MXPA99009086A - Methods and materials for making and using transgenic dicamba-degrading organisms - Google Patents

Abstract

The invention provides isolated and at least partially-purified dicamba-degrading enzymes, isolated DNA molecules coding for dicamba-degrading enzymes, DNA constructs coding for dicamba-degrading enzymes, transgenic host cells comprising DNA coding for dicamba-degrading enzymes, and transgenic plants and plant parts comprising one or more cells comprising DNA coding for dicamba-degrading enzymes. Expression of the dicamba-degrading enzymes results in the production of dicamba-degrading organisms, including dicamba-tolerant plants. The invention further provides a method of controlling weeds in a field containing the transgenic dicamba-tolerant plants of the invention and a method of decontaminating a material containing dicamba comprising applying an effective amount of a transgenic microorganism or dicamba-degrading enzyme of the invention to the material. Finally, the invention provides a method of selecting transformed plants and plant cells based on dicamba tolerance and a method of selecting or screening transformed host cells, intact organisms and parts of organisms based on the fluorescence of 3,6-dichlorosalicylic acid produced as a result of dicamba degradation.

Description

METHODS AND MATERIALS FOR MAKING AND USING TRANSGENIC ORGANISMS DICAMBA DEGRADERS FIELD OF THE INVENTION The present invention relates to transgenic organisms that are capable of degrading dicamba herbicide, including transgenic plants that have been made tolerant to dicamba. The invention also relates to dicamba degrading enzymes and to DNA molecules and DNA constructs encoding dicamba degrading enzymes. The invention also relates to a method for controlling weeds in fields of transgenic plants tolerant to dicamba and to a method for removing the dicamba from materials contaminated with it (bio-remediation). Finally, the invention relates to methods for selecting transformants based on tolerance to dicamba or for detecting the fluorescence of 3,6-dichlorosalicylic acid, which is generated as a result of the degradation of dicamba.

BACKGROUND Herbicides are routinely used in agricultural production. Its effectiveness is often determined by its ability to kill weed growth in the fields and tolerance of crop flow to the herbicide. If the crop is not tolerant to the herbicide, the herbicide will either decrease the productivity of the crop flow or exterminate it. Conversely, if the herbicide is not strong enough, it will allow too much weed growth in the field, which, in turn, decreases the productivity of the crop flow. Accordingly, it is desirable to produce economically important plants which are tolerant to herbicides. To protect the environmental and water quality of agricultural land, it is also desirable to develop technologies to degrade herbicides in case of accidental spills of the herbicide or in cases of unacceptably high levels of soil or water contamination. Genes encoding enzymes which inactivate herbicides and other xenophobic compounds have previously been isolated from a variety of prokaryotic and eukaryotic organisms. In some cases, these genes have been genetically engineered for successful expression in plants. Through this approach, plants that are tolerant to herbicides 2,4-dichlorophenoxyacetic acid (Streber and Willmitzer (1989) Bio / Technology 7:81 1 -816; 2,4-D), bromoxynil (Stalker et al. (1 988) Science 242: 41 9-423, trade name Buctril), glyphosate (Comai et al (1985) Nature 31 7: 741-744; trade name Round-Up) and phosphinothricin (De Block et al. 1987) EMBO J. 6: 2513-2518, trade name Basta). The dicamba (commercial name Banvel) is used as a pre-emergent and post-emergent herbicide for the control of annual and perennial broadleaf weeds and several grass weeds in crops of corn, sorghum, small grains, pasture, hay, cane sugar, asparagus, grass and grass seeds. See Crop Protection Reference, pages 1803-1821 (Chemical &Pharmaceutical Press, Inc., New York, NY, 11th ed., 1995). Unfortunately, the dicamba can injure many commercial crops (including beans, soybeans, cotton, chiles, potatoes, sunflowers, tomatoes, tobacco and fruit trees), ornamental plants and trees, and other broad-leaved plants when it comes in contact with them. . Id. The dicamba is chemically stable and can sometimes be persistent in the environment. The dicamba is in the class of benzoic acid herbicides. It has been suggested that plants tolerant to benzoic acid herbicides, including dicamba, can be produced by incorporating an antisense gene of 1-aminocyclopropane-1-carboxylic acid synthase (ACC), an antisense gene of ACC oxidase, a ACC deaminase, or combinations thereof, in plants. See Canadian patent application 2, 165.036 (published June 16, 1996). However, no experimental data is presented in this application that demonstrates such tolerance. Bacteria that metabolize dicamba are known. See US patent no. 5,445,962; Krueger et al. , J. Agrie. Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl. Microbe!. , 38, 1-66 (1991); Cork and Khalil, Adv. Appl. Microbiol. , 40, 289-320 (1995). It has been suggested that the specific genes responsible for the metabolism of dicamba by these bacteria could be isolated and used to produce plants and other organisms resistant to dicamba. See id. and Yang et al. , Anal. Biochem. , 219: 37-42 (1994). However, prior to the present invention, such genes had not been identified or isolated.

BRIEF DESCRIPTION OF THE INVENTION The invention provides an isolated and at least partially purified degrading dicamba O-demethylase, an isolated and at least partially purified degrading dicamba oxygenase, an isolated and at least partially purified degrading dicamba ferrodoxin, and a degrading reductase. of dicamba isolated and at least partially purified, all as defined and described below. The invention also provides an isolated DNA molecule comprising a DNA sequence encoding a dicamba degrading oxygenase and an isolated DNA molecule comprising a DNA sequence encoding a dicamba-degrading ferredoxin. The invention further provides a DNA construct comprising a DNA sequence encoding a dicamba-degrading oxygenase operably linked to expression control sequences and a DNA construct comprising a DNA sequence encoding a dicamba-degrading ferredoxin operably linked to expression control. The invention further provides a transgenic host cell comprising a DNA encoding a dicamba degrading oxygenase operably linked to expression control sequences and a transgenic host cell comprising DNA encoding a dicamba-degrading ferredoxin operably linked to expression control sequences. The transgenic host cell can comprise DNA encoding a dicamba degrading oxygenase and a dicamba-degrading ferredoxin, both operably linked to expression control sequences. The transgenic host cell can be a plant cell or a procyanidic or eukaryotic microorganism. In addition, the invention provides a plant or part of a transgenic plant comprising one or more cells comprising DNA encoding a dicamba degrading oxygenase operably linked to expression control sequences. One or more cells of the plant or part of the plant may further comprise DNA encoding a dicamba-degrading ferredoxin operably linked to expression control sequences. The plant or part of the transgenic plant is tolerant to dicamba or has its tolerance to dicamba increased as a result of the expression of the degrading oxygenase of dicamba or of the expression of the oxygenase and ferredoxin degraders of dicamba. The invention also provides a method for controlling weeds in a field containing transgenic dicamba tolerant plants according to the invention. The method involves applying a quantity of dicamba to the field, which is effective to control the weed. The invention further provides methods for decontaminating a dicamba containing material. In one embodiment, the method comprises applying an effective amount of a transgenic dicamba-degrading microorganism of the invention to the material. In another embodiment, the method comprises applying an effective amount of a dicamba-degrading O-demethylase or a combination of a dicamba-degrading oxygenase, a dicamba-degrading ferredoxin and a dicamba-degrading reductase to the material.

The invention also provides a method for selecting cells from transformed plants and transformed plants using tolerance to dicamba as the selection marker. In one embodiment, the method comprises transforming at least some of the cells of the plant into a population of cells of the plant with a DNA construct according to the invention and growing the resulting population of plant cells in a medium of culture containing dicamba at a selected concentration so that the transformed cells of the plant will grow and the untransformed cells of the plant will not grow. In another embodiment, the method comprising applying dicamba to a population of plants suspected of comprising a DNA construct according to the invention, which provides degradation of dicamba, the dicamba being applied in a selected amount so that the transformed plants will grow and the growth of the untransformed plants will be inhibited. Finally, the invention provides a method for selecting or classifying transformed host cells, intact organisms, and parts of the organism. The method comprises providing a population of host cells, intact organisms or parts of organisms suspected of comprising a DNA construct according to the invention, providing degradation of dicamba and assessing the presence or level of fluorescence due to acid 3,6- dichlorosalicylic. 3,6-Dichlorosalicylic acid is generated in transformed host cells, intact organisms, or parts of organisms as a result of dicamba degradation, but will not be generated in untransformed host cells, intact organisms or parts of organisms.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. A diagram of the electron transport scheme proposed for Dicamba O-demethylase. The NADH electrons are sequentially transferred from the D? C reductase to the ferredoxin D? C and then to D | C oxygenase. The oxygen reaction with the dicamba substrate to form 3,6-dichlorosalicylic acid is catalyzed by D | C oxygenase. ox, oxidized; network, reduced.

DETAILED DESCRIPTION OF THE CURRENTLY PREFERRED MODALITIES OF THE INVENTION Previous studies (Cork and Kreuger, Advan Appl Microbiol 36: 1-56 and Yang et al. (1 994) Anal Biochem 21 9: 37-42) have shown that the soil bacterium, Pseudomonas maltophilia, strain DI-6, is able to destroy the herbicidal activity of dicamba through a simple step reaction, in which the dicamba (3,6-dichloro-2-methoxybenzoic acid) is converted to 3,6-dichlorosalicylic acid (3,6-DCSA). 3,6-DCSA has no herbicidal activity and has not been shown to have any detrimental effect on plants. In addition, 3,6-DCSA is easily degraded by the normal bacterial flora present in the soil. The experiments described herein confirm the hypothesis of Yang et al. (see id.) that an O-demethylase is responsible for the conversion of dicamba to 3,6-DCSA by P. maltophilia strain DI-6 and states that O-demethylase is a three-component enzyme system consisting of a reductase, a ferredoxin and an oxygenase. See Example 1, which describes in detail the isolation, purification and characterization of O-demethylase from P. maltophilia and its three components. The reaction scheme for the reaction catalyzed by the three components of Dicamba O-demethylase is presented in Figure 1. As illustrated in Figure 1, the NADH electrons are released through a short electron chain consisting of reductase and ferredoxin to the terminal oxygenase, which catalyzes the oxidation of dicamba to produce 3,6-DCSA. In a first embodiment, the invention provides isolated and at least partially purified dicamba degrading enzymes. "Isolated" is used herein to mean that the enzymes have been removed at least from the cells in which they are produced (i.e., they are contained in a cell lysate). "At least partially purified" is used herein to mean that they have been at least partially separated from the other components of the used cell. Preferably, the enzymes have been sufficiently purified so that the enzyme preparations are at least about 70% homogeneous. In particular, the invention provides an isolated and at least partially purified O-demethylase degrading dicamba. "Dicamba degrading o-demethylase" is defined herein as a combination of a dicamba-degrading oxygenase, a dicamba-degrading ferredoxin and a dicamba-degrading reductase, all as defined below. The invention also provides an isolated and partially purified dicamba degrading oxygenase. "Dicamba degrading oxygenase" is defined herein as a purified oxygenase of P. maltophilia strain DI-6 and oxygenases having an amino acid sequence, which is at least about 65% homologous, preferably at least about 85% homologous , to that of the oxygenase of P. maltophilia and which can participate in the degradation of dicamba. "Dicamba degrading oxygenates" include mutant oxygenates having the amino acid sequence of the oxygenase of P. maltophilia, wherein one or more amino acids have been added to, deleted from, or substituted by, the amino acids of the oxygenase sequence of P. maltophilia. The degrading oxygenase activity of dicamba can be determined as described in Example 1. The invention further provides an isolated and at least partially purified degrading ferredoxin of dicamba. "Dicamba degrading ferredoxin" is defined herein as the purified ferredoxin of P. maltophilia strain DI-6 and ferredoxins having an amino acid sequence which is at least about 65% homologous, preferably at least approximately 85% homologous, to that of the ferredoxin of P. maltophilia, and which can participate in the degradation of dicamba. "Dicamba degrading ferredoxins" include mutant ferredoxins having the amino acid sequence of the ferredoxin of P. maltophilia, wherein one or more amino acids have been added to, deleted from, or substituted by, the amino acids of the ferredoxin sequence of P. maltophilia. The activity of dicamba degrading ferredoxins can be determined as described in Example 1. Finally, the invention provides an isolated and at least partially purified degrading dicamba reductase. "Dicamba degrading reductase" is defined herein as the purified reductase of P. maltophilia strain DI-6 and reductases having an amino acid sequence which is at least about 65% homologous, preferably at least about 85% homologous, to that of the reductase of P. maltophilia, and which can participate in the degradation of dicamba. "Dicamba degrading reductases" include mutant reductases having the amino acid sequence of the reductase of P. maltophilia, wherein one or more amino acids have been added to, deleted from, or substituted by, the amino acids of the reductase sequence of P. maltophilia. The activity of dicamba reductase reductases can be determined as described in Example 1. Methods for determining the degree of homology of amino acid sequences are well known in the art. For example, the FASTA program in the Genetics Computing Group (GCG) computer package (University of Wisconsin, Madison, Wl) can be used to compare sequences in several protein databases, such as the Swiss protein database.

