Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
  • C12N9/1088—Glutathione transferase (2.5.1.18)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

• This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding soybean glutathione-S-transferase (GST) enzymes involved in the detoxification of xenobiotic compounds in plants and seeds.
• GST soybean glutathione-S-transferase
• Glutathione-S-transferases are a family of enzymes which catalyze the conjugation of glutathione, homoglutathione (hGSH) and other glutathione- like analogs via a sulfhydiyl group, to a large range of hydrophobic, electrophilic compounds. The conjugation can result in detoxification of these compounds.
• GST enzymes have been identified in a range of plants including maize (Wosnick et al., Gene (Amst) 16 (1) (1989) 153-160; Rossini et al, Plant Physiology (Rockville) 112 (4) (1996) 1595-1600; Holt et al., Planta (Heidelberg) 196 (2) (1995) 295-302), wheat (Edwards et al., Pestic. Biochem. Physiol. (1996) 54(2), 96-104), sorghum (Hatzios et al., J. Environ. Sci.
• Glutathione S-transferases catalyze the nucleophilic attack of the thiol group of GSH to various electrophilic substrates.
• GSTs Glutathione S-transferases
• 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. J Plant Growth Regul 16:95-107, (1997)). They are present at every stage of plant development from early embryogenesis to senescence and in every tissue type examined. The agents that have been shown to cause an increase in GST levels have the potential to cause oxidative destruction in plants, suggesting a role for GSTs in the protection from oxidative damage.
• GSTs 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 (Marrs, 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.
• GST 29 (Shah et al., Plant Mol Biol 6, 203-211 (1986)), GST 27 (Jepson et al. , Plant Mol Biol 26:1855-1866, (1994)), GST 26 (Moore et al., Nucleic Acids Res 14:7227-7235 (1986)).
• GST I a homodimer of GST 29
• GST II a heterodimer of GST 29 and GST 27
• GST III a homodimer of GST 26
• 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, (1995)) and overexpression of GST 27 in tobacco confers alachlor resistance to transgenic tobacco (Jepson, personal communication). Additionally Bridges et al. (U.S. 5589614) disclose the sequence of a maize derived GST isoform II promoter useful for the expression of foreign genes in maize and wheat. In soybean, herbicide compounds conjugated to hGSH have been detected and correlated with herbicide selectivity (Frear et al. , Physiol 20: 299-310 (1983); Brown et al. , Pest 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.
• Glutathione the tripeptide ⁇ -glu-cys-gly, or GSH
• GSH the tripeptide ⁇ -glu-cys-gly, or GSH
• GST glutathione S-transferase
• hGSH Homoglutathione
• nucleic acid fragments encoding soybean GST to use in screening in assays, the creation of herbicide-tolerant transgenic plants, and altered production of GST enzymes depend on the heretofore unrealized isolation of nucleic acid fragments that encode all or a substantial portion of a soybean GST enzyme.
• the present invention provides nucleic acid fragments isolated from soybean encoding all or a substantial portion of a GST enzyme.
• the isolated nucleic acid fragment is selected from the group consisting of (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID 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, SEQ ID NO:20, SEQ ID NO:22, SEQ ID
• nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence sequence selected from the group consisting of SEQ ID 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, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28; and (c) an isolated nucleic acid fragment that is complementary to (a) or (b).
• the nucleic acid fragments and corresponding polypeptides are contained in the accompanying Sequence Listing and described in the Brief Description of the Invention.
• the instant invention relates to chimeric genes encoding soybean GST enzymes or to chimeric genes that comprise nucleic acid fragments as described above, the chimeric genes operably linked to suitable regulatory sequences, wherein expression of the chimeric genes results in altered levels of the encoded enzymes in transformed host cells.
• the present invention further provides a transformed host cell comprising the above described chimeric gene.
• the transformed host cells can be of eukaryotic or prokaryotic origin.
• the invention also includes transformed plants that arise from transformed host cells of higher plants, and from seeds derived from such transformed plants, and subsequent progeny.
• the invention provides methods of altering the level of expression of a soybean GST enzyme in a host cell comprising the steps of; (i) transforming a host cell with the above described chimeric gene and;
• the present invention provides methods of obtaining a nucleic acid fragment encoding all or substantially all of the amino acid sequence encoding a soybean GST enzyme comprising either hybridization or primer-directed amplification methods known in the art and using the above described nucleic acid fragment.
