USOO5882851A United States Patent (19) 11 Patent Number: 5,882,851 K0ch et al. (45) Date of Patent: Mar 16, 1999

54 CYTOCHROME P-450 MONOOXYGENASES Halkier, et al., “The Biosynthesis of Cyanogenic Glucosides in Higher Plants”, The Journal of Biological Chemistry, 75 Inventors: Birgit Maria Koch, Vanløse; Ole 264(33): 19487–19494 (1989). Sibbesen, Roskilde; Barbara Ann Halkier, et al., “BioSynthesis of the Cyanogenic Glucoside Halkier, Copenhagen V; Birger Dhurrin in Seedlings of Sorghum bicolor (L.) Moench and Lindberg Moller, Brønshøj, all of Partial Purification of the Enzyme System Involved”, Plant Denmark Physiol, 90: 1552–1559 (1989). Halkier, et al., “Cyanogenic glucosides: the biosynthetic 73 Assignees: Novartis Finance Corporation, New pathway and the enzyme System involved”, Department of York, N.Y.; Royal Veterinary Plant Physioology, Royal Veterinary and Agricultural Uni Agricultural University, Copenhagen, versity, 40: 49-66 (1988). Denmark Halkier, et al., “The Biosynthesis of Cyanogenic Glucosides 21 Appl. No.: 656,177 in Higher Plants”, The Journal of Biological Chemistry, 265(34): 21114-21121 (1990). 22 PCT Filed: Nov. 28, 1994 Halkier, et al., “Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench”, 86 PCT No.: PCT/EP94/03938 Plant Physiol, 96:10–17 (1991). S371 Date: Aug. 8, 1996 Halkier, et al., “2-Nitro-3-(p-hydroxyphenyl)propionate and aci-1-nitro-2-(p-hydroxyphenyl)ethane, two interme S 102(e) Date: Aug. 8, 1996 diates in the biosynthesis of the cyanogenic glucoside dhur rin in Sorghum bicolor (L.) Moench', Proc. Natl. Acad. Sci., 87 PCT Pub. No.: WO95/16041 88: 487–491 (1991). PCT Pub. Date:Jun. 15, 1995 Koch, et al., “The biosynthesis of cyanogenic glucosides in Seedlings of cassava (Manihot esculenta Crantez).’, 30 Foreign Application Priority Data Archives of Biochemistry and Biophysics, 292: 141-150 Dec. 8, 1993 EP European Pat. Off...... 93810860 (1992). Sibbesen et al., “Purification of the Hydroxylating Enzyme 51 Int. Cl...... C12O 1/00; C12O 1/26; System Involved in the Biosynthesis of the Cyanogenic G01N 33/53; CO7H 19/00 Glucoside Dhurrin in Sorghum Bicolor (1.) Moench', Bio 52 U.S. Cl...... 435/4; 435/7.1; 435/25; chemistry and Biophysics of Cytochrome P450, 232 (1991). 536/22.1 Primary Examiner Ardin H. Marschel 58 Field of Search ...... 536/22.1; 435/7.1, Assistant Examiner-Jezia Riley 435/4, 25; 800/205 Attorney, Agent, or Firm J. Timothy Meigs; Gary M. Pace 56) References Cited 57 ABSTRACT FOREIGN PATENT DOCUMENTS New cytochrome P-450 dependent monooxygenases and O 281 245 A2 2/1988 European Pat. Off.. DNA molecules encoding these monooxygenases are O 522880 A3 7/1992 European Pat. Off.. provided, which are able to catalyze the biosynthetic path WO 91/10745 1/1991 WIPO. way from amino acids to their corresponding cyanohydrins, WO 93/04174 8/1992 WIPO. the preSursors of the cyanogenic glycosides, or to glucosi WO 93/21326 4/1993 WIPO. nolates. Moreover, the invention provides methods for obtaining DNA molecules according to the invention and OTHER PUBLICATIONS methods for obtaining transgenic plants resistant to insects, Gabriac, et al., “Purification and Immunocharacterization of acarids, or nematodes or plants with improved nutritive a Plant Cytochrome P450: The Cinnamic Acid 4-Hydroxy value. lase”, Archives of Biochemistry and Biophysics, 288(1): 302-309 (1991). 42 Claims, No Drawings 5,882,851 1 2 CYTOCHROME P-450 MONOOXYGENASES Biochemistry and Biophysics of cytochrome P-450. Struc ture and Function, Biotechnological and Ecological This application is a S371 of PCT/EP94/03938, filed on Aspects, Archakov, A. I. (ed.), 1991, Koch et al, 8th Int. Nov. 28, 1994, and published on Jun. 15, 1995, as WO Conf. on Cytochrome P450, Abstract PII.053; and Sibbesen 95/16041, which claims priority of European Patent Appli et al, 8th Int. Conf. on Cytochrome P450, Abstract PII.016). cation No. 93810860.2, filed on Dec. 8, 1993. It has been found that L-Tyrosine is converted to p-hydroxy The present invention relates to genetic engineering in mandelo nitrile, the precursor of dhurrin with plants using recombinant DNA technology in general and to N-hydroxytyrosine and supposedly N,N-dihydroxytyrosine, enzymes involved in the biosynthesis of cyanogenic glyco 2-nitroSO-3-(p-hydroxyphenyl)propionic acid, (E)- and (Z)- Sides and glucosinolates and genes encoding these enzymes 1O p-hydroxyphenylace taldehy d o Xime, and in particular. The proteins and genes according to the p-hydroxyphenylacetonitrile as key intermediates. Two invention can be used to improve the nutritive value or pest monooxygenases dependent on cytochrome P-450 have resistance of plants. been reported to be involved in this pathway. A similiar Cyanogenic glycosides constitute Secondary plant pathway also involving cytochrome P-450 dependent metabolites in more than 2000 plant species. In some 15 monooxygenases has been demonstrated for the Synthesis of instances they are the source of HCN which can render a linamarin and lotaustrain from Valine and isoleucine respec plant toxic if it is taken as food. For example the tubers of tively in cassava (Koch et al., Archives of Biochemistry and the cyanogenic crop cassava (Manihot esculenta) constitute Biophysics, 292: 141-150, 1992). an important Staple food in tropical areas. However, the It has now Surprisingly been found that the complex cyanogenic glycosides present in the tuberS may cause pathway from L-tyrosine to p-hydroxy-mandelonitrile Sum cyanide poisoning in humans due to insufficiently processed marized above can be reconstituted by two enzymes only, cassava products. Other plant Species whose enzymatic which turn out to be identical to the cytochrome P-450 production of HCN accounts for their potential toxicity if dependent monooxygenases. This result is very Surprising taken in exceSS as food or used as animal feed include white given the high degree of complexity of the pathway reflected clover (Trifolium repens), sorghum (Sorghum bicolor), linen 25 by its numerous intermediates. Thus the two cytochrome flax (Linum usitatissimum), triglochinin (Triglochin P-450 monooxygenases are multifunctional. A first enzyme, maritima), lima beans (Phaseolus lunatus), almonds designated P-450, converts the parent amino acid to the (Amygdalus) and Seeds of apricot (Prunus), cherries and Oxime. A second enzyme, designated P-450, converts the apple (Malus). The toxic properties could be reduced by oxime to the cyanohydrin. Multifunctional cytochrome blocking the biosynthesis of cyanogenic glycosides in these P-450 enzymes have not previously been found and plants. described in plants. The primary precursors of the naturally occuring cyano Glucosinolates are hydrophilic, non-volatile thioglyco genic glycosides are restricted to the five hydrophobic sides found within several orders of dicotyledoneous protein amino acids Valine, leucine, isoleucine, phenylala angiosperms (Cronquist, 'The Evolution and Classification nine and tyrosine and to a Single non-protein amino acid, 35 of Flowering Plants, New York Botanical Garden, Bronx, cyclopentenylglycine. These amino acids are converted in a 1988). Of greatest economic significance is their presence in Series of reactions to cyanohydrins which are ultimately all members of the Brassicaceae (order of Capparales), linked to a Sugar residue. Amygdalin for example constitutes whose many cultivars have for centuries provided mankind the O-B-gentiobioside and prunasin the O-3-glucoside of with a Source of condiments, relishes, Salad crops and (R)-mandelonitrile. Another example of cyanogenic glyco 40 vegetables as well as fodders and forage crops. More Sides having aromatic aglycones is the epimeric pair of the recently, rape (especially Brassica napuS and Brassica cyanogenic glycosides dhurrin and taxiphyllin which are to campestris) has emerged as a major oil seed of commerce. be found in the genus Sorghum and Taxus, respectively. About 100 different glucosinolates are known possessing the p-Hydroxymandelo-nitrile for example is converted into Same general Structure but differing in the nature of the Side dhurrin by a UDPG-glycosyltransferase. Similiar glycosyl 45 chain. Glucosinolates are formed from protein amino acids transferases are believed to be present in most plants. either directly or after a single or multiple chain extension Vicianin and lucumin are further examples for disaccharide (Underhill et al, Biochem. Soc. Symp. 38:303-326, 1973). derivatives similiar to amygdalin. Sambunigrin contains N-hydroxy amino acids and aldoximes which have been (S)-mandelonitrile as its aglycone and is therefore epimeric identified as intermediates in the biosynthesis of cyanogenic to prunasin. 50 glycosides also serve as efficient precursors for the biosyn Examples of cyanogenic glycosides having aliphatic thesis of glucosinolates (Kindl et al., Phytochemistry aglycones are linamarin and lot australin found in clover, 7:745-756, 1968; Matsuo et al., Phytochemistry 11:697–701, linen flax, cassava and beans. A detailed review on cyano 1972; Underhill, Eur. J. Biochem. 2:61-63, 1967). genic glycosides and their biosynthesis can be found in It has now Surprisingly been found that the cytochrome Conn, Naturwissenschaften 66:28-34, 1979, herein incor 55 P-450 involved in cyanogenic glycoside Synthesis is very porated by reference. Similiar to the corresponding biosynthetic enzyme in glu The biosynthetic pathway for the cyanogenic glucoside coSinolate Synthesis. dhurrin derived from tyrosine has been extensively studied The reduction of the complex biosynthetic pathway for (Halkier et al., 'Cyanogenic glucosides: the biosynthetic cyanohydrins described above to the catalytic activity of pathway and the enzyme System involved in: 'Cyanide 60 only two enzymes, cytochrome P-450 and P-450, allows compounds in biology, Wiley Chichester (Ciba Foundation the introduction of the biosynthetic pathway of dhurrin into Symposium 140), pages 49-66, 1988; Halkier and Moller, plants, which plants in their wildtype phenotype do not Plant Physiol. 90:1552–1559, 1989; Halkier et al, The J. of normally produce cyanogenic glycosides. By transfection of Biol. Chem. 264:19487–19494, 1989; Halkier and Moller, gene constructs coding for one or both of the two cyto Plant Physiol.96:10–17, 1990, Halkier and Moller, The J. of 65 chrome P-450 monooxygenases it will be possible to either Biol. Chem. 265:21114-21121, 1990; Halkier et al, Proc. reconstitute or newly establish a biosynthetic pathway for Natl. Acad. Sci. USA 88:487–491, 1991; Sibbesen et al, in: cyanogenic glycosides. It is therefore an object of the 5,882,851 3 4 present invention to provide genes coding for cytochrome be accomplished by transforming a plant with an additional P-450 monooxygenases active in the biosynthesis of cya gene encoding an expressible cytochrome P-450 monooxy nogenic glycosides. genase with a Substrate Specificity different from the natu The introduction of a biosynthetic pathway for cyano rally occuring enzyme. genic glycosides into plants by methods known in the art, The present invention relates primarily to a DNA mol which in their wildtype phenotype do not express these ecule coding for a cytochrome P-450 monooxygenase, glycosides is of great interest. This is due to the Surprising which catalyzes the conversion of an amino acid to the finding of the present invention that cyanogenic glycosides corresponding N-hydroxyamino acid and the Oxime derived can be toxic to insects, acarids, and nematodes. Therefore, from this N-hydroxyamino acid. Preferably the inventive the introduction or reconstitution of a biosynthetic pathway monooxygenase catalyzes the conversion of an amino acid for cyanogenic glycosides in plants or certain plant tissues Selected from the group consisting of tyrosine, phenylalanine, tryptophan, Valine, leucine, isoleucine and will allow to render plants toxic to insects, acarids or cyclopentenylglycine or an amino acid Selected from the nematodes and thus help to reduce the damage to the crop by group consisting of L-tyrosine, L-Valine and L-isoleucine. pests. In combination with other insecticidal principles Such Additionally the present invention relates to a DNA mol as Bacillus thuringiensis endotoxins the damage to the crop 15 ecule coding for a cytochrome P-450 monooxygenase, by pests could be even further reduced. which monooxygenase catalyzes the conversion of Said Alternatively, the Sequences of the genes encoding the Oxime to a nitrile and the conversion of Said nitrile to the monooxygenases according to the invention can be used to corresponding cyanohydrin. The DNA molecules according design DNA plasmids which upon transfection into a plant to the invention either correspond to naturally occuring containing cyanogenic glycosides Such as cassava, Sorghum genes or to functional homologues thereof which are the or barley eliminate cyanogenic glycosides normally pro result of mutation, deletion, truncation, etc. but Still encode duced in wildtype plants. This can be achieved by expres cytochrome P-450 monooxygenases, which either catalyze sion of antisense or sense RNA or of ribozymes as described the conversion of an amino acid to the corresponding in EP-458. 367 A1, EP-240 208-A2, U.S. Pat. No. 5,231,020, N-hydroxyamino acid and the oxime derived from this WO 89/05852, and WO 90/11682 which RNA inhibits the 25 N-hydroxyamino acid, or the conversion of Said oxime to a expression of monooxygenases according to the invention. nitrile and the Subsequent conversion of Said nitrile to the This is of great interest as in Spite of numerous efforts it has corresponding cyanohydrin. Both monooxygenases are able not been possible through traditional plant breeding to to catalyze more than one reaction of the biosynthetic completely remove the cyanogenic glycosides from for pathway of cyanogenic glycosides but preferably contain a example cassava and Sorghum. On the other hand it has been Single catalytic center. The monooxygenase cytochrome shown that elevated amounts of cyanogenic glycosides in P-450 converting the parent amino acid is also involved in the epidermal cells of barley cultivars confer increased glucosinolate biosynthesis. Because cytochrome P-450 Sensitivity to attack by the mildew fungus Erysiphe graminis determines the Substrate specificity and thus the type of (Pourmohensi, PhD thesis, Göttingen, 1989; Ibenthal et al, glucosinolateS produced and because cytochrome P-450 Angew. Bot. 67:97-106, 1993). A similiar effect has been 35 constitutes the rate limiting Step, the principles already observed in the cyanogenic rubber tree Hevea brasiliensis described above for cyanogenic glucosides can also be used upon attack by the fungus Microcyclus ulei (Lieberei et al., to down- or up-regulate the Synthesis of glucosinolates in Plant Phys. 90:3–36, 1989) and with flax attacked by Col glucosinolate producing plants and to alter the composition letotrichum lini (Llidtke et al, Biochem. Z. 324:433-442, of glucosinolates produced. 1953). In these instances the quantitative resistance of the 40 The inventive DNA molecule encoding cytochrome plants stipulated above and of other plants, where cyano P-450 is obtainable from plants which produce cyanogenic genic glycosides confer increased Sensitivity to attack by glycosides and glucosinolates. These plants include but are microorganisms, can be increased by preventing the produc not limited to plants Selected from the group consisting of tion of cyanogenic glycosides in Such plants. In barley, the the Species Sorghum, Trifolium, Linum, Taxus, Triglochin, cyanogenic glycosides are located in the epidermal cells. 45 Mannihot, Amygdalus and Prunus as well as cruciferous The antisene, Sense or ribozyme constructs are therefore plants. In a preferred embodiment of the invention the DNA preferably but but not necessarily combined with an epider molecule is obtained from Sorghum bicolor (L.) Moench or mis Specific promoter. Manihot esculenta Crantz. The sequence similarity between The presence of even minor amounts of cyanogenic cytochrome P-450 monooxygenases from different plants glycosides in plants may also cause nutritional problems due 50 producing cyanogenic glycosides or glucosinolates is evi to generation of unwanted carcinogens as demonstrated in denced by the Specific croSS-reactivity of antibodies pre barley. Barley malt for example contains low amounts of the pared against cytochrome P-450 isolated from Sorghum, cyanogenic glucoside epiheterodendrin which in the cause with the corresponding cytochrome P-450 enzyme in cas of production of grain-based Spirits can be converted to Sava and with the corresponding enzyme in the glucosinolate ethylcarbamate which is considered to be a carcinogen. 