The dicamba degrading enzymes of the invention can be isolated and purified as described in Example 1 from P. maltophilia or other organisms. Other suitable organisms include bacteria other than P. maltophilia strain DI-6 that degrade dicamba. Several strains of such bacteria are known. See US patent no. 5,445,962; Krueger et al. , J. Agrie. Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl. Microbe/. , 38, 1-66 (1991); Cork and Khalil, Adv. Appl. Microbiol. , 40, 289-320 (1995). Other dicamba degrading bacterial strains can be isolated as were these strains by methods well known in the art. However, preferably, the dicamba degrading enzymes of the invention are prepared using recombinant DNA techniques (see below). In particular, mutant enzymes having the amino acid sequence of the enzyme of P. maltophilia, wherein one or more amino acids have been added to, deleted from, or substituted by, the amino acids of the P. maltophilia sequence are prepared in this using, for example, oligonucleotide-directed mutagenesis, linker screening mutagenesis, mutagenesis using the polymerase chain reaction, and the like. See Ausubel et al. (eds.), Current Protocols In Molecular Biology (Wiley Inteerscience 1990) and McPherson (ed.), Directed Mutagenesis: A Practice! Approach (Directed mutagenesis: a practical approach) (IRL Press, 1991). In a second embodiment, the invention provides isolated DNA molecules encoding dicamba degrading enzymes of the invention. "Isolated" is used in the present to mean that the DNA molecule has been removed from its natural environment or is not a DNA molecule that occurs naturally. Methods for preparing these DNA molecules are well known in the art. See, for example, Maniatis et al. , Molecular Cloning: A Laboratory Manual, (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor, NY (1 982), Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). For example, the DNA molecules of the invention can be isolated cDNA or genomic clones. The identification and isolation of clones encoding dicamba-degrading oxygenase and ferredoxin from P. maltophilia strain DI-6 are described in Examples 2 and 3. Additional clones encoding dicamba degrading enzymes can be obtained, including clones that encode degrading reductases of dicamba, in a similar way. The isolated clones, or portions thereof, can be used as probes to identify and isolate additional clones from organisms other than those from which the clones were originally isolated. Suitable organisms include bacteria that degrade dicamba. As noted above, in addition to P. maltophilia strain DI-6, several strains of bacteria that degrade dicamba are known. See US patent no. 5,445, 962; Krueger et al. , J. Agrie. Food Chem., 37, 534-538 (1989); Cork and Krueger, Adv. Appl. Microbe/. , 38, 1-66 (1 991); Cork and Khalil, Adv. Appl. Microbiol. , 40, 289-320 (1995).

The DNA molecules of the invention can also be chemically synthesized using the sequences of isolated clones. Such techniques are well known in the art. For example, DNA sequences can be synthesized by phosphoamidite chemistry in an automated DNA synthesizer. Chemical synthesis has a number of advantages. In particular, chemical synthesis is desirable because codons preferred by the host in which the DNA sequence will be expressed can be used to optimize expression. Not all codons need to be altered to obtain improved expression, but preferably at least the codons rarely used in the host are changed to codons preferred by the host. High levels of expression can be obtained by changing more than about 50%), most preferably at least about 80%, of the codons to codons preferred by the host. The codon preferences of many host cells are known. See, for example, Maximizing Gene Expression, pages 225-85 (Reznikoff & Gold, eds. , 1986), PCT WO 97/31 1 15, PCT WO 97/1 1086, EP 646643, EP 553494 and US Pat. Nos. 5,689,052, 5,567,862, 5,567,600, 5,552,299 and 5,017,692. The codon preferences of other host cells can be deduced by methods known in the art. In addition, using chemical synthesis, the sequence of the DNA molecule or its encoded protein can be easily changed to, for example, optimize expression (eg, remove secondary mRNA structures that interfere with transcription or translation), add restriction sites only at convenient points, suppress protease cutting sites, etc.

In a third embodiment, the present invention provides DNA constructs comprising DNA encoding a dicamba degrading enzyme operably linked to expression control sequences. "DNA constructs" are defined herein as being constructed DNA molecules (which do not occur naturally) useful for introducing DNA into host cells, and the term includes chimeric genes, expression cartridges, and vectors. As used herein "operably linked" refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative separation of the various sequences) in a manner such that the proteins are expressed. encoded Methods for operably linking expression control sequences to coding sequences are well known in the art. See, for example, Maniatis et al. , Molecular Cloning: A Laboratory Manual, (Molecular Cloning: a laboratory manual), Cold Spring Harbor, NY (1982), Sambrook et al. , Molecular Cloning: A Laboratory Maual, (Molecular cloning: a laboratory manual), Cold Spring Harbor, NY (1989). The "expression control sequences" are DNA sequences involved in any way in the control of transcription or translation in prokaryotes and eukaryotes. Suitable expression control sequences and methods for making and using them are well known in the art. The expression control sequences must include a promoter. The promoter can be any DNA sequence, which shows the transcription activity in the host cell or chosen organism. The promoter can be induced or constitutive. It can occur naturally, it can be composed of portions of several promoters that occur naturally, or it can be partially or totally synthetic. The guide for the design of promoters is provided by promoter structure studies, such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-61 (1987). In addition, the location of the promoter in relation to the start of transcription can be optimized. See, for example, Roberts, et al. , Proc. Nati Acad. Sci. USA, 76, 760-4 (1979). Many promoters suitable for use in prokaryotes and eukaryotes are well known in the art. For example, constitutive promoters suitable for use in plants include: promoters for plant viruses, such as the 35S promoter of cauliflower mosaic virus (Odell et al., Nature 313: 810-812 (1988)). chiorella virus methyltransferase genes (U.S. Patent No. 5,563,328), and the full-length transcript promoter of scrophularia mosaic virus (U.S. Patent No. 5,378.61 9); gene promoters such as rice actin (McEIroy et al., Plant Cell 2: 163-171 (1990)), ubiquitin (Christensen et al., Plant Mol. Biol. 12: 619-632 (1989) and Christensen et al., Plant Mol. Biol. 18: 675 -689 (1992)), pEMU (Last et al., Theor. Appl. Genet. 81: 581-588 (1 991)), MAS (Velten et al., EMBO J. 3: 2723-2730 (1988) )), histone H3 of corn (Lepetit et al., Mol.Gen. Gen. 321: 276-300 (1992)), ALS3 of Brassica napus (PCT application WO 97/41228), and promoters of several Agrobacterium genes (see the patents state Americans us. 4,771, 002, 5, 102, 796, 5, 1 82, 200, 5,428, 147). Suitable inducible promoters for use in plants include: the promoter of the ACE 1 system, which responds to copper (Mett et al., PNAS 90: 4567-4571 (1993)); the promoter of the corn In2 gene, which responds to insurers of benzenesulfonamide herbicide (Hershey et al., Mol.Gen. Genetics 227: 229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243: 32 -38 (1994)), and the promoter of the Tet repressor of Tn 1 0 (Gatz et al., Mol.Gen.Genet., 227: 229-237 (1991)). A particularly preferred inducible promoter for use in plants is one that responds to an inducing agent to which plants normally do not respond. An exemplary inducible promoter of this type is the inducible promoter of a spheroidal hormone gene, the transcription activity of which is induced by a glucocorticosteroid hormone. Schena et al. , Proc. Nati Acad. Sci. USA 88: 1 0421 (1991). Other inducible promoters for use in plants are described in EP 332104, PCT WO 93/21334 and PCT WO 97/06269. Promoters suitable for use in bacteria include the promoter of the maltogenic amylase gene of Bacillus stearothermophilus, the alpha-amylase gene of Bacillus licheniformis, the BAN amylase gene of Bacillus amiloliquefaclens, the alkaline protease gene of Bacillus subtilis, the Bacillus pumllus xylosidase, the PR and PL promoters of lambda phage, and the lac, trp and tac promoters of Escherichia coli. See PCT WO 96/23898 and PCT WO 97/42320.

Promoters suitable for use in yeast host cells include promoters of yeast glycolytic genes, promoters of alcohol dehydrogenase genes, the TPI1 promoter, and the ADH2-4c promoter. See PCT WO 96/23898. Promoters suitable for use in filamentous fungi include the ADH3 promoter, the tpiA promoter, the promoters of genes encoding TAKA amylase from Aspergillus oryzae, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, acid-stable alpha-amylase of A. niger, glucoamylase of A. niger or Aspergillus awamori, lipase of R. miehei, alkaline protease of A. orizae, isomerase of phosphate of triose of A. oryzae and acetamidase of Aspergillus nidulas. See PCT WO 96/23898. Suitable promoters for use in mammalian cells are the SV40 promoter, the metallothionein gene promoter, the murine mammary tumor virus promoter, the Rous sarcoma virus promoter, and the adenovirus major late promoter 2. See PCT WO 96/23898 and PCT WO 97/42320. Suitable promoters for use in insect cells include the polyhedrin promoter, P10 promoter, the autographa californica polyhedrosis virus basic protein promoter, the gene promoter.

Baculovirus immediate early 1 and delayed 39K baculovirus late gene promoter. See PCT WO 96/23898. Finally, promoters composed of portions of other promoters and partially or fully synthetic promoters can be used. See, for example, Ni et al. , Plant J., 7: 661 -676 (1995) and PCT WO 95/14098 describing such promoters for use in plants. The promoter may include, or be modified to include, one or more enhancing elements. Preferably, the promoter will include a plurality of enhancer elements. Promoters that contain enhancer elements provide higher levels of transcription compared to promoters that do not include them. Intensifying elements suitable for use in plants include the 35S enhancer element of cauliflower mosaic virus (U.S. Patent Nos. 5, 106,739 and 5,164,316) and the enhancer element of scleral mosaic virus (Maiti et al., Transgenic Res. ., 6, 143-156 (1997)). Other enhancers suitable for use in other cells are known. See PCT WO 96/23898 and Enhancers and Eukaryotic Expression (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1983). For efficient expression, the coding sequences are also, preferably, operably linked to a 3 'untranslated sequence. The 3 'untranslated sequence contains transcription and / or translation termination sequences. The 3 'untranslated regions can be obtained from the gene flanking regions of bacterial cells, plants or other eukaryotic cells. For use in prokaryotes, the 3 'untranslated region will include a transcription termination sequence. For use in plants and other eukaryotes, the 3 'untranslated region will include a transcription termination sequence and a polyadenylation sequence. The 3 'untranslated sequences suitable for use in plants include those of the cauliflower mosaic virus 35S gene, the phaseolin seed storage protein gene, the small subunit E9 gene of pea ribulosa biphosphate carboxylase, the 7s storage protein genes of soybean, the octopine synthase gene and the nopaline synthase gene. In plants and other eukaryotes, a 5 'untranslated sequence is also used. The 5 'untranslated sequence is the portion of an mRNA which extends from the 5' CAP site to the translation initiation codon. This region of mRNA is necessary for the initiation of translation in eukaryotes and plays a role in the regulation of gene expression. Non-translated 5 'regions suitable for use in plants include those of alfalfa mosaic virus, cucumber mosaic virus and tobacco mosaic virus coating protein gene. As noted above, the DNA construct can be a vector. The vector may contain one or more replication systems, which allows replication in host cells. Self-replicating vectors include plasmids, cosmids and viral vectors. Alternatively, the vector can be an integrating vector, which allows integration into the chromosome of the host cell of the sequence encoding a dicamba degrading enzyme of the invention. The vector also conveniently has unique restriction sites for the insertion of DNA sequences. If a vector does not have unique restriction sites, it can be modified to introduce or eliminate restriction sites to make it more suitable for additional manipulations.

The DNA constructs of the invention can be used to transform a variety of host cells (see below). A genetic marker must be used to select transformed host cells ("a selection marker"). Selection markers typically allow transformed cells to be recovered by negative selection (i.e., inhibiting the growth of cells not containing the selection marker) or by classifying a product encoded by the selection marker. The most commonly used selectable marker gene for plant transformation is the neomycin II phosphotransferase (nptll) gene, isolated from Tn5, which, when placed under the control of plant regulatory signals, confers resistance to kanamycin. Fraley et al. , Proc. Nati Acad. Sci. USA, 80: 4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene, which confers resistance to the hygromycin antibiotic. Vanden Elzen et al. , Plant Mol. Biol., 5: 299 (1985). Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamicin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyl transferase and the bleomycin resistance determinant. Hayford et al. , Plant Physiol. 86: 1216 (1988), Jones et al. , Mol. Gen. Genet. 210: 86 (1987), Svab et al. , Plant Mol. Biol. 14: 197 (1990), Hille et al. , Plant Mol. Biol. 7: 171 (1986). Other selectable marker genes confer resistance to herbicides, such as glyphosate, glufosinate or bromoxynil. Comai et al. , Nature 317: 741-744 (1985), Gordon-Kamm et al. , Plant Cell 2: 603-61 8 (1990) and Stalker et al. , Science 242: 41 9-423 (1988). Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet 13:67 (1987), Shah et al. , Science 233: 478 (1986), Charest et al. , Plant Cell Rep. 8: 643 (1990). Genes commonly used to classify presumptively transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase and chloramphenicol acetyltransferase. Jefferson, R.A. , Plant Mol. Biol. Rep. 5: 387 (1987). , Teeri et al. , EMBO J. 8: 343 (1989), Koncz et al. , Proc. Nati Acad. Sci. USA 84: 131 (1987), De block et al. , EMBO J. 3: 1681 (1984), green fluorescent protein (GFP) (Chalfie et al., Science 263: 802 (1 994), Haseloff et al., 77G 1 1: 328-329 (1995) and application for PCT WO 97/41228). Another approach to the identification of relatively rare transformation events has been the use of a gene encoding a dominant constitutive regulator of the anthocyanin pigmentation pathway of Zea mays. Ludwig et al. , Science 247: 449 (1990). The selection markers suitable for use in prokaryotes and eukaryotes other than plants are also well known. See, for example, PCT WO 96/23898 and PCT WO 42320. For example, antibiotic resistance (ampicillin, kanamycin, tetracycline, chlornaphenicol, neomycin or hygromycin) can be used as the selection marker.