• a primer-amplification-based method uses SEQ ID NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. The product of these methods is also part of the invention.
• Another embodiment of the invention includes a method for identifying a compound that inhibits the activity of a soybean GST enzyme encoded by the nucleic acid fragment and substantially similar and complementary nucleic acid fragments of SEQ ID NOS.: 1-28.
• the method has the steps: (a) transforming a host cell with the above described chimeric gene; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the GST enzyme; (c) optionally purifying the GST enzyme expressed by the transformed host cell; (d) contacting the GST enzyme with a chemical compound of interest; and
• This method may further include conducting step (d) in the presence of at least one electrophilic substrate and at least one thiol donor.
• the isolated nucleic acid fragments of this method are chosen from the group represented by SEQ ID NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27, and the soybean GST enzyme is selected from the group consisting of SEQ ID NOS.: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26 and 28.
• the invention further provides a method for identifying a chemical compound that inhibits the activity of the soybean GST enzyme as described herein, wherein the identification is based on a comparison of the phenotype of a plant transformed with the above described chimeric gene contacted with the inhibitor candidate with the phenotype of a transformed plant that is not contacted with the inhibitor candidate.
• the isolated nucleic acid fragment of this method is selected from the group consisting of SEQ ID NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 25, and 27 and the soybean GST enzyme is selected from the group consisting of SEQ ID NOS.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.
• the invention provides a method for identifying a substrate for the soybean GST enzyme.
• the method comprises the steps of: (a) transforming a host cell with a chimeric gene comprising the nucleic acid fragment as described herein, the chimeric gene encoding a soybean GST enzyme operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell of step (a) under conditions that are suitable for expression of the chimeric gene resulting in production of the GST enzyme; (c) optionally purifying the GST enzyme expressed by the transformed host cell; (d) contacting the GST enzyme with a substrate candidate; and (e) comparing the activity of soybean GST enzyme with the activity of soybean GST enzyme that has been contacted with the substrate candidate and selecting substrate candidates that increase the activity of the sobyean GST enzyme relative to the activity of soybean GST enzyme in the absence of the substrate candidate.
• step (d) of this method is carried out in the presence of at least one thiol donor.
• the isolated nucleic acid fragment of this method is selected from the group consisting of SEQ ID NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and the soybean GST enzyme is selected from the group consisting of SEQ ID NOS.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.
• methods are provided for identifying a soybean GST substrate candidate wherein the identification of the substrate candidate is based on a comparison of the phenotype of a host cell transformed with a chimeric gene expressing a soybean GST enzyme and contacted with a substrate candidate with the phenotype of a similarly transformed host cell grown without contact with a substrate candidate.
• the isolated nucleic acid fragment of this method is selected from the group consisting of SEQ ID NOS.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and the soybean GST enzyme is selected from the group consisting of SEQ ID NOS.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.
• sequence descriptions and sequences listings attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1.821-1.825.
• the Sequence Descriptions contain the 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 Nucleic Acids Research 75:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):3A5-3l3 (1984) which are herein incorporated by reference.
• SEQ ID NO:l is the nucleotide sequence comprising the cDNA insert in clone sel .27b04 encoding a soybean type I GST.
• SEQ ID NO:2 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone sel.27b04.
• SEQ ID NO:3 is the nucleotide sequence comprising the cDNA insert in clone ssm.pk0026.gl 1 encoding a soybean type II GST.
• SEQ ID NO:4 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ssm.pk0026.gl 1.
• SEQ ID NO: 5 is the nucleotide sequence comprising the cDNA insert in clone GSTa encoding a soybean type III GST.
• SEQ ID NO:6 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone GSTa.
• SEQ ID NO: 7 is the nucleotide sequence comprising the cDNA insert in clone se3.03b09 encoding a soybean type III GST.