55 producing plant Tropaeolum majus. Southern blotting using Attempts are being made to introduce mandatory maximum the cDNA clone encoding cytochrome P-450 shows allowable concentrations of ethylcarbamate in fermented Specific and Strong hybridization to genomic DNA isolated food, beverages and spirits (Food Chemical News 29:33.35, from cassava, Tropaeolum majus, and rape. Of all approxi 1988). mately 250 known published Sequences for cytochrome Plants containing cyanogenic glycosides typically con 60 P-450 enzymes, cytochrome P-450 shows the highest tain only a single cyanogenic glycoside or just a few. In Sequence Similarity to the petunia 3'5'-flavonoid hydroxylase certain cases it is of interest to alter the cyanogenic glycoside (30.8%) and 28% sequence similiarity to CYP1A2 from profile of a plant. Since cytochrome P-450 shows broad rabbit. The group of cytochrome P-450 monooxygenases Substrate Specificity this enzyme typically converts the functionally characterized by catalyzing the conversion of aldoxime produced by cytochrome P-450, into the corre 65 an amino acid to the corresponding aldoxime can thus be sponding cyanohydrin. Alteration of the chemical identity of defined as cytochrome P-450 enzymes the amino acid cyanogenic glycosides produced in a Specific plant can thus Sequence of which exhibits a 32% or higher Sequence 5,882,851 S 6 Similarity and preferably a 40% or higher Sequence Similar For other cytochrome P-450 enzymes, the N-terminal ity to that of cytochrome P-450. Cytochrome P-450 gene Sequences may be different. protein families are defined as having less than 40% amino Optionally, a P-450 monooxygenase might also com acid identity to a cytochrome P-450 protein from any other prise one of the following Sequences: family. Consequently, cytochrome P-450, belongs to a 5 new P-450 protein family. The inventive DNA molecule encoding cytochrome -ARLAEIFATI- (SEQ ID NO:13) P-450 is obtainable from plants which produce cyanogenic -EDFTWTTK- (SEQ ID NO: 14) glycosides. In a preferred embodiment of the invention the DNA molecule is obtained from Sorghum bicolor (L.) -OYAALGSVFTVPII- (SEQ ID NO: 15) Moench or Manihot esculenta Crantz. The enzyme isolated from Sorghum bicolor (L.) Moench is designated cyto -XXPFPI-(SEQ ID NO: 16). chrome P-450. The catalytic properties of this enzyme Another embodiment of the present invention deals with resembles those of a cytochrome P-450 activity reported in a method for the preparation of cDNA coding for a cyto microsomes from rat liver (DeMaster et al., J. Org. Chem. 15 chrome P-450 monooxygenase, which either catalyzes the 5074-5075, 1992) which has neither been isolated nor conversion of an amino acid preferably Selected from the further characterized. A characteristic of cytochrome group consisting of tyrosine, phenylalanine, tryptophan, P-450 and of other members belonging to the cytochrome Valine, leucine, isoleucine and cyclopentenylglycine, to the P-450 family is that dehydration of the oxime to the corresponding N-hydroxyamino acid and the Oxime derived corresponding nitrile is dependent on the presence of from this N-hydroxyamino acid (cytochrome P-450); or the NADPH but that this dependence can be overcome by the conversion of Said oxime to a nitrile and the conversion of addition of sodium dithionite or other reductants. Cyto said nitrile to the corresponding cyanohydrin (cytochrome chrome P-450 enzymes able to convert aldoximes into P-450); comprising cyanohydrins might be present in most living organisms. (a) isolating and Solubilizing microSomes from plant For the purposes of gene manipulation using recombi 25 tissue producing cyanogenic glycosides or glucosinolates, nant DNA technology the DNA molecule according to the (b) purifying the cytochrome P-450 monooxygenase, invention may in addition to the gene coding for the (c) raising antibodies against the purified monooxygenase comprise DNA which allows for example monooxygenase, replication and selection of the inventive DNA in microor (d) probing a cDNA expression library of plant tissue ganisms. Such as E. coli, Bacillus, Agrobacterium, Strepto producing cyanogenic glycosides or glycosinolates with Said myces or yeast. It may also comprise DNA which allows the antibody, and monooxygenase genes to be expressed and Selected in (e) isolating clones which express the monooxygenase. homologous or heterologous plants. Such Sequences com MicroSomes can be isolated from plant tissues which prise but are not limited to genes whose codon usage has show a high activity of the enzyme System responsible for been adapted to the codon usage of the heterologous plant as 35 biosynthesis of the cyanogenic glycosides. These tissues described in WO93/07278; to genes conferring resistance to may be different from plant Species to plant Species. A neomycin, kanamycin, methotrexate, hygromycin, preferred Source of microSomes are freshly isolated Shoots bleomycin, Streptomycin, or gentamycin, to harvested 1 to 20 days, preferably 2 to 10 days and most aminoethylcystein, glyophosphate, Sulfonylurea, or phos preferably 2 to 4 days after germination. Etiolated Seedlings phinotricin; to Scorable marker genes Such as galactosidase; 40 are preferred from plant producing cyanogenic glycosides to its natural promoter and transcription termination signals, but light grown Seedlings may also be used. Following to promoter elements such as the 35S and 19S CaMV isolation the microSomes are Solubilized in buffer containing promoters, or tissue specific plant promoterS Such as pro one or more detergents. Preferred detergents are RENEX moters specific for root (described for example in EP-452 690 (J. Lorentzen A/S, Kvistgard, Denmark), reduced Triton 269-A2, WO 91/13992, U.S. Pat. No. 5,023,179), green 45 X-100 (RIX-100) and CHAPS. leaves Such as the maize phosphoenolpyruvate carboxylase The cytochrome P-450 monooxygenases can be purified (PEPC), pith or pollen (described for example in WO applying Standard techniques for protein purification Such as 93/07278), or inducible plant promoters (EP332 104); and ultracentrifugation, fractionated precipitation, dialysis, to heterologous transcription termination signals. SDS-PAGE and column chromatography. Possible columns The present invention also relates to monooxygenases 50 comprise but are not limited to ion exchange columns. Such which catalyze the conversion of an amino acid preferably as DEAE Sepharose, Reactive dye columns such as Ciba Selected from the group consisting of tyrosine, cron yellow 3 agarose, Cibacron blue agarose and Reactive phenylalanine, tryptophan, Valine, leucine, isoleucine and red 120 agarose, and gel filtration columns Such as cyclopentenylglycine to the corresponding N-hydroxyamino Sephacryl S-1000. The cytochrome P-450 content of the acid and the oxime derived from this N-hydroxyamino acid 55 individual fractions can be determined from carbon monox (cytochrome P-450); or the conversion of said oxime to a ide difference spectra. nitrile and the conversion of Said nitrile to the corresponding The purified proteins can be used to elicit antibodies in for cyanohydrine (cytochrome P-450). In a preferred embodi example mice, goats, sheeps, rabbits or chickens upon ment of the invention the monooxygenases are purified and injection. 5 to 50 lug of protein are injected Several times can be used to establish monoclonal or polyclonal antibodies 60 during approximately 14 day intervals. In a preferred which Specifically bind to the monooxygenases. embodiment of the invention 10 to 20 tug are injected 2 to 6 In another preferred embodiment of the invention the times in 14 day intervals. Injections can be done in the cytochrome P-450 monooxygenase can be isolated from presence or absence of adjuvants. Inmunoglobulins are Sorghum, has a molecular weight of 51 kD as determined by purified from the antisera and Spleens can be used for SDS-PAGE and comprises the N-terminal sequence 65 hybridoma fusion as described in Harlow and Lane, Anti bodies: A Laboratory Manual, Cold Spring Harbor MDLADIPKOORLMAGNALVV (SEQ ID NO: 12). Laboratory, Cold Spring Harbor, N.Y., 1988, herein incor 5,882,851 7 8 porated by reference. Antibodies Specifically binding to a desired gene product-in the present case a P-450 cytochrome P-450 monooxygenase can also be used in plant monooxygenase-and is then Spliced into a Suitable expres breeding to detect plants producing altered amounts of Sion vector. The gene libraries So produced can then be cytochrome P-450 monooxygenases and thus altered Screened using Suitable measures, preferably using amounts of cyanogenic glycosides. 5 antibodies, and those clones Selected which comprise the The methods for the preparation of plant tissue cDNA desired gene or at least part of that gene as an insert. libraries are extensively described in Sambrook et al., Alternatively, total DNA from the DNA library, preferably Molecular cloning: A laboratory manual. Cold Spring Har from the cDNA library, can be prepared and used as a bor Laboratory Press, Cold Spring Harbor, N.Y., 1989, the template for a PCR reaction with primers representing low essential parts of which regarding preparation of cDNA degeneracy portions of the amino acid Sequence. Preferably, libraries are herein incorporated by reference. PolyA RNA the primers used will generate PCR products that represent is isolated from plant tissue which shows a high activity of a significant portion of the nucleotide Sequence. The PCR the enzyme System responsible for biosynthesis of the products can be further probed to determine if they corre cyanogenic glycosides or glucosinolates. These tissues may spond to a portion of the P-450 monooxygenase gene using be different from plant Species to plant Species. A preferred 15 a Synthetic oligonucleotide probe corresponding to an amino tissue for polyA RNA isolation is the tissue of freshly acid fragment Sequence located in the interior or middle isolated shoots harvested 1 to 20 days, preferably 2 to 10 region of the P-450 monooxygenase protein. days and most preferably 2 to 4 days after germination. The cDNA clone s an d PCR products prepared as When cDNA libraries are made from glucosinolate produc described above or fragments thereof may be used as a ing plants older or mature plant tissue may also be used. The hybridization probe in a process of identifying further DNA obtained cDNA libraries can be probed with antibodies Sequences from a homologous or a heterologous Source Specifically binding the cytochrome P-450 monooxygenase organism encoding a protein product that exhibits P-450 and clones expressing the monooxygenase can be isolated. monooxygenase activity Such as, for example, a fungi or a An alternative method for the preparation of cDNA cod heterologous plant. A Suitable Source would be tissue from ing for a cytochrome P-450 monooxygenase comprises 25 plants containing cyanogenic glycosides or glucosinolates. (a) isolating and Solubilizing microSomes from plant They may also be used as an RFLP marker to determine, tissue producing cyanogenic glycosides or glucosinolates, for example, the location of the cytochrome P-450 (b) purifying the cytochrome P-450 monooxygenase, monooxygenase gene or a closely lined trait in the plant (c) obtaining a complete or partial protein sequence of the genome or for marker assisted breeding EP-A306,139; WO monoXygenase, 89/07647). (d) designing oligonucleotides specifying DNA coding Using the methods described above it is thus possible to for 4 to 15 amino acids of Said monooxygenase protein isolate a gene that codes for a P-450 monooxygenase. Sequence Genes encoding cytochrome P-450 monooxygenase can (e) probing a cDNA library of plant tissue producing be used in a method for producing a purified recombinant cyanogenic glycosides or glucosinolates with Said 35 cytochrome P-450 monooxygenase which monooxygenase oligonucleotides, or DNA molecules obtained from PCR either catalyzes the conversion of an amino acid preferably amplification of cDNA using Said oligonucleotides, and Selected from the group consisting of tyrosine, (f) isolating clones which encode cytochrome P-450 phenylalanine, tryptophan, Valine, leucine, isoleucine and monoOXygenase. cyclopentenylglycine to the corresponding N-hydroxyamino Amino acid Sequences of internal peptides which are the 40 acid and the oxime derived from this N-hydroxyamino acid; result of protease digestion can be obtained by Standard or the conversion of Said OXime to a nitrile and the conver techniques Such as Edman degradation. Oligonucleotides Sion of Said nitrile to the corresponding cyanohydrine; Specifying DNA coding for partial protein Sequences of the comprising inventive monooxygenases are obtained by reverse transla (a) engineering the gene encoding said monooxygenase to tion of parts of the protein Sequence according to the genetic 45 be expressible in a host organism Such as bacteria, yeast or code. Protein Sequences encoded by DNA sequences of low insect cells, degeneracy are preferred for reverse translation. Their length (b) transforming Said host organism with the engineered ranges from 4 to 15 and preferably from 5 to 10 amino acids. gene, and If necessary the codons used in the oligonucleotides can be (c) isolating the protein from the host organism or the adapted to the codon usage of the plant Source (Murray et al., 50 culture Supernatant. Nucleic Acids Research 17:477-498, 1989). The obtained In a preferred embodiment of the invention the method is oligonucleotides can be used to probe cDNA libraries as used to obtain purified recombinant cytochrome P-450, described in Sambrook et al, Molecular cloning: A labora P-450 or cytochrome P-450 which has been modified tory manual. Cold Spring Harbor Laboratory Press, Cold by known techniques of gene technology. Preferably the Spring Harbor, N.Y., 1989, for clones which are able to 55 modifications lead to increased expression of the recombi basepair with Said oligonucleotides. Alternatively, oligo nant protein or to altered Substrate specificity. nucleotides can be used in a polymerase chain reaction, the The inventive DNA molecules can be used to obtain methodology of which is known in the art, with plant cl)NA transgenic plants resistant to insects or acarids examples of as the template for amplification. In this case the obtained which are listed but not limited to those in Table B as well amplification products are used to probe the cDNA libraries. 