According to another aspect of the present invention, tolerance to dicamba can be used as a selection marker for plants and plant cells. "Tolerance" means that the cells of transformed plants are able to grow (survive and regenerate in plants) when placed in culture medium containing a level of dicamba that prevents untransformed cells from doing so. "Tolerance" also means that the transformed plants are able to grow after the application of a quantity of dicamba that inhibits the growth of untransformed plants. Methods for selecting cells from transformed plants are well known in the art. Briefly, at least some of the cells of the plant in a population of plant cells (eg, an explant or an embryo suspension culture) are transformed with a DNA construct according to the invention providing the degradation of dicamba. The resulting population of plant cells is clochated in culture medium containing dicamba at a selected concentration so that the cells of transformed plants will grow, while the cells of non-transformed plants will not. Suitable concentrations of dicamba can be determined empirically as is known in the art. Methods for selecting transformed plants are also known in the art. Briefly, dicamba is applied to a population of plants suspected of comprising a DNA construct according to the invention, providing degradation of dicamba. The amount of dicamba is selected so that the transformed plants will grow, and the growth of untransformed plants will be inhibited. The level of inhibition should be sufficient, so that transformed and non-transformed plants can be easily distinguished (ie, the inhibition must be statistically significant). Such amounts can be determined empirically as is known in the art. In addition, the generation of 3,6-DCSA as a result of the degradation of dicamba can be used for selection and classification. The generation of 3,6-DCSA can be easily ascertained by observing the fluorescence of this compound, allowing the selection and classification of transformed host cells, intact organisms and parts of organisms (eg, microorganisms, plants, parts of plants and cells). plants) . In this regard, the invention allows the selection and classification of transformed host cells, intact organisms and parts of organisms in the same manner as for the green fluorescent protein (GFP). See US patents nos. 5,162,227 and 5,491,084 and the PCT applications WO 96/27675, WO 97/1 1094, WO 97/41228 and WO 97/42320, all of which are incorporated herein by reference. In particular, 3,6-DCSA can be detected in transformed host cells, intact organisms and parts of organisms using conventional spectrophotometric methods. For example, microscopes can be adjusted with combinations of filters suitable for excitation and fluorescence detection. A hand held lamp can be used for bench work or field work (see Example 1). The classification of fluorescent activated cells can also be used. 3.6-DCSA is excited at a wavelength of 312-313 nm, with a maximum emission wavelength of 424 nm. "Parts" of organisms include organs, tissues or any other part. "Parts of plants" include seeds, pollen, embryos, flowers, fruits, buds, leaves, roots, stems, explants, etc. Selection based on tolerance to dicamba or degradation of dicamba can be used in the production of dicamba tolerant plants or dicamba degrading microorganisms, in which case the use of another selection marker may not be necessary. Selection based on tolerance to dicamba or degradation of dicamba can also be used in the production of transgenic cells or organisms that express other genes of interest. Many such genes are concoded and include genes that encode commercially valuable proteins and genes that confer improved agronomic traits in plants (see, for example, PCT WO 97/41228, the full disclosure of which is incorporated herein by reference) . The DNA constructs of the invention can be used to transform a variety of host cells, including prokaryotes and eukaryotes. The DNA sequences encoding the dicamba degrading enzyme (s) and the selection marker, if a separate selection marker is used, can be in the same or different DNA constructs. Preferably, are arranged in a simple DNA construct as a transcription unit, so that all coding sequences are expressed together. In addition, the gene (s) of interest and the DNA sequences encoding the dicamba degrading enzyme (s), when using the dicamba tolerance or degradation of dicamba as a selection marker, can be in the same or different constructs of DNA Such constructs are prepared in the same manner as described above. Suitable host cells include prokaryotic and eukaryotic microorganisms (e.g., bacteria (including Agrobacterium tumefaciens and Escherichia coli), yeast (including Saccharomyces cerevisiae) and other fungi (including Aspergillus sp.), Plant cells, insect cells and mammalian cells. Preferably, the host cell is one that does not normally degrade dicamba, however, the present invention can also be used to increase the level of degradation of dicamba in host cells that normally degrade dicamba.Thus, "transgenic" cells and organisms of the invention include cells and organisms that normally do not degrade dicamba, but which have been transformed according to the invention, so that they are capable of degrading this herbicide The "transgenic" cells and organisms of the invention also include cells and organisms that normally degrade dicamba, but which have been transformed in accordance with the invention, so that they are able to degrade more of this herbicide or degrade the herbicide more efficiently. Methods for transforming prokaryotic and eukaryotic host cells are well known in the art. See, for example, Maniatis et al. , Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor, NY (1982), Sambrook et al. , Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor, NY (1 989); PCT WO 96/23898 and PCT WO 97/42320. For example, numerous methods for plant transformation have been developed, including biological and physical transformation protocols. See, for example, Miki et al. , "Procedures for Introducing Foreign DNA into Plants", Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Ratón, 1 993) pp. 67-88. In addition, vectors and in vitro culture methods are available for transformation of tissue or plant cells and plant regeneration. See, for example, Gruber et al. , "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Ratón, 1 993) pp. 89-1 19. The most widely used method for introducing an expression vector in plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al. , Science 227: 1229 (1988). A. tumefaciens and A. rhizogenes are pathogenic soil bacteria for plants, which genetically transform the cells of plants. The ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for the genetic transformation of the plant.

See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10: 1 (1991). The descriptions of Agrobacterium vector systems and methods for gene transfer mediated by Agrobacterium are provided by numerous references, including Gruber et al. , supra, Miki et al. , supra, Moloney et al. , Plant Cell Reports 8: 238 (1989), and US Pat. Nos. 4, 940, 838 and 5,464, 763. A generally applicable method of plant transformation is microprojectile-mediated transformation, wherein the DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles at speeds sufficient to penetrate walls and cell membranes of plants. Sanford et al. , Part. Sci. Technol. 5:27 (1987), Sanford, J.C. , Trends Biotech. 6: 299 (1988), Sanford, J.C. , Physiol. Plant 79: 206 (1990), Klein et al. , Biotechnology 10: 268 (1992). Another method for the physical delivery of DNA to plants is the sonication of target cells. Zhang et al. , Bio / Technology 9: 996 (1991). Alternatively, liposomes or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al. , EMBO J., 4: 2731 (1985), Christou et al. , Proc. Nati Acad. Sci. USA 84: 3962 (1987). Direct uptake of DNA into protoplasts has also been reported using pre-excitation with CaCl2, polyvinyl alcohol or poly-L-ornithine. Hain et al. , Mol. Gen. Genet. 199: 161 (1985) and Draper et al. , Plant Cell Physiol. 23: 451 (1982). The electroporation of protoplasts and whole cells and tissues has also been described. Donn et al. , in Abstracts of Vl lth International Congress on Plant Cell and Tissue Culture IAPTC (Abstracts of the 7th International Congress on tissue culture and cells of IAPTC plants), A2-38, p. 53 (1990); D'Halluin et al. , Plant Cell 4: 1495-1505 (1992) and Spencer et al. , Plant Mol. Biol. 24:51 -61 (1994). The transgenic dicamba tolerant plants of any type can be produced according to the invention. In particular, broad-leaved plants (including beans, soybeans, cotton, chickens, potatoes, sunflowers, tomatoes, tobacco, fruit trees and ornamental plants and trees) that are currently known to be injured by dicamba can be transformed so that become tolerant to the herbicide. Other plants (such as corn, sorghum, small grains, sugarcane, asparagus and grass), which are currently considered tolerant to dicamba, can be transformed to increase their tolerance to the herbicide. "Tolerant" means that the transformed plants can grow in the presence of a quantity of dicamba, which inhibits the growth of untransformed plants. It is anticipated that the dicamba degrading oxygenases of the invention can function with endogenous reductases and ferredoxins found in host cells and transgenic organisms to degrade dicamba. The chloroplasts of plants are particularly rich in reductases and ferredoxins. Accordingly, a preferred embodiment for the production of transgenic tolerant transgenic plants is the use of a sequence encoding the peptide that will direct the dicamba degrading oxygenase to chloroplasts ("a sequence that targets chloroplasts"). The DNA encoding the chloroplast focusing sequence is placed, preferably upstream (5 ') of the sequence encoding the dicamba degrading oxygenase, but it can also be placed downstream (3') of the coding sequence, or both. upstream as current below the coding sequence. Exemplary chlorplast focusing sequences include the cab-m7 corn signal sequence (see Becker et al., Plant Mol. Biol. 20:49 (1992) and PCT WO 97-41228) and the glutathione reductase signal sequence of pea (Creissen et al., Plant J. 2: 129 (1992) and PCT WO 97/41228). An alternative preferred embodiment is the direct transformation of chloroplasts using a construct comprising a promoter functional in chloroplasts to obtain the expression of oxygenase in chloroplasts. See, for example, PCT application WO 95/24492 and US patent no. 5,545,818.

In yet another embodiment, the invention provides a method for controlling weeds in a field where dicamba tolerant transgenic plants are growing. The method comprises applying an effective amount of dicamba to the field to control the weed. The methods for applying dicamba and the effective amounts of dicamba to control various types of weeds are known. See, Crop Protection Reference, pages 1803-1821 (Chemical &Pharmaceutical Press, Inc., New York, NY, 11th ed., 1995). In another embodiment, the invention provides a method for degrading dicamba present in a material, such as soil, water or waste products from a dicamba producing facility. Such degradation can be achieved using the dicamba degrading enzymes of the invention. Enzymes can be purified from microorganisms that express them naturally (see Example 1) or can be purified from transgenic host cells that produce them. If the enzymes are used in such methods, then appropriate cofactors should also be provided (see Example 1). The effective amounts can be determined empirically as is known in the art (see Example 1). Alternatively, prokaryotic and eukaryotic transgenic microorganisms can be used to degrade dicamba in such materials. Transgenic prokaryotic and eukaryotic microorganisms can be produced as described above, and the effective amounts can be determined empirically as is known in the art. The dicamba is introduced into the environment in large quantities on a continuous basis. The elimination of dicamba is dependent, in large part, on the action of enzyme systems, which are found in microorganisms that inhabit the soil and water of the planet. An understanding of these enzyme systems, including Dicamba degrading O-demethylases and their three components, is important in the effort to exploit natural and genetically engineered microbes for bioremediation and the restoration of soil, water and other contaminated materials. Thus, dicamba degrading enzymes, DNA molecules, DNA constructs, etc. , of the invention, can be used as research tools for the study of degradation of dicamba and bioremediation. Finally, the dicamba degrading enzymes of the invention can be used in an assay for dicamba. A mixture suspected of containing dicamba is mixed with a Dicamba-degrading O-demethylase or a combination of a dicamba-degrading oxygenase, dicamba-degrading ferredoxin and a dicamba-degrading reductase. Suitable assays are described in Example 1. In particular, detecting or quantifying the fluorescence due to the generation of 3,6-DCSA serves as a convenient assay.

EXAMPLES EXAMPLE 1 . Purification and characterization of the Dicamba O-demethylase components of Pseudomonas maltophilia DI-6 METHODS AND MATERIALS: Growth conditions and bacteria. Pseudomonas maltophilia, strain DI-6 (Kreuger, et al., (1989) J. Agrie. Food Chem., 37: 534-538) was isolated from a soil site persistently contaminated with dicamba. The bacterium was provided by Dr. Douglas Cork of the Illinois Institute of Technology (Chicago, IL), and was maintained in reduced chloride medium (Kreuger, JP, (1989) Ph. D. Thesis, Illinois Institute of Technology , Chicago, IL), supplemented with either dicamba (2 mg / ml) or a mixture of glucose (2 mg / ml) and Casamino Acids (2 mg / ml). The carbon sources were sterilized by filter and added to the medium after it was subjected to an autoclave. The solid media was prepared by the addition of 1% (w / v) Gelrite (Scott Laboratories, West Warwick, R. I.).