• SEQ ID NO: 8 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone se3.03b09.
• SEQ ID NO:9 is the nucleotide sequence comprising the cDNA insert in clone se6.pk0037.h4 encoding a soybean type III GST.
• SEQ ID NO: 10 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone se6.pk0037.h4.
• SEQ ID NO:l 1 is the nucleotide sequence comprising the cDNA insert in clone se6.pk0048.d7 encoding a soybean type III GST.
• SEQ ID NO: 12 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone se6.pk0048.d7.
• SEQ ID NO: 13 is the nucleotide sequence comprising the cDNA insert in clone ses8w.pk0028.c6 encoding a soybean type III GST.
• SEQ ID NO: 14 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ses8w.pk0028.c6.
• SEQ ID NO: 15 is the nucleotide sequence comprising the cDNA insert in clone srl.pkOOl l.d ⁇ encoding a soybean type III GST.
• SEQ ID NO: 16 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone srl.pkOOl l.d ⁇ .
• SEQ ID NO: 17 is the nucleotide sequence comprising the cDNA insert in clone ssl.pk0002.f7 encoding a soybean type III GST.
• SEQ ID NO: 18 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ssl.pk0002.f7.
• SEQ ID NO: 19 is the nucleotide sequence comprising the cDNA insert in clone ssl.pk0005.e6 encoding a soybean type III GST.
• SEQ ID NO:20 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ssl.pk0005.e6.
• SEQ ID NO:21 is the nucleotide sequence comprising the cDNA insert in clone ssl.pk0014.al encoding a soybean type III GST.
• SEQ ID NO:22 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ssl.pk0014.al.
• SEQ ID NO:23 is the nucleotide sequence comprising the cDNA insert in clone ssl.pk0020.bl0 encoding a soybean type III GST.
• SEQ ID NO:24 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ssl.pk0020.bl0.
• SEQ ID NO:25 is the nucleotide sequence comprising the cDNA insert in clone ssm.pk0067.g5 encoding a soybean type III GST.
• SEQ ID NO:26 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone ssm.pk0067.g5.
• SEQ ID NO:27 is the nucleotide sequence comprising the cDNA insert in clone sel.pk0017.f5 encoding a soybean type IV GST.
• SEQ ID NO:28 is the deduced amino acid sequence of the nucleotide sequence comprising the cDNA insert in clone sel.pk0017.f5.
• the transformed E. coli srl.pkOOl Ld6/pET30(LIC)BL21(DE3) comprising the E. coli host BL21(DE3), containing the gene srl.pkOOl l.d ⁇ in a pET30(LIC) vector encoding a soybean type III GST was deposited on 21 August 1997 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 U.S.A. under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The deposit is designated as ATCC 98512.
• the present invention provides novel GST nucleotide sequences and encoded proteins isolated from soybean.
• GST enzymes are known to function in the process of detoxification of a variety of xenobiotic compounds in plants, most notably, herbicides.
• Nucleic acid fragments encoding at least a portion of several soybean GST enzymes have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art.
• sequences of the present invention are useful in the construction of herbicide-tolerant transgenic plants, in the recombinant production of GST enzymes, in the development of screening assays to identify compounds inhibitory to the GST enzymes, and in screening assays to identify chemical substrates of the GSTs.
• Glutathione S-Transferase or “GST” refers to any plant-derived glutathione S-transferase (GST) enzyme capable of catalyzing the conjugation of • glutathione, homoglutathione and other glutathione-like analogs via a sulfhydryl group to hydrophobic and electrophilic compounds.
• GST includes amino acid sequences longer or shorter than the length of natural GSTs, such as functional hybrid or partial fragments of GSTs, or their analogues.
• GST is not intended to be limited in scope on the basis of enzyme activity and may encompass amino acid sequences that possess no measurable enzyme activity but are substantially similar to those sequences known in the art to possess the above- mentioned glutathione conjugating activity.
• class or “GST class” refers to a grouping of the various GST enzymes according to amino acid identity. Currently, four classes have been identified and are referred to as “GST class I” "GST class II", “GST class III” and “GST class IV” . The grouping of plant GSTs into three classes is described by Droog et al. (Plant Physiology 107:1139-1146 (1995)).
• an "isolated nucleic acid fragment” is a polymer- of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases.
• An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
• substantially 'similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology.
• Substantially similar also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate alteration of gene expression by antisense or co-suppression technology " or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary sequences. For example, it is well known in the art that antisense suppression and co- suppression of gene expression may be accomplished using nucleic acid fragments representing less that the entire coding region of a gene, and by nucleic acid fragments that do not share 100% identity with the gene to be suppressed.