60 as nematodes. Preferably the transgenic plants are resistant Clones encoding cytochrome P-450 monooxygenases are to Coleoptera and Lepidoptera Such as western corn root isolated. worm (Diabrotica virgifera virgifera), northern corn root An alternative method of cloning genes is based on the worm (Diabrotica longicornis barberi), southern corn root construction of a gene library composed of expression worm (Diabrotica undecimpunctata howardi), cotton vectors. In that method, analogously to the methods already 65 bollworm, European corn borer, corn root webworm, pink described above, genomic DNA, but preferably cDNA, is bollworm and tobacco budworm. The transgenic plants first isolated from a cell or a tissue capable of expressing a comprise DNA coding for monooxygenases which catalyze 5,882,851 9 10 the conversion of an amino acid to the corresponding After incubation for several days, but preferably after N-hydroxyamino acid and the oxime derived from this incubation for 2 to 3 days at a temperature of 20° C. to 40 N-hydroxyamino acid, or the conversion of Said Oxime to a C., preferably from 23 C. to 35° C. and more preferably at nitrile and the conversion of Said nitrile to the corresponding 25 C. and in diffuse light, the leaf disks are transferred to cyanohydrine. In addition the transgenic plants may com a Suitable medium for the purpose of Shoot induction. prise monooxygenase genes genetically linked to herbicide Especially preferred for the Selection of the transformants is resistance genes. The transgenic plants are preferably mono an LS medium that does not contain auxin but contains cotyledoneous or dicotyledoneous plants examples of which cytokinin instead, and to which a Selective Substance has ar listed in Table A. Preferably they are selected from the been added. The cultures are kept in the light and are group consisting of maize, rice, wheat, barley, Sorghum, transferred to fresh medium at Suitable intervals, but pref cotton, Soybeans, Sunflower, grasses and oil Seed rape. The erably at intervals of one week. Developing green shoots are plants can be obtained by a method comprising cut out and cultured further in a medium that induces the (a) introducing into a plant cell or plant tissue which can shoots to form roots. Especially preferred within the Scope be regenerated to a complete plant, DNA comprising a gene of this invention is an LS medium that does not contain expressible in that plant encoding an inventive auxin or cytokinin but to which a Selective Substance has monoOXygenase, 15 been added for the selection of the transformants. (b) Selecting transgenic plants, and In addition to Agrobacterium-mediated transformation, (c) identifying plants which are resistant to insects, within the Scope of this invention it is possible to use direct acarids, or nematodes. transformation methods for the insertion of the gene con The inventive DNA molecules can also be used to obtain Structions according to the invention into plant material. transgenic plants expressing anti-Sense or Sense RNA or For example, the genetic material contained in a vector ribozymes targeted to the genes of the endogenous P-450 can be inserted directly into a plant cell, for example using monooxygenases the expression of which reduces the purely physical procedures, for example by microinjection expression of cytochrome P-450 monooxygenases. Such using finely drawn micropipettes (Neuhaus et al, Theoretical plants show improved disease resistance or nutritive value and Applied Genetics 74:363-373, 1987), electroporation due to reduced expression of cyanogenic glycosides or 25 (DHalluin et al, The Plant Cell 4:1495–1505, 1992; WO glucosinolates. The plants can be obtained with a method 92/09696), or preferably by bombarding the cells with comprising microprojectiles that are coated with the transforming DNA (a) introducing into a plant cell or tissue which can be (“Microprojectile Bombardment”; Wang et al, Plant regenerated to a complete plant, DNA encoding Sense RNA, Molecular Biology 11:433-439, 1988; Gordon-Kamm et al., anti sense RNA or a ribozyme, the expression of which The Plant Cell 2:603–618, 1990; McCabe et al, Bio/ reduces the expression of cytochrome P-450 monooxyge Technology 11:596–598, 1993; Christou et, Plant Physiol. nases according to claims 1 or 8, 87:671-674, 1988; Kozieletal, Biotechnology 11: 194–200, (b) Selecting transgenic plants, and 1993). Moreover, the plant material to be transformed can (c) identifying plants with improved disease resistance or optionally be pretreated with an osmotically active Sub nutritive value. 35 stance Such as Sucrose, Sorbitol, polyethylene glycol, glu A number of very efficient processes are available for cose or mannitol. introducing DNA into plant cells, which processes are based Other possible methods for the direct transfer of genetic on the use of gene transfer vectors or on direct gene transfer material into a plant cell comprise the treatment of proto proceSSeS. plasts using procedures that modify the plasma membrane, One possible method of inserting a gene construct into a 40 for example polyethylene glycol treatment, heat Shock treat cell makes use of the infection of the plant cell with ment or electroporation, or a combination of those proce Agrobacterium tumefaciens and/or Agrobacterium dures (Shillito et al, Biotechnology 3:1099–1103, 1985). rhizogenes, which has been transformed with the Said gene A further method for the direct introduction of genetic construction. The transgenic plant cells are then cultured material into plant cells, which is based on purely chemical under Suitable culture conditions known to the person skilled 45 procedures and which enables the transformation to be in the art, So that they form shoots and roots and whole carried out very efficiently and rapidly, is described in plants are finally formed. Negrutiu et al, Plant Molecular Biology 8:363-373, 1987. Within the scope of this invention is the so-called leaf disk Also Suitable for the transformation of plant material is transformation using Agrobacterium (Horsch et al, Science direct gene transfer using co-transformation (Schocher et al., 227: 1229–1231, 1985) can also be used. Sterile leaf disks 50 Bio/Technology 4:1093–1096, 1986). from a Suitable target plant are incubated with Agrobacte The list of possible transformation methods given above rium cells comprising one of the chimaeric gene construc by way of example does not claim to be complete and is not tions according to the invention, and are then transferred into intended to limit the Subject of the invention in any way. or onto a Suitable nutrient medium. Especially Suitable, and In another embodiment of the invention target plants are therefore preferred within the scope of this invention, are LS 55 exposed to a pesticidally effective amount of a cyanogenic media that have been solidified by the addition of agar and glycoside to control insects, acarids, or nematodes attacking enriched with one or more of the plant growth regulators a monocotyledonous or dicotyledonous plant Selected from customarily used, especially those Selected from the group the group of plant types consisting of Cereals, Protein CropS, of the auxins consisting of a-naphthylacetic acid, picloram, Fruit Crops, Vegetables and Tubers, Nuts, Oil Crops, Sugar 2,4,5-trichlorophe no Xy a c e tic acid, 2,4- 60 CropS, Forage and Turf Grasses, Forage Legumes, Fiber dichlorophenoxyacetic acid, indole-3-butyric acid, indole Plants and Woody Plants, Drug Crops and Spices and 3-lactic acid, indole-3-Succinic acid, indole-3-acetic acid Flavorings. and p-chlorophenoxyacetic acid, and from the group of the The following examples further describe the materials and cytokinins consisting of kinetin, 6-benzylade nine, methods used in carrying out the invention and the Subse 2-isopentenyladenine and Zeatin. The preferred concentra 65 quent results. They are offered by way of illustration, and tion of auxins and cytokinins is in the range of 0.1 mg/l to their recitation should not be considered as a limitation of 10 mg/l. the claimed invention. 5,882,851 11 12 EXAMPLES which Sometimes results in irreversible aggregation. The column is washed with 150 ml buffer A after which the total Example 1 amount of cytochromes P-450 including cytochrome P-450 and cytochrome P-450 is eluted with buffer B in Preparation of microSomes a total volume of 150 ml. During this procedure, NADPH Seeds of Sorghum bicolor (L.) Moench (hybrid S-1000) cytochrome P-450-oxidoreductase and Cytochrome bs are obtained from Seedtec International Inc. (Hereford,Tex.) remain bound to the column and may Subsequently be eluted and germinated in the dark for 2 days at 28 C. on metal and separated with buffer B and a gradient of 0-300 mM Screens covered with gauze. Transfer of the Seeds to germi KC1. nation trays is carried out under dim green light. The cytochrome P-450 eluate is adjusted to 1.0% CHAPS, MicroSomes are prepared from approximately 3 cm tall stirred for 30 minutes and then directly applied to a 25 ml etiolated Seedlings. The Seedlings are harvested and homog (2.6x5 cm) column of Reactive yellow 3 Sepharose equili enized using a mortar and pestle in 2 volumes (V/w) of 250 brated in buffer C+10% RENEX 690. The flow rate used is mM sucrose, 100 mM tricine (pH 7.9), 50 mM NaCl, 2 mM 25 ml/h. The column is washed with buffer C until the EDTA and 2 mM DTT Polyvinylpolypyrrolidone is added 15 absorbance Also shows that RENEX 690 is washed out. (0.1 g/g fresh weight) prior to homogenization. The homo Cytochrome P-450 does not bind to this column, and is genate is filtered through 22 um nylon cloth and centrifuged obtained in the run-off and wash. Subsequently the column 20 minutes at 48000 g. The Supernatant is centrifuged for 1 is eluted with 400 mM KCl in buffer C. The cytochrome hour at 165000 g. The microsomal pellet is resuspended and P-450 containing fractions are combined yielding homogenized in isolation buffer using a Potter-Elvehjem approximately 60 ml and diluted with 5 volumes of buffer C homogenizer fitted with a teflon pestle. After recentrifuga to lower the KCl Strength and permit rebinding of cyto tion and rehomogenization, the homogenate is dialyzed chrome P-450 on a second Reactive yellow 3 column. overnight against 50 mM Tricine (pH 7.9), 2 mM DTT under This column is eluted with a KCl gradient (0–500 mM) in a a nitrogen atmosphere. total volume of 100 ml in buffer C. This serves to elute Example 2 25 cytochrome P-450. The cytochrome P-450 pool from the yellow agarose is Enzyme assays: Determination of total cytochrome P-450 diluted 5 times with buffer C to 20-25 mM KCl and applied Quantitative determination of total cytochrome P-450 is to a Cibachron blue agarose column (0.9x6 cm) equilibrated carried out by difference spectroScopy using an extinction in buffer C. The flow rate used is 8 ml/h. The column is difference coefficient of 91 mMcm for the complex washed with 20 ml buffer C at the same flow rate. Cyto between reduced cytochrome P-450 and carbon monoxide chrome P-450 is eluted with a gradient of KC1, 0-2.0M in (Also ) (Omura et al., J. Biol. Chem. 239:2370-2378, buffer C in a total volume of 30 ml. 1964). Cytochrome P-450 substrate binding spectra are The runoff from the first yellow 3 agarose column is recorded with Stepwise increased Substrate concentration applied (flow rate 40 ml/h) to a column (2.8x8 cm) of until Saturating conditions are reached. 35 Cibachron Blue Agarose equilibrated in buffer C. The col Example 3 umn is subsequently washed with buffer C and the cyto chrome P-450 is eluted with a 0-500 mM linear KCI Purification of cytochrome P-450, and P-450 gradient (2x100 ml) in buffer C. The combined cytochrome P-450 fractions are diluted 5 times with buffer C and applied 40 (flow rate 7 ml/h) to a column (0.9x5 cm) of Reactive red Buffer A Buffer B Buffer C 120 agarose equilibrated in buffer C. The column is washed 8.6% glycerol 8.6% glycerol 8.6% glycerol with 25 ml buffer C and cytochrome P-450 is eluted with 10 mM 40 mM 40 mM. KHPO/KHPO a 0-1.0M KCl linear gradient (2x30 ml) in buffer C. KHPO/KHPO, KHPO/KHPO (pH 7.9) Optionally the eluate is gelfiltrated through a Sephadex G-50 (pH 7.9) (pH 7.9) 45 O.20 nM EDTA 5.0 nM EDTA 5.0 nM EDTA column, equilibrated in a buffer composed of 50 mM potas 2.0 nM DTT 2.0 nM DTF 2.0 nM DTT sium phosphate (pH 7.9)/400 mM KCl/0.1% CHAPS/2 mM 1.0% RENEX 690 10% RENEX 690 10% CHAPS DTT. The eluted cytochrome P-450 is dialyzed for 2 hours against 50 mM potassium phosphate (pH 7.9)/2 mM O.2% CHAPS DTT, diluted 4 fold with dialysis buffer in an Amicon 50 ultrafiltration cell fitted with a YM-30 membrane and con Buffers are degassed three times by Stirring in vacuo centrated to 1.45 nmols/ml. before detergent and DTT are added. Between each All procedures are carried out at 4 C. The total cyto degassing, the buffer is flushed with argon. chrome P-450 content of the individual fractions is deter Microsomes (400 mg protein in 20 ml) are diluted to 100 mined from the carbon monoxide difference spectrum. The ml with a buffer composed of 8.6% glycerol, 10 mM 55 absorption spectrum of the oxidized cytochrome P-450 is KHPO/KHPO (pH 7.9). The microsomes are solubilized also recorded. The presence of a specific cytochrome P-450 by slow addition of 100 ml of the same buffer containing 2% is monitored by Substrate binding spectra RENEX 690 and 0.2% RTX-100 and constant stirring for 30 Example 4 minutes. Solubilized cytochrome P-450 is obtained as the Supernatant after centrifugation for 30 minutes at 200 000 g 60 Antibody preparation in a Beckman 70:Ti rotor. The Supernatant (190 ml) is Polyclonal antibodies are elicited in rabbits by six applied (flow rate 100 ml/h) to a column (5x5 cm) of DEAE repeated Subcutaneous injections (approx. 15 lug protein per Sepharose fast flow/S-100 Sepharose (20:80 wet volumes) rabbit per injection) at 15 day intervals of cytochrome equilibrated in buffer A. The ion exchange resin DEAE P-450, or P-450 isolated by dye column chromatogra Sepharose is diluted with the gel filtration material 65 phy or denatured enzyme purified by preparative SDS Sephacryl S-100 in the ratio 1:4 to avoid too high concen PAGE. Freunds complete adjuvant is included in the first trations of the cytochrome P-450 enzymes upon binding, injection whereas Freunds incomplete adjuvant is used in 5,882,851 13 14 Subsequent injections. The immunoglobulin fractions of the antisera are purified by ammonium Sulfate precipitation TABLED (Harboe et al., “A Manual of Quantitative Immunoelectro phoresis: Methods and Applications, Universitetsforlaget, Addition of N-hydroxytyrosine until saturation concentration is reached, Oslo, 1973). The antiobides are monospecific as demon followed by addition of tyrosine strated by Western blotting. initial resulting Added substrate A390-420 dilution factor A390-420 50 ul 20 mM 0,0689 550/500 0.0758 Example 5 N-hydroxytyrosine 120 ul 5 mM O,0919 62O/5OO 0,1140 Characterization of P-450, N-hydroxytyrosine 5.1. Substrate binding spectra of P-450, 140 ul 5 mM 0,0911 640/500 0,1166 Cytochrome P-450 in the oxidized State has a Strong N-hydroxytyrosine absorption peak at 420 nm, representing the low Spin State + 90 ul 5 mM tyrosine 0.0726 730/500 0,1060 of the iron of the heme group. The binding of a ligand to the 15 heme group shifts the absorption maximum by changing the Both tyrosine and N-hydroxytyrosine produce a type I Spin State of the iron. Binding of tyrosine at the catalytic Site binding spectrum. The data show, that tyrosine and of cytochrome P-450 induces a change of the Spin State N-hydroxytyrosine bind to the same active site, that is the of the oxidized iron from low to high spin, and thereby Same cytochrome P-450, thus demonstrating that cyto changes the absorption maximum from 420 nm to 390 nm chrome P-450, is multifunctional. From the amounts of producing a type I spectrum (Jefcoate, Methods in Enzy cytochrome P-450 used the absorption coefficient mology 52:258-279, 1978). The following experimental (esoo lo) is calculated to be 67 cmmM'. A complete procedure is used to obtain the Substrate binding Spectrum: transition from a low Spin State to a high Spin State would two identical cuvettes containing a buffered Solution of the have resulted in an absorption coefficient of 138 cmmM. isolated cytochrome P-450 are prepared. The Substrate of the 25 5.2. Molecular weight and Amino acid Sequence data enzyme is added to the Sample cuvette whereas the same The molecular weight of P-450, as determined by volume of buffer is added to the other cuvette. The difference SDS-PAGE is 57 kD. spectrum is then recorded in an SLM-Aminco DW2c spec Amino acid Sequences are obtained by automated Edman trophotometer. The absorption difference, A390-420, is pro degradation. The internal polypeptides are obtained by portional to the concentration of cytochrome P-450 with trypsin digestion of the purified protein and Subsequent a bound Substrate at its active site. If a Saturating concen Separation of the peptides using reverse phase HPLC. tration of Substrate is added to the Sample cuvette, the N-terminal Sequence: absorption difference is proportional to the concentration of the substrate specific cytochrome P-450 in the cuvettes. The -MATMEVEAAAATVLAAP- (SEQ ID NO:3) Saturating concentration of the Substrate is determined by 35 titrating the cytochrome P-450 Sample with increasing Internal Sequences: amounts of Substrate and monitoring Asoo to. If a cytochrome P-450 sample can be saturated with two different substrates, there may be two different cytochrome -VWDEPLR- (SEQ ID NO: 4) P-450 enzymes in the sample, or there may be one cyto 40 chrome P-450 enzyme able to bind to both substrates. To - YWYNLATK- (SEQ ID NO:5) discriminate between these possibilities, Saturating amounts -SDTFMATPLVSSAEPR- (SEQ ID NO: 6) of the two Substrates are added Sequentially and the Asoo to absorption change is monitored. If, independent of the order -AQSQDITFAAVDNPSNAVEXALAEMVNNPESMAKD NO: 7) of addition, the addition of the Second Sample gives rise to 45 an increased Asoo to Value compared to the Value after the -AOGNPLLTIEEVK- (SEQ ID NO:8) addition of the first Substrate, the two Substrates are bound -LVOESDIPK- (SEQ ID NO: 9) by different enzymes. If Asoo to remains unchanged upon addition of the Second Substrate, independent of the order of -ISFSTG- (SEQ ID NO: 10) addition, both Substrates bind to the same active site, i.e. to 50 the same cytochrome P-450 enzyme. The data shown in -LPAHLYPSISLH- (SEQ ID NO: 11) Tables C and D below represent results of a typical experi 5.3. Reconstitution of cytochrome P-450 activity: ment. Reconstitution of the enzyme activity of a microSomal P-450 enzyme is accomplished by insertion of the cyto TABLE C 55 chrome P-450 enzyme and the corresponding NADPH cytochrome P-450 oxidoreductase into appropriate lipid To 500 ul of isolated cytochrome P-450 dissolved in 50 mM Tricine pH 7.9 tyrosine is added until saturation concentration is reached micelles made from different commercially available lipids. followed by addition of N-hydroxytyrosine: Isolation of NADPH-cytochrome P-450 oxidoreductase is done according to Halkier and Moller, Plant Physiol. initial resulting 60 96:10-17, 1990. A mixture of lipids can be used but with Added substrate A390-420 dilution factor A390-420 cytochrome P-450 di-lauroyl-phosphatidyl choline 30 ul 5 mM tyrosine 0.0437 530/500 0,0463 (DLPC) provides the best enzymatic activity. One rate 60 ul 5 mM tyrosine 0,0496 560/500 0.0556 limiting factor of this rate limiting reaction is the number of 90 ul 5 mM tyrosine 0,0486 590/500 0.0573 + 100 ul 20 mM 0.04.09 690/500 0,0564 correctly formed complexes of cytochrome P-450 and N-hydroxytyrosine 65 NADPH-cytochrome P-450 oxidoreductase. Excess amounts of the oxidoreductase and concentrated enzyme Solutions ensure a Sufficient number of active complexes. 