Chemicals and reagents. Dicamba, 3,6-DCSA and [14C] dicamba (U-phenyl-14C, 42.4 mCi / mmol, radiochemical purity greater than 98%) were supplied by Sandoz Agro, Inc. (Des Plaines, IL). To increase the solubility, the stock solutions of dicamba and 3,6-DCSA were prepared by titration with KOH at pH 7.0. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), unless stated otherwise. Superose 12 column packings, Mono Q, Q-Sepharose (Fast Flow) and Phenyl Sepharose (CL-4B) for FPLC apparatus (fast performance liquid chromatography) were obtained from Pharmacia (Milwaukee, Wl). Ampholyte pH 4-6 and ampholyte pH 4-9 were purchased from Serva (Heidelberg, FTG). Acrylamide, β-mercaptoethanol, N, N, N ', N'-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) were from Biol-Rad Laboratories (Hercules, CA). The thin layer chromatography plates were silica gel (250 μm thick) with UV indicator 254, and were purchased from J .T. Baker Chemical Co. (Phillipsburg, NJ). Enzyme assays Dicamba O-demethylase activity was assayed by measuring the formation of [1 C] 3,6-DCSA of [1 C] dicamba. Briefly, the activity in mixtures of enzyme components was measured at 30 ° C in a reaction mixture containing 300 ul standard phosphate buffer 25 mM potassium (pH 7.0), MgCl 2 1 0 m M, 0.5 mM NADH (beta nicotinamide adenine dinucleotide, reduced) form, 0.5 mM ferrous sulfate, 50 uM cold dicamba, [14C] dicamba 2.5 uM (final radioactive dicamba specific activity was 0.9 mCi 1 / mmol) and different amounts of cell lysate or enzyme partially purified. All enzyme assays during the final purification steps were conducted in phosphate buffer because it was found that the optimum pH for dicamba O-demethylase activity is in the middle range of the phosphate buffers, and activity was observed of major enzyme with phosphate buffer compared to Tris-HCl [tris (hydroxymethyl) aminomethane hydrochloride] at pH 7.0. The reactions were initiated by the addition of the substrate, dicamba. At specific times, the reactions were stopped by adding 50 μl of 5% H2SO4 (vol / vol). Then dicamba and dicamba metabolites were extracted twice with an ether volume, and the extracts were evaporated to dryness. The recovery efficiencies (averages ± standard deviations) for extraction production were 87% ± 2% for dicamba and 85% ± 3% for 3.6-DCSA (Yang et al., Anal. Biochem. 21 9: 37- 42 (1 994)). The [4C] dicamba and the 14C-labeled metabolites were separated by thin layer chromatography (TLC). The dicamba extracted with ether and its metabolites were redissolved in 50 μl of ether before being stained on a TLC plate. The solvent system for running the TLC was chloroform-ethanol-acetic acid (85: 10: 5 [vol / vol / vol]). The resolved reaction products were visualized and quantified by exposing the TLC plate to a phosphor screen for 24 hours and then tracking the screen on a Phosphorlmager SF (Molecular Dynamics, Sunnyvale, CA). Estimates of the amount of radioactivity in a particular spot on the TLC plate were determined by comparing the total pixel count at that spot with respect to a spot on the same plate containing a known amount of [14C] dicamba. One unit of activity was defined as the amount of enzyme that catalyzes the formation of 1 nmol of 3,6-DCSA of dicamba per minute at 30 ° C. The specific activities were based on the total protein concentration of the assay mixture. The activity of the Dicamba demethylase reductase component was tested by measuring the reduction of 2,6-dichlorophenolindolfenol (DCI P) with a Hitachi U-200 spectrophotometer. The reaction contained in 0.5 mM NADH, 0.2 mM FAD (flavin adenine dinucleotide), 50 μM DCIP, 20 mM Tris buffer (pH 8.0), and 1 0-1 00 μl of enzyme sample in a total volume of 1 ml. The change in absorbance at 600 nm was measured over time at room temperature. The specific activity was calculated using an extinction coefficient at 600 nm of 21.0 μM "1cm" 1 for reduced DCI P. The specific activity was expressed as DCI P nmol reduced min "mg" 1 protein. further, an in situ CDI P assay was used to detect and locate the reductase activity in separate protein preparations in isoelectric focusing (IEF) gels. After electrophoresis of the proteins in an EF I gel, sliced lanes of the gel were washed with 20 ml of cold buffer of 20 mM Tris-HCl (pH 8.0). Low melt agarose was dissolved on heating in 10 ml of 20 mM Tris-HCl buffer (pH 8.0) at a final concentration of 1.5% (w / v). when the agarose was cooled to almost room temperature, it was supplemented with 0.2 mM FAD, 50 μM DCI P and 0.5 mM NADH. The test mixture was emptied on a glass plate and allowed to solidify. The piece of gel was placed on top of the solidified reaction mixture and allowed to settle at room temperature for 15 minutes. If the gel slice contained a protein with reductase activity, an incipient band of reduced DCI P was generated in the blue support of DCI P. Cell lysates. The cells were grown at an optical density at 550 nm of 1.3 to 1.5 in a liquid medium of reduced chloride containing a mixture of glucose and Casamino acids on a rotary shaker (250 rpm at 30 ° C). The cells were harvested by centrifugation, washed twice with cold 1 00 mM MgCl 2, and centrifuged again. The cell pastes were used either immediately or were rapidly frozen in liquid nitrogen and stored at -80 ° C. At the time of enzyme purification, 25 g of frozen cells were thawed and resuspended in 50 ml of buffer containing 25 mM Tris buffer (pH 7.0), 10 mM MgCl 2 and 0.5 mM EDTA. Phenylmethylsulfonyl fluoride and dithiothreitol were added at final concentrations of 0.5 mM and 1 mM, respectively. After the addition of 10 mg of lysozyme and 1 mg of DNase, the cells were stirred for 10 min on ice and ruptured by sonication (sonicator model XL2020; Heat Systems) on ice at a medium setting (setting 5) with 1 2 20-second bursts and 40-second rest intervals. The resulting cell lysates were diluted to 90 ml with isolation buffer and centrifuged at 76,000 x g for 1 h at 4 ° C. The supernatant was used as the source of clarified cell lysate. Purification of enzymes. All procedures were performed at 4 ° C, unless stated otherwise. The solid ammonium sulfate was added slowly to a volume of 90 ml of clarified cell lysate at 40% (w / v) saturation, with constant agitation. After 15 minutes of stirring, the mixtures were centrifuged at 1 5,400 x g for 15 minutes, and the precipitate was discarded. Additional solid ammonium sulfate was added at 70% (w / v) saturation, with constant stirring of the supernatant. After 1.5 min of stirring, the mixtures were centrifuged under the conditions described above. The supernatant was discarded, and the precipitate was resuspended in a minimum volume of buffer A (20 mM Tris (pH 8.0), 2.5 mM MgCl2, 0.5 mM EDTA, 5% (v / v) glycerol, and dithiothreitol 1 mM). Then the 40% -70% ammonium sulfate cut was loaded onto a Phenyl-Sepharose column (2.5 by 10 cm) connected to an FPLC apparatus (Pharmacia) and levigated with a decreasing linear gradient of ( NH4) 2S04 from 10% (w / v) to 0% (w / v) The column was pre-equilibrated with buffer A containing 10% (w / v) of ammonium sulfate.The flow rate was 1 ml / The protein concentrations were continuously monitored at A280 during the column levigation, the column was washed with 1 20 ml of buffer A containing 10% (w / v) of ammonium sulphate until A280 readings were obtained. The bound proteins were levigated with a decreasing gradient of (NH4) 2SO4 in a total volume of 21 0 ml.] Fractions of 2 ml were collected. 1 μl counts of each fraction were added to the standard dicamba O-demethylase assay mixture (see above), except that the non-radioactive dicamba was used as the substrate. Dicamba O-demethylase activity was detected by monitoring the conversion of dicamba to the highly fluorescent reaction product 3,6-DCSA with a UV lamp held by hand (312 nm, Fotodyne) in a dark room. This procedure allowed the resolution of Dicamba O-demethylase in three tanks containing the separated components (designated components I, I I and I I I). Each component was essential for the Dicamba O-demethylase activity (see below). When a single component was tested, the other two components were provided in excess. Fractions containing a simple type of activity were deposited (Component I, fractions 128-145, component I I, unbound fractions 1 2-33, component I I, fractions 62-69). (i) Purification of component I. Fractions containing the activity of component I (levigating from a column of Phenyl-Sepharose at 0 M (NH) 2S0, fractions 128-145) were deposited to provide a total volume of 34 ml. The deposited samples were concentrated at 10 ml by centrifugation in a Centriprep-10 device (Amicon) and then applied to a FPLC column of Q-Sepharose (Fast Flow) (Pharmacia) (2.5 by 6 cm) equilibrated with buffer A and was washed with 80 ml of buffer A. The proteins bound to the column were levigated with a linear gradient of 1 00 ml from 0 to 0.6 M KCI in buffer A at a flow rate of 1 ml / min. The fractions were collected in 1.5 minute intervals. Fractions exhibiting the activity of component I (fractions 29-37) were deposited, dialyzed against absorber A overnight at 4 ° C and applied to an FPLC anion exchange column Mono Q HR 5/5 in buffer A. The proteins were levigated at 1 ml / min when using a gradient of 50 ml of increasing KCI concentration () to 0.5 M). Fractions showing activity of component I (fractions 1 to 25) were deposited and concentrated to 0.4 ml by centrifugation in a Centricon-10 device. The concentrated sample was then subjected to chromatography on a Superlose 12 FPLC column (1.6 by 50 cm) at a flow rate of 0.2 ml / min with buffer A containing 100 mM KCI. Fractions 7-10 showing activity of component I were deposited and concentrated by centrifugation in a Centricon-1 0 device. Partially purified component I was diluted with cold glycine at 1% >; (p / v) and concentrated by centrifugation in a Centricon-10 device three more times to desalt in the preparation for IEF electrophoresis. The desalted and concentrated sample was then applied to a 6% (w / v) I EG gel (pH 4-6) and subjected to electrophoresis for 1.5 hours at 4 ° C (see below). After electrophoresis, the gel was washed with 25 mM cold phosphate buffer (pH 7.0) for 5 minutes and then each slice of the gel lane was cut into small pieces (6 mm x 4 mm). Proteins were levigated from the cut gel fragments by grinding them with a pipette tip in the presence of 10 μl of 25 mM phosphate buffer (pH 7.0). The protein from each segment was mixed with an excess of components I I and I I I and tested for dicamba O-demethylase activity. The gel segment, which showed the activity of component I (which was also reddish brown in color) was loaded onto a 12.5% (w / v) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) to verify The purity of the sample. (ii) Purification of component II. Component I I obtained by Phenyl-Sepharose column chromatography was dialysed against buffer A overnight at 4 ° C and applied to a FPLC Q-Sepharose column (2.5 by 6 cm). The levigation conditions of the sample were identical to those described above for component I, except that the levigation gradient was 0 to 1 M KCI in buffer A. Fractions exhibiting component II activity (fractions 30-37) were deposited, dialyzed against buffer A, concentrated to 0.4 ml and applied to a FPLC Superóse column (1.6 by 50 cm). The procedures for the application and levigation of the sample were identical to those described above for component I. Fractions exhibiting component II activity (fractions 3-6) were deposited, diluted with an equal volume of buffer A and applied to a Mono Q FPLC column. The proteins were levigated from the column using the same KCL gradient as for component I. Fractions 20-25 showed activity of component II. The partially purified component II was further purified by I EF electrophoresis (pH 4-6) using the same conditions as described in component I. The gel segment, which showed the activity of component I I was loaded on a SDS-PAGE at 12.5% (w / v) for further analysis. (iii) Purification of component III. Component I I I obtained by Phenyl-Sepharose column chromatography was dialysed against buffer A overnight at 4 ° C and applied to a FPLC Q-Sepharose column (2.5 by 6 cm). The conditions were identical to those described above for component I. Fractions exhibiting component III activity (fractions 26-38) were dialysed against buffer B [10 mM Tris-HCl (pH 7.5), 2.5 M MgCl2, 5% glycerol (v / v), 1 mM dithiothreitol] and concentrated to 5 ml. The blue dye affinity matrix [Cibacron Blue 3GA type 3000 (Sigma)] was packed on an FPLC column (1 x 5 cm) and pre-equilibrated with 20 ml of buffer B. The concentrated component III was loaded onto the column of blue dye and was washed with 20 ml of buffer B at a flow rate of 0.2 ml / min until A280 of the baseline levels reached column effluent. The ligated protein was then levigated with 5 mM NADH in Buffer B. Fractions containing reductase activity were detected when assayed for Dicamba O-demethylase activity in the presence of an excess of components I and II and also by the ability of each fraction to reduce DCI P in the presence of NADH. Fractions having strong reductase activity in both assays were deposited, dialyzed against buffer A containing 100 mM KCL, concentrated to 0.2 ml, and applied to a column of FPLC Super 12. The same conditions were used to run the Superóse column as described for component I. The fractions containing proteins, which catalyzed the reduction of DCIP were deposited, dialyzed against buffer A and applied to a Mono Q FPLC column. The proteins were levigated using the same conditions as for component I. The partially purified component I I was further purified by gel electrophoresis I EF (pH 4-6). The protein reductase activity within the EF gel I was detected by assaying for the reduction of DCI P in an agarose gel envelope as described above. The gel segment, which showed activity of component I I, was loaded onto a 12.5% (w / v) SDS-PAGE for further analysis. Determination of the NH2-terminal amino acid sequences. The protein bands identified on IEF gels as having activities of component I, component I I, or component III were cut and placed in the cavities of a 12.5% (w / v) SDS polyacrylamide gel. After electrophoresis, the gel slices containing the purified proteins was transmancharon onto a PVDF (polyvinylidene difluoride) membrane (Millipore) in a Trans-Blot (Bio-Rad, Richmond, CA) cell at 25 volts for 1 6 hours. The stain absorber was a 20% (v / v) methanol solution with 10 mM CAPS [3- (cyclohexylamino) -1-propanesulfonic acid], pH 1 0.0. sequencing was performed using an Applied Biosystems Inc. 420 H machine by Edman degradation (Edman and Henschen (1 975) pages 232-279, in SB Needleman (ed.), Protein sequence determination, 2a ed., Springer-Verlage, New York). Determination of protein concentration. Protein concentrations were determined by the method of Bradford (1976) Anal. Biochem. 72: 248-254, with bovine serum albumin as the standard. SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to modified Laemmli methods (Laemmli (1970) Nature, 227: 680-685). SDS gels were made at 12.5% of 85 x 65 x 0.75 mm as follows: running gel: 2.5 ml of acrylamide solution / 40% bis (w / v) (37.5: 5: 1), 1 ml of solution of run buffer Tris-HCl 3M (pH 8.8), SDS at 0.8% (w / v), 4.5 ml H2O, 5 μl TEMED, and 40 μl 10% APS (w / v); accumulation gel: 0.5 ml of acrylamide / bis at 40% (w / v), 0.5 ml of buffer accumulation solution [Tris-HCl 1 M (pH 6.8), SDS at 0.8% (w / v)], 3 mi of H2O, 5 μl of TEMED, and 12.5 μl of 10% APS (w / v). The composition of the run buffer was 25 mM Tris-HCl (pH 8.3), 0.2 M glycine, and 0.1% SDS) (w / v). The sample buffer contained 0.25 ml of accumulation buffer, 0.6 ml of 20% SDS (w / v), 0.2 ml of β-mercaptoethanol, and 0.95 ml of bromophenol blue 0.1% (w / v) in glycerol 50 % or (v / v). Electrophoresis was performed at 80 volts in a Bio-Rad Mini Gel apparatus until the tracking dye was 0.5 cm from the anode end of the gel. The proteins were stained with Coomassie Brilliant Blue R-250 at 0.1% (w / v) in a mixture of isopropanol, water and acetic acid at a ratio of 3: 6: 1 (v / v / v). The stain removal was carried out in a mixture of methanol, water and acetic acid at a ratio of 7:83: 10 (v / v / v). standard proteins (Gibco BRL) included: myosin (214.2 kDa), phosphorylase B (1 1 1 .4 kDa), bovine serum albumin (74.25 kDa), ovalbumin (45.5 kDa), carbonic anhydrase (29.5 kDa), ß- lactoglobulin (18.3 kDa), and lysozyme (15.4 kDa). Determination of molecular weight. The molecular weight (Mr) of peptides under denaturing conditions was estimated using SDS-PAGE analysis. The molecular weights of the natural components I, II and III were estimated by gel filtration through a column of FPLC 1 0/30 of Superóse 12 HR (Pharmacia) at a flow rate of 0.2 ml / min in buffer A containing KCI 1 00 mM. The calibration proteins were gel filtration standards from Bio-Rad. These were: bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (1.7 kDa) and vitamin B-12 (1.35 kDa). The empty volume of the Superosa 12 column was calculated using Blue Dextran (Mr 2,000, 000, Sigma). IEF. Isoelectric focusing gel electrophoresis (I EF) was performed in a vertical mini-gel apparatus (Model # MGV-1 00) of C. B.S. Scientific Co. (Del Mar, CA). IEF gels with 6% (w / v) polyacrylamide (70 x 90 x 1 mm) were made by mixing the following: 1.6 m 30% (w / v) acrylamide / bis (37: 5: 1), 0.8 g of glycerol, 0.32 ml of ampholyte pH 4-6 (Serva), 0.08 ml of ampholyte pH 4-9 (Serva), 5.2 ml of H2O, 1 0 μl of TEMED and 80 μl of APS at 10% or ( p / v). The cathode buffer was β-alanine 1 00 mM and the anode buffer was 100 mM acetic acid. Protein samples in approximately 1 to 10 μl of 1% (w / v) glycine were mixed with an equal volume of sample buffer [glycerol 50% (v / v), ampholyte pH 4-0 to 1. 6% (v / v), ampholyte from pH 4-6 to 2.4% (v / v)]. Samples were loaded at the cathode end of the gel and allowed to migrate at 200 volts for 1.5 hours and 400 volts for another 1.5 hours. The proteins were stained with Coomassie Brilliant Blue R-250 using the procedure described above for SDS polyacrylamide gels. The IEF markers (Sigma) were: amyloglucosidase, pl 3.6; glucose oxidase, pl 4.2; trypsin inhibitor, pl 4.6; β-lactoglobulin A, pl 5.1; carbonic anhydrase I I, pl 5.4; carbonic anhydrase I I, pl 5.9 and carbonic anhydrase I, pl 6.6. Kinetic analysis. The kinetics of the demethylation reaction catalyzed by Dicamba O-demethylase were studied by analyzing the initial rates of the reaction in the presence of a constant concentration of the enzyme and increasing concentrations of the substrate, dicamba. The reaction mixtures contained 25 mM potassium phosphate buffer (pH 7.0), 10 mM MgCl 2, 0.5 mM NADH, 0.5 mM FeSO 4, 25 μg partially purified O-demethylase enzyme (the fraction of 40% -70% or (p / v) of (NH4) 2SO4 of a clarified cell lysate], various concentrations (0.5 to 50 μM) of dicamba and various concentrations (0.025 to 2.5 μM) of [14C] dicamba (U-phenyl-14C, 42.4 mCi / mmol ) in a total volume of 300 μl For assays with dicamba concentrations of 0.5 μM and 1 μM, the reaction volume was increased to 900 μl to ensure that sufficient quantities of radioactive dicamba and its metabolites were present. Amounts of all other components in the reaction were tripled The conversion of [14C] dicamba to [14C] 3,6-DCSA was determined for different times at each concentration of dicamba using a Phosphorlmager SF to trace radioactivity on phosphor screens , which had been exposed to T plates LC for 24 hours. One unit of activity was defined as the amount of enzyme that forms 1 nmol of 3,6-DCSA per minute at 30 ° C. The initial rates of each reaction were determined by plotting the reaction rate against time at each substrate concentration. Data were modeled on Michaelis-Menten kinetics and Km and Vmax values were determined by fitting to Lineweaver-Burk plots using SigmaPlot® (Jandel Scientific, Corte Madera, CA). Oxygen requirement Preliminary experiments using a Clark oxygen electrode indicated oxygen consumption during a standard dicamba O-demethylase assay with dicamba as a substrate. To verify an oxygen requirement in the O-demethylation of dicamba by Dicamba O-demethylase, reactions were conducted in an anaerobic chamber, which contained less than 1 ppm of oxygen. The procedures for oxygen displacement of the reaction mixture were carried out at 4 ° C. The reaction mixtures lacking enzyme were placed in a flask and sealed with a rubber stopper. For displacement of oxygen, the bottle was evacuated twice by vacuum and flushed with nitrogen each time. After a third evacuation, the flask was flushed with 90% nitrogen) plus 10% hydrogen. The enzyme solution was similarly purged of oxygen (being careful not to bubble the enzyme solution). Both the reaction mixtures and the enzyme solutions were transferred to an anaerobic chamber (atmosphere of 95% N2-5% H2). Two hundred forty microliters of clarified cell lysate was injected through the rubber stopper with a microsyringe and mixed gently with 960 μl of oxygen-free reaction mixture. The reactions were carried out at 30 ° C. An examination of the reaction products on TLC plates showed that the production rate of [14C] 3,6-DCSA from [14C] dicamba under anaerobic conditions was significantly lower than the rate of reactions with the same amount of enzyme under aerobic conditions. Under anaerobic conditions, there was virtually no conversion from dicamba to 3,6-DCSA within 1 hour. However, when a parallel reaction mixture was taken from the anaerobic chamber after 30 min and incubated with air, a significant amount of one of the components of the O-demethylase enzyme complex of dicamba was an oxygenase. It can be noted that the in vitro conversion of [14C] dicamba to [14C] 3,6-DCSA mimics the previously described in vivo conversion pathway (Cork and Kreuger, Adv. Appl. Microbiol. 36: 1-66 (1 991); Yang et al. , Anal. Biochem. 21 9: 37-42 (1994)). In these studies, DCSA was identified as a reaction product by both TLC and capillary electrophoresis. The severe identification of the first major degradation product of dicamba as DCSA both in vivo and in vitro has been obtained by gas chromatography-mass spectrometry analysis. Requirements for components and cofactors. After the initial separation of the three components of O-demethylase by phenyl-Sepharose column chromatography, the partially purified preparations were taken individually through further purification on a Q-Sepharose column (2.5 by 6 cm). Samples were applied to a liquid chromatography column of rapid protein Q-Sepharose (Fast Flow) (Pharmacia) in buffer A and levigated with a 100 ml linear gradient from 0 to 0.6M KCI (for the oxygen component) or 0 to 1.0 M of KCI (for the ferredoxin and reductase components) in fractions of 1.5 mi. The appropriate deposited fractions of separate columns for oxygenase purification (fractions 29 to 37), for purification of ferredoxin (fractions 30 to 37) or for reductase purification (fractions 26 to 38) were used for the determination of requirements of components and cofactors . The three components were tested for O-demethylase activity in various combinations to determine component requirements. To determine the cofactor requirements, the O-demethylase activity was tested using a mixture of the three components with [14 C] for 30 minutes at 30 ° C. The amounts of partially purified protein (provided in an optimized ratio) in the reaction mixtures were 85 μg of oxygenase, 55 μg of ferredoxin and 50 μg of reductase. The concentration of cofactors used in the reaction mixtures were 0.5 mM NADH, 0.2 mM FAD, 0.5 mM FeSO3, 10 mM MgCI2, 0.5 mM NADPH and 0.2 mM FMN.