• a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine).
• a codon encoding another less hydrophobic residue such as glycine
• a more hydrophobic residue such as valine, leucine, or isoleucine
• changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid
• one positively charged residue for another such as lysine for arginine
• nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
• Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
• substantially similar sequences encompassed by this " invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65 °C), with the sequences exemplified herein.
• Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein.
• nucleic acid fragments are at least 90% identical to the identical to the DNA sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are at least 95%) identical to the DNA sequence of the nucleic acid fragments reported herein.
• a "substantial portion" of an amino acid or nucleotide sequence comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer- automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
• BLAST Basic Local Alignment Search Tool
• a sequence often 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.
• gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
• short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
• a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.
• the instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular fungal proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
• nucleotide bases that are capable to hybridizing to one another.
• adenosine is complementary to thymine and cytosine is complementary to guanine.
• the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.
• 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.
• 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 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, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.
• the skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
• “Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically ' synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
• Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
• “Native gene” refers to a gene as found in nature with its own regulatory sequences.
• “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged ' in a manner different than that found in nature.
• Endogenous gene refers to a native gene in its natural location in the genome of an organism.
• a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced ' into the host organism by gene transfer.
• Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
• a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
• Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
• Suitable regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
• Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence.
• the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
• an "enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a ⁇ promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
• Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) Biochemistry of Plants 75:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
• the “translation leader sequence” refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G.D. (1995) Molecular Biotechnology 3:225).
• the "3" non-coding sequences refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
• the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
• RNA transcript refers to the product resulting from RNA polymerase- catalyzed transcription of a DNA sequence.
• RNA transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
• Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
• cDNA refers to a double-stranded DNA that is complementary to and derived from mRNA.
• Sense RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell.
• Antisense RNA refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Patent No. 5,107,065).
• the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
• “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.
• 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.
• a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
• Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
• expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
• Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
• Overexpression refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
• Co-suppression refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231,020).
• Altered levels refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non- transformed organisms.
• “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
• "Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
• a "chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made.
• Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast transit peptide.