5,882,851 15 16 A reconstituted enzyme is obtained using the following described for example in Sambrook et al, Molecular cloning: components: A laboratory manual. Cold Spring Harbor Laboratory Press, Cytochrome P-450: 100 tug/ml in 50 mM potassium Cold Spring Harbor, N.Y., 1989 or can be ordered by phosphate buffer pH 7.9 commercial companies Such as Strategene (La Jolla, Calif.). Oxidoreductase, purified from Sorghum bicolor: 100 Antibodies obtained against cytochrome P450 are used lug/ml in 50 mM potassium phosphate buffer pH 7.9 to Screen the expression libraries as described by Young et Lipid: 10 mg/ml di-lauroyl-phosphatidyl choline, Soni al, Procl. Natl. Acad. Sci USA 82:2583–2587. Antigen cated in 50 mM potassium phosphate buffer pH 7.9 antibody complexes are detected enzymatically with alka NADPH: 25 mg/ml HO line phosphatase-conjugated antibodies (Dakopatts). DNA 'C-tyrosine: commercially available from Amersham; from 4 positive plaques is prepared according to U-'C-L-tyrosine 0.5 uCi Grossberger, Nucleic Acid Research 15:6737, 1987. Inserts 10 ul of the lipid Suspension is mixed in a glass vial with from 2 phages are subcloned into pBluescript II SK 50 ul of the cytochrome P-450 (0–1.5 pmol) solution. 50 (Strategene). Comparison of the deduced amino acid ul of the oxidoreductase (0-0.15 U) solution is added and Sequence from one of the four inserts with the amino acid then 10 ul tyrosine solution and 10 ul NADPH Solution are 15 Sequences obtained from protein Sequencing of P450 added and the mixture is Sonicated in a Branson 5200 shows that this clone is a partial cDNA clone for P450. Sonication bath for one minute. The reaction mixture is The partial cDNA clone is used as a probe for a new screen Subsequently incubated for 1 hour at 30° C. At the end of the of the 2gt10 and 2gt11 libraries. The insert sizes of 45 incubation period the reaction is Stopped by transferring the positive clones are determined by Southern blotting. Addi glass Vials onto ice. Radioactively labelled intermediates tionally the 45 positive clones are examined for hybridiza formed are extracted into 50 ul ehtyl acetate, applied to a tion with two different mixtures of oligonucleotides by Silica coated TLC plate and developed using an ethyl Southern blotting. The Sequences of the oligonucleotide acetate/toluene (1:5 V/v) mixture as mobile phase. The mixtures are based on the partial amino acid Sequence data resultant product, p-hydroxyphenylacetaldehyde oXime is and specify a sequence near the N-terminal end (amino acids Visualized by autoradiography of the TLC plate. 25 4 to 9) and a sequence near the C-terminal end (amino acids Alternatively, the intermediates are analyzed by reverse 533–538). Oligonucleotide synthesis is carried out on a phase HPLC coupled to a Berthold radioactiviy monitor. The Cyclone Plus DNA Synthesizer. Sequencing of one clone HPLC separation was carried out using a nucleosil 100 derived from the 2gt10 library showing the expected size 10Cs column isocratically eluted with 1,5%. 2-propanol in and hybridizing with the two oligonucleotide mixtures 25 mM Hepes pH 7.9 (Halkier et al., J. Biol. Chem. shows that the clone is a full-length cDNA clone encoding 264:19487–19494, 1989). Control samples may be made by cytochrome P450. omitting either cytochrome P-450 oxidoreductase or Oligonucleotide specifying amino acids 4 to 9 (MEVEAA) NADPH. (SEQ ID NO: 17) When reconstituted into micelles cytochrome P-450, 5'-ATG-GAG.A-GTIC,G,TA-GAG.A-GCCGTA catalyzes the conversion of L-tyrosine all the way to 35 GC-3' p-hydroxyphenyl-acetaldehyde oXime. The K, and turn Oligonucleotide specifying amino acids 533 to 538 over number of the enzyme are 0.14 mM and 198 min', (DFTMAT) (SEQ ID NO: 18) respectively, when assayed in the presence of 15 mM NaCl, 5'-GAC,T-ACC,G,TA-TTC,T-ATG-GCC,G,TA whereas the values are 0.21 mM and 228 min' when AC-3' assayed in the absence of added Salt. 40 5.6. DNA sequencing The formation of p-hydroxyphenyl-acetaldehyde Oxime DNA sequencing is carried out by the dideoxy chain demonstrates that cytochrome P-450 is a multi method (Sanger et al, Proc. Natl. Acad. Sci. USA functional heme-thiolate protein catalyzing reactions in 74:5463-5467, 1977) using Sl-dATP. T7 DNA poly addition to the initial N-hydroxylation of L-tyrosine. The merase and deoxynucleotides are obtained from Pharmacia, E/Z ration of the parahydroxyphenyl-acetaldehyde Oxime 45 dideoxynucleotides from Boehringer Mannheim. Sequenc produced by the reconstituted cytochrome P-450, and ing of the full-length cDNA clone is done partly by Sub determined by HPLC chromatography is 69/31. Using the cloning and partly by using Synthetic oligonuecleotides as TLC/autoradiography System, minute amounts of radiola primers. The oligonucleotide primers can be ordered with belled products co migrating with authentic commerical companies. p-hydroxybenzaldehyde and 1-nitro-2(p-hydroxyphenyl) 50 5.7. Southern blotting ethane are detected in the reaction mixtures. 2DNA isolated from the positive clones is digested with 5.4. Inhibitory effect of antibodies against cytochrome Eco RI. The inserts are separated from DNA by electro P-450, phoresis on a 0.7% agarose gel. After electrophoresis, DNA The experiments are carried out using monospecific anti is capillary blotted onto a Zetaprobe membrane (Biorad) bodies against P-450 as described in Section 6.4. which 55 using 10 mM NaOH for the transfer. Hybridization is uses antibodies against cytochrome P-450. The results are performed at 68° C. in 1.5xSSPE (270 mM NaCl, 15 mM Similiar to those obtained with the antibody against cyto NaHPO pH 7.0, 1.5 mM EDTA, 1% sodium dodecyl chrome P-450 except that the cytochrome P-450, anti sulphate) 10% dextransulphate, 0.5% skim milk and 0.1 body exerts a stronger inhibitory effect (up to 60%) on mg/ml salmon sperm DNA for 16 hours. When the partial cyanide production. 60 cDNA clone is used as probe for hybridization it is labeled 5.5. cDNA libraries and colony screening with O.--PdCTP using a random prime labelling kit Poly A RNA is isolated from 3 cm high etiolated (Amersham International plc.). The oligonucleotide mix seedlings of Sorghum bicolor (L.) Moench grown as tures are 5'end labeled according to Okkels et al. (Okkels et described for Seedlings used for preparation of microSomes. al, FEBS Letters 237: 108-112, 1988). The filters are washed The poly A RNA is used for the construction of a 2gt11 65 first in 2XSSC (0.9M NaCl, 0.09M trisodium citrate, 0.1% expression library and a .gt10 library. The construction of SDS) at 47 C. for 15 min., then in a fresh solution of the the libraries can be done according to the procedures same composition at 56° C. for 15 min. and finally in 5,882,851 17 18 0.1XSSPE, 0.5% SDS for 30 minutes at 65° C. The presence the reconstitution experiments described in Section 5.3. of radioactively labelled hybridization bands on the filter is above. The expressed cDNA clone encoding P-450 monitored by X-ray autoradiography. specifies the synthesis of a single cytochrome P-450 5.8. Characterization of a full-length cDNA clone enzyme. Since this enzyme catalyzes the conversion of 2DNA isolated from the positive clones is digested with tyrosine all the way to p-hydroxyphenylacetaldehyde oXime, EcoRI. The insert is separated from 2 DNA by electrophore this unambigeously demonstrates that cytochrome P-450 sis on a 0.7% agarose gel and subcloned into the EcoRI site is multifunctional. of the vector pBluescript SK (Strategene) contained within The following oligonucleotides are used: TYROL1b (SEQ ID NO: 19) the sequence GCAGGAATTCCGG (SEQ ID NO: 24). The 5'-CGG GAT CCA TAT GCT GCT GTTATTAGC AGT four last bases of this Sequence are listed as the first four TTTTCT GTC GTA-3' bases in SEQ ID NO: 1. A clone comprising the described TYROL2 (SEQ ID NO: 20) cDNA has been deposited with the Agricultural Research 5'-GAC CGG CCGAAG CCTTAATTAGAT GGAGAT Culture Collection (NRRL), 1815 N. University Street, GGA-3' Peoria, Ill. 61604 U.S.A. under the accession number NRRL TYROL3 (SEQ ID NO: 21) B-21168. 15 5'-AGT GGA TCC AGC GGAATG CCG GCT T-3' The orientation of the insert in the vector is determined as TYROL4 (SEQ ID NO: 22) the polylinker restriction site for Pst I being adjacent to the 5'-CGT CAT GCT. CTT CGG AA-3' 5'end of the cytochrome P-450 sequence. The sequence of 5.10. Expression of truncated and modified cytochrome the insert is shown in SEQ ID NO: 1. The sequence P-450tyr in E. coli comprises an open reading frame (ORF) starting at nucle A modified cytocrome P-450, in which the 35 otide 188 and ending at nucleotide 1861 of SEQ ID NO: 1. N-terminal amino acids are replaced by the nine N-terminal It encodes a protein of 558 amino acids and a molecular amino acids from bovine 17C. hydroxylase is introduced into mass of 61887 Da shown in SEQ ID NO: 2. The sequence the expression vector pSP19g10L which can be obtained comprises the sequences of SEQ ID NO: 3 to SEQ ID NO: from Dr. Henry Barnes (La Jolla, Calif.). This plasmid 11. The protein is not Subject to post-translational modifi 25 contains the lac Z promoter fused with the known short cation at the N- and C-terminal ends except for the removal leader Sequence (g10L) of gene 10 from bacteriophage T., of the N-terminal methionine residue. The N-terminal region (Olin et al., 1988). A construct containing the N-terminal of cytochrome P-450, however, shows four motifs which amino acids from bovine 17C. hydroxylase and a truncated in animals are known to target heme-thiolate proteins to the form of the P-450 gene is designed using PCR mutagen endoplasmatic reticulum. esis: Oligonucleotide TYROL 1d (5'-CGG GAT CCA TAT Searches for Sequence Similarity are made using the GGCTCT GTTATTAGC AGTTTTTCT GTC GTA CCT programmes BLAST and FASTA in the nucleotide sequence GGC CCG-3'; SEQID NO 23) containing a 5' mutant cDNA databases provided by the EMBL. Pairwise comparisons of Sequence as well as a BamHI and Nde restriction site is cytochrome P-450 with other cytochrome P-450 used together with oligonucleotide TYROL 3 comprising Sequences are performed using the programme GAP of the 35 the Sequence Surrounding the unique BamHI restriction site Genetics Computer Group GCG Software package. Multiple downstream of the ATG start codon of the cDNA coding for alignments were made using the GCG programme PILEUP. P-450 to amplify a modified N-terminal Sequence of 5.9. Expression of native cytochrome P-450 in E. coli P-450. The amplification product is cut with Nde and Plasmid pCWOri+ (Gegner et al, Prod. Natl. Acad. Sci. BamHI restriction enzymes. To introduce a HindIII site USA 88:750-754, 1991) is used to express the wildtype 40 immediately downstream of the Stop codon of the P-450 cytochrome P-450, cDNA sequence as described by Bar gene, oligonucleotides TYROL 2 and TYROL 4 are used in nes et al, Prod. Natl. Acad. Sci. USA 80:5597-5601, 1991. a polymerase chain reaction to obtain a C-terminal fragment cDNA sequences are introduced into the expression plasmid of the P-450 gene comprising a HindIII restriction site using polymerase chain reaction (PCR) mutagenesis. A immediately downstream of the Stop codon. The amplifica synthetic oligonucleotide (TYROL1b) containing an amino 45 tion product is cut with Pst and HindIII restriction enzymes. acid-conserving and nucleotide modifying 5' cDNA The complete expression plasmid is constructed by Simul Sequence is used in conjunction with a downstream oligo taneous ligation of the N-terminal NdeI/BamHI fragment, nucleotide (TYROL3) to amplify the N-terminal sequence the BamHI/Pst fragment of the P-450 gene and the between the ATG initiator codon (contained within an NdeI C-terminal PstI/HindIII fragment into NdeI/HindIII cleaved Site) and a unique BamHI restriction site within the cyto 50 pSP19g10L vector DNA. The expression vector obtained is chrome P-450 Sequence. A Synthetic oligonucleotide transformed into E. coli strain JM109. Transformed E. coli (TYROL2) is used in conjunction with an oligonucleotide produce 300 nmol to 500 nmol cytochrome P-450 per (TYROL4) complementary to a unique PstI restriction site liter cell culture upon growth at 28 C. in the presence of 1 to introduce a HindIII restriction site immediately down mM isopropyl-B-D-thiogalactopyranoside and at 125 rpm. Stream of the TGA stop codon. The expression plasmid 55 Expression levels as high as 900 nmol per liter, equivalent pCWtyr is constructed by simultaneous ligation of the 278 to 55 mg P-450 per liter, have been obtained. basepair PCR NdeI/BamHI fragment, the 1257 basepair Administration of tyrosine to the cell culture results in the BamHI/Pst fragment of cytochrome P-450 and the 146 production of p-hydroxyphenylacetaldehyde oXime, basepair PCR PstI/HindIII fragment with the NdeI/HindIII whereas a cell culture transformed with pSP19g10L alone cleaved vector DNA. E. coli strain JM 109 transformed with 60 does not produce the oXime. plasmid pCWtyr is grown in LB/ampicilline medium at 37 Reconstitution experiments with E. cole-expressed cyto C. Expression of cytochrome P-450 is obtained by grow chrome P-450 and Sorghum NADPH cytochorme P450 ing the cells in a medium containing 1 mM isopropyl reductase in dilaurylpohsphatidylcholine micelles is per beta-D-thiogalactopyranoside (IPTG) and shifting the cells formed as described in section 5.3. above. Turnover rates of to growth at 28 C. at 125 rpm. E. coli produces a func 65 349 nmol oXime per nmol P-450 per minute can be tionally active cytochrome P-450 enzyme which con demonstrated, which is equivalent to the values obtained verts tyrosine into OXime. The analytical procedures are as in with Sorghum P-450. 