AU728817B2 - Method of controlling insect pests - Google Patents

Description

WO 97/46105 PCT/EP97/02737 Method of Controlling Insect Pests The present invention relates to a method of controlling species of the Lepidoptera genus Ostrinia species preferably Ostrinia furnacalis (Asian Corn Borer), in crop plants by use of toxin proteins obtainable from Bacillus thuringiensis and/or other Bacillus species.

Bacillus thuringiensis belongs to the large group of gram-positive, aerobic, endospore-forming bacteria. Unlike other very closely related species of Bacillus such as B. cereus or B. anthracis, the majority of the hitherto known Bacillus thuringiensis species produce in the course of their sporulation a parasporal inclusion body which, due to its crystalline structure, is generally referred to as a crystalline body. This crystalline body is composed of insecticidally active crystalline protoxin proteins, the so-called 8-endotoxins.

The protein crystals are responsible for the toxicity to insects of Bacillus thuringiensis. The 8-endotoxin does not exhibit its insecticidal activity until after oral ingestion of the crystalline body, when the latter is dissolved in the intestinal juice of the target insects. In most cases the actual toxic component is released from the protoxin as a result of proteolytic cleavage caused by the action of proteases from the digestive tract of the insects.

The 8-endotoxins of the various Bacillus thuringiensis strains are characterized by high specificity toward certain target insects, especially with respect to various Lepidoptera, Coleoptera and Diptera larvae, and by a high degree of activity against such succeptible larvae. A further advantage of Bacillus thuringiensis 8-endotoxins resides in the fact that the toxins are harmless to humans, other mammals, birds and fish.

The various insecticidal crystal proteins from Bacillus thuringiensis have been classified based upon their spectrum of activity and sequence similarity. The classification put forth by H6fte and Whiteley, Microbiol. Rev. 53: 242-255 (1989) placed the then known insecticidal crystal proteins into four major classes. Generally, the major classes are defined by their spectrum of activity, with the Cryl proteins being active against Lepidoptera, Cryll proteins against both Lepidoptera and Diptera, Crylll proteins being active against Coleoptera, and CrylV proteins against Diptera.

Within each major class, the 8-endotoxins are grouped according to sequence similarity.

The Cryl proteins are typically produced as 130-140 kDa protoxin proteins which are proteolytically cleaved to produce insecticidally active toxin proteins about 60-70 kDa P:\OPER\MKR\SPECI\30296-97 235.doc-2208AX) -2in size. The active portion of a 8-endotoxin residues in the NH 2 -terminal portion of the full-length molecule. H6fte and Whiteley, supra, classified the then known Cryl proteins into six groups, IA(a), IA(b), IA(c), IB, IC and ID. Since then, proteins classified as CrylE, CrylF, CrylG, CrylH and CrylX have also been characterized.

The spectrum of insecticidal activity of an individual 8-endotoxin from Bacillus thuringiensis tends to be quite narrow, with a given 5-endotoxin being active against only a few insects. Specificity is the result of the efficiency of the various steps involved in producing an active toxin protein and its subsequent ability to interact with the epithelial cells in the insect digestive tract.

10 It is one of the objects of this invention to provide a method of controlling Ostrinia fumacalis (Asian Corn Borer) species in plants, preferably cereal crops, including, but not limited to the species of maize, wheat, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo and sugar cane. This objection **could surprisingly be achieved within the scope of the invention by administering a toxin protein of Bacillus thuringiensis such as a Cryl-type toxin protein, to the crop plant to be protected. In another embodiment of the invention toxin proteins obtainable from vegetative cultures of Bacillus species, so-called Vegetative Insecticidal Proteins (VIPs) such as VIP3.

[EP-A 0 690 916; International Application No. EP 95/03826, the disclosure of S* 20 which is incorporated herein by reference in its entirety], can also be used to control Ostrinia fumacalis (Asian Corn Borer) pests.

Thus, in one embodiment the present invention relates to a method for protecting plants including progeny thereof against damage caused by Ostrinia fumacalis species comprising directly or indirectly applying to the plant or the plant seed or the growing area of the plant as an active ingredient a toxin protein of Bacillus species, wherein the toxin protein is a VIP-type protein.

PAOPER\MKRISPECI30296-97 235.doc-22A)8f0 -2A- The present invention thus relates to a method for protecting plants including progeny thereof against damage caused by Ostrinia fumacalis (Asian Corn Borer) species comprising directly or indirectly administering to the plant or the plant seed or the growing area of the plant to be protected a toxin protein of Bacillus species, wherein the toxin protein is a VIP-type protein. Preferably the VIP-type protein is a VIP1-type protein, such as a VIP1A(a) protein or a VIP1A(b) protein, or a VIP2-type protein, such as a VIP2A(a) protein, or a VIP2A(b) protein, or a VIP3-type protein, such as a VIP3A(a) protein or a VIP3A(b) protein, or a combination of a VIP1-type protein and VIP2-type protein or a VIP3-type protein. Preferably the toxin protein of Bacillus species is a Cryl-type or a VIP-type protein mentioned above, either purely or in the form of an entomocidal composition comprising at least one of said proteins or a microorganism, preferably a Bacillus thuringiensis and/or a Bacillus cereus S: strain, containing at least one toxin gene encoding the toxin protein. Said microorganisms used in the method according to the invention may either be S 15 naturally occurring strains or, in the alternative, recombinant strains comprising a recombinant gene encoding the toxin.

In a preferred embodiment, transgenic plants are used to administer the toxin 00 to the plants to be protected against damage caused by Ostrinia fumacalis (Asian Corn WO 97/46105 PCT/EP97/02737 -3- Borer) species. Such plants are obtained by transformation with a toxin gene encoding an insecticidal toxin protein from a Bacillus species such as a Cry-type, preferably a Cryltype toxin protein or a VIP-type protein, and expressing said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species upon planting the so transformed plant in an area where said insect pest occurs.

Entomocidal compositions to be used in the method according to the invention for protecting crop plants against Ostrinia furnacalis (Asian Corn Borer) pests for example comprise as an active ingredient at least one Cry-type toxin protein, more preferred at least one Cryl-type toxin protein, even more preferred at least one CrylA-type toxin protein, particularly preferred at least one CrylA(b)-type toxin protein and most particularly preferred at least one crylA(b) type toxin protein according to SEQ ID NOS: 53 to 55, even more preferred of Bacillus thuringiensis or a microorganism containing at least one gene encoding said toxin protein, preferably a Bacillus thuringiensis strain containing at least one gene encoding said toxin protein, or a derivative or mutant thereof, together with an agricultural adjuvant such as a carrier, diluent, surfactant or application-promoting adjuvant.

The active ingredient contained in the entomocidal composition may also be a VIP-type toxin protein as disclosed in EP-A- 0 690 916 and the PCT International Application No EP95/03826 or a combination of Cryl-type and VIP-type proteins.Preferred within the scope of protection is aVIP1-type protein, such as a VIP1A(a) protein or a VIP1A(b) protein, or a VIP2- type protein, such as a VIP2A(a) protein or a VIP2A(b) protein or a combination of a VIP1-type protein and a VIP2-type protein or aVIP3-type protein, such as a VIP3A(a) protein or a VIP3A(b) protein.

More preferred within the scope of protection are VIP-type toxin proteins as shown in SEQ ID NOS: 1,2, 4-7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52.

The composition may also contain a further biologically active compound. Said compound can be both a fertilizer or micronutrient donor or other preparations that influence plant growth. It can also be a selective herbicide, insecticide, fungicide, bactericide, nematicide, molluscide or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily WO 97/46105 PCT/EP97/02737 -4employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

The composition may comprise from 0.1 to 99% by weight of the active ingredient, from 1 to 99.9% by weight of a solid or liquid adjuvant, and from 0 to 25% by weight of a surfactant. The active ingredient or the composition containing said active ingredient, may be administered to the plants or crops to be protected together with certain other insecticides or chemicals (1993 Crop Protection Chemicals Reference, Chemical and Pharmaceutical Press, Canada) without loss of potency. It is compatible with most other commonly used agricultural spray materials but should not be used in extremely alkaline spray solutions if a Cryl-type toxin is involved. It may be administered as a dust, a suspension, a wettable powder or in any other material form suitable for agricultural application.

The active ingredient, that is preferably a Cryl-type toxin protein of Bacillus thuringiensis and/or one of the VIP-type proteins mentioned previously, or the composition comprising said active ingredient may be applied to an environment in which the insect pest may occur, a plant or plant part in order to protect said plant or plant part from damage caused by an insect pest, or seed in order to protect a plant which develops from said seed from damage caused the pest.

A preferred method of application in the area of plant protection is application to the foliage of the plants (foliar application), with the number of applications and the rate of application depending on the plant to be protected and the risk of infestation by the pest in question.

The compositions to be used in a method according to the invention are also suitable for protecting plant propagating material, e.g. seed, such as fruit, tubers or grains, or plant cuttings, from insect pests. The propagation material can be treated with the formulation before planting: seed, for example, can be dressed before being sown. The active ingredient of the invention can also be applied to grains (coating), either by impregnating the grains with a liquid formulation or by coating them with a solid formulation.

The formulation can also be applied to the planting site when the propagating material is being planted, for example to the seed furrow during sowing. The invention relates also to those methods of treating plant propagation material and to the plant propagation material thus treated.

WO 97/46105 PCT/EP97/02737 Within the scope of the invention the compositions may be applied in any method known for treatment of seed or soil with bacterial strains. For example, see US Patent No.4,863,866. The strains are effective for biocontrol even if the microorganism is not living.

Preferred is, however, the application of the living microorganism.

Target crops to be protected within the scope of the present invention are those that are host plants for Ostrinia furnacalis (Asian Corn Borer) species and include but are not limited to the species of maize, wheat, barley, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo and sugar cane.

The active ingredient according to the invention may be used in unmodified form or together with any suitable agriculturally acceptable carrier. Such carriers are adjuvants conventionally employed in the art of agricultural formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances. Like the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objective and the prevailing circumstances. Advantageous rates of application range from about 50 g to about 5 kg of active ingredient per hectare approximately 2.471 acres), and preferably from about 100 g to about 2 kg a.i./ha. Preferred rates of application are 200 g to about 1 kg a.i./ha or 200 g to 500 g a.i./ha.

For seed dressing advantageous application rates range from 0.5 g to 1000 g a.i. per 100 kg seed, preferably from 3g to 100 g a.i. per 100kg seed. Most preferred are application rate from 10 g to 50 g a.i. per 100 kg seed.

Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. The formulations, i.e. the entomocidal compositions, preparations or mixtures thereof with other active ingredients, and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, by homogeneously mixing and/or grinding the active ingredients with extenders, solvents, solid carriers, and in some cases surface-active compounds (surfactants).

WO 97/46105 PCT/EP97/02737 -6- Suitable solvents are: aromatic hydrocarbons, preferably the fractions containing 8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethylsulfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils such as epoxidised coconut oil or soybean oil; or water.

The solid carriers used, for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.

Depending on the nature of the active ingredients to be formulated, suitable surfaceactive compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds. Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C sub 10 -C sub 22), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained, e.g.

from coconut oil or tallow oil. Further suitable surfactants are also the fatty acid methyltaurin salts as well as modified and unmodified phospholipids.

More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates. The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and generally contain a C sub 8 -C sub 22 alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. the sodium or calcium salt of lignosulfonic acid, of dodecylsulfate, or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated WO 97/46105 PCT/EP97/02737 -7benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing about 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

Non-ionic surfactant are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups.

These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol.

Fatty acid esters of polyoxyethylene sorbitan, such as polyoxyethylene sorbitan trioleate, are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which contain, as Nsubstituent, at least one C sub 8 -C sub 22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or hydroxyl-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g., stearyltrimethylammonium chloride or benzyldi-(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described, in "McCutcheon's Detergents and Emulsifiers Annual", MC Publishing Corp. Ridgewood,

N.J.,

1979; Dr. Helmut Stache, "Tensid Taschenbuch" (Handbook of Surfactants), Carl Hanser Verlag, MunichNienna.

Another particularly preferred characteristic of an entomocidal composition of the present invention is the persistence of the active ingredient when applied to plants and soil.

Possible causes for loss of activity include inactivation by ultra-violet light, heat, leaf exudates and pH. For example, at high pH, particularly in the presence of reductant, 8endotoxin crystals are solubilized and thus become more accessible to proteolytic WO 97/46105 PCTEP97/02737 -8inactivation. High leaf pH might also be important, particularly where the leaf surface can be in the range of pH 8-10. Formulation of an entomocidal composition to be used in a method according to the present invention can address these problems by either including additives to help prevent loss of the active ingredient or encapsulating the material in such a way that the active ingredient is protected from inactivation. Encapsulation can be accomplished chemically (McGuire and Shasha, J Econ Entomol 85: 1425-1433, 1992) or biologically (Barnes and Cummings, 1986; EP-A 0 192 319). Chemical encapsulation involves a process in which the active ingredient is coated with a polymer while biological encapsulation involves the expression of the -endotoxin genes in a microbe. For biological encapsulation, the intact microbe containing the toxin protein is used as the active ingredient in the formulation. The addition of UV protectants might effectively reduce irradiation damage. Inactivation due to heat could also be controlled by including an appropriate additive.

Preferred within the present application are formulations comprising living microorganisms as an active ingredient either in form of the vegetative cell or more preferable in form of spores, if available. Suitable formulations may consist, for example, of polymer gels which are crosslinked with polyvalent cations and comprise these microorganisms. This is described, for example, by D.R. Fravel et al. in Phytopathology, Vol. 75, No. 7, 774-777, 1985 for alginate as the polymer material. It is also known from this publication that carrier materials can be co-used. These formulations are as a rule prepared by mixing solutions of naturally occurring or synthetic gel-forming polymers, for example alginates, and aqueous salt solutions of polyvalent metal ions such that individual droplets form, it being possible for the microorganisms to be suspended in one of the two or in both reaction solutions. Gel formation starts with the mixing in drop form. Subsequent drying of these gel particles is possible. This process is called ionotropic gelling. Depending on the degree of drying, compact and hard particles of polymers which are structurally crosslinked via polyvalent cations and comprise the microorganisms and a carrier present predominantly uniformly distributed are formed. The size of the particles can be up to 5 mm.

Compositions based on partly crosslinked polysaccharides which, in addition to a microorganism, for example, can also comprise finely divided silicic acid as the carrier material, crosslinking taking place, for example, via Ca ions, are described in EP-A1-O 097 571. The compositions have a water activity of not more than 0.3. W.J. Cornick et al.

describe in a review article [New Directions in Biological Control: Alternatives for Suppressing Agricultural Pests and Diseases, pages 345-372, Alan R. Liss, Inc. (1990)] WO 97/46105 PCT/EP97/02737 -9various formulation systems, granules with vermiculite as the carrier and compact alginate beads prepared by the ionotropic gelling process being mentioned. Such compositions are also disclosed by D.R.Fravel in Pesticide Formulations and Application Systems: 1 lth Volume, ASTM STP 1112 American Society for Testing and Materials, Philadelphia, 1992, pages 173 to 179 and can be used to formulate the recombinant microorganisms according to the invention. Further methods for formulating living microorganism are described in W096/02638.

The compositions according to the invention are valuable for preventive and/or curative treatment in the field of pest control even at low rates of application while being well tolerated by and non-toxic to warm-blooded species, fish and plants and have a very favourable biocidal spectrum. The compositions according to the invention are active against all or individual development stages of Ostrinia furnacalis (Asian Corn Borer) pests.