• a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys.100:1621-1632).
• Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:211) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Patent No. 4,945,050).
• Herbicide-tolerant plant as used herein is defined as a plant that survives and preferably grows normally at a usually effective dose of a herbicide.
• Herbicide tolerance in plants according to the present invention refers to detoxification mechanisms in a plant, although the herbicide binding or target site is still sensitive.
• Thiol donor refers to a compound that contains the structure RSH (where R is not equal to H).
• suitable thiol donors may include, but are not limited to, Glutathione and homoglutathione.
• Electrophilic substrate refers to a compound that is amenable to conjugation with glutathione or homoglutathione via a sulfhydryl group. Electrophilic substrates include a wide variety of compounds including pesticides, anti-pathogenic compounds such as fungicides and profungicides, pheramones, and herbicides.
• electrophilic substrates with herbicidal activity may include, but are not limited to, chlorimuronethyl, alachlor, and atrazine, l-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid, t-stilbene oxide, and l,2-epoxy-3-(p-nitrophenoxy)propane.
• Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis”).
• the nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous enzymes from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
• genes encoding other GST enzymes could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant using methodology well known to those skilled in the art.
• Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis).
• the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
• 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 amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
• two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
• the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes.
• the second primer sequence may be based upon sequences derived from the cloning vector.
• the skilled artisan can follow the RACE protocol (Frohman et al., (1988) PNAS USA 55:8998) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5 1 end.
• Primers oriented in the 3' and 5' directions can be designed from the instant sequences.
• 3' RACE or 5' RACE systems BBL
• specific 3' or 5' cDNA fragments can be isolated (Ohara et al., (1989) PNAS USA 86:5613; Loh et al., (1989) Science 243:211).
• 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 7:165).
• Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner, R.A. ( ⁇ 98A) Adv. Immunol. 36:1; Maniatis).
• nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed GST enzymes are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of GST enzyme available as well as the herbicide-tolerant phenotype of the plant.
• Overexpression of the GST enzymes of the instant invention may be accomplished by first constructing chimeric genes in which the coding region are operably linked to promoters capable of directing expression of a gene in the desired tissues at the desired stage of development.
• the chimeric genes may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals must also be provided.
• the instant chimeric genes may also comprise one or more introns in order to facilitate gene expression.
• any combination of any promoter and any terminator capable of inducing expression of a GST coding region may be used in the chimeric genetic sequence.
• Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes.
• One type of efficient plant promoter that may be used is a high level plant promoter.
• Such promoters, in operable linkage with the genetic sequence for GST, should be capable of promoting expression of the GST such that the transformed plant is tolerant to an herbicide due to the presence of, or increased levels of, GST enzymatic activity.
• High level plant promoters that may be used in 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 App. Gen., 1:483-498 1982)), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light- induced in plant cells (See, for example, Genetic Engineering of Plants, an
• Plasmid vectors comprising the instant chimeric genes can then constructed.
• the choice of plasmid vector depends upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985)
• 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 5(5:247-253 (1989)), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels, J.J., Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53 (1991)), or nuclear localization signals (Raikhel, N.
• 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.
• 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.
• Proteins transformed with the present GST genes will have a variety of phenotypes corresponding to the various properties conveyed by the GST class of proteins.