5,882,851 19 20 Purified Sorghum cytochrome P-450 can be shown to section 5.3. A typical assay contains 10 ul DLPC (10 form type I Substrate binding Spectra with tyrosine and mg/ml); 50 ul cytochrome P-450 (24 mg/ml); 50 ul N-hydroxytyrosine (compare section 5.1.). Using P-450, NADPH-cytochrome P-450 oxidoreductase; 20 ul of either expressed in E. coli it can be shown that in addition to 10 mM p-hydroxyphenylace to nitrile or tyrosine and N-hydroxytyrosine P-450, is also able to p-hydroxyphenylacetaldehyde oxime; 10 ul NADPH (25 form a type Ispectrum with p-hydroxyphenylacetaldehyde mg/ml); and 60 ul potassium phosphate buffer (pH 7.9). o Xime, 2-nitro -(p-hydroxyphenyl)ethane, The reconstitution assay demonstrates that cytochrome P-450 converts p-hydroxyphenylacetaldehyde oxime to p-hydroxyphenylacetonitrile as well as phenylalanine. The p-hydroxymandelonitrile. The analytical procedures used molar extinction coefficient Eo-soo for tyrosine and are those described for cytochrome P-450. N-hydroxytyrosine as genuine Substrates of P-450 are 6.4. Inhibitory effect of antibodies against cytochrome 75.8 cmmM and 64.6 cmmM, respectively, whereas P-450 the extinction coefficients of the other compounds vary from The effect of antibodies raised against cytochrome 20–40 cmmM'. P-450 on the biosynthetic activity is measured as the Reconstitution experiments using phenylalanine as Sub decrease in cyanide production upon incubation of the Strate do not result in the production of the corresponding 15 Sorghum microSomes with p-hydroxyphenylacetaldehyde Oxime. This indicates, that cytochrome P-450, has a Oxime and p-hydroxybenzylcyanide as Substrates. The com narrow Substrate Specificity with respect to its enzymatic position of the 150 ul total volume reaction mixtures is: activity although it is able to bind many tyrosine analogues. microSomes containing 33 ug protein, 1.5 limol Substrate, Administration of 'C-tyrosine directly to E. coli cells 7.5 umol tricine pH 8.0, 0,33 umol NADPH, 0–255 lug expressing cytochrome P-450 results in the production of antibodies and 0-255 ug reference immunoglobulin. The p-hydroxyphenylacetaldehyde Oxime, indicating that E. coli total amount of immunoglobulin in the assay is in each is able to provide the reducing equivalents for cytochrome Sample adjusted to 225 ug using purified immunoglobulin P-450. from a nonimmunized rabbit. The antibodies are preincu The following oligonucleotides are used: bated with the microsomes for 15 minute at 30° C. before TYROL1d (SEQ ID NO. 23) 25 5'-CGG GAT CCATAT GGCTCT GTTATTAGC AGT substrate and NADPH are added. Subsequently the reaction TTTTCT GTC GTA CCT GGC CCG-3 is incubated at 30° C. for 30 minutes. Cyanide is determined TYROL2 (SEQ ID NO: 20) by the König reaction (König, Z. Angew. Chem. 18:115, 5'-GAC CGG CCGAAG CTTTAATTAGAT GGAGAT 1905) using methodology described in Halkier and Moller, GGA-3' Plant Physiol. 90:1552–1559, 1989. A value of Assss=1.5 TYROL3 (SEQ ID NO: 21) corresponds to 10 nmoles cyanide. Protein concentration 5'-AGT GGA TCC AGC GGAATG CCG GCT T-3' was determined using the method of Bradford (Bradford, TYROL4 (SEQ ID NO: 22) Anal. Biochem. 72:248-254, 1976). A typical result of such 5'-CGT CAT GCT. CTT CGG AA-3' an inhibition experiment is shown in Table E. 35 Example 6 TABLE E Characterization of P-450 Substrate p-hydroxyphenylacetaldehyde Oxime 6.1. Substrate binding spectra of P450 Similiar experiments as reported in Section 5.1 are carried pug antibody O 15 3O 60 12O 225 out using isolated cytochrome P-450 with 40 A680-585 1,00 O.96 0.92 0.87 0.88 0.66 p-hydroxyphenylace taldo Xime and inhibition O% 4% 8% 13% 12% 34% p-hydroxyphenylacetonitrile as Substrate. Cytochrome A680-sss 107 1,03 1,03 0.88 0.87 O,70 P-450 is found to be multifunctional as P-450. Isolated inhibition O% 4% 4% 18% 18% 35% cytochrome P-450 resembles the cytochrome P-450 reported to convert OXimes to nitriles in rat liver microSomes 45 The data show, that the antibody inhibits the reactions to (DeMaster et al., J. Org. Chem. 5074-5075, 1992). the same extent whichever Substrate is added to the microSo 6.2. Molecular weight and Amino acid Sequence data mal preparation. The molecular weight of P-450 as determined by SDS Example 7 PAGE is 51 kD. Amino acid Sequences are obtained by automated Edman 50 Induction of glucosinolate production in Tropaeolum majus. degradation. The internal polypeptides are obtained by Seeds of Tropaeolum majus L. cv Empress of India trypsin digestion of the purified protein and Subsequent (Dansk Havefroforsyning, Kolding, DK) are allowed to Separation of peptides using reverse phase HPLC. imbibe and germinate in complete darkness for one week at N-terminal Sequence: 25 C. In vivo biosynthesis experiments are performed 55 wherein 1 uCi of the tracer "C-labelled phenylalanine is -MDLADIPKOORLMAGNALVV- (SEQ ID NO:12) administered to excised dark-grown Seedlings for 24 hours Additional peptide Sequences: followed by boiling of the plant material in 90% methanol and analysis of the extracts by HPLC as described by -ARLAEIFATII- (SEQ ID NO:13) Lykkesfeldt and Moller, 1993. Prior to administration of the 60 tracer to the excised Seedling or leaf, the intact plant is -EDFTWTTK- (SEQ ID NO:14) Subjected to a potential inducer for 24 hours. Administration -OYAALGSVFTVPII- (SEQ ID NO: 15) of 10 mM phenylalanine or 2% ethanol to the vermiculate in which the etiolated Seedlings are grown results in a threefold - XXPFP- (SEQ ID NO: 16) increase in glucosinolate production as compared to control 6.3. Reconstitution of cytochrome P-450-activity: 65 experiments with water. Spraying with 100 uM jasmonic The reconstitution assay used for cytochrome P-450 is acid followed by incubation for 24 hours results in a fivefold Similiar to that used for cytochrome P-450, described in induction in etiolated Seedlings and green leaves. 5,882,851 21 22 Example 8 lanine is administered to etiolated tropaeolum Seedlings in Preparation of biosynthetically active microsomes from the presence and absence of 1 mM of the cytochrome P450 glucosinolate-producing plant meterial inhibitors enilketoconazol and tetcyclacis demonstrate that The biosynthetic pathways of glucosinolates, and cyano cytochrome P450 inhibitors cause a reduction of glucosino genic glucosides share homology by having amino acids as late without causing a reduction in the uptake of phenyla precursors and OXimes as intermediates. The assignment of lanine as measured by ethanol eXtraction of the plant mate amino acids and OXimes as precursors and intermediates in rial. This indicates that the biosynthesis of glucosinolates is the glucosinolate biosynthetic pathway is based on in vivo dependent on cytochrome P450. experiments demonstrating that these compounds are effi Direct demonstration of the involvment of cytochrome cient precursors for glucosinolates. In vitro biosynthetic P450 in glucosinolate biosynthesis can be obtained using the studies have hitherto not been possible due to the detrimen in vitro microSomal enzyme System from tropaeolum to tal effect of the degradation products of glucosinolates on demonstrate photoreversible carbon monoxide inhibition of enzyme activities. The degradation products are formed Oxime production. The microSomal reaction mixtures are upon disruption of the cellular Structure. In the disrupted incubated using different experimental conditions. The reac tissue, the glucosinolate-degrading enzyme myrosinase gets 15 tion mixtures are analyzed by HPLC. in contact with the glucosinolates resulting in the generation of isothiocyanates inactivating the enzymes. We demon Experimental Condition % inhibition of oxime production Strate that microSomal preparations isolated form one week old plants of either Sinapis alba or Trapaeolum majus are O without light O O with light 11 able to convert tyrosine and phenylalanine, respectively, to CO/O without light 65 the corresponding oximes. The enzymatically active CO/O, with light 23 microSomal preparations are obtained by using an isolation buffer fortified with 100 mMascorbic acid known to inhibit the activity of my roSinase and by inducing the The possibility to reactivate the microSomal enzyme glucosinolate-producing enzyme System prior to the prepa 25 system upon irradiation with 450 nm light shows, that the ration of microSomes. The glucosinolate-producing enzyme conversion of phenylalanine to the corresponding oxime in Systems are induced by taking 7-dayS-old dark-grown Sina the biosynthetic pathway of glucosinolate is dependent on pis plants or 3-4 weeks old light-grown Tropaeolum plants cytochrome P450. and placing them in the light for 3 days. During this three Example 11 day period, the young plants are sprayed with 50 uM jasmonic acid once a day. After 3 days of induction, the Toxicity of cyanogenic glycosides for insects. plants are harvested and microSomes are prepared as Insects or insect larvae are fed on a diet containing added described in Section 5.1, except that the homogenisation cyanogenic glycoside, a diet containing added cyanogenic buffer consists of 250 mM Tricine pH 7.9, 250 mM sucrose, glycoside, and callus, or a diet Supplemented with the 50 mMSodium bisulfite, 100 mMascrobic acid, 4 mM DDT, 35 Supernatant of callus ground-up in the presence of the 2 mM EDTA, 1 mM PMSF, and 5 mg/ml BSA. The cyanogenic glycoside. Mortality is compared to the mortal microSomal preparation is dialysed against homogenization ity of insects or insect larvae fed on the diet only. buffer for 1 hour, followed by dialysis against 50 mM Tricine pH 7.9 and 2 mM DTT for another hour. Example 12 40 Activity of Amygdalin on larval mortality of Western Corn Example 9 Root Worm (WCRW): In vitro biosynthesis of oxime by extracts from WCRW larvae are fed on a diet with added amygdalin, a glucosinolate-containing plants diet with added amygdalin and Black Mexican Sweet (BMS) The microsomal reaction mixture consists of 80 ul 45 callus or on a diet Supplemented with the Supernatant of microSomes (10 mg protein per ml), 10 ul "C-phenylalanine BMS-callus ground-up in the presence of amygdalin. Larval (0.5 uCi, 464 mCi/mmol, Amersham) or 'C-tyrosine (0.5 mortality is compared to the mortality of larvae fed on the uCi, 450 mCi/mmol, Amersham) and 10 ul NADPH (75 diet only. mg/ml). The reaction mixtures are incubated for 1 hour at The results show that amygdalin is lethal in the presence 37 C. At the end of the incubation period, the reaction 50 of BMS-callus with an LCs of ~1 mg/ml, and that it is lethal mixtures are extracted with 1500 ul ethyl acetate. The ethyl at 2 mg/ml in the absence of BMS-callus. There is signifi acetate phase is evaporated to dryneSS, redissolved in a Small cantly leSS lethality at amygdalin concentrations of less than Volume and analyzed. The production of Oximes in the 1 mg/ml when BMS-callus is absent. microSomal reaction mixtures can be demonstrated by thin Example 13 layer chromatography as well as by HPLC analysis as 55 described in section 5.3. Activity of Dhurrin on larval mortality of Western Corn Root Worm (WCRW): Example 10 The activity of dhurrin on larval mortality of WCRW was Involvement of cytochrome P450-dependent monooxygena determined as described for amygdalin in example 12. SeS in the glucosinolate pathway 60 The results show that the LCs of dhurrin is 368 ug/ml Based on the similarity between the first part of the with 95% confidence limits of 0.28-0.48 ug/ml. The slope of biosynthetic pathways of glucosinolates and cyanogenic the regression line is 2.5. glucosides, it was anticipated that the conversion of amino acid to OXime in the glucosinolate pathway is catalyzed by Example 14 a multifunctional cytochrome P450 monooxygenase 65 Transfection of maize by direct Bombarding of Immature homologous to P450 in the cyanogenic glucoside pathway. Zygotic Embryos and Isolation of Transformed Callus In Vivo experiments, where radioactively labelled phenyla with the Use of Phosphinothricin as a selection agent. 5,882,851 23 24 Immature embryos are obtained approximately 14 days after Self-pollination. The immature Zygotic embryos are TABLE A-continued divided among different target plates containing medium capable of inducing and Supporting embryogenic callus PLANT CLASSIFICATION ACCORDING TO USE formation at 36 immature embryos per plate. The immature 5 P. coccineius Scarlet runner bean Zygotic embryos are bombarded with plasmids encoding a (multiflorus) P. nungo Black gram cytochrome P-450 monooxygenase and a chimeric gene P. vulgaris French, common, kidney or dwarf bean coding for resistance to phosphinothricin using the PDS Vicia faba Horse bean, broad bean Vigna angularis Adzuki bean 1000/He device from DuPont. The plasmids are precipitated V. Sesquipedalis Asparagus (yard-long bean) onto 1 um gold particles essentially according DuPont's V. Sinensis Cowpea procedure. Each target plate is shot one time with the FRUIT CROPS plasmid and gold preparation and phosphinothricin is used to Select transformed cells in vitro. Selection is applied at 3 Dicot mg/l one day after bombardment and maintained for a total 15 Amygdalus communis Almond of 12 weeks. The embryogenic callus So obtained is regen Ananas COmoSuS Pineapple Artocarpus communis Breadfruit erated in the absence of the Selection agent phosphinothri Carica papaya Papaya cin. The regenerated plants are tested for their resistance to Citrulius vulgaris Watermelon insects, acarids or nematodes. Citrus grandis Pummelo C. medica Citron, lemon Although the foregoing invention has been described in 20 C. nobilis Tangerine Some detail by way of illustration and example for purposes C. reticulata Mandarin of clarity of understanding, it will be obvious that certain C. Sinensis Orange Cydonia oblonga Quince changes and modifications may be practiced within the Diospyros kaki Japanese persimmon Scope of the appended claims. Ficus carica Fig 25 Fragaria chiloensis Wild strawberry TABLE A F virginiana Strawberry Litchi chinensis Litchi PLANT CLASSIFICATION ACCORDING TO USE Maius asiatica Chines apple M. pumila Appple CEREALS Mangifera indica Mango Morus rubra Red mulberry Monocot Musa cavendishi Banana M. paradisiaca Banana Avena nuda (chinensis) Chines naked oat Passiflora edulis Passion fruit, purple granadilla A. Sativa Common oats P ligularis Passion flower Eleusine coracan African millet PerSea annericana Avocado pear Eragrostis tef Tef grass 35 Phoenix dactylifera Date palm Fagopyrun esculentum Buckwheat Prunus armeniaca Apricot F. tataricium Rye buckwheat Pavium Sweet cherry, mazzard Hordeum distichun Two-row barley P. cerasifera Cherry plum H. vulgare Barley (divaricata) Oryza Sativa Rice P. ceraSuS Cherry Panicum italicium Italian millet P domestica European plum or prune P. miliaceum Broomcorn millet 40 P. maheleb Maheleb cherry Pennisetum glaucun Spiked millet P. persica Peach and nectarine P. spicatum (americanum) Perl millet P. pseudoceraSuS Cherry Secale cereale Rye P. Salicinia Japanese peach Sorghum vulgare Grain sorghums P Serotina Wild black cherry X Triticosecale Triticale Psidium guaiava Guava Triticium aestivium Common wheat 45 Punica granatun Pomegranate T. dicoccunn Emmer Pyrus communis Pear T. durun Abyssinian hard wheat Pils.SurienSis Chinese pear T. monococcium Einkorn wheat Ribes grossularia Gooseberry Zea mayS Corn, Sweet corn R. nigrum Black currant Dicot R. rubrun Red and white currant 50 Rubus idaeus European raspberry Anaranthus paniculatus Rispenfuchsschwanz R. StrigOSuS American raspberry Fagopyrun esculentum Buchweizen Tamarindus indica Tamarind F. tataricium Vaccinium angustifolium Sugarberry PROTEIN CROPS W. ashei Rabbiteye blueberry V. corymbosum Highbush blueberry Dicot 55 V. myrtilloides Canada blueberry V. OxycoccoS Cranberry Arachis hypogea Groundnut, peanut Viburnum trilobum American cranberry bush Caianus indicus Pigeon pea Vitris labrusca Fox grape Cicer arietinium Chickpea Grape DolichoS labiab Hyacinth bean V. vinifera Glycine gracilis Manchurian Soya VEGETABLES AND TUBERS G. max Soyabean 60 G. ii.S.SurienSis Wild soya Monocot Lathyrus Sativus Grass pea Lens culinaris Lentil Allium ascaionicum Shallot, breen onion Mucuna pruriens Cowitch, Florida velvet bean A. cepa Onion Phaseolus acutifolius Tepary bean A. chinense Onion P. aureus Mung, green gram 65 A. fistuloSun Welch onion P. lunatus Lima bean, Sieva A. porrun Leek 5,882,851 25 26