The insecticidal action of the compounds according to the invention can become obvious either directly, i.e. by destroying the pests immediately or only after some time has elapsed.

The said composition can be provided in form of a chemical mixture comprising the toxin proteins in an essentially pure form or in form of a mixture comprising at least one of the toxin proteins as part of a microorganism or a transgenic plant.

In a specific embodiment of the invention, one of the active ingredients may be applied to the plant directly by, for example, leaf application as described herein previously, whereas the second active principle may be provided by the plant itself upon expression of a previously transformed gene encoding the said second principle.

The entomocidal compositions to be used in the method according to the invention usually contain from about 0.1 to about 99%, preferably from about 0.1 to about and most preferably from about 3 to about 90% of active ingredient; from about 1 to about 99.9%, preferably from about 1 to about 99%, and most preferably from about 5 to about 95% of a solid or liquid adjuvant; and from about 0 to about 25%, preferably about 0.1 to about 25%, and most preferably from about 0.1 to about 20% of a surfactant.

Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations of substantially lower concentration. The entomocidal compositions may also contain further ingredients, such as stabilizers, WO 97/46105 PCT/EP97/02737 antifoams, viscosity regulators, binders, tackifiers as well as fertilizers or other active ingredients in order to obtain special effects.

The present invention also relates to formulations comprising living microorganisms as an active ingredient which are present in the form of vegetative cells or more in the form of spores, if available.

A further object of the invention relates to the use of recombinant microorganisms comprising a toxin gene encoding a toxin protein of Bacillus thuringiensis such as a Cryl-type protein, in a method of controlling crop plants against damages caused by Ostrinia furnacalis (Asian Corn Borer) species, which recombinant organisms are either applied directly to the plant to be protected or the recombinantly produced toxin protein is first isolated from the recombinant microorganism and formulated as described above before being applied to the crop plant to be protected. The recombinant microorganisms may also contain a toxin gene encoding a VIP-type toxin protein as disclosed in the EP-A-690 916 and the International Application No EP95/03826 or a combination of genes encoding at least a Cry-type toxin and a VIP-type toxin, respectively.

For recombinant production of the toxin protein in a host organism, the coding sequence may be inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced. The choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the practioneer in the art. The resultant molecule, containing the individual elements linked in the proper reading frame, are inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli (see, e.g. Studier and Moffatt, J. Mol. Biol. 189:113 (1986); Brosius, DNA 8:759 (1989)), yeast (see, Schneider and Guarente, Meth. Enzymol. 194: 373 (1991)) and insect cells (see, Luckow and Summers, Bio/Technol. 6: 47 (1988)). Specific examples include plasmids such as pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, La Jolla, CA), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus WO 97/46105 PCT/EP97/02737 -11 (AcMNPV). A preferred baculovirus/insect system is pVI11392/Sf21 cells (Invitrogen, La Jolla, CA).

The recombinantly produced toxin protein can be isolated and purified using a variety of standard techniques. The actual techniques which may be used will vary depending upon the host organism used, whether the toxin protein is designed for secretion, and other such factors a skilled artisan is aware of (see, e.g. chapter 16 of Ausubel, F. et al., "Current Protocols in Molecular Biology", pub. by John Wiley Sons, Inc. (1994).

A preferred object of the invention relates to the use of transgenic plants comprising and expressing a toxin gene encoding a toxin protein of Bacillus thuringiensis, especially a Cryl-type toxin protein, in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, in a method of protecting crop plants against damages caused by Ostrinia furnacalis (Asian Corn Borer) pests. The plants can be the result of nuclear transformation or plastid transformation (see WO 95/24492).

Especially preferred are transgenic plants expressing a CrylA(b) toxin protein of Bacillus thuringiensis. The invention also relates to the use of transgenic plants comprising a toxin gene encoding a VIP-type protein as described in EP-A-690 916 and International Application No EP95/03826, herein incorporated by reference in its entirety. The invention also relates to the use of transgenic plants comprising and expressing a toxin gene encoding a toxin protein of Bacillus thuringiensis, but especially a Cry-type toxin protein, and also comprising and expressing a toxin gene encoding a VIP-type protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species. A host plant expressing said toxin genes will have enhanced resistance to insect attack of Ostrinia furnacalis (Asian Corn Borer) species and will be better equipped to withstand crop losses associated with such attack.

In one preferred embodiment, expression of one or more Bt 8-endotoxins in a transgenic plant is accompanied by the expression of one or more VIP-type proteins. This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary.

Alternatively, a plant, Parent 1, can be genetically engineered for the expression of VIP-type proteins. A second plant, Parent 2, can be genetically engineered for the expression of Bt 8-endotoxin. By crossing Parent 1 with Parent 2, progeny plants are obtained which WO 97/46105 PCT/EP97/02737 -12express all the genes introduced into Parents 1 and 2. Particularly preferred Bt endotoxins are those disclosed in EP-A 0618976, herein incorporated by reference.

Also comprised by the present invention is the use of recombinant microorganisms or transgenic plants comprising a gene encoding DNA molecules which hybridizes to a DNA molecule encoding a toxin protein of Bacillus species, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said toxin protein at least 10 nucleotides in length, under moderately stringent conditions. The invention preferably comprises the use of recombinant microorganisms or transgenic plants comprising a gene encoding DNA molecules which hybridizes to a DNA molecule encoding a toxin protein of Bacillus thuringiensis or B cereus especially to a DNA molecule encoding a Cry-type protein or to a toxin gene encoding a VIP-type toxin protein, preferably to a CrylA(b) protein.

Factors that effect the stability of hybrids determine the stringency of the hybridization. One such factor is the melting temperature Tm which can be easily calculated according to the formula provided in DNA PROBES, George H. Keller and Mark M. Manak, Macmillan Publishers Ltd, 1993, Section one: Molecular Hybridization Technology; page 8 ff.

The preferred hybridization temperature is in the range of about 250C below the calculated melting temperature Tm and preferably in the range of about 12-150C below the calculated melting temperature Tm and in the case of oligonucleotides in the range of about 5-10°C below the melting temperature Tm.

The invention further relates to a commercial bag comprising seed of a transgenic plant comprising at least a toxin gene encoding a toxin protein of Bacillus thuringiensis, preferably a Cry-type toxin protein, more preferably a Cryl-type toxin protein, but most preferably a CrylA-type toxin protein and expressing the said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, together with lable instructions for the use thereof for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants. Preferred within this invention is a commercial bag comprising seed of a transgenic plant comprising as an active ingredient a gene encoding at least a Cry-type WO 97/46105 PCT/EP97/02737 -13toxin protein and a VIP-type protein. Especially preferred is a combination of a CrylA(b) toxin protein with a VIP-type protein.

The further object of the invention is a commercial bag comprising an insecticidal composition according to the invention together with lable instructions for the use thereof for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants.

By plant is meant any plant species which can be genetically transformed by methods known in the art, but especially those plants that are host plants for Ostrinia furnacalis (Asian Corn Borer) species including, but not limited to, the following species of plants: maize, wheat, barley, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo (orchardgrass, fescue, and the like), and sugar cane.

Methods known in the art for plant transformation are discussed below. Host plants include, but are not limited to, those species previously listed as target crops.

The invention further relates to seed of a transgenic plant comprising a gene encoding a toxin protein of Bacillus thuringiensis and expressing said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, and a commercial bag containing said seed.

By plant is meant any plant species that is a host for Ostrinia furnacalis (Asian Corn Borer) including, but not limited to, the species of maize, wheat, barley, rye, oats, rice, sorghum, millet and related crops, forage grasses, bamboo and sugar cane.

It has been discovered that the codon usage of a native Bacillus thuringiensis toxin gene is significantly different from that which is typical of a plant gene. In particular, the codon usage of a native Bacillus thuringiensis gene is very different from that of a maize gene. As a result, the mRNA from this gene may not be efficiently utilized. Codon usage might influence the expression of genes at the level of translation or transcription or mRNA processing. To optimize a toxin gene for expression in plants, for example in maize, the codon usage is optimized by using the codons which are most preferred in maize (maize preferred codons) in the synthesis of a synthetic gene which encodes the same protein as found for the native toxin gene sequence. The optimized maize preferred codon usage is effective for expression of high levels of the Bt insecticidal WO 97/46105 PCT/EP97/02737 -14protein. Further details for constructing maize-optimized synthetic toxin genes can be found in WO 93/07278, herein incorporated by reference in its entirety.

Toxin genes derived from microorganisms may also differ from plant genes. Plant genes differ from genes found in microorganisms in that their transcribed RNA does not possess defined ribosome binding site sequence adjacent to the initiating methionine.

Consequently, microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation initiator at the ATG. Clontech (1993/1994 catalog, page 210) has suggested the sequence GTCGACCATGGTC as a consensus translation initiator for the expression of the E. coli uidA gene in plants. Further, Joshi (Nucl Acids Res 15: 6643- 6653 (1987)) has compared many plant sequences adjacent to the ATG and suggests the consensus TAAACAATGGCT. In situations where difficulties are encountered in the expression of microbial ORFs in plants, inclusion of one of these sequences at the initiating ATG may improve translation. In such cases the last three nucleotides of the consensus may not be appropriate for inclusion in the modified sequence due to their modification of the second amino acid residue. Preferred sequences adjacent to the initiating methionine may differ between different plant species. By surveying the sequence of maize genes present in the GenBank/EMBL database it can be discerned which nucleotides adjacent to the ATG should be modified to enhance translation of the toxin gene introduced into maize.

In addition, it has been shown that removal of illegitimate splice sites can enhance expression and stability of introduced genes. Genes cloned from non-plant sources and not optimized for expression in plants may contain motifs which can be recognized in plants as 5' or 3' splice sites. Consequently, the transcription process can be prematurely terminated, generating truncated or deleted mRNA. The toxin genes can be engineered to remove these illegitimate splice sites using techniques well known in the art.

Many 8-endotoxin proteins from Bacillus thuringiensis are expressed as protoxins.

These protoxins are solubilized in the alkaline environment of the insect gut and are then proteolytically converted by proteases into a toxic core fragment (Hofte and Whiteley, Microbiol. Rev. 53: 242-255 (1989)). For 5-endotoxin proteins of the Cryl class, the toxic core fragment is localized in the N-terminal half of the protoxin. It is within the scope of the present invention that genes encoding either the full-length protoxin form or the WO 97/46105 PCT/EP97/02737 truncated toxic core fragment of the novel toxin protein can be used in plant transformation vectors to confer insecticidal properties upon the host plant.

The recombinant DNA molecules can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e. monocot or dicot, targeted for transformation.

Suitable methods of transforming plant cells include microinjection (Crossway et al., BioTechniques 4:320-334 (1986)), electroporation (Riggs et al, Proc. Natl. Acad. Sci.

USA 83:5602-5606 (1986), Agrobacterium-mediated transformation (Hinchee et al., Biotechnology 6:915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J.

3:2717-2722 (1984)), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent 4,945,050; and McCabe et al., Biotechnology 6:923-926 (1988)). See also, Weissinger et al., Annual Rev. Genet. 22:421-477 (1988); Sanford et al., Particulate Science and Technology 5:27-37 91987)(onion); Christou et al., Plant Physiol. 87:671-674 (1988)(soybean); McCabe et al., Bio/Technology 6:923-926 (1988)(soybean); Datta et al., Bio/Technology 8:736-740 (1990)(rice); Klein et al., Proc.

Natl. Acad. Sci. USA, 85:4305-4309 (1988)(maize); Klein et al., Bio/Technology 6:559-563 (1988)(maize); Klein et al., Plant Physiol. 91:440-444 (1988)(maize); Fromm et al., Bio/Technology 8:833-839 (1990); and Gordon-Kamm et al., Plant Cell 2:603-618 (1990)(maize); Svab et al. Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Koziel et al. (Biotechnology 11: 194-200 (1993)) (maize); Shimamoto et al.

Nature 338: 274-277 (1989) (rice); Christou et al. Biotechnology 9: 957-962 (1991) (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (Biotechnology 11: 1553-1558 (1993) (wheat); Weeks et al. (Plant Physiol. 102: 1077- 1084 (1993) (wheat); Wan et al (Plant Physiol 104: 37-48 (1994) (barley); Umbeck et al, (Bio/Technology 5: 263-266 (1987) (cotton).

One particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectile bombardment can be found in WO 93/07278, herein incorporated by reference in its entirety. An additional preferred embodiment is the protoplast transformation method for maize as disclosed in Application EP-A-292 435, hereby incorporated by reference in its entirety.

The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be WO 97/46105 PCT/EP97/02737 -16maintained and propagated in progeny plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.

Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties.

Alternatively new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment", yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.

In seeds production germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by WO 97/46105 PCT/EP97/02737 -17the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof.

Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD®), methalaxyl (Apron'), and pirimiphos-methyl (Actellic®). If desired these compounds are formulated together with further carriers, surfactants or applicationpromoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.

It is a further aspect of the present invention to provide new agricultural methods such as the methods examplified above which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention to provide control against Ostrinia furnacalis (Asian Corn Borer).

To breed progeny from plants transformed according to the method of the present invention, a method such as that which follows may be used: maize plants produced as described in the examples set forth below are grown in pots in a greenhouse or in soil, as is known in the art, and permitted to flower. Pollen is obtained from the mature tassel and used to pollinate the ears of the same plant, sibling plants, or any desirable maize plant.

Similarly, the ear developing on the transformed plant may be pollinated by pollen obtained from the same plant, sibling plants, or any desirable maize plant. Transformed progeny obtained by this method may be distinguished from non-transformed progeny by the presence of the introduced gene(s) and/or accompanying DNA (genotype), or the phenotype conferred. The transformed progeny may similarly be selfed or crossed to other plants, as is normally done with any plant carrying a desirable trait. Similarly, tobacco or other transformed plants produced by this method may be selfed or crossed as is known in WO 97/46105 PCTIEP97/02737 -18the art in order to produce progeny with desired characteristics. Similarly, other transgenic organisms produced by a combination of the methods known in the art and this invention may be bred as is known in the art in order to produce progeny with desired characteristics.

WO 97/46105 PCT/EP97/02737 -19-

EXAMPLES

The following examples further describe materials and methods used to obtain specific embodiments of the present invention. They are offered by way of illustration, and should not be interpreted as limitating the disclosure of the specification.

EXAMPLE 1: General Methods DNA manipulations were done using procedures that are routinely practized in the art.

These procedures can often be modified and/or substituted without substantively changing the result. Except where other references are identified, the procedures are described in general text books such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989.

EXAMPLE 2: Plant Transformation Vectors Plant transformation is accomplished using the transformation vectors pCIB 4431 and pCIB 3064 described in WO 93/07278 and Koziel et al (1993) [Biotechnology Vol 11, 194-200], both disclosures being incorporated herein by reference.

pCIB4431 is a vector designed to transform maize. It contains two chimeric synthetic Bt crylA(b) endotoxin genes expressible in maize the one of them constituting a PEP carboxylase promoter/synthetic-crylA(b) gene, the other one a pollen promoter/syntheticcrylA(b) gene.

pCIB4431 contains the synthetic crylA(b) gene provided in SEQ ID NO: 1 and was deposited on September 21, 1992 with the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A. under accession no NRRL B-18998.

pCIB3064 contains a plant expressible bar gene (615 bp), which was originally cloned from Streptomyces hygroscopicus [Thompson et al. (1987) EMBO J 6, 2519-2523]. It encodes a phosphinotricin acetyltransferase (PAT), conferring tolerance to phosphinotricin. The bar gene is under the control of the CaMV 35S promoter and terminator [OW et al (1987) Proc NatI Acad Sci USA 84, 4870-4874] to provide resistance to phosphinotricin.