• Glutathione conjugation catalyzed by GSTs are known to result in sequestration and detoxification of a number of herbicides and other xenobiotics (Marrs et al, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:127-58 (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 glutathione S-transferase.
• transgenic plants tolerant to a wide variety of stresses, may be produced by the present method by expressing foreign GST genes in suitable plant hosts.
• the instant GST enzymes produced in heterologous host cells can be used to prepare antibodies to the enzymes by methods well known to those skilled in the art.
• the antibodies are useful for detecting the enzymes in situ in cells or in vitro in cell extracts.
• Preferred heterologous host cells for production of the instant GST enzymes are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of the instant GST enzymes. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the enzymes.
• Vectors or cassettes useful for the transformation of suitable host cells are well known in the art.
• the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
• Suitable vectors comprise a region 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 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 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, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, W ⁇ , ⁇ P ⁇ , T7, tac, and trc (useful for expression in E. coli).
• Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.
• An example of a vector for high level expression of the instant GST enzymes in a bacterial host is provided (Example 5).
• the instant soybean GST enzymes can be used as a targets to facilitate design and/or identification of inhibitors of the enzymes that may be useful as herbicides or herbicide synergists. This is desirable because the enzymes described herein catalyze the sulfhydryl conjugation of glutathione to compounds toxic to the plant. Conjugation can result in detoxification of these compounds. It is likely that inhibition of the detoxification process will result in inhibition of plant growth or plant death. Thus, the instant soybean GST enzymes could be appropriate for new herbicide or herbicide synergist discovery and design.
• nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to expression of the instant enzymes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes or in the identification of mutants.
• the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers.
• RFLP restriction fragment length polymorphism
• the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et at., Genomics 7:174-181 (1987)) in order to construct a genetic map.
• the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al., (1980) Am. J. Hum. Genet. 32:314-331).
• nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al., In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press, pp. 319-346 (1996), and references cited therein).
• nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization ⁇ (FISH) mapping.
• FISH direct fluorescence in situ hybridization ⁇
• nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification, polymorphism of PCR-amplified fragments (CAPS), allele-specific ligation, nucleotide extension reactions, Radiation Hybrid Mapping and Happy Mapping.
• the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art.
• EXAMPLE 1 Composition of cDNA Libraries: Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various soybean tissues were prepared. The characteristics of the libraries are described in Table 1.
• soybean seedling 5-10 day ssl III ssl.pk0002.f7 soybean seedling 5-10 day ssl III ssl.pk0005.e6 soybean seedling 5-10 day ssl III ssl.pk0014.al soybean seedling 5-10 day ssl III ssl.pk0020.bl0 soybean seedling 5-10 day ssm III ssm.pk0067.g5 soybean shoot meristem sel IV sel.pk0017. 5 Soybean embryo,
• cDNA libraries were prepared in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). The Uni-ZAPTM XR libraries were converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences.
• Soybeans (cv Williams 82) were germinated in vermiculite in a controlled growth room at 23 °C with 14-h light/10-h dark cycle at 330 ⁇ E ⁇ r 2 s _1 .
• One week old seedlings were treated with 1 mM 2,4-D for 24 h before harvest. Seedlings were frozen in liquid nitrogen and ground with a mortar and pestle and RNA was prepared using TriZol reagent (Life Technologies Bethesda, MD). Approximately 1.5 ⁇ g of total RNA was reverse transcribed using the GeneAmp Kit (Perkin Elmer, Branchburg, NJ) and oligo dT primer.
• the resulting first strand cDNA was used as a template for PCR amplification with AmpliTaq (Perkin Elmer) and the following primers: primer 1: (GAY GAR GAN CTN CTN GAY TTY TGG) (SEQ ID NO:29) and primer 2: (GAC TCG AGT CGA CAT GCT T 16 ) (SEQ ID NO:30).
• primer 1 and primer 3 were designed based on N-terminal protein sequence previously described (Flury et al., 1995, supra).
• a Perkin-Elmer Thermal Cycle was allowed to cycle at 95 °C for 30 sec, 52 °C for 30 sec and 72 °C for 30 sec for 30 cycles.
• PCR product was cloned in pCR2.1 (Invitrogen, San Diego, CA) according to the manufacturer's instructions, named pBDl ⁇ and sequenced using an ABI sequencer.