TABLE A-continued TABLE A-continued

PLANT CLASSIFICATION ACCORDING TO USE PLANT CLASSIFICATION ACCORDING TO USE

A. Sativum Garlic NUTS A. Schoenopra Sun Chives Asparagus Officinalis Asparagus (var. attilis) Dicot Zea mayS Sweet corn Dicot Anacardium occidentale Cashew Arachis hypogaea Peanut Amoracia lapathifolia Horseradish Carya illinoinensis Pecan Apium graveolens Celery C. ovata Shagbark hickory Arabidopsis thaliana Common wall cress Castanea Sativa Chestnut Beta vulgaris Sugar, mangold or garden beet CovoS nucifera coconut palm Brassica alboglabra Chinese kale Coryius annericana American hazel, filbert campestris Turnip rape C. aveliana European hazel, cobnut carinata Ambyssian mustard 15 Jugians nigra Black walnut Ceet Karashina J. regia English walnut chinensis Chinese mustard or pak-choi J. Sinensis Walnut hirta White mustard Litchi chinensis Litchi ... juncea Macadamia integrifolia Queensland nut Pai, brown mustard, Indian mustard Pistacia vera Pistachio nut kaber Charlock Prunus amygdalus Almond napobrassica Swede or rutabaga OIL CROPS naptis Rape, oil rape, kale nigra Black mustard Monocot Oileracea Cole, kale, collards, brussels sprouts, cauliflower, cabbage, kohlrabi, broccoli Zea mayS Corn B. pekinensis Chines cabbage or celery cabbage Dicot B. rapa Turnip 25 Caianus cajan (indicus) Pigeon pea Aleurites cordata Tung, China wood oil Canavalia ensiformis Jack bean A. moluccana (triloba) Candlenut Canna edulis Edible canna Arachis hypogea Ground nut, penut Capsicum annuum Common cultivated pepper brassica campestris Rapeseed oil, canola oil C. chinense Pepper B. napuS Rapeseed oil, canona oil C. frutescens Cayenne pepper Cannabis Sativa Hampseed oil C. pendulum Pepper Carthamus tinctorius Safflower oil CocoS nucifera Coconut palm C. pubescens Pepper Elaeis guineensis Oil palm Cichorium endivia Endive Glycine gracilus Manch, soya C. intybus Chicory G. max Soybean Taro Colocasia antiquorum 35 G. ii.S.SurienSis Wild soya Cranbe maritina Sea kale CoSSypium hirsutiun Cottonseed oil Cucunnis meio Melon, cantaloupe Helianthus annus Sunflower C. Sativius Cucumber Linum usitatis.Simum Flax Cucurbitaficifolia Malabar gourd Olea europaea Olive C. foetidissima Calabazilla, buffalo gourd Papaver SOmniferum Poppy seed C. maxina Pumpkin 40 Ricinius communis Castor bean C. noSchata Winter pumpkin Sesamun indicum Sesame C. pepO Summer squash, vegetable marrow SUGAR CROPS Cynara Scolynus Globe artochoke Daucus carota Carrot Monocot DioScorea alata Yarn D. batatas Chines yarn 45 Saccharum officinarum Sugarcane D. cavennensis Attoto yam (officinarum X Eruca Sativa Mill. Rocket salad, rocket or roquette spontaneum) Ipomea batatas Sweet potato S. robustun Lactuca Sativa Lettuce S. Sinense Sugarcane Lepidium Sativum Garden cress S. Spontaneun Kans grass Lycopersicon cerasiforne Cherry tomato 50 Sorghum dochna Sorgo syrup, Sugar sorghun L. esculentum Tomato Dicot Manihot esculenta Manioc, cassava Nasturtium officinale Water cress Acer Saccharun Sugar maple Pastinaca Sative Parsnip Beta vulgaris Sugar or mangold beet Petroselinum crispum Parsley FORAGE AND TURF GRASSES (Sativum) 55 Physalis peruviana Ground cherry Monocot Pisun Sativium Pea Raphanus Sativus Radish Agropyron cristatun Crested wheatgrass Rheum officinale Rhubarb desertorum Crested wheatgrass R. rhaponticum English rhabarb elongatun Tall wheatgrass Scorzonera hispanica Black salsify 60 intermedium Intermediate wheatgrass Sechium edule Chayote Smithii Western wheatgrass Soianum andigenun Andean potato Spicatun Blue bunch wheatgrass S. melongena Eggplant trachycaulum Slender wheatgrass S. nitricatum Pepino ... trichophorum Pubescen wheatgrass S. phureja Potato Alopecurus pratensis Meadow foxtail S. tuberoSun Common potato 65 Andropogon gerardi Big bluestem Psinacia oleracea Spinach Arrhenatherun elatius Tall oat grass 5,882,851 27 28