WO 97/46105 PCT/EP97/02737 EXAMPLE 3: Production of transgenic maize plants containing the synthetic maize CrylA(b) gene The example below utilizes a biolistic device to introduce DNA coated particles into maize cells, from which transformed plants are generated.

3.1 Tissue Immature maize embryos, approximately 1.5-2.5 mm in length, were excised from an ear of genotype 6N615 14-15 days after pollination. The mother plant was grown in the greenhouse. Before excision, the ear was surface sterilized with 20% Clorox for minutes and rinsed 3 times with sterile water. Individual embryos were plated scutellum side up in a 2 cm square area, 36 embryos to a plate, on the callus initiation medium, 2DG4 5 chloramben medium (N6 major salts, B5 minor salts, MS iron, 2% sucrose, with mg/I chloramben, 20 mg/I glucose, and 10 ml G4 additions (Table 1) added after autoclaving.

Table 1: G4 Additions Ingredient per liter medium Casein hydrolysate 0.5 g Proline 1.38 g Nicotinic acid 0.2 mg Pyridoxine-HCI 0.2 mg Thiamine-HCI 0.5 mg Choline-HCI 0.1 mg Riboflavin 0.05 mg Biotin 0.1 mg Folic acid 0.05 mg Ca pantothenate 0.1 mg p-aminobenzoic acid 0.05 mg B12 0.136 pg WO 97/46105 PCT/EP97/02737 -21 3.2 Preparation of DNA for delivery The microcarrier was prepared essentially according to the instructions supplied with the Biolistic device. While vortexing 50 pl 1.0 pm gold microcarrier, 5 pl of pCIB4431 (1.23 pg/pl) [#898] 2 pl pCIB3064 (0.895 pg/pl) [#456] was added followed by 50 pl 2.5 M CaCI2, then 20 pi 0.1 M spermidine (free base, TC grade). The resulting mixture was vortexed 3 minutes and microfuged for 10 sec. The supernatant was removed and the microcarriers washed 2 times with 250 pl of 100% EtOH (HPLC grade) by vortexing briefly, centrifuging and removing the supernatant. The microcarriers are resuspended in pl 100% EtOH.

3.3 Bombardment Tissue was bombarded using the PDS-1000He Biolistics device. The tissue was placed on the shelf 8 cm below the stopping screen shelf. The tissue was shot one time with the DNA/gold microcarrier solution, 10 pl dried onto the macrocarrier. The stopping screen used was hand punched using 10x10 stainless steel mesh screen. Rupture discs of 1550 psi value were used. After bombardment, the embryos were cultured in the dark at 250 C.

3.4 Callus formation Embryos were transferred to callus initiation medium with 3 mg/l PPT 1 day after bombardment. Embryos were scored for callus initiation at 2 and 3 weeks after bombardment. Any responses were transferred to callus maintenance medium, 2DG4 2,4-D medium with 3 mg/L PPT. Callus maintenance medium is N6 major salts, minor salts, MS iron, 2% sucrose, with 0.5 mg/l 2,4-D, 20 mg/l glucose, and 10 ml G4 additions added after autoclaving. Embryogenic callus was subcultured every 2 weeks to fresh maintenance medium containing 3 mg/L PPT. All callus was incubated in the dark at The Type I callus formation response was 15%. Every embryo which produced callus was cultured as an individual event giving rise to an individual line.

WO 97/46105 PCT/EP97/02737 -22 Reqeneration After 12 weeks on selection, the tissue was removed from callus maintenance medium with PPT and was placed on regeneration medium. Regeneration medium is 0.25MS3S5BA (0.25 mg/I 2,4 D, 5 mg/I BAP, MS salts, 3% sucrose) for 2 weeks followed by subculture to MS3S medium for regeneration of plants. After 4 to 10 weeks, plants were removed and put into GA 7's.

EXAMPLE 4: Analysis of transgenic maize plants 4.1 ELISA Assay Detection of crylA(b) gene expression in transgenic maize is monitored using Asian corn borer insect bioassays and ELISA analysis for a quantitative determination of the level of crylA(b) protein obtained.

Quantitative determination of crylA(b) insecticidal protein in the leaves of transgenic plants is performed using enzyme-linked immunosorbant assays (ELISA) as disclosed in Clark M F, Lister R M, Bar-Joseph M: ELISA Techniques. In: Weissbach A, Weissbach H (eds) Methods in Enzymology 118:742-766, Academic Press, Florida (1986). Immunoaffinity purified polyclonal rabbit and goat antibodies specific for the B. thurinqiensis subsp. kurstaki insecticidal protein are used to determine the amount of insecticidal protein per mg soluble protein from crude extracts of leaf samples. The sensitivity of the double sandwich ELISA is ng insecticidal protein per mg soluble protein using 50 gg of total protein per ELISA microtiter dish well.

Corn extracts are made by grinding leaf tissue in gauze lined plastic bags using a hand held ball-bearing homogenizer (AGDIA, Elkart IN.) in the presence of extraction buffer (50 mM Na2CO3 pH 9.5, 100 mM NaCI, 0.05% Triton, 0.05% Tween, 1 mM PMSF and 1 pM leupeptin). Protein determination is performed using the Bio-Rad (Richmond, CA) protein assay.

4.2 Asian Corn Borer Assay One to four 4 cm sections are cut from an extended leaf of a corn plant. Each leaf piece is placed on a moistened filter disc in a 50 x 9 mm petri dish. Five neonate Asian corn borer larvae are placed on each leaf piece (making a total of 5-20 larvae per plant). The petri WO 97/46105 PCT/EP97/02737 -23 dishes are incubated at 29.5 Leaf feeding damage and mortality data are scored after 24, 48, and 72 hours.

EXAMPLE 5: Ostrinia furnacalis (Asian Corn Borer) Field Testing Assay Small peat pots containing transgenic seedlings which were first tested for the presence and the expression of the transgene, are transplanted into the field. Non-transgenic inbred lines are planted in the same field over a six week period, to serve as controls and for pollinations.

When plants in the field reach 40 cm of extended leaf height, infestation with laboratoryreared ostrinia furnacalis (Asian Corn Borer) larvae begins on both the transgenic and nontransgenic control plants. About 300 neonate larvae mixed with corn cob grits are introduced into the whorl of each plant using a Davis inoculator. Infestations continues on a weekly basis for four weeks to stimulate first generation Asian Corn Borer. Starting two weeks after the initial infestation, each plant is rated weekly for four weeks using a 1 to 9 scale no visible leaf injury; 9=most leaf with long lesions, several leaves with broken mid ribe, possibly stunted plants due to Asian Corn Borer feeding). A mean Asian Corn Borer damage rating score is calculated for each transgenic and non-transgenic control plant. As each plant reaches anthesis, 300 larvae/plant are applied weekly for four weeks to stimulate second generation infestation. One hundred of neonate larvae in corn cob grits are introduced into the leaf axil at the primary ear and at the leaf axil one node above and below the primary ear node. Therefore a total of approximatively 2400 larvae are applied to each plant. About 50 days after the initial second generation infestation, stalks from all transplanted and some non-transgenic plants are harvested. The extent of internal second generation infestation tunneling damage in the whole plants is determined.

EXAMPLE 6: Assay of extract from transformed protoplasts for insecticidal activity against Ostrinia furnacalis (Asian Corn Borer) Western blot analysis is performed using extracts obtained from maize cells which had been transiently transformed with DNA to express the maize optimized gene.

Qualitative insect toxicity testing is carried out using harvested protoplasts. Suspensions are prepared for each replicate tested in the bioassays. A replicate is considered positive if it causes significantly higher mortality than the controls. For example, replicates are tested for their activity against insects in the order Lepidoptera by using the Asian corn borer, WO 97/46105 PCT/EP97/02737 -24 Ostrinia furnacalis. One-hundred pl of a protoplast suspension in 0.1% Triton X-100 is pipetted onto the surface of artificial Black cutworm diet, (Bioserv, Inc., Frenchtown, NJ; F9240) in 50 mm x 10 mm snap-cap petri dishes. After air drying 10 neonatal larvae are added to each plate. Mortality is recorded after about 4 days.

EXAMPLE 7: Ostrinia furnacalis (Asian Corn Borer) Plant Dipping Assay 7.1 Bacillus thuringiensis (Bt) crystals Bacillus thuringiensis (Bt) crystals are prepared for stock suspension with 22 ml of distilled water. The suspension is kept in the refrigerator.

7.2 Parameters recorded 3 days old larvae of Ostrinia furnacalis (Asian Corn Borer) are allowed to feed on maize leaves. Larvae had previously been fed with untreated leaves. 120 hours later the number of larvae dead is recorded. The kind of feeding injuries on leaf plants is observed in each case.

7.3 Method for testing Plants of two homozygous inbred lines of Zea mays susceptible to Asian Corn Borer are used (Lines A and Seedling plants aged 9-10 days are dipped in various concentrations of Bt protein suspension and are used in feeding experiments, wherein larvae are released on dried leaves of seedling plants, 5-10 larvae per plant. The seedling plants are covered with nylon mesh bags and kept in a nylon mesh case. concentrations with 4 replications are tested and mortality is determined. The temperature is kept at 21-300C.

WO 97/46105 PCT/EP97/02737 7.4 Results Table 2: Results for Line A Dose [ppm] Number of insects Number of dead insects Mortality 40 31 77.5 40 29 72.5 40 15 37.5 40 13 32.5 40 5 12.5 0 40 0 0.00 Table 3: Results for Line B Dose [ppm] Number of Number of Mortality insects dead insects 40 38 94.74 40 33 81.58 40 28 68.42 40 19 44.74 40 10 21.05 0 40 2 5.00 Two kinds of injuries were clearly distinguished in maize leaves: Bt dipped leaves of the seedling plants were lightly damaged, whereas the leaves of control seedlings were severely damaged.

The following LCso-Values were obtained: Line A: LCso 23.412 ppm (range from 17.834 to 30.734) Line B: LC 5 o 12.234 ppm (range from 9.547 to 15.676) WO 97/46105 PCT/EP97/02737 -26 EXAMPLE 8: Ostrinia furnacalis (Asian Corn Borer) Plant Dipping Assay (VIP3A) 8.1 VIP3A protein of VIP3A protein were prepared with 50ml of distilled water in order to prepare varying concentration of VIP3 protein: 100ppm, 50ppm, 25ppm, 12.5ppm, 6.25ppm and Oppm (check).

8.2 Ostrinia furnacalis (Asian Corn Borer) The pupae collected from farmers field at Racha Buri by Entomology and Animal Science Division DOA is order to prepare Larvae (L2) for testing.

8.3 Parameters recorded Data were collected after incubation 5 days by counting number of died larvae and then analyzed percentage of mortality of larvae by probit Analysis Program.

7.3 Method for testing Plants of two homozygous inbred lines of Zea mays susceptible to Asian Corn Borer are used (Lines B and Line B was conducted for potted plant test (4 replications 5 rated concentation and check) and Line C was conducted for leaf dipping test (4 replications with 100, 50, 25ppm and check). Seedling plants aged 10-14 days are dipped in various concentrations of VIP3A protein suspension and are used in feeding experiments, wherein larvae are released on dried leaves of seedling plants, 5-10 larvae per plant. The seedling plants are covered with nylon mesh bags and kept in a nylon mesh case. Cut leaves were put in platic blocks and kept in control room four replications were applied for this experiment.

WO 97/46105 PCT/EP97/02737 -27- 7.4 Results Table 4: Results for Line B Table 4a Dose [ppm] Number of insects Number of dead insects Mortality 100 40 31 76.92 40 26 64.10 40 16 38.46 12.5 40 14 33.33 6.25 40 9 20.51 0 40 1 2.50 Table 4b Replication number of dead larvae after 5 days concentration (ppm) Check 6.25 12.5 25 50 100 1(10) 0 3 3 5 8 6 II (10) 0 2 2 3 6 9 111(10) 1 3 5 4 5 9 IV (10) 0 1 4 4 7 7 Total 1 9 14 16 26 31 Table 5: Results for Line C Table Replication number of dead larvae after 24 hours concentration (ppm) Check 25 50 100 1(10) 0 0 0 1 11(10) 0 0 0 1 111(10) 0 0 0 0 IV (10) 0 0 0 1 Total 0 0 0 3 WO 97/46105 PCT/EP97/02737 -28 Table Replication number of dead larvae after 48 hours concentration (ppm) Check 25 50 100 1(10) 0 0 0 1 11(10) 0 0 0 1 111(10) 0 0 1 0 IV (10) 0 0 0 1 Total 0 0 1 3 Table Replication number of dead larvae after 72 hours concentration (ppm) Check 25 50 100 1(10) 0 1 0 2 11(10) 0 1 0 1 111(10) 0 0 2 3 IV (10) 0 0 2 1 Total 0 2 4 7 Table Replication number of dead larvae after 96 hours concentration (ppm) Check 25 50 100 0 1 1 4 11(10) 0 2 1 3 111 (10) 0 1 3 IV (10) 0 1 4 3 Total 0 5 1 P:\OPER\MKR\SPECRl30296-97 235.doc-2208AD -29- Table Replication number of dead larvae after 120 hours concentration (ppm) Check 25 50 100 1(10) 1 3 2 7 11(10) 0 2 1 4 111(10) 0 3 5 IV (10) 0 2 4 9 Total 1 10 12 0..

0 0.0.

S S a 0

*SSS

00

S

S

S.