RESULTS Component I. The O-demethylase component of dicamba, which was ligated very strongly to the Phenyl-Sepharose column (initially designated as component I and subsequently identified as an oxygenase) was clearly reddish-brown in color. This indicated the potential presence of one or some proteins containing one or several iron-sulfur groupings or one or several heme groups. The fractions with activity of component I of the Phenyl-Sepharose column were subjected to further purification by Q-Sepharose (Fast Flow) and Mono Q chromatography and then to separation in an oversize size 12. The protein of component I it was then purified further in an IEF gel. The protein from the largest band in the EF I gel (with an apparent pl of approximately 4.6) was cut and separated from any remaining minor contaminant by SDS-PAGE. The major component I protein obtained after purification by I EF was greater than 90%) pure as judged by densitometric analysis of this SDS-polyacrylamide gel stained with Coomassie Blue. The N-terminal amino acid sequence of the dominant protein with an apparent molecular mass of approximately 40,000 Daltones was determined. Results of amino acid sequencing indicated that the first 29 amino acids of the N-terminal region were present in the following sequence (residues in parentheses are the best assumptions): Thr Phe Val Arg Asn Wing Trp Tyr Val Wing Wing Leu Pro Glu Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr lie Leu Asp (Asp or Thr) (Pro) [SEQ I D NO: 1] Comparison with amino acid sequences in several bases indicated little or no homology with NH2-terminal sequences reported for other monoxigenases or dioxygenases.