• Primer 1 was designed to take advantage of the lack of degeneracy for encoding tryptophan.
• the clone did not include the entire coding region and a second round of PCR was performed using the following primers: Primer 3: CAT ATG AGT GAT GAG GTA GTG TTA TTA GAT TTC TGG (SEQ ID NO:31) and Primer 4: TTA TTA CAC AAA TAT TAC TTA TTT GAA AGG CTA A (SEQ ID NO:32) and using .002 ⁇ g of linearized pBD16 as a template.
• the resulting PCR product was cloned into pCR2.1 and named pBD17 and sequenced using an ABI sequencer. Additional gene specific primers were made and used to determine the complete sequence. All regions were sequenced at least two times in both directions. The nucleotide sequence and encoded protein sequence are shown in
• 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).
• NCBI National Center for Biotechnology Information
• Each cD ⁇ A identified encodes at least a portion of either a GST Class I, II, III, or IV.
• Example 5 describes the strategy for sequencing the above described clones.
• Soybean heat-shock protein (Gmhsp26-A) gene complete cds se6.pk0048.d7 III Y10820
• Soybean heat-shock protein (Gmhsp26-A) gene complete cds. srl.pkOOl l.d ⁇ III U20809
• Soybean heat-shock protein (Gmhsp26-A) gene complete cds ssl.pk0020.bl0 III M20363
• Soybean heat-shock protein (Gmhsp26-A) gene complete cds. SEQ ID NO.
• a chimeric gene comprising a cDNA encoding a soybean GST enzyme in sense orientation can be constructed by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pMLl 03 as described below.
• PCR polymerase chain reaction
• Amplification is then performed in a 100 uL volume in a standard PCR mix consisting of 0.4 mM of each oligonucleotide and 0.3 pM of target DNA in 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 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 ThermocyclerTM for 30 cycles comprising 1 min at 95 °C, 2 min at 55 °C and 3 min at 72 °C, with a final 7 min extension at 72 °C after the last cycle.
• the amplified DNA is then digested with restriction enzymes Ncol and Smal and fractionated on a 0.7% low melting point agarose gel in 40 mM Tris-acetate, pH 8.5, 1 mM EDTA.
• the appropriate band can be excised from the gel, melted at 68 °C and combined with a 4.9 kb Ncol-Smal fragment of the plasmid pML103.
• Plasmid pML103 has been deposited under the terms of the Budapest Treaty with the ATCC and bears accession number ATCC 97366.
• the DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega Corp., 7113 Benhart Dr., Raleigh, NC).
• Vector and insert DNA can be ligated at 15 °C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL 1 -Blue (Epicurian Coli XL-1; Stratagene).
• Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (DNA Sequencing Kit, U. S. Biochemical).
• the resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding a plant gst enzyme, and the 10 kD zein 3' region.
• the chimeric gene so constructed can then be introduced into corn cells by the following procedure.
• Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132 (Indiana Agric. Exp. Station, Indiana, USA). The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos Eire then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al., Set Sin. Peking 18:659-668 (1975)). The embryos are kept in the dark at 27 °C.
• Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos.
• the embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
• the plasmid, p35S/Ac obtained from Dr. Peter Eckes, Hoechst Ag, v Frankfurt, ' Germany
• This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT).
• the enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin.
• the pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. Nature 313:810-812 (1985)) and the 3M region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
• the particle bombardment method (Klein et al., Nature 327:70-73 (1987)) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 ⁇ m in diameter) are coated with DNA using the following technique.
• plasmid DNAs are added to 50 ⁇ L of a suspension of gold particles (60 mg per mL).
• Calcium chloride 50 uL of a 2.5 M solution
• spermidine free base 20 ⁇ L of a 1.0 M solution
• the suspension is vortexed during the addition of these solutions.
• the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.
• the particles are resuspended in 200 ⁇ L of absolute ethanol, centrifuged again and the supernatant removed.
• the ethanol rinse is performed again and the particles resuspended in a final volume of 30 uL of ethanol.
• An aliquot (5 ⁇ L) of the DNA-coated gold particles 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, 861 Ridgeview Dr., Medina, OH), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
• the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covers a circular area of about 5 cm in diameter.
• the petri dish containing the tissue can be placed in the chamber of the PDS-1000/ ⁇ e approximately 8 cm from the stopping screen.
• the air in the chamber is then evacuated to a vacuum of 28 inches of Hg.