The following LC 50 Values were obtained after 120 hours 5 Line B: LC 5 0 29.558 ppm (range from 21.298 to 41.022) Line C: LC 5 0 78.498 ppm (range from 53.644 to 114.866) Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as 10 "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

WO 97/46105 PCT/EP97/02737 30 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Novartis AG STREET: Schwarzwaldallee 215 CITY: Basel COUNTRY: Switzerland POSTAL CODE (ZIP): 4002 TELEPHONE: +41 61 69 11 11 TELEFAX: 41 61 696 79 76 TELEX: 962 991 (ii) TITLE OF INVENTION: Method of Controlling Insect Pests (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC conpatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFIWARE: PatentIn Release Version #1.30B INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 6049 base pairs TYPE: nucleic acid STRANDEENESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: ENA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Bacillus cereus STRAIN: AB78 INDIVIDUAL ISOLATE: NRRL B-21058 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1082..2467 OTHER INFORMATION: /product= "VIP2A(a)" (ix) FEATURE: NAME/KEY: misc_feature WO 97/46105 WO 9746105PCT/EP97/02737 31 LOCATION: 2475. .5126 OTHER INFORMvATION: /note= "Coding sequence for the 100 kd VIPlA~a) protein. This coding sequence is repeated in SEQ ID NO: and translated separately." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATCGATACAA D3TWITITA C'ITAGACCGG TAGThWqr= AATrI',T=A MITACTPI ATACATFITA ATAGCCAMT CICTFFFT CCACGAGCT TACITIC TAA

T

rrCIGCA TAGAGGTA1C AAITIGTCA CACTAAT AATTAGGTGT 'I ITrATGCC 'FITACGTAAT TI CC=TAT IICCIGA GCATA TCGAG A TPPItrT CACr=CCAT CrAAATATAT GGAACTIT 'FITIGGATAT GTAAAAAAC AAACAAGTGC TITATrAAC CTAACCFI~T ATATGAAAAC AACATGAAG CITAGG'TrA TAGTAAArT T=ATAAT TIACAGGCT ATCTATCTAT AAT1TIIC ACAGACTAAA TCCAGATIM3 A A IG AAA AGA ATG~ GAG Met LYS Arg Met Glu

CTAGCTGYCGT

ATACTTGCCA

TGAATAGAAA

TIr=AGAGC

ACFITAGCA

TATAGCCIT

TCTGTGIA

CTA'ITAAAAT

GGATIITGGT

CATAAACGGT

ATCCIACAG

TCATAAAA

ATAAGAAAAA

CFITAATAAG

TTWTACAATA

ATA ETCXTT

CAACCIATC

TTAATCCTT

TArTCGAAA TAAAATCrAC GAGAAAThG

AMXCTF\GG

CTACACCATA

CAGGCATA

AGCACCAAAA

ACTCAATA'IG

CGq7I'ITGG

CCCAGLVITA

TI'IATATATT

AACAAACGGA

AAGGGGGGAG

ACITATCTAA

AGTATGITIT

TIMGTACGT

GAAGAArM ACCTAGCMtT

ATCAAGITIG

rTrGAGGGCGC TITU 3AA

ACCAAAAA'IC

ACCTrATrAA

GATGAGTIT

AMACATAAT

T1

T

IGTACTM'

TICCATAACG

GGGAGIGAAA

ATrAGATAAT

CFITCTATA

CTITATAAAA

ATCTATATr 'ItGCTAATAA

CCCATAGGAT

TIGAATrmrI

TAAACAACI'A

TCIITITA

GCATCTArTrr AAATIfGGM

T

A ETAAAATAA TAACGCrFI

AAATAATCIM

AACIACI~

GA IGC'TTAT

AAAAGCAT

AAATATGXAAT

CAACAACAAA

MT ITCATA 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1126 1174 1222 GGA AAG TIG TIT AM GM T(C A AAA AAA WTA Gly Lys Leu Phe Met Val Ser Lys Lys Leu CAA GTA WTF ACTr AAA ACT GTA TMI MI AGT ACA GrT TMC =C ATA =C Gin Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser le Ser 25 TTA. TA AAT AAT GAA GTG ATA AAA GCT GAA CAA TA AAT ATA AAT Leu Leu Asn Asn Giu Val Ile Lys Ala Glu Gin Leu Asn Ile Asn Ser 40 CPA AGT AAA TAT ACT AAC TM' CAA PAT OTA AAA ATrx ACT GAG PAG GTA 1270 WO 97/46105 PCT/EP97/02737 -32- Gln Ser Lys Tyr Thr Asn Leu Gin Asn Leu 55

GAG

Glu

AAA

Lys

AAT

Asn

ACT

Thr

GAA

Glu

ACC

Thr

ACA

Thr 160

CAA

Gin

PTA

Leu

ACG

Thr

ATI

Ile

GTC

Val 240

TTA

Leu GAT PIT Asp Phe GAA AAA Glu Lys TT TTA Phe Leu PIT TCT Phe Ser ATIT GAT Ile Asp 130 TAT AAA Tyr Lys 145 GAA GGT Glu Gly TIT PTA Phe Leu ACT GCT Thr Ala GT CCG Val Pro 210 'ITA AAT Leu Asn 225 CAT GTA His Val CAA APT Gin Ile AAA GAA GAT Lys Glu Asp GAG GG AAA Glu Trp Lys 85 GAT AAT AAA Asp Asn Lys 100 ATG GCA GGC Met Ala Gly 115 AAG ATG PIT Lys Met Phe AAT GTG GAA Asn Val Glu AAT ACG ATT Asn Thr Ile 165 GAT AGG GAT Asp Arg Asp 180 CAA CAA GT Gin Gin Val 195 AGT GGG AAA Ser Gly Lys AAT AGT GAA Asn Ser Glu GAT AAG GTA Asp Lys Val 245 GAA GGG ACT Glu Gly Thr 260

AAG

Lys 70

CTA

Leu

AAT

Asn

'CA

Ser

GAT

Asp

CCG

Pro 150

AAT

Asn

AT

Ile

WCC

Ser

GGT

Gly

TAC

Tyr 230

TCA

Ser

PTA

Leu GAA AAA GCG Glu Lys Ala ACT GCT ACT Thr Ala Thr GAT ATA AAG Asp Ile Lys 105 TIT GAA GAT Phe Glu Asp 120 AAA ACC AAT Lys Thr Asn 135 ACA ACA AT Thr Thr Ile TCT GAT GCA Ser Asp Ala AAG TIT GAT Lys Phe Asp 185 AGT AAA GAA Ser Lys Glu 200 'CT ACT ACT Ser Thr Thr 215 AAA AIG CTC Lys Met Leu AAA GIG GTG Lys Val Val AAA AAG AGT Lys Lys Ser 265 Lys Ile AAA GAA Lys Glu GAA AAA Glu Lys 90 ACA AAT Thr Asn GAA ATA Glu Ile CTA TCA Leu Ser GGA TIT Gly Phe 155 ATG GCA Met Ala 170 AGT TAT Ser Tyr AGA GIT Arg Val CCA ACA Pro Thr AT GAT Ile Asp 235 AAA AAA Lys Lys 250 CIT GAC Leu Asp Thr Asp TGG GGG Trp Gly GGA AAA Gly Lys TAT AAA Tyr Lys AAA GAT Lys Asp 125 AAT TCT Asn Ser 140 AAT AAA Asn Lys CAG TIT Gin Phe CTA GAT Leu Asp AT TIG Ile Leu 205 AAA GCA Lys Ala 220 AAT GGG Asn Gly GGG GIM Gly Val TT AAA Phe Lys Lys Val AAA GAA Lys Glu ATG AAT Met Asn GAA AIT Glu Ile 110 TA AAA Leu Lys ATT ATC Ile Ile TCT TA Ser Leu AAA GAA Lys Glu 175 ACG CAT Thr His 190 AAG GIT Lys Val GGT G=C Gly Val TAT ATG Tyr Met GAG 'IGC Glu Cys 255 AAT GAT Asn Asp 270 1318 1366 1414 1462 1510 1558 1606 1654 1702 1750 1798 1846 1894 WO 97/46105 WO 9746105PCT/EP97/02737 33 ATA AAT COT Ile Asn Ala GCa AAA GAT Ala Lys Asp 290

GAA

Ciu 275 C CAT AGO TCC Ala His Ser Trp COT A IG Gly Met 280 AAG PAT TAT Lys Asn Ty~r GAA GAG RiC Giu Glu Trp 285 CCC TAT CI Cly Tyr Ala 1942 1990 'TrA ACC CAT TCG Leu Thr Asp Ser

CAA

Gin 295 ACC CAA C? 'TrA Arg Glu Ala Leu

GAT

Asp 300 AGO CAA Arg Gin 305 CAT TAT AAA Asp Tyr Lys CAA ATC Glu Ile 310 CPA GAT Leu Asp 325 PAT PAT TAT MIT AGA PAT CAA CCC CGA Asn Asn Tyr Leu Arg Asn Gin Gly Cly 315 2038

ACT

Ser 320 CCA PAT CPA AAA Cly Asn Glu Lys CTI CPA ATA Ala Gin le PAT ATT 'TCT Asn le Ser CAT CT A4sp, Ala 335 Trp Cys 350 2086 2134 UTA CCC AC AAA Leu Cly Lys Lys

CCA

Pro 340 ATA CCC CPA PAT le Pro Ciu Asn ACT iTG TAT ACA Thr Val 'lYr Arg CCC ATC CCC Cly Met Pro AAA CAT FTr Lys Asp Phe 370

CA

Giu 355 CCT TAT CA Phe Cly Ty~kr Gin ACT GAT CCG 'TA Ser Asp Pro Leu CPA GAA CAA TIr Ciu Ciu Gin Phe PAT ACA A EC Asn Thr Ile AAA CA Lys Ciu 380 CCA C Ala Ala 395 CCT 'TTr TrA Pro Ser Leu 365 CAC AAA CGA Asp Lys Ciy TT CCA =It Phe Cly Ser 2182 2230 TAT ATG Tyr Met 385 AGA AA Arg Lys 400 ACT ACA AC Ser Thr Ser AT ATA 'ITA Ile Ile Leu TTA 'ICC Leu Ser 390 ACT GAA COT CT Ser Clii Arg Leu 2278 2326 Ara TA CPA Tr CCC Leu Gin Val Pro CCA ACT ACG COT CC Gly Ser Thr Cly Ala TAT IIA ACT CC Tyr Loeu Ser Ala 405 ATr COT Ile Cly 420 CCA Trr CCA Ciy Phe Ala CPA AAA GAG ATC Ciu Lys Ciu Ile CrA MI Loeu Leu 430 2374 GAT AAA CAT Asp Lys Asp AAA GOT T Lys Gly Vai 450

ACT

Ser 435 AAA TAT CAT AT Lys Tlyr His Ile AAA CPA ACA GAG LYS Vai Thr Ciu GrA A Tr A T Val Ile Ile 445 2422 AC CCA TAT CPA Lys Arg Tyr Val CAT OCA ACA =IT MT ACA PAT Asp Ala* Thr Leu Leu Thr Asn 460 2467 TAACCAATGX AAAAATATGXA AGAAAAAGI' AGCAACGT CTA~CCGtA CG'TAITAGCc =ICAT'T TIGCATGGAA ArIXgTGAATrCC TO'FIACGCA GACACCAAAA CAAA ICAAAT 'IWACAACA CAGAAAAATC PACACAAACA GATrCCACCGA AAAGGArETAC FICCCA'ITA TITICAAAGCA AAACATFITA GTAAT ITAC TATG IX3~2A CCGACACGTG ATAGTAC=~ 2527 2587 2647 2707 WO 97/46105 WO 9746105PCT/EP97/02737 34 TA ITATrAT CAACAAAGAG TA FIXCGTI ATIYGITIWIA RGN3GATGlAA CAGGCAArTA AAAGCAAr GWCCATITAG AGATACAAAA TIAATATTG TAGWCAAAAC CTAACCCCAGC GAAAGAATCA CAGGAAT=t GAAAAGGGAA ATIGAGAA3 AGAAATGG TATACGA'1C TAAAGGTAT ACGAAATTIG TACAGATrAT GAAAASGCAG CCCA PTtGTA GCTGCT=.

AAATAAAAT TrATCCAATA CAAATAAACT A'1NAGATAAA TTCAGAGTAA AGAAACGGGA TAGAAATCAA 1GCGAAAA~r AAAAAGGAAA ATAGTICCA ACAGTAAAAC A =~AAAGAA AAGIrCAGCA AGATGAAM~ TAGCGMAACC ATCGAAAATA ACA~CGGATAC GGATGGGGAC ACAATAGAAT CCGTAAAG PFICAAATCC ACrAGAAAGT CAAGAGAW= AGATrI

T

CA

CAAGTIGIGAA =~AGTAG GTGTAGAGC TCATICATCC

AAACAACAAG

GATIXCAT

APT=AATA

ATCAAAATAG

CTAAAITrAT

AGAAATCCI

AAaTCTwrA

T(TATICCI

TGGACGAVr

CACACAGTIM

AATCAAAGG

GAAAAGGTGA

ACGAAT=tm

AATATCAGTR

TAACITAC

AAGGGAAAGA

AGTATCAATC

'I'rMAATAGA

AATITAACAA

CqCAAAAAAT ACCrIIGGGA

CTAGCAAG

GTAT=A

AAACG=IAA

TATATCACC

CTTATACAAA

TACAGAAGGT GC i7CTG AAGCGGGGAT TGACCAAAA CGTAAACTAT CAAcAZ1cI' AAAcAGTIrC ACAAj2AATG TCGCAATrc AATACGGCI cAGCGGGATA T=AAATGCA AGGAA~CITGGr GCCATOACG ATGTAAAACC TACAA~CAAGT TA TCGCAACr ArTAcGGcGA AA IcTAATIC TAC2AGCCITA TrACCCGAAA AAAGGACA1AA A IXGAATCGC AATAACACA TCCGATACA TrAAMTAAAA AAcAAGTAGA TAATGP3CA GGAPACAAAC CAAAcAGATG G TrrATAA GATAAAAGAT 'B3GCGGAGAA TGGAATGGTG TCATAcAACA AAWCAAGGCF GGTGA=c GAACG'rGTAG cA&3AAAAACG IMAGCGGCA AGATAAAACA CCG T=AA CIIAAAAGA W-XCCCK G AAMAGAAATA GAGGGAITAT TATA'FrATAA AAACAAACO3, GAMTAM~A GATGAAAATA CAGCMAAAGAk AG1XGACCAAA GAAA=IAAA GATrGTAAGc A PFrATATGA 1GTAAAA=T GGTAlIq=G TCGGAGTAG GGAACAT~rA CAGGAAATAC AATGFI7GAT ATAACAA'I= 7=rITTAA ATAACGATAC AATATAWI CTGGAGAAAG A EGGATGlATT TAA FICCA AATAATAAAC CA1GAT ACACATGGAA ATATAGTAAC AAAACAGCGT CTA~rA=r AAAGA

T

rATG AAAAWCCAGA CIATAIC CAGAXGAAAT ATATACGAAT CGAGCG=AT CAA I

T

FAAA[G ATACCACP= A=TCAAAAA TGXAATcn'PAC 2767 2827 2887 2947 3007 3067 3127 3187 3247 3307 3367 3427 3487 3547 3607 3667 3727 3787 3847 3907 3967 4027 4087 4147 4207 4267 4327 WO 97/46105 WO 9746105PCTIEP97/02737 35 AATCAAATIM TCTATACTT GACAAACACA AATAr,=I TCCGGATGxCT AATIWACAT CT'ATrATATA AG'F=ATATA IGGGGAGATr TATccGATcA A'ITAGATAT ATAGCIr-TA GAATGAT GAA ATAACFIAT ACCG3GAAAAT TAACAGATr AGAAGAGGA ATCITATIM AGCTAGTFI AATA PD2AAC TAG'IGAGTI'A GGACCAAACG GACAA~rAAA TITGATI'ITA TGGATrAAAT TGGGAaI=A TICTASA TATAATAAAT AAGTIAATAT ACI'GTAGGAT GGGGATACFiT [TAAATAI3T CTIGAAAATG AAGAAAC AAATATrAAA CI=IATGT GCITICCCAC AAAATrAGAC GGIC'=AT TATI=ACGT CA'fGATIGAA TCwICGAAG FPATGG IA GATAATGAAG A'TrIGGCGAT TCACA'IT= CAGCITFPF CTIATAA CCCATGCCCA TCAAC~rAAG A1ACCI\AAAT TAGGACA=~ CArT=AAA AA FFA RC ACCCATAATG TECAAAGACrG ATGATAATGC IGAGTCrAAT CAGTGGAAA TAACGGAAAA TAAATACAGA RXNTCAAGAA TAAGTCAGA AAAAAACACA CTACAAAAAC AGTAAqrI, ATATAMAAAG TAATCCAATT ITGGGATGA TATIWATA CAGAAATrAA ACAGA~PrAT ATAAAAMOGG IXGGATTCAT

