or (b).--

2. The isolated nucleic acid fragment of Claim 1 selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19

3. A polypeptide encoded by the isolated nucleic acid fragment of Claim 1.

4. The polypeptide of Claim 3 selected from the group consisting of SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.

5. A chimeric gene comprising the isolated nucleic acid fragment of Claim 1 operably linked to suitable regulatory sequences.

6. A transformed host cell comprising a host cell and the chimeric gene of Claim 5.

7. The transformed host cell of Claim 6 wherein the host cell is a plant cell.

8. The transformed host cell of Claim 6 wherein the host cell is E. coli.

9. A method of altering the level of expression of a Glutathione S-Transferase enzyme in a host cell comprising:
(a) transforming a host cell with the chimeric gene of Claim 5 and;
(b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene result in production of altered levels of a Glutathione S-Transferase enzyme in the transformed host cell relative to expression levels of an untransformed host cell.

10. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a Glutathione S-Transferase enzyme comprising:

(a) probing a cDNA or genomic library with the nucleic acid fragment of Claim 1;

(b) identifying a DNA clone that hybridizes with the nucleic acid fragment of Claim 1; and (c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b);

wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a Glutathione S-Transferase enzyme

11. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a Glutathione S-Transferase enzyme comprising:
(a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO: 17, and SEQ ID NO:19 (b) amplifying a cDNA insert present in a loaning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the cf cDNA insert encodes a portion of an amino acid sequence encoding a Glutathione S-Transferase enzyme.

12. The product of the method of Claim 10 ar 11.

13. A method for identifying a chemical compound that inhibits the activity of a GST enzyme encoded by the nucleic acid fragment of Claim 1, the method comprising a steps of:
(a) transforming a host cell with a chimeric gene comprising the nucleic acid fragment of Claim 1 encoding a Glutathione S-Transferase enzyme, the chimeric gene operably linked to at least one suitable regulatory sequence;

(b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in production of the Glutathione S-Transferase enzyme;
(c) optionally purifying the Glutathione S-Transferase enzyme expressed by the transformed host cell;

(d) contacting the Glutathione S-Transferase enzyme with a chemical compound of interest; and (e) identifying the chemical compound of interest that reduces the activity of the Glutathione S-Transferase enzyme relative to the activity of the Glutathione S-Transferase enzyme in the absence of the chemical compound of interest.

14. The method of Claim 13 wherein step (d) is carried out in the presence of at least one electrophilic substrate and at least one thiol donor.

15. The method of Claim 13 wherein said nucleic acid fragment is selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3; SEQ ID
N0:5, SEQ ID N0:9;, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, and SEQ ID NO:I9, and wherein the Glutathione S-Transferase enzyme is selected from the group consisting of SEQ ID N0:2, SEQ ID N0:4, SEQ ID
N0:6, SEQ ID NO:10, SEQ ID N0:12, SEQ ID NO:14, SEQ ID N0:16, SEQ ID
NO:18, and SEQ ID NO:20.

16. A method for identifying a chemical compound that inhibits the activity of a Glutathione S-Transferase enzyme encoded by the nucleic acid fragment of Claim 1, the method comprising the steps of:
(a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment of Claim 1 encoding a Glutathione S-Transferase enzyme, the chimeric gene operably linked to at least one regulatory sequence;
(b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in production of the Glutathione S-Transferase enzyme;
(c) contacting the transformed host cell of step (b) with an inhibitor candidate; and (d) comparing the phenotype of the transformed host cell contacted with an inhibitor candidate with the phenotype of the transformed host cell that was not contacted with an inhibitor candidate to identify the chemical compound that inhibits the activity of the Glutathione S-Tranferase enzyme.

17. The method of Claim 16 wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO:l; SEQ ID N0:3, SEQ ID N0:5, SEQ
ID N0:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID N0:15, SEQ ID NO;17, and SEQ ID N0:19, and wherein the Glutathione S-Transferase enzyme is selected from the group consisting of SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ

ID N0:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID N0:16, SEQ ID N0:18, and SEQ ID N0:20.

18. A method for identifying a substrate fox a Glutathione S-Transferase enzyme, the Glutathione S-Transferase enzyme encoded by the isolated nucleic acid fragment of Claim 1, the method comprising the steps of (a) transforming a host cell with a chimeric gene comprising an isolated nucleic acid fragment of Claim 1 encoding a Glutathione S-Transferase enzyme, the chimeric gene operably linked to at least one suitable regulatory sequence;
(b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in production of the Glutathione S-Transferase enzyme;
(c) optionally purifying the Glutathione S-Transferase enzyme expressed by the transformed host cell;
(d) contacting the Glutathione S-Transferase enzyme with a substrate candidate; and (e) comparing the activity of Glutathione S-Transferase enzyme that has been contacted with the substrate candidate with Glutathione S-Transferase enzyme that has not been contacted with the substrate candidate, selecting substrate candidates that increase the activity ofthe Glutathione S-Transferase enzyme relative to the activity of the Glutathione S-Transferase enzyme in the absence of a substrate candidate.

19. The method of Claim 18 wherein step (d) is corned out in the presence of at least one thiol donor.

20. The method of Claim 18 wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3, SEQ ID NO:5, SEQ
ID N0:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID N0:15, SEQ ID NO:17, and SEQ ID N0:19, and wherein the GST enzyme is selected from the group consisting of SEQ ID N0:2, SEQ ID NO:4, SEQ ID N0:6, SEQ ID NO:10, SEQ
ID N0:12, SEQ ID N0:14, SEQ ID N0:16, SEQ ID N0:18, and SEQ ID NO:20.

21. A method far identifying a substrate for a Glutathione S-Transferase enzyme, the method comprising the steps of (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment of Claim 1, the chimeric gene operably linked to at least one suitable regulatory sequence;

(b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in production of the Glutathione S-Transferase enzyme;
(c) contacting the transformed host cell of step (b) with a Glutathione S-Transferase substrate candidate; and (d) comparing the phenotype of the transformed host cell contacted with the substrate candidate with the phenotype of the transformed host cell that was not contacted with the substrate candidate to identify a Glutathione S-Transferase enzyme substrate.

22. The method of Claim 21 wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ. ID NO:19, and wherein the GST enzyme is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ
ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.