Component II. The protein fraction, which did not bind to a Phenyl-Sepharose column, was designated as component I I. Because a yellowish fraction could be replaced by Clostridium pasterurianum ferredoxin (but with slower reaction rates) when assayed in combination with components I and I I, it was tentatively designated as a fraction containing ferredoxin. Clostridium ferredoxin clearly worked in place of component I I, but given the highly impure nature of component I I used in these experiments, the efficiency of the Clostridium enzyme was significantly lower than that of the putative ferredoxin of strain DI-6. In particular, 55 μg of partially purified component II mixed with excess amounts of components I and III catalyzed the conversion of dicamba to 3,6-DCSA at a rate of approximately 5 nmol min "1 mg" 1, while 100 μg of Highly purified ferredoxin from Clostridium resulted in an activity of only 0.6 nmol min "1 mg.'1 The steps of urification involving chromatography on Q-Sepharose (Fast Flow), Superóse 12 gel filtration and Mono Q chromatography yielded approximately one milligram of purified protein from about initial 25 grams of cell paste This fraction was purified in a manner similar to the oxygenase component mediated electrophoresis in an EF I gel and electrophoresis suggested by the active IEF fraction in an SDS-polyacrylamide gel. of activity of component II in levigated proteins from gel segments I EF indicated that a fraction with a pl of about 3.0 contained the protein active in component I I. The protein from this gel slice was levigated and subjected to SDS-PAGE. Gel staining with Coomassie Blue revealed a promising band of protein along with a smear of lower molecular weight proteins. The prominent protein with an apparent molecular weight of about 28,000 Daltones was stained on a PVDF membrane. The amino acid sequencing revealed the following N-terminal sequence of 20 amino acids: Thr Tyr Val Val Thr Asp Ala Xaa lie Lys Xaa Lys Ty Met Asp Xaa Val Glu Val Xaa [SEQ ID NO: 2] Component III. The I I I component of O-demethylase from dicamba was retained on a Phenyl-Sepharose column at high concentrations of (NH4) 2S0 and was levigated at approximately 4% (w / v) of (NH4) 2SO4. This light yellow fraction was tentatively identified as a fraction containing reductase based on its ability to reduce oxidized cytochrome c and DCI P in the presence of NADH and because it could be replaced by porcine heart cytochrome c reductase (Type 1, Sigma) in assays with components I and II. In this set of reactions, the use of 50 μg of partially purified I I I component produced a reaction rate of approximately 5 nmol min "1 mg" 1, when mixed with an excess of components I and I I. The highly purified cytochrome c reductase showed a specific activity of approximately 2.5 nmol min "1 mg" 1 in the reaction, in activity well below that provided by component III, when one considers the impurity of the crude component III used in these essays. In addition, component I II exhibited reductase activity when incubated with cytochrome c or 2,6-dichlorophenol-indophenol (DCPI P) in the presence of NADH. Neither component I nor component I I showed activity in either of these two reductase assays. Further purification of this fraction by chromatography on columns containing Q-Sepharose (Fast Flow), blue dye affinity matrix, packages of Supersec 1 2, and Mono Q resulted in low amounts of protein in the fractions with reductase activity. The component I I I protein was approximately 70% pure as judged by densitometric analysis of the active protein fraction after separation by SDS-PAGE and staining with Coomassie Blue. To further exacerbate the purification of component III, two different protein fractions from the Mono Q column passage were found to contain activity when tested with the ferredoxin and oxygenase components. Further purification of these two fractions by electrophoresis in an EF I gel revealed that the reductase activities of the two fractions had clearly different isoelectric points. This was demonstrated by cutting lanes containing each of the two reductase fractions of the I EF gel and placing the slices on top of a low melting agarose pad containing a DCI P reaction mixture. The reductase activity in both Slices of gel was identified by the NADH-dependent reduction of DCI P to its colorless, reduced form. The reductase in fraction 35 had an apparent pl of about 5.6 while the reductase in fraction 27 had an apparent pl of about 4.8. Both reductase activities isolated from the IEF gel slices were unstable and were present in low amounts. In fact, only the reductase of Fraction 35 of the Mono Q column fractionation retained sufficient concentration of protein and activity to allow further purification and characterization. A slice of an I EF gel containing this reductase activity was levigated and separated from contaminating proteins to SDS-PAGE. The predominant protein in this gel was one with a mass of approximately 45,000 Daltones. Size exclusion chromatography had indicated an approximate molecular mass of 50,000 Daltons for component III in its natural state. Biochemical characteristics of Dicamba O-demethylase. Dicamba O-demethylase activity was measured during in vitro incubations at temperatures ranging from 20 ° C to 50 ° C and at pH values of about 6 to 9. Activity pecked sharply at 30 ° C and broadly at pH values between 6.5 and 7.5. The enzymatic activity was dependent on the type of pH buffer employed. At pH 7, for example, activity was approximately 40% lower in Tris-containing buffers than in buffers containing phosphate. Values for Km and Vmax for Dicamba O-demethylase were estimated using SigmaPlot® to generate better adjusted Michaelis-Menten curves and Lineweaver-Burk plots of duplicate experiment data. The Km of dicamba was estimated to be approximately 9.9 ± 3.9 μM and Vmax for the reaction was estimated to be approximately 108 ± 12 nmol / min / mg. The three components were tested for Dicamba O-demethylase activity in various combinations. None of the components showed enzymatic activity when tested alone. Actually, a significant amount of O-demethylase activity could be detected only when all three components were combined. A mixture of components I and I I exhibited small amounts of enzymatic activity, probably due to traces of component I I I contaminating the fractions of component I. Both NADH and NADPH supported enzymatic activity, NADH being markedly more effective than NADPH. Mg2 + was necessary for enzymatic activity. Fe2 +, flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) produced little or no stimulation of enzyme activity with partially purified protein preparations in these experiments. The highest activity was obtained using a combination of NADH, Fe2 +, Mg2 +, and FAD.

DISCUSSION The Dicamba O-demethylase from Pseudomonas maltophilia, strain Dl-6, is a three-component oxygenase (Wang, XZ (1996) Ph. D. thesis, University of Nebraska-Lincoln, Lincoln, NE), responsible for the conversion of the herbicide, dicamba (2-methoxy-3,6-dic! orobenzoic acid), to 3,6-dichlorosalicylic acid (3,6-DCSA, 2-hydroxy-3,6-dichlorobenzoic acid). Several purification schemes have been devised, which have allowed the isolation of each of the three components to a homogeneous or almost homogeneous state. The initial separation of the three components was achieved by chromatography on a Phenyl-Sepharose column. The enzymatic activities and other characteristics of the partially purified components allowed a tentative identification of the components such as a reductase, a ferredoxin and an oxygenase - a composition similar to that found in a number of other oxygenates of multiple components that contain heme and that do not contain previously studied heme (Batie, et al. (1 992) pages 543-565, in F. Müller (ed.), Chemistry and biochemistry of flavoenzymes, vol.III, CRC Press, Boca Raton; Harayama, et al. (1 992) Annu Rev. Microbiol 46: 565-601; Mason and Cammack (1992) Annu Rev. Microbiol 46: 277-305; Rosche et al. (1995) Biochem. Biophys. Acta 1252: 177-179) . The isolated component III of the Phenyl-Sepharose column catalyzed the NADH-dependent reduction of both cytochrome c and the dye, DCI P. In addition, its ability to support the conversion of dicamba to 3,6-DCSA, when combined with the Components I and II could be replaced in part by cytochrome c reductase. Component II could be replaced by the addition of ferredoxin from Clostridium pasteurianum to reactions containing components I and I I I. The absolute need for molecular oxygen to support the O-demethylation reaction indicated that the remaining component was an oxygenase. Oxygenase, c. The I component of Dicamba O-demethylase (designated as oxygenase D? C) has been purified to homogeneity and subjected to N-terminal amino acid sequencing. The resulting sequence of twenty-nine amino acid residues did not show any significant homology to other protein sequences in the various data banks. However, the information obtained from this amino acid sequence allowed the design of degenerate oligonucleotide probes, which have been used successfully to detect and clone the gene of component I (see Example 2). Additionally, a comparison of the amino acid sequence derived from the nucleotide sequence of this clone with that of the protein sequences in the database showed strong homology to other oxygenases (see Example 2). The apparent molecular mass of the D | C oxygenase, estimated from its migration in SDS-polyacrylamide gels, is approximately 40,000 Daltons. Purified preparations of oxygenase exhibited only a larger band in SDS-polyacrylamide gels with Coomassie Blue and Edman degradation of the protein contained in this band indicated the presence of only one N-terminal species. Estimates derived from the behavior of natural D? C oxygen in size exclusion columns suggest a molecular size of approximately 90,000 Daltones. All these results suggest that natural oxygenase exists as a homodimer. The oxygenase / hydroxylase component of a number of multicomponent systems is composed of an array of subunit type (aß) n, in which the largest subunit is approximately 50,000 Daltons in size and the smallest β subunit is approximately 20 , 000 Daltons in molecular mass (Harayama, et al (1 992) Annu Rev. Microbiol 46: 565-601). In contrast, the O-demethylase oxygenase component of dicamba appears to possess a simple subunit of approximately 40 kDa in molecular mass, which can exister as a dimer in its natural state. This subunit arrangement type (a) n, is similar to that found in other well characterized oxygenases, such as 4-chlorophenylacetamide 3,4-dioxygenase from Pseudomonas sp. CBS strain (Markus, et al (1986) J. Biol. Chem. 261: 12883-12888), phthalate dioxygenase from Pseudomonas cepacia (Batie, et al. (1987) J. Biol. Chem. 262: 1510-1 51 8), 4-sulfobenzoate 3,4-dioxygenase from Comamonas testoteroni (Locher, et al (1 991) Biochem J., 274: 833-842), 2-oxo-1,2-dihydroquinoline 8-monooxiganase from Pseudomonas putida 86 (Rosche et al. (1995) Biochem Biophys. Acta 1252: 177-179), 4-carboxydiphenyl ether dioxygenase from Pseudomonas pseudoalcaligenes (Dehmel, et al. (1995) Arch. Microbiol. 163: 35-41) , and 3-chlorobenzoate 3,4-dioxygenase from Pseudomonas putida, (Nakatsu, et al (1995) Microbiology (Reading) 141: 485-495). Ferredoxin D? C- The I I component of O-demethylase from dicamba (designated ferredoxin D? C) was almost homogeneously purified by several steps of column chromatography and I EF. The final purification by SDS-PAGE yielded a larger protein band (Mr ~ 28,000) and a smear of slightly smaller proteins, which may represent partial breakdown products of the purified ferredoxin. A comparison of the N-terminal sequence of 20 amino acid residues obtained from the highest protein band to other amino acid sequences in the various protein data banks using the computer program package of Genetics Computing Group (GCG) (University of Wisconsin, Madison, Wl) revealed strong homology to a number of bacterial ferredoxins "dicluster". For example, an alignment and comparison of the first 20 amino acids of ferredoxins from Pseudomonas stutzeri, Pseudomonas putida, Rhodobacter capsulatus and Azotobacter vinelandii showed these respective ferredoxins to be 65% >;, 65%, 65% and 60% identity with the N-terminal sequence of the protein of Pseudomonas maltophilia, strain DI-6. The identity of the four residues designated in the sequence of putative ferredoxin as Xaa was uncertain. Based on the position of the extra peaks in the chromatography of the Edman degradation products, it is likely that these Xaa residues are actually residues of propionyl-cysteine formed by the alkylation of the cysteine residues with acrylamide during SDS-PAGE ( Bruñe (1992) Anal Biochem 207: 285-290). If the four Xaa residues are all cysteine residues, the identities of the bacterial ferredoxin sequences with the ferredoxin of Pseudomonas maltophilia become 85%, 85% > , 85% > and 80% > , respectively. The four dicluster ferredoxins, which show strong homology to ferredoxin D? C > they have a stacking of [3Fe-4S] followed by a stacking of [4Fe-4S] at the N-terminus of the protein. This suggests that ferredoxin D? C is distinctly different from the ferredoxin components with [2Fe-2S] clumps, which are usually associated with non-haeme multi-component oxygenates (Harayama, et al. (1992) Annu Rev. Microbiol 46: 565-601; Mason and Cammack (1992) Annu., Rev. Microbiol., 46: 277-305; Rosche, et al. (1 995) Biochem. Biophys., Acta 1252: 177-179). In fact, an analysis of the EPR spectrum (paramagnetic electron resonance) [to be reported in detail elsewhere, Qiao, F., X-Z. Wang, PL Herman, DP Weeks, and JH Golbek (submitted for publication)] suggest that the stacking of [3Fe-4S] at the N-terminus of ferredoxin D? C is the redox center, which is active in electron transport . Ferredoxin D | C is normal for other bacterial ferredoxins since it has a low isoelectric point (ie, a pl of 3 or slightly higher). Low pl% frequently leads to the aberrant migration of these proteins in SDS-polyacrylamide gels and during size exclusion chromatography (O'Keefe, et al (1 991) Biochemistry 30: 447-455). In the case of ferredoxin D? C, the molecular mass estimates based on protein migration during SDS-PAGE were approximately 28,000 Daltones. Similarly, size exclusion chromatography indicated an apparent molecular mass for natural ferredoxin D? C of approximately 28,000 daltons. This molecular mass is significantly greater than that of the other ferredoxins found in oxygenates of multiple bacterial components [ie, 8-13 kDa] (Batie, et al. (1 992) pages 543-565, in F. Müller (ed. .), Chemistry and biochemistry of flavoenzymes, vol II, CRC Press, Boca Raton, Harayama, et al (1 992) Annu Rev. Microbiol 46: 565-601). ReductaseD, c- The component I I I of O-demethylase from dicamba (designated as reductase D? C) has been the most recalcitrant of the three components to be purified. This is partly due to its apparent instability and low abundance in lysates of strain DI-6. However, enough protein has been purified to assign a tentative molecular mass of 45,000 Daltones. This is similar to the molecular mass of approximately 50,000 Daltones obtained from size exclusion chromatography and suggests that reductase c exists in its natural form as a monomer. The purification of the reductase component has been further complicated by the fact that chromatography on a Mono Q and I EF column resolves the purified reductase preparations in two activities with apparently different p-values. Both fractions of the Mono Q column worked in combination with purified ferredoxin D? C and D? C oxygenase to produce the activity of Dicamba O-demethylase. The presence in Sphingomonas sp. strain RW1 of two similar flavoproteins, which also function as reductase components in the three-component dibenzofuran 4,4a-dioxygenase has recently been reported by Bünz and Cook (1993) J. Bacteriol. 175: 6467-6475). Interestingly, both reductasas were 44,000 Daltones in molecular mass, quite similar to that of the reductase D? c of 45,000 Daltones. Multiple components of leghemoglobin reductase have also been observed in lupine root nodules using isoelectric focusing techniques (Topunov, et al. (1982) Biockhimiya (English Edition) 162: 378-379). In this case, IEF revealed four separate components with NADH-dependent reductase activity. The resolution of the question of whether there is only one D reductase that exists in two or two different reductases in the DI-6 strain will depend on the development of improved procedures to isolate larger amounts of the proteins and / or in the cloning and sequencing of the or genes involved. Characteristics of Dicamba O-demethylase. In addition, the physical and biochemical properties of the individual components noted above, enzymatic activity analyzes have shown that the O-demethylase system has a strong affinity (Km = ~ 10μM) for its substrate and a Vmax of approximately 100-1 10. nmol / min / mg. As expected for a soil bacterium collected in a temperate climate zone, the maximum enzymatic activity was observed at temperatures near 30 ° C. while the optimum pH for the enzyme system was in the range of pH 6.5 to pH 7.5, the activity measured with a given enzyme preparation was strongly affected by the type of buffer system employed. The activity in the presence of Tris shocks was at least 40% lower than with phosphate buffers at the same pH. The reaction scheme for the reaction catalyzed by the three components of Dicamba O-demethylase is presented in Figure 1. The electrons of NADH are released through a short electron chain consisting of reductase and ferredoxin to the terminal oxygenase, which catalyzes the oxidation of dicamba. The similarities between Dicamba O-demethylase and several multi-component dioxygenases suggest that Dicamba O-demethylase may potentially possess cryptic dioxygenase activity. However, it is clear that this enzyme is not in the class of dioxygenases which divide O2 and incorporate one oxygen atom in the larger substrate and the other in a small organic substrate, such as α-ketoglutarate (Fujumori and Hausinger (1). 993) J. Biol. Chem. 268: 2431 1-2431 7). In fact, the combinations of reductase D? C, ferredoxin D? C, and oxygenase D? C require only O2, NADH, Mg2 +, Fe2 +, and dicamba for activity.