CATIGCAAAA

BGAGTGACAC

CCAAATATAG

AAATrAATGC AG'TrA rATA TIX3AATAAAA

TCI'ATICAGAA

IGNITACA

TAATACTGTA

TGATrATCrA GGICACvrAAA

AAAGACFTCA

TAAI=CTCG

T1TI'CWIAG A~TAAArTTAG TrPTAAAAAT 'ITI~3Cr TrATGTACC ACrIAAAGr

TAAAAAMGAA

TA'ITACITAT

TCrATGAAGC

GTAATGGAAT

ACATrAGACT

CAACCMMIA

ATATAAAGAG

ATGlAAATAAT

AGATABZTAA

TIMATITATr C=rCAATr

ATAAAACATA

CGTACGAAAT

TAA B3AAM~

AAGCAGACCA

TITINDAAAT

ATATCGATAA

GATAACI'CAA

AAACAATATr

AAAPAAATA

CAATGTGAGA

AATAAAGACA

ACAGAEGrAG

AGTAGGTG

TA IGGTGAAT AAATA IGAAG

GATAAANI'IT

CAAGGATTAT

GA=AAAG

TGrG=AAA

TATATCGTA

AAGAAAAG'I

AATATrATAA

TIAATGTA

CAGTrAATr TA~rATInGG TAAG3AAGGAT

CTGAG

AATAAGCCTA

CCATTATACT

CTGATATC

CIT=AATAA

AAANIWIXMA

'ITACTATAGA

A FrACAAAAG ATITrAAAAC

CATCAATAAA

GrATrAAG'T

PTAITAATGA

ITACTTATAG

ACAAGGAT[GG

TITATACAG

AGAGA~r

GATAG'I~TAA

C7IvPGGAG'lr,

ACTACCCCA

GGTFITAACA

TCAAATGXAA

EWTAGAAQA

CAAGGCG=r TGI A

ATAAATCAAA

CTTAAMAAAT

GGATrAAAAT

GAATAGGTAT

CATAAGCAAC

GAATh2CGC 4387 4447 4507 4567 4627 4687 4747 4807 4867 4927 4987 5047 5107 5167 5227 5287 5347 5407 5467 5527 5587 5647 5707 5767 5827 5887 5947 6007 WO 97/46105 PCT/EP97/02737 -36- AGCCCGGGGG ATCCACTAGT TCTAGAGCGG CCGCCACCGC GG 6049 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 462 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys Leu Gin 1 5 10 Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser Ile Ser Leu 25 Leu Asn Asn Glu Val Ile Lys Ala Glu Gin Leu Asn Ile Asn Ser Gin 40 Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys Ile Thr Asp Lys Val Glu 55 Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys 70 75 Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn 90 Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr Asn Tyr Lys Glu Ile Thr 100 105 110 Phe Ser Met Ala Gly Ser Phe Glu Asp Glu Ile Lys Asp Leu Lys Glu 115 120 125 Ile Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser Ile Ile Thr 130 135 140 Tyr Lys Asn Val Glu Pro Thr Thr Ile Gly Phe Asn Lys Ser Leu Thr 145 150 155 160 Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Ala Gin Phe Lys Glu Gin 165 170 175 Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Tyr Leu Asp Thr His Leu 180 185 190 Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val Ile Leu Lys Val Thr 195 200 205 Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val Ile WO 97/46105 PCT/EP97/02737 -37- 210 Leu Asn 225 His Val Gin Ile Asn Ala Lys Asp 290 Gin Asp 305 Gly Asn Gly Lys Met Pro Asp Phe 370 Met Ser 385 Lys Ile Leu Ser Lys Asp Gly Val 450 Asn Ser Glu Tyr Lys Met Leu Ile Asp Glu Glu 275 Leu Tyr Glu Lys Glu 355 Glu Thr Ile Ala Ser 435 Lys Lys Gly 260 Ala Thr Lys Lys Pro 340 Phe Glu Ser Leu Ile 420 Lys Arg Lys Lys Trp Gin 295 Asn Ala Glu Gin Leu 375 Ser Gin Phe Ile Val 455 Lys 250 Leu Lys Ala Leu Lys 330 Thr Asp Ile Leu Lys 410 Glu Val Thr 220 Asp Asn Gly Tyr 235 Lys Gly Val Glu Asp Phe Lys Asn 270 Asn Tyr Glu Glu 285 Leu Asp Gly Tyr 300 Arg Asn Gin Gly 315 Asn Ile Ser Asp Val Tyr Arg Trp 350 Pro Leu Pro Ser 365 Lys Glu Asp Lys 380 Ala Ala Phe Gly 395 Gly Ser Thr Gly Lys Glu Ile Leu 430 Thr Glu Val Ile 445 Leu Leu Thr Asn 460 Met Cys 255 Asp Trp Ala Gly Ala 335 Cys Leu Gly Ser Ala 415 Leu Ile Val 240 Leu Ile Ala Arg Ser 320 Leu Gly Lys Tyr Arg 400 Tyr Asp Lys INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARAC

T

ERISTICS:

LENGTH: 20 amino acids TYPE: amino acid STRANDEENESS: single TOPOLOGY: linear WO 97/46105 PCT/EP97/02737 -38- (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Peptide LOCATION: 1..20 OTHER INFORMATION: targetting" /note= "Signal peptide for vacuolar (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro Ile Arg Val Thr Asp Arg 1 5 10 Ala Ala Ser Thr INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 2655 base pairs TYPE: nucleic acid STRANDENESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: INA (genomic) (iii) HYPOTHEI'ICAL: NO (iv) ANI'I-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bacillus cereus STRAIN: AB78 INDIVIDUAL ISOLATE: NRRL B-21058 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2652 OTHER INFORMATION: /product= "100 kDa protein VIP1A(a)" /note= "This sequence is identical to the portion of SEQ ID NO:1 between and including nucleotide 2475 to 5126.' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: A'G AAA AAT Met Lys Asn 465 ITA GCT CCT Leu Ala Pro 480 AG AAG AAA AAG TA GCA AGT GT Met Lys Lys Lys Leu Ala Ser Val 470 ATG TIT TTG AAT GGA AAT GTG AAT Met Phe Leu Asn Gly Asn Val Asn 485 GTA ACG Val Thr 475 GCT GIT Ala Val 490 TGT ACG TIA Cys Thr Leu TAC GCA GAC ITyr Ala Asp WO 97/46105 PCT/EP97/02737 -39- AAA AGA AAT CAA Lys Thr Asn Gin TCT ACA ACA GAG AAA Ser Thr Thr Gin Lys 505 AAT GAA GAG AAA Asn Gin Gin Lys ATG GAC CGA AAA Met Asp Arg Lys TTA arT GGG Leu Leu Gly TAT TAT TC Tyr Tyr Phe 520 AAA GGA AAA Lys Gly Lys GAT T Asp Phe 525 AGT ANT CTT Ser Asn Leu CAT CAA CAA Asp Gin Gin 545

ACT

Thr 530 AIXT PIT =CA Met Phe Ala CG ACA CT CAT AGT ACT CT ATT TAT Pro Thr Arg Asp Ser Thr Leu Ile Tyr 535 540 ACA GCA AAT AAA Thr Ala Asn Lys CTA TrA CAT AAA AAA CAA Leu Leu Asp Lys Lys Gin 550 555 CAA GAA TAT Gin Giu Tyr CAG TCT Gin Ser 560 ATT CGT TGG AT Ile Arg rp Ile

GGT

Gly 565 TIM APT CAG AGT Leu Ile Gin Ser

AAA

Lys 570 GAA ACG GGA GAT Glu Thr Oly Asp

PTC

Phe 575 ACA AAC PTA Thr Phe Asn Leu Ter Ser 580 GAG GAT GAA CAG GCA A=T ATA GAA AM Glu Asp Giu Gin Ala Ile Ile Glu Ile 585 GGG AAA Apr Apr Gly Lys Ile Ile TCT AAT AAA GGG Ser Asn Lys Oly 595 TrA Ir CGA Aw Leu Vai Pro Ile AAA GAA AAG Lys Giu Lys 600 CAA GT GTC Gin Val Val CAT PTA His Leu 605 GAA AAA GGA Glu Lys Oly AAA =T AAT Lys Phe Asn 625 ATA CAT AGT Ile Asp Ser 640

AAA

Lys 610 ATA GAG TAT CAA Ile Giu Tyr Gin APT CAC ACT AAA Ile Asp Ser Lys TIT AAA GAA CTT Phe Lys Glu Leu TrA GAT ACA Ser Asp Thr 620 PTA T AAA Leu Phe Lys GAA Cr AGA Glu Leu Arg CAA AAC CAA CCC AG CAA =C GAG Gin Asn Gin Pro Gin Gin Vai Gin 645 CAA CAT Gin Asp 650

AAT

Asn 655 CCT GAA IT Pro Giu Phe AAC AAG AAA Asn Lys Lys 660 GAA TCA GAG Glu Ser Gin TC TA GCG AAA Phe Leu Ala Lys TC AAA ATA PAT Ser Lys Ile Asn 'r Leu 675 =IC ACT CAA AAA ATG Phe Thr Gin Lys Met 680 AAA AGO GM APT Lys Arg Glu Ile CAT GAA Asp Glu 685 CAC ACG GAT Asp Thr Asp GGG TAT AG Gly Tyr Thr 705

ACG

Thr 690 CAT GGG GAC TT Asp Gly Asp Ser ATr CCT Ile Pro 695 GAG CIT TGG Asp Leu Trp GAA GAA AAT Glu Giu Asn 700 CAT TCT OTA Asp Ser Leu AT CAA AAT AGA Ile Gin Asn Arg OCT GTA AAG TGG Ala Val Lys Trp WO 97/46105 WO 9746105PCTIEP97/02737 40 OCA ACT Ala Ser 720 AAA CCC TAT AG Lys Cly Tyr Thr

A

Lys 725 =r CT A MAT Phe Val Ser Asn ccA Pro 730 cTA CPA ACT GAG Leu Ciu Ser His

ACA

Thr 735 (GT GCT CAT CCT Val Gly Asp Pro ACA CAT TAT GAA Thr Asp Ty'r Clu GCA GCA AGA CAT Ala Ala. Arg Asp CrA Leu 750 CAT TIX TWA MAT Asp Leu Ser Msn CCA ACT GI'& AAT Pro Ser Val Msn 770 MAT 'TrA WC AAT Msn Leu Ser Msn 785 MCG CPA ACO TT MC Lys Giu Thr Phe Msn 760 CcA 'TOG GTA Gar Pro Leu Val Ala CT TITr Ala Phe 765 CTr ACT A IX GMA Val Ser Met Glu MCG GM Lys Val 775 ATA 'TA 'rCA le Leu Ser CCA MAT GAA Pro Msn Giu 780 TGG TCT TAT Trp Ser Tryr ACT GTA GAG Ser Vai Giu =r Ser 790 CAT TCA TCC ACG His Ser Ser Thr

MAT

Asn 795 1008 ACA MAT Thr Msn 800 ACA GMA GCT C Thr Giu Gly Ala A'IT TCG TIC GGA GfT AGC le Ser Phe Gly Val Ser 815 820 CAA CM TGG GCA ACA TOT Gin Ciu Trp Gly Thr Ser 835 Tr GlT GMA CC Ser Val Ciu Ala 805 GTA MAC TAT CMA Val Asn Tyr Gin ACA GGA MAT ACT Thr Gly Msn Thr 840 CCC ATr Gly Ile 810 GCA CCA MAA GOT Gly Pro Lys Cly IC'r GMA ACA GIT Ser Glu Thr Val

GCA

Ala 830 1056 1104 1152 'IcC CM TIC MAT Ser Gin Phe Msn ACG CT Thr Ala 845 'rCA C GGA TAT TIA MAT GCA MAT Ser Ala Gly Tyr Leu Msn Ala Msn 850 OCT CCC ATIC TAC CAT CTA MAA Car Cly Ala Ile Tyr Asp Val Lys Pro 865 870 alIT Val 855 CGA TAT MAC MAT Arg Ty~r Msn Mn GTA GGA ACT Val Gly Thr 860 'TrA MT MAC Leu Msn Msn ACA ACA ACT TIT Thr Thr Ser Phe

GTA

Val 875 1200 1248 1296 1344 CAT AOT ATC OCA AOT Asp Thr Ile Ala Thr 880- ATT AG le Thr 885 CC MAA TCT MAT Ala Lys Ser Msn wcr Ser 890 ACA CCC TrA MAT Thr Ala Leu Msn

ATA

Ile 895 TOT COT GGA GMA Ser Pro Cly Giu

ACT

Ser 900 TAC CCC MAA AMA Ty r Pro Lys Lys CAA MAT GCA AWC Gin Msn Gly Ile

GCA

Ala 910 ATA ACA WCA A'IG le Thr Ser Met

CAT

Asp 915 CAT TIT MAT TcG Asp Phe Msn Ser

CAT

His 920 COG NET ACA Pro Ile Thr 'TrA MAT AMA Leu Msn Lys 925 1392 MA A G TA CAT MAT CTGX OTA MAT MAT AAA COT ATG ATG 'TM GAA ACA Lys Cmn Val Asp Asn Leu Leu Msn Msn Lys Pro Met Met Leu Glu Thr 1440 WO 97/46105 PCT/EP97/02737 -41 930 AAC CAA ACA CAT OCT GTT TAT Asn Gin Thr Asp Cly Val Tyr ATA AAA CAT ACA Ile Lys Asp Thx GGA AAT ATA Cly Asn Ile 1488 1536 GTA ACT Val Thr 960 GGC GGA CAA TGG3 Cly Gly Giu Trp

AAT

Asn 965 GGT GIC ATA CAA Cly Val Ile Gin

CAA

Gin 970 ATC AAG GCT AAA Ile Lys Ala Lys ACA GCG Tar ATT AT Thr Ala Ser Ile Ile 975

GTG

CAT CAT GGG GAA CGT CTA GCG GCA AAA Vai Ala Ala Lys Val Asp Asp Gly 980 TAT GAA AAT CCA Tyr Glu Asn Pro Glu Arg 985 GAA CAT Clu Asp 1000 GTA GCA CAA Val Ala Glu AAA CGT Lys Arg 990 ACT TA AAA Thr Leu Lys CAT GCC Asp Ala 1010 C=l AAC CT TCA TAT CCA Leu Lys Leu Ser Tyr Pro 1015 AAA ACA CCC TCT TA Lys Thr Pro Ser Leu 1005 CAT GAA ATA AAA GAA Asp Glu Ile Lys Glu i020 ATA TAC GAA TCC AGC Ile IYr Glu Ser Ser 1035 1584 1632 1680 1728 ATA GAG GGA TTA Ile Giu Cly Leu 1025 GT ATG ACT TAC Val Met Thr Tyr 1040 TA TAT TAT AAA AAC AAA CC Leu Tyr TYr Lys Asn Lys Pro 1030 TA CAT GAA AAT Leu Asp Glu Asn 1045 ACA GCA AAA Thr Ala Lys GAA GTG Glu Vai 1050 ACC AAA CAA Thr Lys Gin T'IA TAT CAT Leu Tyr Asp 1070 1776 1824 TrA AAT Leu Asn 1055 CAT ACC ACT Asp Thr Thr GGG AAA