TABLE A-continued TABLE A-continued

PLANT CLASSIFICATION ACCORDING TO USE PLANT CLASSIFICATION ACCORDING TO USE 5 Bothrichloa barbinodis Cane blestem T. ingrescens Ball clover B. ischaemun King ranch bluestem T. pratense Red clover B. Saccharoides Silver bluestem T. repens White clover Bouteloua curipendula Side oats grama T. resupinatium Persian clover B. eriopoda Black grama T. Subterraneun Subterranean clover B. gracilis Blue grama Trigonelia foenungraecum Bronius erectus Upright brome Fenugreek B. inermis Smooth brome Vicia Sative Common vetch B. riparius Meadow brome V. villosa Hairy vetch Cenchrus ciliaris Buffel grass V. atropurpurea Purple vetch Chloris gayana Rhodes grass V angustifolia Narrowleaf vetch Cymbopogon nardus Citronella grass V. da Sycarpa Wooly pod vetch Cynodon dactylon Bermuda grass 15 Wervilia Monantha (bitter) vetch Dactylis glomerata Cocksfoot V pannonica Hungarian vetch Dichanthium annulatum Kleberg bluestem W. calcarata Bard vetch D. aristatum Angleton bluestem FIBER PLANTS AND WOODY PLANTS D. Sericetum Silky bluestem Digitaria decumbens Pangola grass Monocot D. Smutsii Elymus angustus Altai wild rye Banbulsa vulgaris Bamboo E. iunceus Russian wild rye Eragrostis curvula Weeping love grass Dicot Festuca arundinacea Tall fescue F. ovina Sheeps fescue Agave Sisaiana Sisal hemp F. piratensis Meadow fescue Boehneria nivea Rhea fiber, ramie F. rubra Red fescue 25 Cannabis indica Hemp Lolium multiflorum Italian ryegrass C. Sativa Hemp L. perenne Perennial ryegrass Ceiba pentandra Silk cotton tree, kapok tree Panicum maximum Guinea grass Corchorus mucronata Hemp P purpurascens Para grass (striata) P. virgatum Switchgrass Gossypium arboreun Tree cotton Paspalum dilatatum Dallis grass, large water grass G. barbadense Egyptian cotton P notatium Bahia grass G. herbaceum Cotton Pennisetum clandestinum Kikuyu grass G. hirsutumn Upland cotton P purpureum Dry napier grass G. nanking Oriental cotton Phalaris a Fundinacea Reed canary grass Wild flax Phleum bertolini Timothy Linum angustifolium L. usitatissinium Flax P. pratense Timothy 35 Poa fendleriana Mutton grass Musa textiles Manila hemp, abaca P nemoralis Wood meadow grass DRUG CROPS P. pratensis Kentucky bluegrass Setaria Sphaceiata Rhodesian timothy Dicot Sorghastrum nutans Indian grass Sorghum halepense Johnson grass Angelica archangelica Angelica S. Sudanense Sudan grass 40 Sorghum vulgare Great millet Chrysanthemum cinerariifolium Palm pyrethrum FORAGE LEGUMES Cannelia Sinensis Chinese tea C. coccineun Pyrethrum Dicot Coffea arabica Coffee C. canephora Quillow coffee Coronilla varia Crown vetch 45 Cola acuminata Kola nut Crotaiaria juncea Sun hemp Nicotiana rustica Tobacco Lespedeza Stipulacea Korean lespedeza N. tabacunn Tobacco L. Striata Common lespedeza L. Sericea Papaver dubium Poppy Lotus corniculatus Birdsfoot trefoil P. Somniferum Opium poppy L. iliiginosus 50 Theobroma cacao COCOa Lupinus albuS Wolf bean, white lupin SPICES AND FLAVORINGS L. angustifolius Blue lupin L. luteus European yellow lupin Monocot L. mutabilis South American lupin Medicago arabica Spotted burr-clover Vanilla fragrans Vanilla M. arborea Tree alfalfa 55 Dicot M. falcata Yellow lucerne M. hispida California burr-clover M. Satiya Alfalfa Artemisa dracunculus Tarragon M. tribuloides Barrel medic Cinnamomum zeylanicum Cinnamon tree Melilotus albus White sweet clover Hibiscus esculentus Okra Yellow sweet clover M. officinalis 60 Salvia Officinalis Sage Onobrychis viciifolia Sainfoin Thymus vulgaris Thyme Ornithopus sativus Serradella Pimpinella anisum Anise Kudzu vine Pueraria thunbergiana Mentha arvensis Menthol Trifolium alexandrinum Egyptian clover T. augustifolium Fineleaf clover M. piperita Peppermint T. diffusum Rose clover M. viridis Spearmint T. hybridum Alsike clover 65 Coriandrum Sativum Coriander T. incarnatium Crimson clover 5,882,851 29 30 Halkier et al, Proc. Natl. Acad. Sci. USA 88:487–491, TABLE B 1991

REPRESENTATIVE PLANT PESTS DHalluin et al, The Plant Cell 4:1495–1505, 1992 Harlow and Lane, Antibodies A Laboratory Manual, Coleoptera: Cold Spring Harbor Laboratory, 1988 Diabrotica, Melanotus, Agriotes, Limonius, Dalopius, Eleodes, Chaetocnema, Macrodactylus, Sphenophorus, Sitophilus, Lisorhoptrus, Harboe et al., “A Manual of Quantitative Immunoelectro Oulema, Rhyzopertha, Prostephanus, Phyllophage, Cyclocephala, Popillia, phoresis: Methods and Applications, Universitetsforlaget, Anthonomus, Zabrotes, Leptinotarsa Oslo, 1973 Lepidoptera: Horsch et al, Science 227:1229–1231, 1985 Heliothis, Ostrinia, Diatraea, Elasmopalpus, Papaipema, Agrotis, LOXagrotis, Euxoa, Peridroma Saucia, Chorizagrotis, Spodoptera, Ibenthal et al, Angew. Bot. 67:97-106, 1993 Pseudaletia, Chilo, Busseola, Sesamia, Eldana, Maliarpha, Scirpophaga, Jefcoate, Methods in Enzymology 52:258-279, 1978 Duataea, Rupela, Sitotroga cerealella, Sitroga, Plodia interpunctella, Crambus, Mythimna, Nola, Pectinophora, Acontia, Trichoplusia, 15 Kindl et al., Phytochemistry 7:745-756, 1968 Anticarsia, Pseudoplusia, Manduca, Leptinotarsa, Lema Thysanoptera: Koch et al, 8th Int. Conf. on Cytochrome P450, Abstract PII.053 Frankliniella, Anaphothrips, Hercothrips, Stenothrips Koch et al, Archives of Biochemistry and Biophysics, Homoptera: 292:141-150, 1992 Dalbulus, Cicadulina, Rhopalosiphum, Melanaphis, Anuraphis, Prosapia, Nilaparvata, Sogatella, LaodelphaX, Sogatodes, Nephotettix, Recilian, König, Z. Angew. Chem. 18:115, 1905 Cofana, Empoasca, Poophilus, Schizaphis, Sipha, Paratrioza, Empoasca, Koziel et al, Biotechnology 11: 194–200, 1993 Ophilia. Scleroracus, Macrosteles, Circulifer, Aceratagallia, Agallia, Myzus, Macrosiphum, Aphis Lieberei et al, Plant Phys. 90:3–36, 1989 Diptera: 25 Ludtke et al, Biochem. Z. 324:433–442, 1953 Delia platura, Euxesta, Diopsis, Atherigona, Hydrellia, Orseolia, Lykkesfeldt et al, Plant Physiology 102: 609-613, 1993 Chironomus, Contarinia Orthoptera: Matsuo et al., Phytochemistry 11:697–701, 1972 Murray et al, Nucleic Acids Research 17:477-498, 1989 Melanoplus, Schistocerca, Sphenarium, Aneolamia Isoptera: Nebert et al, DNA Cell Biol. 10:1-14, 1991 Negrutiu et al, Plant Molecular Biology 8:363-373, 1987 Microtermes, Macrotermes, Allodontermes, Odontotermes Heteroptera: Neuhaus et al, Theoretical and Applied Genetics 74:363-373, 1987 Nezara, Acrosternum, Euschistus, Blissus Acarina: 35 Okkels et al, FEBS Letters 237:108-112, 1988 Tetranychus, Paratetranychus, Oligonychus Olin et al, Gene 73: 227-235, 1988 Omura et al., J. Biol. Chem. 239:2370-2378, 1964 Pourmohensi, PhD thesis, Göttingen, 1989 LIST OF REFERENCES CITED 40 Sambrook et al, Molecular cloning: A laboratory manual. Barnes et al, Prod. Natl. Acad. Sci. USA 80:5597-5601, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1991 N.Y., 1989 Bradford, Anal. Biochem. 72:248-254, 1976 Sanger et al, Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977 Christou et, Plant Physiol. 87:671-674, 1988 45 Conn, Naturwissenschaften 66:28-34, 1979 Schocher et al, Bio/Technology 4:1093–1096, 1986 Cronquist, 'The Evolution and Classification of Flower Shillito et al, Biotechnology 3:1099–1103, 1985 ing Plants, New York Botanical Garden, Bronx, 1988 Sibbesen et al., in: Biochemistry and Biophysics of cyto chrome P-450. Structrue and Function, Biotechnological DeMaster et al., J. Org. Chem. 5074-5075, 1992 50 and Ecological Aspects, Archakov, A. I. (ed.), 1991 Food Chemical News 29:33.35, 1988 Sibbesen et al, 8th Int. Conf. on Cytochrome P450, Gegner et al, Prod. Natl. Acad. Sci. USA 88:750-754, Abstract PII.016 1991 Underhill, Eur. J. Biochem. 2:61-63, 1967) Grossberger, Nucleic Acid Research 15:6737, 1987 55 Halkier et al, 'Cyanogenic glucosides: the biosynthetic Underhill et al, Biochem. Soc. Symp. 38:303-326, 1973 pathway and the enzyme System involve in: 'Cyanide Young et al., Procl. Natl. Acad. Sci USA 82:2583–2587 compounds in biology, Wiley Chichester (Ciba Foundation Wang et, Plant Molecular Biology 11:433–439, 1988 Symposium 140), pages 49-66, 1988 60 Halkier and Moller, Plant Physiol. 90:1552–1559, 1989 EP-24O 208-A2 WO 89/O5852 US 5,023,179 Halkier et al, The J. of Biol. Chem. 264:19487–19494, EP-306, 139-A WO 89/07647 US 5,231,020 EP 332 104 WO 91/13992 1989 EP-452 269-A2 WO 93/07278 Halkier and Moller, Plant Physiol. 96:10–17, 1990 EP-458 367 A1 WO92fO9696 65 WO 90/11682 Halkier and Moller, The J. of Biol. Chem. 265:21114-21121, 1990

5,882,851 35 36 -continued

As n Le u Th r A r W a As n L e u A Th S A 1 a A a Th r G 1 9 5 2 O O 2 O 5

A s p V a 1 A a W a W a His Wa A A r Gl 21 O o

Wa I e A rig A. r Me t Ph As in Arg A r G 1 y Gl G 2 25 2 3 O s 2 4 A la As p G 1 y G 1 G Me t G 1 u Me t A W a 25 25

Ph e Thr S e r Le Le Le Ty r A 1 a Ph W a S e r Le 2 6 2 6 5 R

Pro Tr p Le u A r Le Le u As p Gl L y S I W a 2 7 5 28 O

G u. A 1 a As in W a W a A rig L e u Th I 29 O 3 O O

A rig Tr p A rig Gl S e G 1 y G 1 u G Me t Gl Ph 3 O 5 o 3 2 Le u As p Wa Le Le Ly is As p A G Le 3 3

Th r I e G u G A G in S e r Gl I Th r Ph 3 4 3 4 5 3 5

V a l As p As in P. r A Wa G u Le A 1 a Gl Me W a 3 5 5 3 6 O 3 65

As in As in Pro Gl W a Me A Ly is A 1 a Me l A r W a 3 7 O 3 7 38

V a 1 G 1 y A rig Gl W a G in G 1 u S e I 38 5 39

Ty r V a 1 Lys Al I A r G u. A a Ph Le 4 1

P r o Phe As in W a W a A l a Le u A Th Th r G 4 2 4 25

Ty r A rig Wa P. r Gl S e His Wa I Le S e Le 43 5 4 4 O 4 4 5

G l y Arg As in P. r W a A s p G 1 u Le P he 4 5 O 4 5 4 6

A rig H is Le u A Th A A S e r A sp W a A Le Th r 4 6 5 4 7 4 7

Le u Arg Ph e I Ph Thr G 1 y Gl 49

S e r Le u G 1 y T h Me Wa Me t Le Ph Gl A rig Gl 5 O 5 O 5 5 1.