EXAMPLE 2: Identification and sequencing of a clone encoding the O-demethylase oxygenase of Dicamba from Pseudomonas maltophilia DI-6 As noted in Example 1, the first 29 amino acids of the N-terminal amino acid sequence of the D | C oxygenase have been determined to be (the residues in parentheses are the best assumptions): Thr Phe val Arg Asn Wing Trp Tyr Val Wing Wing Leu Pro Glu Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Me Leu Asp (Asp or Thr) (Pro) [SEQ I D NO: 1] This sequence allowed the design of degenerate oligonucleotide probes, which were synthesized by Operon, Alameda, CA. In particular, a mixture of 32 probes, each of which was 17 nucleotides in length, and containing all possible nucleotide sequences, which could encode the amino acid sequence highlighted in bold before, was used. The oligonucleotide probes were labeled at the 3 'end with digoxigenin (DIG) according to instructions provided by Boehringer Mannheim, Indianapolis, I N. The DIG-labeled probes were first hybridized to genomic DNA of P. maltophilia DI-6, which had been digested with various combinations of restriction enzymes, resolved on a 1% agarose gel > , and they were stained to a nylon filter. Based on these results, a genomic library fractionated by size was constructed in a pBluescript I I KS + vector and transformed into competent DH5a cells of Escherichia coli. The genomic library contained fragments of 1 -2 kb Xho l / Hind III. The oligonucleotide probes labeled with DIG were hybridized to an array of bacterial colonies traced in strips on nylon filters. The plasmid DNA was isolated from positive colonies and subcloned. Both strands of each subclone were sequenced by DNA Sequencing Facility at the University of Nebraska-Lincoln. Hybridization and detection of DIG-labeled probes were performed according to protocols provided by Boehringer Mannheim. A genomic DNA clone was identified encoding the oxygenase. The nucleotide sequence and deduced amino acid sequence of the whole oxygenase are given in the Sequence Listing below as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. A comparison of the amino acid sequence derived from the nucleotide sequence of this clone with that of the protein sequences in the Swiss protein database showed homology with other oxygenases. The homology was determined using the FASTA program of the GCG software package. The strongest homology was with the oxygenase component of vanillate desmethylase (from Pseudomonas sp., Strain ATCC 19151) which showed 33.8% > of identity.

EXAMPLE 3: Identification and sequencing of a clone encoding the Dicamba O-demethylase ferredoxin from Pseudomonas maltophilia DI-6 As noted in Example 1, it was determined that the first 20 amino acids of the N-terminal amino acid sequence of ferredoxin D | C were: Thr Tyr Val Val Thr Asp Ala Xaa Me Lys Xaa Lys Tyr Met Asp Xaa Val Glu Val Xaa [SEQ I D NO: 2] This sequence allowed the design of degenerate oligonucleotide probes. In particular, we used a mixture of 16 probes, each of which was 17 nucleotides in length, and contained all possible nucleotide sequences, which could encode the amino acid sequence highlighted in bold before (taking Xaa is Cys ). DIG labeled probes were used to classify a genomic library as described in Example 2, except that the genomic library contained fragments of 2-3 kb Xho I / Eco Rl. The nucleotide sequence of the ferredoxin D | C clone and the deduced amino acid sequence of the complete ferredoxin D | C are given in SEQ ID NO: 5 and SEQ I D NO: 6, respectively, in the Sequence Listing below. A comparison of the amino acid sequence derived from the nucleotide sequence of this clone was made with that of the protein sequences in the Swiss protein database using the FASTA program of the GCG program package. This comparison showed strong homology to other ferredoxins, including ferredoxins from Pseudomonas stutzeri, Pseudomonas putida, Rhodobacter capsulatus, Azotobacter vinelandii and Rhodospirillum rubrum (see discussion in Example 1 above).

LIST OF SEQUENCES (1) GENERAL NFORMATION: (i) APPLICANT: Weeks, Donald P. Wang, Xiao-Zhuo Herman, Patricia L. (ii) TITLE OF THE INVENTION: METHODS AND MATERIALS TO MAKE AND USE ORGANIC ISMS TRANSGENIC DICAMBA DEGRADERS (iii ) NUMBER OF SEQUENCES: 6 (iv) DI RECTION FOR CORRESPONDENCE: (A) DESTI NARIO: Sheridan Ross PC (B) STREET: 1 700 Lincoln St. Suite 3500 (C) CI UDA: Denver (D) STATE: Colorado (D) COUNTRY: EU (E) POSTAL CODE: 80203 (v) LEGI BLE COMPUTER FORM: (A) IT PO OF MEDIUM: FLEXI BLE DISK (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PACKAGE: patent in release # 1 .0, Version # 1 30 (vi) DATA CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASI FICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 60 / 042,666 (B) SUBMISSION DATE: April 4, 1997 (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 60 / 042,941 (B) SUBMISSION DATE: 04-abrii-1 997 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME : Crook, Wannell M. (B) REGISTRATION NUMBER: 31, 071 (C) REFERENCE NUMBER / CASE: 3553-1 8-PCT (ix) I NFORMATION OF TELECOMU NICATIONS: (A) TELEPHONE: (303) 863- 9700 (B) TELEFAX: (303) 863-0223 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 amino acids (B) TI PO: amino acid (C) FI LAMENT: simple (D) TOPOLOGY: linear (ii) IT POINT OF MOLECULE: protein (ix) CHARACTERISTIC: (A) NAME / KEY: Region (B) LOCATION: 28 (D) OTHER INFORMATION: / note = "Better assumptions for Xaa = Asp or Thr "(ix) FEATURE: (A) NAME / KEY: Region (B) LOCATION: 29 (D) OTHER INFORMATION: / note =" Best guess for Xaa = Pro " (xi) SEQUENCE DESCRITION: SEQ I D NO: 1 Thr Phe Val Arg Asn Wing Trp Tyr Val Wing Wing Leu Pro Glu Glu Leu 1 5? O 15 Ser Glu Lys Pro Leu Gly Arg Thr He Leu Asp Xaa Xaa 20 25 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SECU ENCIA: (A) LENGTH: 20 amino acids (B) TI PO: amino acid (C) FI LAMENT: simple (D) TOPOLOGY: linear (ii) TI PO OF MOLECULE: protein (ix) CHARACTERISTICS: (A) NAME / KEY: Region (B) LOCATION: 8 (D) OTHER INFORMATION: / note = "Best guess for Xaa = Cys" (ix) FEATURE: (A) NAME / KEY: Region (B) LOCATION: 1 1 (D) OTHER I NFORMATION: / note = "Best guess for Xaa = Cys" (ix) CHARACTERISTICS: (A) NAME / KEY: Region (B) LOCATION: 16 (D) OTHER INFORMATION: / note = "Best guess for Xaa = Cys" (ix) FEATURE: (A) NAME / KEY: Region (B) LOCATION: 20 (D) OTHER I NFORMATION: / note = "Best guess for Xaa = Cys" (xi) DESCRITION OF THE SEQUENCE: SEQ I D NO: 2: Thr Tyr Val Val Thr Asp Ala Xaa lie Lys Xaa Lys Tyr Met Asp Xaa 1 5 lo 15 Val Glu Val Xaa 20 (2) I NFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1 020 base pairs (B) TI PO: nucleic acid (C) FI LAMENT: simple (D) TOPOLOGY: linear (ii) TI MOLECULE PO: cDNA (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 1 .. 120 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: ATG ACC TTC GTC CGC AAT GCC TGG TAT GTG GCG GCG CTG CCC GAG GAA 48 Met Thr Phe Val Arg Asn Wing Trp Tyr Val Wing Wing Leu Pro Glu Glu 1 5 10 15 CTG TCC GAA AAG CCG CTC GGC CGG ACG ATT CTC GAC AC CCG CTC GCG 96 Leu Ser Glu Lys Pro Leu Gly Arg Thr He Leu Asp Thr Pro Leu Wing 20 25 30 CTC TAC CGC CAG CCC GAC GGT GTG GTC GCG GCG CTG CTC GAC ATC TGT 144 Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Wing Leu Leu Asp He Cys 35 40 45 CCG CAC CGC TTC GCG CCG CTG AGC GAC GGC ATC CTC GTC AAC GGC CAT 192 Pro His Arg Phe Ala Pro Leu Ser Asp Gly He Leu Val Asn Gly His 50 55 60 CTC CAÁ TGC CCC TAT CAC GGG CTG GAA TTC GAT GGC GGC GGG CAG TGC 240 Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln Cys 65 70 75 80 GTC CAT AAC CCG CAC GGC AAT GGC GCC CGC CCG GCT TCG CTC AAC GTC 288 Val His Asn Pro His Gly Asn Gly Ala Arg ro Wing Ser Leu Asn Val 85 90 95 CGC TCC TTC CCG GTG GTG GAG CGC GAC GCG CTG ATC TGG ATC TGG CCC 336 Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu He Trp He Trp Pro 100 105 110 GGC GAT CCG GCG CTG GCC GAT CCT GGG GCG ATC CCC GAC TTC GGC TGC 384 Gly Asp Pro Wing Leu Wing Asp Pro Gly Wing Pro Asp Phe Gly Cys l5 120 125 CGC GTC GAT CCC GCC TAT CGG ACC GTC GGC GGC TAT GGG CAT GTC GAC 432 Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val Asp 130 135 140 TGC AAC TAC AAG CTG CTG GTC GAC AAC CTG ATG GAC CTC GGC CAC GCC 480 Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His Wing 145 150 155 160 CAA TAT GTC CAT CGC GCC AAC GCC CAG ACC GAC GCC TTC GAC CGG CTG 528 Gln Tyr Val His Arg Wing Asn Wing Gln Thr Asp Wing Phe Asp Arg Leu 165 170 175 GAG CGC GAG GTG ATC GTC GGC GAC GGT GAG ATA CAG GCG CTG ATG AAG 576 Glu Arg Glu Val He Val Gly Asp Gly Glu He Gln Ala Leu Met Lys 180 185 190 ATT CCC GGC GGC ACG CCG AGC GTG CTG ATG GCC AAG TTC CTG CGC GGC 624 He Pro Gly Gly Thr Pro Ser Val Leu Met Wing Lys Phe Leu Arg Gly 195 200 205 GCC AAT ACC CCC GTC GAC GCT TGG AAC GAC ATC CGC TGG AAC AAG GTG 672 Wing Asn Thr Pro Val Asp Wing Trp Asn Asp He Arg Trp Asn Lys Val 210 215 220 AGC GCG ATG CTC AAC TTC ATC GCG GTG GCG CCG GAA GGC ACC CCG AAG 720 Be Wing Met Leu Asn Phe He Wing Val Wing Pro Glu Gly Thr Pro Lys 225 230 235 240 GAG CAG AGC ATC CAC TCG CGC GGT ACC CAT ATC CTG ACC CCC GAG ACG 768 Glu Gln Ser He His Ser Arg Gly Thr His He Leu Thr Pro Glu Thr 245 250 255 GAG GCG AGC TGC CAT TAT TTC TTC GGC TCC TCG CGC AAT TTC GGC ATC 816 Glu Wing Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly He 260 265 270 GAC GAT CCG GAG ATG GAC GGC GTG CTG CGC AGC TGG CAG GCT CAG GCG 864 Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Wing Gln Wing 275 280 285 CTG GTC AAG GAG GAC AAG GTC GTC GTC GAG GCG ATC GAG CGC CGC CGC 912 Leu Val Lys Glu Asp Lys Val Val Val Glu Ala He Glu Arg Arg Arg 290 295 300 GCC TAT GTC GAG GCG AAT GGC ATC CGC CCG GCG ATG C TG TCG TGC GAC 960 Wing Tyr Val Glu Wing Asn Gly He Arg Pro Wing Met Leu Ser Cys Asp 305 310 315 320 GAA GCC GCA GTC CGT GTC AGC CGC GAG ATC GAG AAG CTT GAG CAG CTC 1008 Glu Ala Wing Val Arg Val Ser Arg Glu He Glu Lys Leu Glu Gln Leu 325 330 335 GAA GCC GCC TGA 1020 Glu Ala Ala * 340 (2) I NFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 340 amino acids (B) TI PO: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Met Thr Phe Val Arg Asn Wing Trp Tyr Val Wing Wing Leu Pro Glu Glu 1 5 10 15 Leu Ser Glu Lys Pro Leu Gly Arg Thr He Leu Asp Thr Pro Leu Wing 20 25 30 Leu Tyr Arg Gln Pro Asp Gly Val Val Wing Ala Leu Leu Asp He Cys 35 40 45 Pro His Arg Phe Wing Pro Leu Ser Asp Gly He Leu Val Asn Gly His 50 55 60 Leu Gln Cys Pro Tyr His Gly Leu Glu phe Asp Gly Gly Gly Gln Cys 65 70 75 80 Val His Asn Pro His Gly Asn Gly Wing Arg Pro Wing Ser Leu Asn Val 85 90 95 Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu He Trp He Trp Pro 100 105 110 Gly Asp Pro Wing Leu Wing Asp Pro Gly Wing Pro Asp Phe Gly Cys 115 120 125 Arg Val Asp Pro Wing Tyr Arg Thr Val Gly Gly Tyr Gly His Val Asp 130 135 140 Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His Wing 145 150 155 160 Gln Tyr Val His Arg Wing Asn Wing Gln Thr Asp Wing Phe Asp Arg Leu 165 170 175 Glu Arg Glu Val He Val Gly Asp Gly Glu He Gln Ala Leu Met Lys 180 185 190 He Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg Gly 195 200 205 Wing Asn Thr Pro Val Asp Wing Trp Asn Asp He Arg Trp Asn Lys Val 210 215 220, Ser Wing Met Leu Asn Phe He Wing Val Wing Pro Glu Gly Thr Pro Lys 225 230 235 240 Glu Gln Ser He His Ser Arg Gly Thr His He Leu Thr Pro Glu Thr 245 250 255 Glu Wing Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly He 260 265 270 Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Wing Gln Wing 275 280 285 Leu Val Lys Glu Asp Lys Val Val Val Glu Ala He Glu Arg Arg Arg 290 295 300 Wing Tyr Val Glu Wing Asn Gly He Arg Pro Wing Met Leu Ser Cys Asp 305 310 315 320 Glu Ala Ala Val Arg Val Ser Arg Glu He Glu Lys Leu Glu Gln Leu 325 330 335 Glu Ala Al-a * 340 (2) I NFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 339 base pairs (B) TI PO: nucleic acid (D) FILAMENTO: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION: 1 ..339 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: ATG ACC TAT GTC GTC ACC GAC GCC TGC ATC AAG TGC AAG TAC ATG GAC 48 Met Thr Tyr Val Val Thr Asp Ala Cys lie Lys Cys Lys Tyr Met Asp 1 5 10 15 TGC GTG GAA GTC TGC CCT GTG GAC TGC TTC TAC GAA GGC GAG AAC ATG 96 Cys Val Glu Val Cys Pro Val Asp Cys Phe Tyr Glu Gly Glu Asn Met 20 25 30 CTC GTC ATC AAT CCC AGT GAA TGC ATC GAC TGC GGC GTC TGC GAA CCG 144 Leu Val He Asn Pro Ser Glu Cys He Asp Cys Gly Val Cys Glu Pro 35 40 45 GAA TGC CCA GCC GAA GCC ATC CTT CCC GAC ACC GAA AGC GGT CTC GAG 192 Glu Cys Pro Ala Glu Ala He Leu Pro Asp Thr Glu Ser Gly Leu Glu 50 55 60 CAG TGG ATG GAA CTG AAC ACG AAG TAC TCG GCC TGG CCG AAT CTC 240 Gln Trp Met Glu Leu Asn Thr Lys Tyr Ser Ala Glu Trp Pro Asn Leu 65 70 75 80 ACG TCC AAG AAA GAT TCG CCG GAA GAT GCC GAC GAG TAC AAG GGC GTG 288 Thr Ser Lys Lys Asp Ser Pro Glu Asp Wing Asp Glu Tyr Lys Gly Val 85 90 '95 GAA GGC AAG TTC GAG AAG TTC TTC TCG CCC GAG CCC GGC GAG GGC GAC 336 Glu Gly Lys Phe Glu Lys Phe Phe Ser Pro Glu Pro Gly Glu Gly Asp 100 105 110 TGA 339 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1 13 amino acids (B) TI PO: amino acid (D) TOPOLOGY: linear (ii) TI PO OF MOLECULE: protein ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Met Thr Tyr Val Val Thr Asp Ala Cys He Lys Cys Lys Tyr Met Asp 1, - 5 10 15 Cys Val Glu Val Cys Pro Val Asp Cys Phe Tyr Glu Gly Glu Asn Met. 20 25 30 Leu Val He Asn Pro Ser Glu Cys He Asp Cys Gly Val Cys Glu Pro 35 40 45 Glu Cys Pro Wing Glu Wing He Leu Pro Asp Thr Glu Ser Gly Leu Glu 50 55 60 Gln Trp Met Glu Leu Asn Thr Lys Tyr Ser Wing Glu Trp Pro Asn Leu 65 70 75 80 Thr Ser Lys Lys Asp Ser Pro Glu Asp Wing Asp Glu Tyr Lys Gly Val 85 90 95 Glu Gly Lys Phe Glu Lys Phe Phe Ser Pro Glu Pro Gly Glu Gly Asp 100 105 110