T

T

Gly Lys Phe 1060 AAA GAT GTA AGT CAT Lys Asp Val Ser His 1065 GTA AAA CTG ACT Val Lys Leu Thr CCA AAA ATG AAT CIT Pro Lys Met Asn Val 1075 ACA ATC Thr Ile 1080 AAA TG TCT LYS Leu Ser ATA apr Ile Leu 1085 1872 1920 TAT GAT Tyr Asp AAT CCT GAG Asn Ala Clu 1090 TCr AAT CAT AAC TCA Ser Asn Asp Asn Ser 1095 ATT GGT AAA TGC ACA AAC Ile Cly Lys Trp Thr Asn 1100 ACA AAT AIT CIT Thr Asn Ile Vai 1105 AAT AAT CCG GAT Asn Asn Pro Asp 1120 TrA AAT AAA AAT Leu Asn Lys Asn 1135 TCA GGT GGA MT MC GGA AM AM CAA TAT rT =r Ser Cly Gly Asn Asn Cly Lys Lys Gin Tyr Ser Ser 1110 1115 1968 2016 CT AAT TIM ACA A MT ACA CAT GCT CAA GA M AAA Ala Asn Leu Thr Leu Asn Thr Asp Ala Gin Glu Lys 4 j) 1130 CGT CAC TAT TAT ATA Arg Asp Tyr Tyr Ile 1140 AGT TA TAT AM AAG TCA CAA Ser Leu Tyr Met Lys Ser Glu 2064 1145 1150 AAA AAC ACA CAA =lT GAG ATT ACT ATA CAT GGG GAG AT TAT CCC AM 2112 WO 97/46105 PCTIEP97/02737 -42- Lys Asn Thr Gin Cys Glu Ile Thr Ile Asp Gly Glu Ile Tyr Pro Ile 1155 1160 1165 ACT ACA AAA ACA G'ITG AAT GTG AAT AAA GAC AAT TAC AAA AGA TTA GAT 2160 Thr Thr Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp 1170 1175 1180 AT ATA GCT CAT AAT ATA AAA AGT AAT CCA ATT TCT TCA CIT CAT ATT 2208 Ile Ile Ala His Asn Ile Lys Ser Asn Pro Ile Ser Ser Leu His Ile 1185 1190 1195 AAA ACG AAT GAT GAA ATA ACT TA TIT TGG GAT GAT ATT 'ITCT ATA ACA 2256 Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp Asp Asp Ile Ser Ile Thr 1200 1205 1210 GAT GTA GCA TCA ATA AAA CCG GAA AAT TA ACA GAT TCA GAA AT AAA 2304 Asp Val Ala Ser Ile Lys Pro Glu Asn Leu Thr Asp Ser Glu Ile Lys 1215 1220 1225 1230 CAG ATT TAT AGT AGG TAT GGT APT AAG TTA GAA GAT GGA ATC CIT ATT 2352 Gin Ile Tyr Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile 1235 1240 1245 GAT AAA AAA GGT GGG ATT CAT TAT GGT GAA TIT ATT AAT GAA GCT AGT 2400 Asp Lys Lys Gly Gly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala Ser 1250 1255 1260 TIT AAT ATT GAA CCA TIM CAA AAT TAT GTG ACC AAA TAT GAA GT ACT 2448 Phe Asn Ile Glu Pro Leu Gin Asn Tyr Val Thr Lys Tyr Glu Val Thr 1265 1270 1275 TAT AGT AGT GAG TA GGA CCA AAC GTG AGT GAC ACA CTT GAA AGT GAT 2496 Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu Ser Asp 1280 1285 1290 AAA ATT TAC AAG GAT GGG ACA APT AAA TIT GAT TIT ACC AAA TAT AGT 2544 Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys Tyr Ser 1295 1300 1305 1310 AAA AAT GAA CAA GGA TTA TIT TAT GAC AGT GGA TTA AAT IGG GAC IT 2592 Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe 1315 1320 1325 AAA APT AAT GCT ATT ACT TAT GAT GGT AAA GAG A'G AAT GIT TIT CAT 2640 Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His 1330 1335 1340 AGA TAT AAT AAA TAG 2655 Arg Tyr Asn Lys 1345 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: WO 97/46105 PCT/EP97/02737 -43 LENGTH: 884 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Leu Ser Met Ser Asp Gin Phe Gly Glu 145 Lys Ile Asn Ser Asp 225 (xi) SEQUENCE Lys Asn Met Lys 5 Ala Pro Met Phe Lys Thr Asn Gin Asp Arg Lys Gly Asn Leu Thr Met Gin Gin Thr Ala Ser Ile Arg Trp 100 Thr Phe Asn Leu 115 Lys Ile Ile Ser 130 Lys Gly Lys Leu Phe Asn Ile Asp 165 Asp Ser Gin Asn 180 Pro Glu Phe Asn 195 Lys Ile Asn Leu 210 Thr Asp Thr Asp DESCRIPTION: SEQ ID Lys Lys Leu Ala Ser 10 Leu Asn Ile Ser Leu Leu 55 Phe Ala 70 Asn Lys Ile Gly Ser Glu Asn Lys 135 Val Pro 150 Ser Lys Gin Pro Lys Lys Phe Thr 215 Gly Asp 230 Gly Thr 40 Gly Pro Leu Leu Asp 120 Gly Ile Thr Gin Glu 200 Gin Ser Asn Val 25 Thr Gin Tyr Tyr Thr Arg Leu Asp 90 Ile Gin 105 Glu Gin Lys Glu Lys Ile Phe Lys 170 Gin Val 185 Ser Gin Lys Met lie Pro Val Asn Lys Phe Asp 75 Lys Ser Ala Lys Glu 155 Glu Gin Glu Lys Asp 235 Val Ala Asn Lys Ser Lys Lys Ile Gin 140 Tyr Leu Gin Phe Arg 220 Leu Thr Cys Thr Leu Val Tyr Ala Asp Gin Gin Lys Glu Gly Lys Asp Phe Thr Leu Ile Tyr Gin Gin Glu Tyr Glu Thr Gly Asp 110 Ile Glu Ile Asn 125 Val Val His Leu Gin Ser Asp Thr 160 Lys Leu Phe Lys 175 Asp Glu Leu Arg 190 Leu Ala Lys Pro 205 Glu Ile Asp Glu Trp Glu Glu Asn 240 Gly Tyr Thr Ile Gin Asn 245 Arg Ile Ala Val 250 Lys Trp Asp Asp Ser Leu 255 WO 97/46105 PCT/EP97/02737 -44- Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His 260 265 270 Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu 275 280 285 Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe 290 295 300 Pro Ser Val Asn Val Ser Met Glu Lys Val Ile Leu Ser Pro Asn Glu 305 310 315 320 Asn Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr 325 330 335 Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gly Ile Gly Pro Lys Gly 340 345 350 Ile Ser Phe Gly Val Ser Val Asn Tyr Gin His Ser Glu Thr Val Ala 355 360 365 Gin Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn Thr Ala 370 375 380 Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr 385 390 395 400 Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn 405 410 415 Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn 420 425 430 Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly Ile Ala 435 440 445 Ile Thr Ser Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys 450 455 460 Lys Gin Val Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr 465 470 475 480 Asn Gin Thr Asp Gly Val Tyr Lys Ile Lys Asp Thr His Gly Asn Ile 485 490 495 Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gin Gin Ile Lys Ala Lys 500 505 510 Thr Ala Ser Ile Ile Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg 515 520 525 Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu 530 535 540 WO 97/46105 PCT/EP97/02737 Thr 545 Ile Val Leu Val Tyr 625 Thr Asn Leu Lys Leu Lys Glu Gly Met Thr Asn Asp 595 Lys Leu 610 Asp Asn Asn Ile Asn Pro Asn Lys 675 Asn Thr Asp Ala Leu 550 Leu Leu Tyr 565 Tyr Leu Asp 580 Thr Thr Gly Thr Pro Lys Ala Glu Ser 630 Val Ser Gly 645 Asp Ala Asn 660 Asn Arg Asp Gin Cys Glu Thr Val Asn 710 His Asn Ile 725 Asp Glu Ile 740 Ser Ile Lys Ser Arg Tyr Gly Gly Ile 790 Glu Pro Leu 805 Glu Leu Gly 820 Lys Asp Gly Lys Tyr Glu Lys Met 615 Asn Gly Leu Tyr Ile 695 Val Lys Thr Pro Gly 775 His Gin Pro Thr Leu Lys Asn Phe 600 Asn Asp Asn Thr Tyr 680 Thr Asn Ser Leu Glu 760 Ile Tyr Asn Asn Ile Ser Tyr Pro 555 Asn Lys Pro 570 Thr Ala Lys 585 Lys Asp Val Val Thr Ile Asn Ser Ile 635 Asn Gly Lys 650 Leu Asn Thr 665 Ile Ser Leu Ile Asp Gly Lys Asp Asn 715 Asn Pro Ile 730 Phe Trp Asp 745 Asn Leu Thr Lys Leu Glu Gly Glu Phe 795 Tyr Val Thr 810 Val Ser Asp 825 Lys Phe Asp Asp Glu Ile Lys Ile Tyr Glu Ser 575 Glu Val Thr Lys 590 Ser His Leu Tyr 605 Lys Leu Ser Ile 620 Gly Lys Trp Thr Lys Gin Tyr Ser 655 Asp Ala Gin Glu 670 Tyr Met Lys Ser 685 Glu Ile Tyr Pro 700 Tyr Lys Arg Leu Ser Ser Leu His 735 Asp Ile Ser Ile 750 Asp Ser Glu Ile 765 Asp Gly Ile Leu 780 Ile Asn Glu Ala Lys Tyr Glu Val 815 Thr Leu Glu Ser 830 Phe Thr Lys Tyr 690 Thr 705 Ile Lys Asp Gin Asp 785 Phe Tyr Lys Thr Lys Ile Ala Thr Asn Val Ala 755 Ile Tyr 770 Lys Lys Asn Ile Ser Ser Ile Tyr WO 97/46105 PCT/EP97/02737 -46- Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe 850 855 860 Lys 865 Ile Asn Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His 870 875 880 Arg Tyr Asn Lys INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 2004 base pairs TYPE: nucleic acid STRANDEENESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Bacillus cereus STRAIN: AB78 INDIVIDUAL ISOLATE: NRRL B-21058 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2001 OTHER INFORMATION: /product= "80 kDa protein VIP1A(a)" /note= "This sequence is identical to that found in SEQ ID NO:1 between and including nucleotide positions 3126 and 5126" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

ATG

Met 885 AAA AGG GAA ATI Lys Arg Glu Ile

GAT

Asp 890 GAA GAC ACG Glu Asp Thr GAT ACG Asp Thr 895 ACG ATIT Thr Ile 910 GAT GGG GAC 'ITCT AT Asp Gly Asp Ser Ile 900 CAA AAT AGA AC GCT Gin Asn Arg Ile Ala 915 CCT GAC CT 'IGG GAA GAA AAT GGG TAT Pro Asp Leu Trp Glu Glu Asn Gly Tyr 905 GTA AAG Val Lys TGG GAC GAT TCT CTA GCA Trp Asp Asp Ser Leu Ala 920

AGT

Ser 925 AAA GGG TAT Lys Gly Tyr ACG AAA T'IT GIT Thr Lys Phe Val 930 TAT ACA GAT TAT Tyr Thr Asp Tyr 945 TCA AAT CCA CTA GAA AGT CAC ACA Ser Asn Pro Leu Glu Ser His Thr 935 940 GT T G GAT CCT Val Gly Asp Pro WO 97/46105 PCTIEP97/02737 -47 CAA AAG Glu Lys 950 GCA GCA AGA GAT Ala Ala Arg Asp CAT TqG TCA PAT CCA AAG CAA ACG IT Asp Leu Ser Asn Ala Lys Giu Thr Phe 960

AAC

Asn 965 CCA IM3 GTA GCr Pro Leu Val Ala

GCT

Ala 970 TIr CCA ACT GTG Phe Pro Ser Val arr ACT ATG GAA Val Ser Met Glu

AAG

Lys 980 GTG ATA TA TCA Val Ile Leu Ser

CCA

Pro 985 AAT CPA PAT TTA Asn Glu Asn Leu

TCC

Ser 990 AAT ACT GTA GAG Asn Ser Val Glu TT CAT Ser His 995 TCA TCC ACG Ser Ser Thr GCG GGG A'T Ala Cly Ile 1015 CAA CAC Tar Gin His Ser 1030 2 2

\AT

sn LOO0 TGC T=r TAT ACA PAT ACA GAA Trp Ser Tyr Thr Asn Thr Glu 1005 GGT GOT Tr GTI GAA Gly Ala Ser Val Glu 1010 qIT ACC GTA AAC TAT Vai Ser Vai Asn Tyr 1025 GGA CCA AAA GCT AT WG T=C GGA Cly Pro Lys Gly Ile Ser Phe Gly 1020 GAA ACA GT GCA CPA GAA IGG Glu Thr Val Ala Gin Glu Trp 1035 GGA ACA Tar Cly Thr Ser 1040 ACA CGA PAT Thr Gly Asn ACT TCG CAA TI AAT AC GCT Thr Ser Gin Phe Asn Thr Ala 1045 1050 TCA GCG GGA TAT TrA Ser Ala Cly Tyr Leu 1055 PAT CCA AAT Asn Ala Asn arr Val 1060 CGA TAT PAC PAT Arg Tyr Asn Asn GTA GGA ACT GOT Val Cly Thr Gly 1065 CCC AWC TAC Ala Ile Tyr 1070 ACT ATC GCA Thr Ile Ala 1085 CAT GTA AAA CCT ACA Asp Val Lys Pro Thr 1075 ACT ATr ACG GCG AAA Thr Ile Thr Ala Lys 1090 ACA A&T rT GTA WA PAT WC CAT Thr Ser Phe Val Leu Asn Asn Asp 1080 T AAT TCT ACA GCC Ser Asn Ser Thr Ala 1095 TrA PAT ATA T=r Leu Asn Ile Ser 1100 CCT CGA GAA ACT TAC CC AAA Pro Gly Glu Ser Tyr Pro Lys 1105 AAA CGA CPA PAT Lys Cly Gin Asn 1110 CGA AIC CCA ATA Cly Ile Ala Ile 1115 ACA TCA ATG CAT CAT Thr Ser Met Asp Asp 1120 TIT AAT TCC Phe Asn Ser CAT CCC His Pro 1125 ATr ACA TrA Ile Thr Leu PAT AAA Asn Lys 1130 AAA CAA OTA Lys Gin Val CAT PAT Asp Asn 1135 CTG CTA AAT PAT Leu Leu Asn Asn 1140 ITT TAT AAG ATA Val Tyr Lys Ile 1155 AAA CCT AMR AT TI GAA ACA AC CAA Lys Pro Met Met Leu Clu Thr Asn Gin 1145 ACA CAT GGT Thr Asp Cly 1150 AAA CAT ACA CAT CGA PAT ATA TA ACT GGC GCA CPA TGG AAT GOT =TC Lys Asp Thr His Cly Asn Ile Val Thr Cly Gly Clu Trp Asn Giy Vai WO 97/46105 PCT/EP97/02737 -48- 1160 1165 ATA CAA CAA ATC AAG GOT AAA ACA GCG =lT T Ile Gin Gin Ile Lys Ala Lys Thr Ala Ser Ile 1170 AT CT= GAT GAT GGG Ile Val Asp Asp Cly 1185 1175 1180 GAA CGT GTA GCA Glu Arg Val Ala 1190 GAA AAA CGT GTA Glu Lys Arg Val 1195 GCG GCA AAA Ala Ala Lys CAT TAT Asp Tyr 1200 GAA AAT CCA Glu Asn Pro 960 1008 GAA GAT Glu Asp 1205 AAA ACA CCC Lys Thr Pro TCT TA ACT Ser Leu Thr 1210 TA AAA GAT GCC Leu Lys Asp Ala 1215 CTC AAG CTT Leu Lys Leu

TCA

Ser 1220 TAT CCA CAT GAA Tyr Pro Asp Glu ATA AAA Ile Lys 1225 GAA ATA GAG GGA 'TA Glu Ile Glu Cly Leu 1230 TTA TAT TAT Leu Tyr Tyr AAA AAC Lys Asn 1235 1056 AAA CCC ATA Lys Pro Ile TAC GAA TCG ACC GTT ATG ACT Tyr Glu Ser Ser Val Met Thr 1240 1245 TAC TIA CAT Tyr Leu Asp GAA AAT ACA Glu Asn Thr 1250 AAA TIT AAA Lys Phe Lys CCA AAA GAA GM.