• the macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
• the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks, the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium. Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks, the tissue ' can be transferred to regeneration medium (Fromm et al., Bio/Technology 8:833-839 (1990)).
• Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pBI121 (Clonetech Inc., 6500 Donlon Rd, Somis, CA) or other appropriate transformation vector.
• Amplification could be performed as described above and the amplified DNA would then be digested with restriction enzymes Xbal and Smal and fractionated on a 0.7% low melting point agarose gel in 40 mM Tris- acetate, pH 8.5, 1 mM EDTA.
• the appropriate band can be excised from the gel, melted at 68 °C and combined with a 13 kb Xbal-Smal fragment of the plasmid pBI121 and handled as in Example 3.
• the resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, right border region, the nos promoter linked to the NPT II gene and a nos terminator region followed by a cauliflower mosaic virus 35S promoter linked to a cDNA fragment encoding a plant GST enzyme and the nos terminator 3' region flanked by the left border region.
• the resulting plasmid could be mobilized into the Agrobacterium strain LBA4404/pAL4404 (Hoekema et al.
• N ⁇ twre 303:179-180, (1983) using tri- parental matings (Ruvkin and Ausubel, Nature 289:85-88, (1981)).
• the resulting Agrobacterium strains could be then cocultivated with protoplasts (van den Elzen et al. Plant Mol. Biol, 5:149-154 (1985)) or leaf disks (Horsch et al. Science 227:1229-1231, (1985)) of Nicotiana tabacum cv Wisconsin 38 and kanamycin- resistant transformants would be selected. Kanamycin-resistant transformed tobacco plants would be regenerated.
• Example 5 illustrates the expression of isolated full length genes encoding class I, II, III or IV GST proteins in E. coli.
• cDNA from full length clones listed in Table 2 encoding the instant soybean GST enzymes were inserted into the ligation independent cloning (LIC) pET30 vector (Novagen, Inc., 597 Science Dr, Madison, WI) under the control of the T7 promoter, according to the manufacturer's instructions (see Novagen publications "LIC Vector Kits", publication number TB163 and U.S. 4952496).
• the vector was then used to transform BL21(DE3) competent E. coli hosts.
• Primers with a specific 3' extension designed for ligation independent cloning were designed to amplify the GST gene (Maniatis). Amplification products were gel-purified and annealed into the LIC vector after treatment with T4 DNA polymerase (Novagen).
• Insert-containing vectors were then used to transform NovaBlue competent E. coli cells and transformants were screened for the presence of viable inserts.
• Expressed protein was purified using the HIS binding kit (Novagen) according to the manufacturer's instructions. Purified protein was examined on 15-20% SDS Phast Gels (Bio-Rad Laboratories, 861 Ridgeview Dr, Medina, OH) and quantitated spectrophotometrically using BSA as a standard. Protein data is tabulated below in Table 3.
• 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, l-chloro-2, 4-dinitro- benzene (CDNB), ethacrynic acid, t-stilbene oxide, and l,2-epoxy-3-(p-nitro- phenoxy) propane. The enzymes were purified as described in Example 5 and used in the following assay.
• the conjugation reaction with each electrophilic substrate was performed by incubating 0.3 to 30 ⁇ g enzyme in 0.1 M MOPS (pH 7.0) containing 0.4 mM of the electrophilic substrate.
• the reaction was inititated by the addition of glutathione to a final concentration of 4 mM. After 5 to 30 min, the reaction was terminated by the addition of 45 ⁇ L acetonitrile, microfuged for 10 min to remove precipitated protein, and then the supernatent was removed and added to 65 ⁇ L of water.
• 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 uncatalyzed 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 ⁇ 'mg - 1 with the seven different substrates. Activities are related to the activity of a known and previously isolated and purified GST enzyme, GH2/4 (also called GST 26) (Czarnecka et al., Plant Molecular Biology 3:45-58 (1984); Ulmasoz et al., Plant Physiol 108:919-927 (1995)).

Landscapes

• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Medicinal Chemistry (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Priority Applications (4)

Applications Claiming Priority (1)

Publications (1)

Family

ID=22267979

Family Applications (1)

Country Status (4)

Cited By (4)

Citations (3)

• 1998
  • 1998-09-30 AU AU11854/99A patent/AU1185499A/en not_active Abandoned
  • 1998-09-30 WO PCT/US1998/020501 patent/WO2000018936A1/en not_active Application Discontinuation
  • 1998-09-30 EP EP98954930A patent/EP1117811A1/de not_active Withdrawn
  • 1998-09-30 CA CA002340791A patent/CA2340791A1/en not_active Abandoned

Patent Citations (3)

Non-Patent Citations (7)

Also Published As

Similar Documents

Legal Events