G 1 y P he Th r T. r P r A a G 1 y W a Wa 5 15 5 2 O 5 25

G u S e r Lys Se Th Ph Me t A 1 a Th Wa A 53 O 5 3

G u Pro A rig Le A Le u Ty r 5 4 5 5 5

( 2) INFORMATION FOR SEQ ID NO:3: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein

( i i i ) HYPOTH ETICAL: NO 5,882,851 37 38 -continued

( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: N-terminal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Me t A a Thr Me t G u Wa G u. A a A a A a A a Thr Wa Le u A a A a 1. 5 1 O 1 5

P ro

( 2) INFORMATION FOR SEQ ID NO: 4: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 4: V a l T r p A s p G 1 u Pro Le u Arg 1. 5

( 2) INFORMATION FOR SEQ ID NO: 5: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Ty r V a 1 Ty r A s in Le u A la Thr Lys 1. 5

( 2) INFORMATION FOR SEQ ID NO: 6: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 6: S e r A s p Thr Phe Me t A a Thr Pro Le u V a l S e r S e r A a G 1 u Pro Arg 1. 5 1 O 1 5

( 2) INFORMATION FOR SEQ ID NO: 7: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids 5,882,851 39 40 -continued (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 7: A a G 1 in Ser G 1 in A s p I le Thr Ph e A 1 a. A 1 a V a 1 A s p As n Pro Ser As n 1. 5 1 O 1 5

A a Wa G u Xa a A a Le u A a G u Me t W a As in As in Pro G 1 u Wa Me t 2 O 25 3 O A la Lys

( 2) INFORMATION FOR SEQ ID NO:8: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:8: A a G 1 in G 1 y As in Pro Le u Le u Thir I le G 1 u G 1 u Val Lys 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO: 9: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Le u V a 1 G | n G 1 u Se r A s p I le Pro Lys 1. 5

( 2) INFORMATION FOR SEQ ID NO: 10: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 5,882,851 41 42 -continued

I le Ser P h e Ser Thr G 1 y

( 2) INFORMATION FOR SEQ ID NO: 11: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Le u Pro A 1 a His Le u T y r Pro Se r I le Ser Le u H is 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO: 12: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: N-terminal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Me t A s p Le u A 1 a. A s p I le Pro Lys G 1 n G | n Arg Le u Me t A a G 1 y As n 1. 5 1 O 1 5

A a Le u Wa W a 2 O

( 2) INFORMATION FOR SEQ ID NO: 13: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 13: A 1 a. A rig L e u A a G 1 u I le Ph e A a Thr I I e I le 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO: 14: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein 5,882,851 43 44 -continued ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14: G 1 u A s p Phe Thr V a 1 T hir Thr Lys 1. 5

( 2) INFORMATION FOR SEQ ID NO:15: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:15: G 1 in Ty r A 1 a. A 1 a Le u G 1 y Ser V a 1 P he Thr V a 1 Pro I le I e 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO: 16: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i i i ) HYPOTHETICAL: NO ( i i i ). ANTI-SENSE: NO ( v ) FRAGMENT TYPE: internal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Xa a Xa a P r o Phi e Pro I e 1. 5

( 2) INFORMATION FOR SEQ ID NO: 17: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( v ii) IMMEDIATE SOURCE: (B) CLONE: Oligonucleotide specifying AA sequence MEVEAA ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

A T G GAR GTNG ARG CNGC 1 7

( 2) INFORMATION FOR SEQ ID NO: 18: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 17 base pairs 5,882,851 45 46 -continued (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( v ii) IMMEDIATE SOURCE: (B) CLONE: Oligonucleotide specifying AA sequence DFTMAT ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GAYA CNT TYA T G G CNAC 1 7

( 2) INFORMATION FOR SEQ ID NO: 19: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( v ii) IMMEDIATE SOURCE: (B) CLONE: TYROL1b ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

C G G GAT C CAT A T G C T G C T G T TAT TAG CA GT TTTT C T G T C G T A 4 2

( 2) INFORMATION FOR SEQ ID NO: 20: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH:33 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( v ii) IMMEDIATE SOURCE: (B) CLONE: TYROL2 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2O:

GA C C G GCC GA A G CTT TAATT AGA T G GAGA T G GA 3 3

( 2) INFORMATION FOR SEQ ID NO: 21: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( v ii) IMMEDIATE SOURCE: (B) CLONE: Tyrol3 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

A G T G GAT C C A G C G GAA T G C C G G CTT 25

( 2) INFORMATION FOR SEQ ID NO: 22: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 17 base pairs 5,882,851 47 48 -continued (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear i) MOLECULE TYPE: DNA (genomic) i ) HYPOTHETICAL: NO i ) IMMEDIATE SOURCE: (B) CLONE: TYROL4 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

C GT CAT G C T C T T C G GAA 1 7

( 2) INFORMATION FOR SEQ ID NO: 23: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear i) MOLECULE TYPE: DNA (genomic) ( i ) HYPOTHETICAL: NO ( i ) ANTI-SENSE: NO i ) IMMEDIATE SOURCE: (B) CLONE: TYROL1d ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

C G G GAT C CAT A T G G C T C T G T TAT TAG CA GT TTTT C T G T C G T A C C T G G C C C G 5 1

( 2) INFORMATION FOR SEQ ID NO:24: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid ( A ) DESCRIPTION: desc = “oligonucleotide with an EcoRI site” ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:24:

G CAG GAATT C C G G 13

What is claimed is: e) isolating clones that express the cytochrome P-450 1. An isolated DNA molecule coding for a cytochrome monooxygenase. P-450 monooxygenase that catalyzes the conversion of an 4. A method of isolating a cDNA molecule coding for a amino acid to the corresponding N-hydroxyamino acid and cytochrome P-450 monooxygenase that catalyzes the con the conversion of Said N-hydroxyamino acid to the corre 50 Version of an amino acid to the corresponding sponding Oxime. N-hydroxyamino acid and the conversion of Said 2. An isolated DNA molecule according to claim 1, N-hydroxyamino acid to the corresponding oxime, compris wherein said DNA molecule comprises SEQ ID NO: 1. Ing: 3. A method of isolating a cDNA molecule coding for a a) isolating and Solubilizing microSomes from plant tissue cytochrome P-450 monooxygenase that catalyzes the con 55 that produces cyanogenic glycosides or glucosinolates, Version of an amino acid to the corresponding N-hydroxyamino acid and the conversion of Said b) purifying the cytochrome P-450 monooxygenase; N-hydroxyamino acid to the corresponding oxime, compris c) obtaining a protein sequence of the cytochrome P-450 Ing: monooxygenase, a) isolating and Solubilizing microSomes from plant tissue 60 d) designing oligonucleotides that specify DNA coding that produces cyanogenic glycosides or glucosinolates, for 4 to 15 amino acids of said cytochrome P-450 b) purifying the cytochrome P-450 monooxygenase; monooxygenase protein Sequence; c) raising antibodies against the purified cytochrome e) probing a cDNA library of plant tissue producing P-450 monooxygenase; cyanogenic glycosides or glucosinolates with Said d) probing with said antibody a cDNA expression library 65 oligonucleotides, or DNA molecules obtained from of plant tissue that produces cyanogenic glycosides or PCR amplification of cDNA using said oligonucle glucosinolates, and otides, and 5,882,851 49 SO f) isolating clones that encode the cytochrome P-450 f) isolating clones that encode the cytochrome P-450 monooxygenase. monooxygenase. 5. A method for producing a transgenic plant resistant to 10. A method for producing a transgenic plant resistant to insects, acarids, or nematodes, comprising: insects, acarids, or nematodes, comprising: a) introducing into a plant cell or plant tissue that can be 5 a) introducing into a plant cell or plant tissue that can be regenerated into a complete plant, DNA comprising a regenerated into a complete plant, DNA comprising a gene expressible in Said plant that encodes a cyto gene expressible in Said plant that encodes a cyto chrome P-450 monooxygenase that catalyzes the con chrome P-450 monooxygenase that catalyzes the con Version of an oxime to the corresponding nitrile and the version of an amino acid to the corresponding conversion of Said nitrile to the corresponding cyano N-hydroxyamino acid and the conversion of Said hydrine; N-hydroxyamino acid to the corresponding oxime; b) selecting transgenic plants, and b) selecting transgenic plants, and c) identifying transgenic plants that are resistant to c) identifying transgenic plants that are resistant to insects, acarids, or nematodes. insects, acarids, or nematodes. 11. A method for producing a transgenic plant having 6. A method for producing a transgenic plant having 15 improved disease resistance or nutritive value, comprising: improved disease resistance or nutritive value, comprising: a) introducing into a plant cell or plant tissue that can be a) introducing into a plant cell or plant tissue that can be regenerated into a complete plant, DNA encoding Sense regenerated into a complete plant, DNA encoding Sense RNA, antisense RNA, or a ribosome, the expression of which reduces the expression of a cytochrome P-450 RNA, antisense RNA, or a ribosome, the expression of monooxygenase that catalyzes the conversion of an which reduces the expression of a cytochrome P-450 Oxime to the corresponding nitrile and the conversion monooxygenase that catalyzes the conversion of an of Said nitrile to the corresponding cyanohydrine; amino acid to the corresponding N-hydroxyamino acid b) selecting transgenic plants, and and the conversion of said N-hydroxyamino acid to the c) identifying transgenic plants having improved disease corresponding oxime; 25 resistance or nutritive value. b) selecting transgenic plants, and 12. An isolated DNA molecule according to claim 1, c) identifying transgenic plants having improved disease wherein said DNA molecule is isolated from a plant that resistance or nutritive value. produces cyanogenic glycosides or glucosinolates. 7. An isolated DNA molecule coding for a cytochrome 13. An isolated DNA molecule according to claim 1, P-450 monooxygenase that catalyzes the conversion of an wherein said DNA molecule is isolated from a plant selected Oxime to the corresponding nitrile and the conversion of Said from the group consisting of the genera Sorghum, Trifolium, nitrile to the corresponding cyanohydrine. Linum, Taxus, Triglochin, Mannihot, Amygdalus, Prunus, 8. A method of isolating a cDNA molecule coding for a and cruciferous plants. cytochrome P-450 monooxygenase that catalyzes the con 14. An isolated DNA molecule according to claim 13, version of an OXime to the corresponding nitrile and the 35 wherein said DNA molecule is isolated from Sorghum conversion of Said nitrile to the corresponding bicolor. cyanohydrine, comprising: 15. An isolated DNA molecule according to claim 1, wherein Said amino acid is Selected from the group consist a) isolating and Solubilizing microSomes from plant tissue ing of tyrosine, phenylalanine, tryptophan, Valine, leucine, that produces cyanogenic glycosides or glucosinolates, isoleucine, and cyclopentenylglycine isoleucine. b) purifying the cytochrome P-450 monooxygenase; 40 16. An isolated DNA molecule according to claim 15, c) raising antibodies against the purified cytochrome wherein Said amino acid is tyrosine. P-450 monooxygenase; 17. An isolated DNA molecule according to claim 1, wherein Said cytochrome P-450 monooxygenase has a d) probing with said antibody a cDNA expression library molecular weight of 57 kD, as determined by SDS-PAGE. of plant tissue that produces cyanogenic glycosides or 45 18. An isolated DNA molecule according to claim 1, glucosinolates, and wherein Said cytochrome P-450 monooxygenase comprises e) isolating clones that express the cytochrome P-450 an amino acid sequence as shown in SEQ ID NO: 2. monooxygenase. 19. An isolated DNA molecule according to claim 1, 9. A method of isolating a cDNA molecule coding for a wherein Said cytochrome P-450 monooxygenase comprises cytochrome P-450 monooxygenase that catalyzes the con 50 an N-terminal amino acid sequence as shown in SEQ ID version of an OXime to the corresponding nitrile and the NO: 3. conversion of Said nitrile to the corresponding 20. An isolated DNA molecule according to claim 1, cyanohydrine, comprising: wherein Said cytochrome P-450 monooxygenase comprises a) isolating and Solubilizing microSomes from plant tissue an internal amino acid Sequence Selected from the group that produces cyanogenic glycosides or glucosinolates, 55 consisting of SEQ ID NOs: 4-11. 21. An isolated DNA molecule according to claim 20, b) purifying the cytochrome P-450 monooxygenase; wherein Said cytochrome P-450 monooxygenase comprises c) obtaining a protein sequence of the cytochrome P-450 an internal amino acid sequence as shown in SEQ ID NO: monooxygenase, 4. d) designing oligonucleotides that specify DNA coding 60 22. An isolated DNA molecule according to claim 20, for 4 to 15 amino acids of said cytochrome P-450 wherein Said cytochrome P-450 monooxygenase comprises monooxygenase protein Sequence; an internal amino acid sequence as shown in SEQ ID NO: e) probing a cDNA library of plant tissue producing 5. cyanogenic glycosides or glucosinolates with Said 23. An isolated DNA molecule according to claim 20, oligonucleotides, or DNA molecules obtained from 65 wherein Said cytochrome P-450 monooxygenase comprises PCR amplification of cDNA using said oligonucle an internal amino acid sequence as shown in SEQ ID NO: otides, and 6. 5,882,851 S1 52 24. An isolated DNA molecule according to claim 20, 33. An isolated DNA molecule according to claim 32, wherein Said cytochrome P-450 monooxygenase comprises wherein Said amino acid is Selected from the group consist an internal amino acid sequence as shown in SEQ ID NO: ing of tyrosine, phenylalanine, tryptophan, Valine, leucine, 7. isoleucine, and cyclopentenylglycine isoleucine. 25. An isolated DNA molecule according to claim 20, 34. An isolated DNA molecule according to claim 33, wherein Said cytochrome P-450 monooxygenase comprises wherein Said amino acid is tyrosine. an internal amino acid sequence as shown in SEQ ID NO: 35. An isolated DNA molecule according to claim 7, 8. wherein the ability of said cytochrome P-450 monooxyge 26. An isolated DNA molecule according to claim 20, nase to convert an OXime to the corresponding nitrile wherein Said cytochrome P-450 monooxygenase comprises depends on the presence of NADPH and wherein this an internal amino acid sequence as shown in SEQ ID NO: dependency can be overcome by the addition of reductants. 9. 36. An isolated DNA molecule according to claim 7, 27. An isolated DNA molecule according to claim 20, wherein Said cytochrome P-450 monooxygenase has a wherein Said cytochrome P-450 monooxygenase comprises molecular weight of 51 kD, as determined by SDS-PAGE. an internal amino acid sequence as shown in SEQ ID NO: 15 37. An isolated DNA molecule according to claim 7, 10. wherein Said cytochrome P-450 monooxygenase comprises 28. An isolated DNA molecule according to claim 20, an N-terminal amino acid sequence as shown in SEQ ID wherein Said cytochrome P-450 monooxygenase comprises NO: 12. an internal amino acid sequence as shown in SEQ ID NO: 38. An isolated DNA molecule according to claim 7, 11. wherein Said cytochrome P-450 monooxygenase comprises 29. An isolated DNA molecule according to claim 7, an amino acid Sequence Selected from the group consisting wherein said DNA molecule is isolated from a plant that of SEO ID NOS: 13-16. produces cyanogenic glycosides or glucosinolates. 39. An isolated DNA molecule according to claim 38, 30. An isolated DNA molecule according to claim 7, wherein Said cytochrome P-450 monooxygenase comprises wherein said DNA molecule is isolated from a plant selected 25 an amino acid sequence as shown in SEQ ID NO: 13. from the group consisting of the genera Sorghum, Trifolium, 40. An isolated DNA molecule according to claim 38, Linum, Taxus, Triglochin, Mannihot, Amygdalus, Prunus, wherein Said cytochrome P-450 monooxygenase comprises and cruciferous plants. an amino acid sequence as shown in SEQ ID NO: 14. 31. An isolated DNA molecule according to claim 30, 41. An isolated DNA molecule according to claim 38, wherein said DNA molecule is isolated from Sorghum wherein Said cytochrome P-450 monooxygenase comprises bicolor: an amino acid sequence as shown in SEQ ID NO: 15. 32. An isolated DNA molecule according to claim 7, 42. An isolated DNA molecule according to claim 38, wherein said oxime is obtained by the conversion of an wherein said cytochrome P-450 monooxygenase comprises amino acid to the corresponding N-hydroxyamino acid and an amino acid sequence as shown in SEQ ID NO: 16. the conversion of said N-hydroxyamino acid to the oxime by 35 another cytochrome P-450 monooxygenase. k k k k k