Ala Lys Glu Val 1255 ACC AAA CAA TA AAT Thr Lys Gin Leu Asn 1260 CAT ACC ACT Asp Thr Thr CT ACT CCA Leu Thr Pro 1280

GC

G

1 y 1265 1104 1152 1200 CAT GTA ACT CAT TTA TAT Asp Val Ser His Leu Tyr 1270 CAT GTA AAA Asp Val Lys 1275 AAA ATG AAT CIT Lys Met Asn Val ACA ATC Thr Ile 1285 AAA TIG TCT Lys Leu Ser ATA CT TAT CAT AAT Ile Leu Tyr Asp Asn 1290 GCT GAG TCT AAT CAT Ala Clu Ser Asn Asp 1295

AAC

Asn 1300 1248 TCA AT GGT AAA Ser Ile Cly Lys

TGG

Trp 1305 ACA AAC ACA AAT ATT =T TCA GGT GGA AAT AAC Thr Asn Thr Asn Ile Val Ser Cly Cly Asn Asn 1296 1310 1315 CGA AAA AAA Cly Lys Lys AAT ACA CAT Asn Thr Asp 133E CAA TAT TCT TT AAT AAT CCC Gin Tyr Ser Ser Asn Asn Pro 1320 1325 GCT CAA GAA Ala Gin Glu AAA TrA AAT AAA Lys Leu Asn Lys 1340 CAT GCT AAT IG ACA TTA Asp Ala Asn Leu Thr Leu 1330 AAT CT CAC TAT TAT ATA Asn Arg Asp Tyr Tyr Ile 1345 CAA Tar GAG AF ACT ATA Gin Cys Clu Ile Thr Ile 1360 1344 1392 AGT TTA Ser Leu 1350 CAT GGG Asp Cly 1365 TAT ATG AAG Tyr Met Lys GAG AT TAT Clu Ile Tyr TCA GAA AAA AAC ACA Ser Glu Lys Asn Thr 1355 CCG ATC ACT ACA AAA Pro Ile Thr Thr Lys 1370 1440 1488 ACA CTG AAT GIG AAT Thr Val Asn Val Asn 1375

AAA

Lys 1380 CAC AAT TAC AAA AGA TA CAT NT ATA CCT CAT AAT ATA AAA ACT AAT 1536 WO 97/46105 PCTIEP97/02737 -49- Asp Asn Tyr Lys Arg Leu Asp Ile Ile Ala His Asn Ile Lys Ser Asn 1385 1390 1395 CCA ATP TCT TCA CT CAT ATT Pro Ile Ser Ser Leu His Ile 1400 TGG GAT GAT ATT TCT ATA ACA Trp Asp Asp Ile Ser Ile Thr 1415 AAA ACG AAT Lys Thr Asn 1405 GAT GAA ATA ACT TTA TIT Asp Glu Ile Thr Leu Phe 1410 1584 GAT GTA Asp Val 1420 GCA TCA ATA AAA CCG GAA AAT Ala Ser Ile Lys Pro Glu Asn 1425 1632 TIA ACA GAT TCA Leu Thr Asp Ser 1430 TIA GAA GAT GGA Leu Glu Asp Gly 1445 GAA ATT AAA Glu Ile Lys 1435 ATC CTT ATr Ile Leu Ile 1450 AG ATI TAT AGT G1n Ile Tyr Ser AGG TAT Arg Tyr 1440 GGT ATT AAG Gly Ile Lys 1680 1728 GAT AAA AAA Asp Lys Lys GGT GGG ATT CAT Gly Gly Ile His 1455 TAT GGT Tyr Gly 1460 AAT TAT Asn Tyr 1475 GAA TIT ATT AAT GAA GCT AGT Glu Phe Ile Asn Glu Ala Ser 1465 GTG ACC AAA TAT GAA GIT ACT Val Thr Lys Tyr Glu Val Thr 1480 AGT GAC ACA CIT GAA AGT GAT Ser Asp Thr Leu Glu Ser Asp 1495 TIT AAT AT r Phe Asn Ile 1470 GAA CCA TIM CCA Glu Pro Leu Pro 1776 TAT AGT AGT GAG TA GGA CCA AAC GIG Tyr Ser Ser Glu Leu Gly Pro Asn Val 1824 1485 1490 AAA ATT TAC Lys Ile Tyr 1500 AAG GAT GGG ACA AT AAA Lys Asp Gly Thr Ile Lys 1505 1872 TIT GAT TIT ACC AAA TAT AGT AAA AAT GAA CAA Phe Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gin GGA TTA Gly Leu 1520 TIT TAT GAC Phe Tyr Asp 1510 1515 1920 1968 AGT GGA Ser Gly 1525 ITA AAT TGG GAC TIT AAA ATT AAT GCT AT ACT TAT GAT GGT Leu Asn Trp Asp Phe Lys 1530 Ile Asn Ala 1535 Ile Thr Tyr Asp Gly 1540 AAA GAG ATG AAT Lys Glu Met Asn GTI TIT CAT Val Phe His 1545 AGA TAT AAT AAA TAG Arg Tyr Asn Lys 1550 2004 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 667 amino acids TYPE: amino acid ITOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: WO 97/46105 PCT/EP97/02737 Met Lys Arg Glu Ile Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser Ile 1 5 10 Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr Ile Gin Asn Arg Ile Ala 25 Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe Val 40 Ser Asn Pro Leu Glu Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr 55 Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr Phe 70 75 Asn Pro Leu Val Ala Ala Phe Pro Ser Val Asn Val Ser Met Glu Lys 90 Val Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu Ser His 100 105 110 Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Ser Val Glu 115 120 125 Ala Gly Ile Gly Pro Lys Gly Ile Ser Phe Gly Val Ser Val Asn Tyr 130 135 140 Gin His Ser Glu Thr Val Ala Gin Glu Trp Gly Thr Ser Thr Gly Asn 145 150 155 160 Thr Ser Gin Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val 165 170 175 Arg Tyr Asn Asn Val Gly Thr Gly Ala Ile Tyr Asp Val Lys Pro Thr 180 185 190 Thr Ser Phe Val Leu Asn Asn Asp Thr Ile Ala Thr Ile Thr Ala Lys 195 200 205 Ser Asn Ser Thr Ala Leu Asn Ile Ser Pro Gly Glu Ser Tyr Pro Lys 210 215 220 Lys Gly Gin Asn Gly Ile Ala Ile Thr Ser Met Asp Asp Phe Asn Ser 225 230 235 240 His Pro Ile Thr Leu Asn Lys Lys Gin Val Asp Asn Leu Leu Asn Asn 245 250 255 Lys Pro Met Met Leu Glu Thr Asn Gin Thr Asp Gly Val Tyr Lys Ile 260 265 270 Lys Asp Thr His Gly Asn Ile Val Thr Gly Gly Glu Trp Asn Gly Val 275 280 285 Ile Gin Gin Ile Lys Ala Lys Thr Ala Ser Ile Ile Val Asp Asp Gly WO 97/46105 PCT/EP97/02737 -51 290 Glu Arg 305 Glu Asp Tyr Pro Lys Pro Ala Lys 370 Asp Val 385 Thr Ile Ser Ile Gly Lys Asn Thr 450 Ser Leu 465 Asp Gly Asp Asn Pro Ile Trp Asp 530 Leu Thr 545 Leu Glu Val Lys Asp Ile 355 Glu Ser Lys Gly Lys 435 Asp Tyr Glu Tyr Ser 515 Asp Asp Asp Ala Thr Glu 340 Tyr Val His Leu Lys 420 Gin Ala Met Ile Lys 500 Ser Ile Ser Gly Glu Pro 325 Ile Glu Thr Leu Ser 405 Trp Tyr Gin Lys Tyr 485 Arg Leu Ser Glu Ile 565 295 Lys Arg 310 Ser Leu Lys Glu Ser Ser Lys Gin 375 Tyr Asp 390 Ile Leu Thr Asn Ser Ser Glu Lys 455 Ser Glu 470 Pro Ile Leu Asp His Ile Ile Thr 535 Ile Lys 550 Leu Ile Val Thr Ile Val 360 Leu Val Tyr Thr Asn 440 Leu Lys Thr Ile Lys 520 Asp Gin Asp Ala Lys 330 Gly Thr Asp Leu Asn 410 Ile Pro Lys Thr Lys 490 Ala Asn Ala Tyr Lys 570 Lys 315 Asp Leu Tyr Thr Thr 395 Ala Val Asp Asn Gin 475 Thr His Asp Ser Ser 555 Gly 300 Asp Ala Leu Leu Thr 380 Pro Glu Ser Ala Arg 460 Cys Val Asn Glu Ile 540 Arg Gly Tyr Glu Leu Lys Tyr Tyr 350 Asp Glu 365 Gly Lys Lys Met Ser Asn Gly Gly 430 Asn Leu 445 Asp Tyr Glu Ile Asn Val Ile Lys 510 Ile Thr 525 Lys Pro Tyr Gly Ile His Pro 320 Ser Asn Thr Lys Val 400 Asn Asn Leu Ile Ile 480 Lys Asn Phe Asn Lys 560 Gly Glu Phe Ile Asn Glu Ala Ser Phe Asn Ile Glu Pro Leu Pro Asn Tyr WO 97/46105 PCT/EP97/02737 -52 Val Thr Lys 595 Tyr Glu Val Thr Ser Ser Glu Leu Gly Pro Asn Val 605 Gly Thr Ile Lys Ser Asp 610 Thr Leu Glu Ser Lys Ile Tyr Lys Asp 620 Asp Phe Thr Lys Ser Lys Asn Glu Gin Gly Leu Phe Tyr 635 Asp 640 Ser Gly Leu Asn Asp Phe Lys Ile Asn Ala 650 Ile Thr Tyr Asp Gly 655 Lys Glu Met Val Phe His Arg Tyr Asn Lys 665 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Bacillus cereus STRAIN: AB78 INDIVIDUAL ISOLATE: NRRL B-21058 (ix) FEATURE: NAME/KEY: Peptide LOCATION: 1..16 OTHER INFORMATION: /note= "N-terminal sequence of protein purified from strain AB78" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Lys Arg Glu Ile Asp Glu Asp Thr Asp Thr Asx Gly Asp Ser Ile Pro 1 5 10 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDENESS: single TOPOLOGY: linear WO 97/46105 PCT/EP97/02737 -53 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..21 OTHER INFORMATION: /note= "Oligonucleotide probe based on amino acids 3 to 9 of SEQ ID NO:8, using codon usage of Bacillus thuringiensis" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GAAATIGATC AAGATACNGA T 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Bacillus thuringiensis STRAIN: AB88 (ix) FEATURE: NAME/KEY: Peptide LOCATION: 1..14 OTHER INFORMATION: /note= "N-terminal amino acid sequence of protein known as anion exchange fraction 23 (smaller) (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Glu Pro Phe Val Ser Ala Xaa Xaa Xaa Gln Xaa Xaa Xaa 1 5 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids WO 97/46105 PCTIEP97/02737 -54- TYPE: amino acid STRANDEDNESS: single TOPOLOGY: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Bacillus thuringiensis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Xaa Glu Tyr Glu Asn Val Glu Pro Phe Val Ser Ala Xaa 1 5 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Bacillus thurigiensis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Asn Lys Asn Asn Thr Lys Leu Pro Thr Arg Ala Leu Pro 1 5 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Bacillus thuringiensis STRAIN: AB88 (ix) FEATURE: NAME/KEY: Peptide LOCATION: 1..15 W0 97/46105 PCTEP97/02737 OTHER INFORMATION: /note= "N-terminal amino acid sequence of 35 kDa VIP active against Agrotis ipsilon" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Ala Leu Ser Glu Asn Thr Gly Lys Asp Gly Gly Tyr Ile Val Pro 1 5 10 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single IOPOLOGY: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Bacillus thuringiensis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Asp Asn Asn Pro Asn Ile Asn Glu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single IOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (ix) FEATURE: NAME/KEY: Peptide LOCATION: 1. .9 OTHER INFORMATION: /note= kDa delta-endotoxin" "N-terminal sequence of (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asp Asn Asn Pro Asn Ile Asn Glu ,,WO 97/46105 PTE9/23 PCT/EP97/02737 56 INFORMATION FOR SEQ ID NO:l6: (1 SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid STRANDEDNjESS: single TIOPOLOJGY: linear (ii) MOLECZULE TYPE: peptide (iii) HYPOTHEICAL:

NO

FRAGM'ENT TYPE: N-term-inal (vi) ORIGINAL SOURCE: ORGANISM: Bacillus thuringiensis (ix) FEATURE: NAME/KEY: Peptide LOCATION: 1. .11 OTHER INFORMATION: /note= "N-terminal sequence from kDa. delta-endotoxin" (xi) SEQUENCE DESCRIPTION: SEQ, ID NO:16: Met Asn Val Leu Asn Ser Gly Arg Thr Th-r Ile 1 5 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 2655 base pairs TYPE: nucleic acid STRANDEEN ESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: EMJ (genom-ic) (iii) HYPOTHEICAL:

NO

(iv) A~rI-SENSE:

NO

(ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .2652 OTHER INFORMATION: /note= "Maize optim-ized

IEA

sequence for 100 kd vip1-A(a) protein from AB78" (xi) SE)QUENCE DESCRIPTION: SEQ ID NO:17: A B3AAGAACA TIXAAGAAGAA GCTG~CCAGC G1XG-TGACCT GCACL)L-nGCT