Claims (49)

1. REIVI NDICATIONS 1 . An isolated DNA molecule comprising a DNA sequence encoding a dicamba degrading oxygenase.
2. 2. The DNA molecule of claim 1, comprising a DNA sequence encoding a dicamba degrading oxygenase having the amino acid sequence of SEQ I D NO: 4.
3. 3. The DNA molecule of claim 2, comprising the nucleotide sequence of SEQ ID NO: 3.
4. 4. A DNA construct comprising a DNA sequence encoding a dicamba degrading oxygenase operably linked to expression control sequences.
5. 5. The DNA construct of claim 4, comprising a DNA sequence encoding a dicamba degrading oxygenase having the amino acid sequence of SEQ I D NO: 4.
6. 6. The DNA construct of claim 5, comprising the nucleotide sequence of SEQ ID NO: 3.
7. 7. The DNA construct of claim 4, which is a vector.
8. 8. An isolated and at least partially purified degrading dicamba oxygenase.
9. 9. The dicamba degrading oxygenase of claim 8, having the amino acid sequence of SEQ I D NO: 4. 1 0.
10. An isolated DNA molecule comprising a DNA sequence encoding a dicamba-degrading ferredoxin. eleven .
11. The DNA molecule of claim 10 comprising a sequence of DNA encoding a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 6.
12. 12. The DNA molecule of claim 1 comprising the nucleotide sequence of SEQ I D NO: 5.
13. 13. A DNA construct comprising a DNA sequence encoding a dicamba-degrading ferredoxin operably linked to expression control sequences.
14. The DNA construct of claim 13 comprising a DNA sequence encoding a dicamba-degrading ferredoxin having the amino acid sequence of SEQ I D NO: 6.
15. 15. The DNA construct of claim 14 comprising the nucleotide sequence of SEQ I D NO: 5.
16. 16. The Dna construct of claim 13, which is a vector.
17. 17. A degradable ferredoxin of dicamba isolated and at least partially purified.
18. 18. The dicamba degrading ferredoxin of claim 18, which has the amino acid sequence of SEQ ID NO: 6.
19. 1 9. A degrading degratase of dicamba isolated and at least partially purified.
20. 20. An O-demethylase degrading dicamba isolated and at least partially purified. twenty-one .
21. A transgenic host cell comprising DNA encoding a dicamba degrading oxygenase operably linked to expression control sequences.
22. 22. The transgenic host cells of claim 21, wherein the DNA encodes a dicamba degrading oxygenase having the amino acid sequence of SEQ ID NO: 4.
23. 23. The transgene host cell of claim 22, wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 3.
24. 24. The transgenic host cell of claim 21, which is a plant cell.
25. 25. The transgenic host cell of re-excitation 21, which is a microorganism.
26. 26. A transgenic host cell comprising DNA encoding a dicamba-degrading ferredoxin operably linked to expression control sequences.
27. 27. The transgenic host cell of claim 26, wherein the DNA encodes a dicamba-degrading ferredoxin having the amino acid sequence of SEQ I D NO: 6.
28. 28. The transgenic host cell of claim 27, wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 5.
29. 29. The transgenic host cell of claim 26, which is a plant cell.
30. 30. The transgenic host cell of claim 26, which is a microorganism.
31. 31 The transgenic host cell of claim 21, further comprising DNA encoding a dicamba-degrading ferredoxin operably linked to expression control sequences.
32. 32. The transgenic host cell of claim 31, wherein the DNA encodes a dicamba-degrading ferredoxin having the amino acid sequence of SEQ I D NO: 6.
33. 33. The transgenic host cell of claim 32, wherein the DNA comprises the nucleotide sequence of SEQ I D NO: 5.
34. 34. The transgenic host cell of claim 31, which is a plant cell.
35. 35. The transgenic host cell of claim 31, which is a microorganism.
36. 36. The plant or part of a transgenic plant comprising one or more cells comprising DNA encoding a dicamba-degrading oxygenate operably linked to expression control sequences.
37. 37. The transgenic plant or plant part of claim 36, wherein the DNA encodes a dicamba degrading oxygenhaving the amino acid sequence of SEQ ID NO: 4.
38. 38. The transgenic plant or plant part of claim 37, wherein the DNA comprises the nucleotide sequence of SEQ I D NO: 3.
39. 39. The plant or part of the transgenic plant of claim 36, wherein the plant is a broad-leaved plant, which is tolerant to dicamba as a result of the expression of the degrading oxygenof dicamba and the plant part is a part of a broadleaf plant, which is tolerant to dicamba as a result of the expression of dicamba degrading oxygen
40. 40. The plant or part of the transgenic plant of claim 36, wherein one or more cells further comprise DNA encoding a dicamba-degrading ferredoxin operably linked to expression control sequences.
41. 41 The transgenic plant or plant part of claim 40, wherein the DNA encodes a dicamba-degrading ferredoxin having the amino acid sequence of SEQ ID NO: 6.
42. 42. The transgenic plant or plant part of claim 41, wherein the DNA encodes the nucleotide sequence of SEQ ID NO: 5.
43. 43. The plant or part of the transgenic plant of claim 40, wherein the plant is a broad-leaved plant, which is tolerant to dicamba as a result of the expression of dicamba's oxygenand ferredoxin degraders, and the part of The plant is part of a broad-leaved plant, which is tolerant to dicamba as a result of the expression of dicamba's oxygenand ferredoxin degraders.
44. 44. A method for controlling weeds in a field containing a transgenic plant according to any of claims 36-43, which comprises applying an amount of dicamba to the effective field to control the weed in the field.
45. 45. A method for decontaminating a material containing applying an amount of a transgenic microorganism according to claim 25 or 35 to the material, the amount being effective to degrade at least some of the dicamba in the material.
46. 46. A method for decontaminating a dicamba-containing material, comprising applying an amount of a dicamba-degrading O-demethylor a combination of a divane-degrading oxygen a dicamba-degrading ferredoxin and a dicamba-degrading reductto the material, effective the amount to degrade at least some of the dicamba in the material.
47. 47. A method for selecting cells from transformed plants, comprising: providing a population of plant cells; transforming at least some of the plant cells into the cell population of plants with a DNA construct according to any of claims 4-16; and growing the resulting population of plant cells in a culture medium containing dicamba at a selected concentration so that the plant cells will grow and the cells of untransformed plants will not grow.
48. 48. A method for selecting transformed plants comprising: providing a population of plants suspected of comprising a DNA construct according to any of claims 4-16; and apply a quantity of dicamba to the selected plants so that the transformed plants will grow and the growth of untransformed plants will be inhibited.
49. 49. A method for selecting, or classifying, host cells, intact organisms and parts of transformed organisms, the method comprising: providing a population of host cells, intact organisms, or parts of organsimos that are suspected to comprise a DNA construct in accordance to any of claims 4-16; contact host cells, intact organisms, or parts of organisms with dicamba; and investigating the presence or level of fluorescence due to 3,6-dichlorosalicylic acid, 3,6-dichlorosalicylic acid being generated in host cells, intact organisms, or parts of transformed organisms as a result of degradation of dicamba.