GGCCCCCATGX

WO 97/46105 W097/6105PCT/EP97/02737 57 TWXY1XACG

ACCCAGAAGA

GGCAAGGACT

GACCAGCAGA

TGXGACGGCC

GAGCAGGCCA

GTGG=~CCC

AAG~rCAACA

AACCAGCCCC

AGCCAGGAGT

GAGAINCGACG

GGCTACACCA

TACAQCAAGT

TACGAGAAGG

GTGGCCGCCT

AACCTGAGCA

GGCGCCAGCG

TACCAGCACA~

TTCAkCACCG

GGCGCCAW=

ACCNI'CACCG

AAGAAGGGCC

ACcC1ACA

AACCAGACOG

GAGTIGGAACG

GGCGAGCGCG

ACCCCCAGCC

A IXGAGGGCC

GCAACGGAA

ACCAGCAGAA

WCAGCAACCT

CQGCCAACAA

TGATCCAGAG

TGGAGAAGGG

TCGACAGCAA

AGCAGG'PGCA

TCCTGCCAA

AGGACACCGA

TCCAGAACOG

WCGTGAGCAA

CCGCCCCGCGA

WCCCCAGCGT

ACAGCG'IGGA

TGAGGCCGG

GCGAGACCGT

CCAGCGCCGG

ACGACGTGAA

CCAAGWCGAA

AGAACGGCAT

AGAAS3CAGGar

ACGGCG=CA

TGCCGAGAA

TACOCC1AA 1XC1=ACrA

CGCCG=E~AC

GGAGATGGAC

GACCAT=

GCIGCIGGAC

CAAGGAGACC

CAACGGCAAG

CAAGCflG GACCFTrAAG

GCAGGACGAG

GCCCAGCAAG

CACCGACGGC

CATCGCCGTG

CCCCI)GAG

CCTGACP

GAACGIGAGC

GAGCCA=1C

CATCGGTCCC

GGCC-AGGAG

CrACVIGAC

GCCCACCACC

TrCACCGcC

CGCCATCACC

GGACAACCTG

CAAGATCAAG

GCAGA'TCAAG

GCGCGTGGCC

GGACGCCT

CAAGAACAAG

GCCGACAGCA

CGCAAGGGCC

GCCCCCACGC

AAGAAGCAGC

GCCGACTr-CA

ATWACAGCA

CCCATCAAGA

CGkGC'IGAAGC CrGCGCAACC

ATCAACI

GACAGCA=C

AAGTGGGACG

AGCCACACCG

AGCAACGCCA

ATGGAAAGG

AGCACCAACT

AAGGGCATCA

TGGGCACCA

GCCAA~CGM

CIMAACAM~A

AGCATGGACG

CIGAACAACA

GACACCCACG

GCCAAGACCG

GCCAAGGACT

AAGCTGAC

CCCA T~rACG

AGACCAACCA

TGCTG1GGCrA

GTIGACAGCAC

AGGAGTACCA

CC'IqVAACCT

ACAAGGGCAA

'1CGA~rACCA I1ITI 2AAGAT

CCGAGTICA

WCACCCAGCA

CCGACCIG*IG

ACAGCCI'GGC

TGGCGACCC

AGGAGACCTr 1GAICCIGAG GGAGCrACAC

GCMTCGCG

GCACCGGCAA

GCTACCA

7AACAACGA

GCCCCGGCGA

ACIqVAACAG

AGCCCABGAT

GCAACA=T

CCAG3CATAT

ACGAGAACCC

ACCCCGACGA

AGAGCAGCcGp

GATCAGCACC

CTrACqICAG CCBGA TCTAC

GAGCAWCOGC

GAGCGAGGAC

GGAGAAGCAG

GAGCGACACC

CGAC-AGCCAG

CAAGAAGGAG

GATGAAGCGC

GGAGGAGAAC

TAGCAAGGGC

CTACACCGAC

CAACCCCIG

CCCCAACGAG

CAACACCGAG

GAGCGIr.AAC

CACCAGCCAG

C~lGGCACC

CACCATCGCC

GAkGCTACCC

CCACCCCAI

GCIGAGACC

GACCGGCGOC

C)G'GACGAC

CGAGGACAAG

GATCAAGGAG

GA IGACCTAT 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 9 4. WO 97/46105 PTE9/23 PCT/EP97/02737 58 CFAGACGAGA ACACCGCCAA AAGGACIA GCCACCI'GTA CIGAGCATCC TGTACGACAA ACCAACAWCG

IGAGCGGCGG

GCCAACCWGA

CCCIGAACAC

ATCAGC=I3 ACA GAAG ATATACCCCA. TCACCACCAA.

ATCAWCGCCC

ACAACAWCAA

GAGATCACCC TGItrGGGA 'AACCIG~C

ACAGCGAGAT

GGCATCcyIGA TCGACAAGAA TTC-AACATOG AGCCCCTGCA CIUGGCCCA ACGTGAGCGA AAGTrCGACI'

TACCAAGTA

AACIGGCT

TCAAGATCAA

CGCTACAACA

AGTAG

GGAGGTGACC

CGACGTGAAG

CGCCGAGAGC

CAACAACGGC

CGACGCCCAG

CXAGAAGAAC

GACGAAC

GAGCAACCCC

CGACATAWCG

CAAGCAGATA

GGGCGGCAWC

GAACTACGIG

CACCC7GGAG

CAGCAAGAAC

CGCCATCACC

AAGCAGC'IrGA ACGACACCAC CGGCAAGThr CIMACCCCCA AGAaGAAcGT GACCAW~aAG AACGACAACA GCATCGGCAA GTXGACCAAC AAGAAGCAGT ACAGCAGCAA

CMACCCCGAC

GAGAAGC'IGA ACAAGAACCG CGACTACTAC ACCCAGIrC0. AGAmIACmA CGACGGCGAG GTAACAAGG ACAACTACAA GCGCC'IWGAC AWAOCAGCC TCACATCAA

GACCAACGAC

ATrACCGACG TCGCCAGCAT

CAAGCCCGAG

TACAGTCGcT AcGGCATCA GC'PGGAGGAC CACrACGGCG AG1TCAWCAA

CGAGGCCASC

ACCAA~rACG AGGIGACCTA

CAGCAGCGAG

AGCXGACAAGA TTACAAGGA CGGC:ACCATC GAGCAGGGCC 'GTmrACGA CAGCGGCCwM TACGACGGCA AGGA.GATGAA CGI'GrCc' 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2655 INFORMATION FOR SEQ ID NO:18: Wi SEQUENCE CHARACTERIST'ICS: LENGTH: 2004 base-pairs TIYPE: nucleic acid STRANDEJNESS: single TODPOLOGY: linear (ii) MOLECULE TYPE: UIA (genomnic) (iii) HYPOTHETICAL:

NO

(iv) ANni-SENSE: NO (ix) FEATURE: NAME/KEY: misc -feature LOCATION: 1. .2004 OTHER INFORMATION: /note= "Maize optimized IA sequence for VIPlA(a) 80 kd protein from AB3781' WO 97/46105 PCT/EP97/02737 59 (xi) SEQUEN\CE DESCRIPTION: SEQ ID NO:18: ATIAAGCCG AGATMCA WGICACmGAC ACCGACGGCG ACAGCAWCC CGACClI= GAGGAGAAmG GCTACACCyAT CCAGAACGGC A TG rmj AG(fl3GGAMAJ CA(CI o 120 AGCAAGGGCTj ACACCAAGIT C91AGCAAC CCcIGA GCACC GGGCCCC 180 TACACCGACT ACGZCAAGGC~ CGCCCGCGAcCIGcIJ GCAAcXCM GGAGAC~lpW 240 AACCCCC'IrGc DGCCGCCcr CCCCAGCy3 AACG-IAc AAAGpG1l2'Gc 0 CCCAACGAG ACO kGCAA CACGGGAG AGICCAIGh G CA CA] cm ACACC 360 AACACCGAGG GCXGCCAGCT GGAGGCOG A ECGX cc A:GGCA'IG CIItCGCG 420 AGCmGTGAAC ACCAGCIAC CGAGACCa GCCCAGGAG? GGGCAcc CACCGGC C 480 ACCGCCAGJp TCAACACCGC CAGCGCCG TACLCIG~y CCAACGTGCj CTACAACAAC 540 GTGGCACCGcj GCGCCATCrA CGACOIA CCCAACC CCt rG ACT CMM 600 ACCACCAcy CCATCG MCA AT TCACCGCCCy- TGAA CCCCGGCxGC 660 AGCTACCCCAJ AGAAGGGCCA GAAMCGC GCCAWlr.CCA GCA~GAcM C1XAACAGC 720 CACCCCATrCA CCC-IGAACAA GAAGAGM GA(2C'crLI ACAW GCCAM 780 C'TG1GACrJA ACCAGACG CGCGTCTrAC AAGATr- A ACCC CPCr~ 4 ACCGGCGGCc AGTGGAACGG CGTGATCCAG CAGATC A CA CACXC 800 -ICC C r C G G C h3 c J C A CA. y C AGCA 'Ac 9 00 GGACGACG CCCCXCCT GCCcIApGAAGCCG'CCGI AGGACTA C c~ WG G AX M A GcIMCTrAC ACAACAATA CC CWCACcGwm 100 A I A C A z y T C C M A G C A C C G A C A A C G C C A C1 4 AMCCAAG IACACGICC ACT'AC AGA CAAGC CC"CCG. GCAGAC= 12080 ACC~ ry r~ A c C J c p r C AC AA C GA C A~ A G ACAA2 CGAC 2 C C 1 260 ATACC ACCX AACGAGAA CCA C C C c c G A 1 8 GA~ r C'-r CA T A G T C A C Ar Cc G A C A A G C A C C I AcGJ k C A C2 A ;c 1 400 CAGCC AGTATCCAT CACA CCAG AG lCY AGAkCAG CATGTCAAG 1500 CCC~ GCAC~ W r C O C CAA A ACCAA CCCCA AQWr CAG A T CACAGC IxC 1 560 ACCAAA TAcy-TCCCC ACA A= I~T~ GA ATC TGACCAAGGA CACTAG r 1600 WO 97/46105 PCT/EP97/02737 AAGCCCGAGA ACCIACCGA CAGCGAGATC AAGCAGATAT CI'GGAGGACG GCATCCIUAT CGACAAGAAG GGCGGCATCC GAGCCCAGCP TCAACATCGA GCCCCIGCAG AACTACGTGA AGCAGCGAGC 'TGGCCCCAA CGIGACGAC

ACCCTGGAGA

GGCACCAICA AG'ITCGA=T CACCAAGTAC AGCAAGAACG AGCGGCCIGA ACTGGGA=Cr CAAGATCAAC GCCATCACCr GTGTTCCACC GCTACAACAA

GTAG

INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 4074 base pairs TYPE: nucleic acid STRANDEIDESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: ENA (genomic) ACAGTCGCrA CGGCATCAAG ACTACGGCGA GPKr-ATCAAC CCAAGTACGA GGIGACCrAC GCGACAAGAT ITACAAGGAC AGCAGGGCCr GTItTACGAC ACGACGGCAA GGAGATGAAC 1680 1740 1800 1860 1920 1980 2004 (ix) FEIURE: NAME/KEY: CDS LOCATION: 1..1386 OTHER INFORMATION: /product= "VTP2A(b) from Btt' (ix) FEATURE: NAME/KEY: CDS LOCATION: 1394..3895 OTHER INFORMATION: /product= "VIPlA(b) from Btt" (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..4074 OTHER INFORMATION: /note= "Cloned EMIA sequence from Btt which contains the genes for both VIPlA(b) and VIP2A(b) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ATG CAA AGA ATG GAG GGA AAG TO Trr G1Xai GT TCA AAA ACA TA CAA Met Gin Arg Met Glu Gly Lys Leu Phe Val Val Ser Lys Thr Leu Gin 670 675 680 GTA GIT ACT AGA ACT GTA TIG CIT ACT ACA GIT TAC TCT ATA ACT TA Val Val Thr Arg Thr Val Leu Leu Ser Thr Val Tyr Ser Ile Thr Leu 685 690 695 TA AAT AAT GTA GTG ATA AAA GCT GAC CAA 'TA AAT ATA PAT TCT CAA Leu Asn Asn Val Val Ile Lys Ala Asp Gin Leu Asn Ile Asn Ser Gin WO 97/46105 PCT/EP97/02737 -61 700 AGT AAA TAT ACT Ser Lys Tyr Thr I'M CAA AAT CTA Leu Gin Asn Leu ATC CCT GAT AAT Ile Pro Asp Asn CCA GAG Ala Glu 730 GAT TI T

AAA

Asp Phe Lys GGG GAA GAG Gly Giu Glu 750

GAA

Glu 735 CAT AAG GGG AAA Asp Lys Gly Lys

GCG

Ala 740 AAA GAA TG GGG Lys Giu Trp Gly TGG AGG CCT CCT Trp Arg Pro Pro

GCT

Ala 755 ACT GAG AAA GGA Thr Giu Lys Cly AAA GAG AAA Lys Glu Lys 745 ATG AAT AAT Met Asn Asn GAA ATT ACT Glu Ile Thr TT TA Phe Leu 765 GAT AAT AAA AAT Asp Asn Lys Asn ATA AAG ACC Ile Lys Thr AAT TAT AAA Asn Tyr Lys 775 TCT ATG GCA GCT TCA Ser Met Ala Giy Ser 785 TGT GAA GAT GAA Cys Giu Asp Glu ATA AAA CAT TA GAA GAA Ile Lys Asp Leu Giu Glu 790 795 ATT CAT AAG ATC Ile Asp Lys Ile =VT CAT Phe Asp 800 AAA GCC AAT Lys Ala Asn TCG ACT TCT AT Ser Ser Ser Ile ATC ACC Ile Thr 810 TAT AAA AAT Tyr Ls Asn GAA GGT AAT Glu Gly Asn 830 Trr TA GGT Phe Leu Gly 845 GTG GAA CCA GCA ACA ATr Val Glu Pro Ala Thr Ile 815 820 OGA Trr AAT Gly Phe Asn ACG ATr AAT TCT Thr Ile Asn Ser GAT CCA ATG GCA CAG Asp Ala Met Ala Gin 835 AAA TCT TA ACA Lys Ser Leu Thr 825 TrAAA GAA CAA Phe Lxs Glu Gin 840 CAT ACT CAT TrA Asp Thr His Leu 528 576 AAG GAT ATG Lys Asp Met

AAG

Ls 850 T1'r GAT ACT TAT Phe Asp Ser Tyr

CTA

Leu 855 ACT GCT CAA CAA GT CC Thr Ala Gin Gin Vai Ser 860 865 AGT AAA AAA AGA Ser Ls Lxs Arg AT TI AAG Gr Ile Leu Lys Val GTr CCC ACT GGG Val Pro Ser Gly GGT TCT ACT ACT Gly Ser Thr Thr

CCA

Pro 885 ACA AAA CCA Thr Lys Ala TrA AAC AAT Leu Asn Asn CAT CTA CAT His Val Asp 910 GAA TAC AAA ATG Glu Tyr Lys Met

CTC

Leu 900 AT CAT AAT GGG Ile Asp Asn Gly GGT GIC ATT Gly Val Ile 890 TAT GTG CIC Tyr Val Leu 905 GAG TGC TA Glu Cxs Leu AAG GTA TCA AAA Lys Vai Ser Lys CTA AAA AAA GGG Val Lys Lys Gly CAA CT GAA GGG ACT TA AAA AAG ACT CC GAC Tf AAA AAT CAT ATA j. W097/46105 PCT/EP97/02737 -62- Gln Val Giu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp Ile 925 930 935 AAT GCT GAA GCG CAT AGC TGC GGG ATG AAA NT TAT GAA CAC TGG GCT 864 Asn Ala Glu Ala His Ser Tip Gly Met Lys Ile Tyr Glu Asp Tip Ala 940 945 950 955 AAA AAT TA ACC GCT TCG CAA AGG GAA GCT TTA CAT GGG TAT GCT AGC 912 Lys Asn Leu Thr Ala Ser Gin Arg Glu Ala Leu Asp Cly Tyr Ala Arg 960 965 970 CAA CAT TAT AAA GAA ATC AAT AAT TAT TG CCC AAT CAA GGC GGG AG'r 960 Gin Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 975 980 985 GGA AAT GAA AAG cM GAT CCC CAA TA AAA AAT ATr =r GAT GCT TTA 1008 Gly Asn Glu Lys Leu Asp Ala Gin Leu Lys Asn Ile Ser Asp Ala Leu 990 995 1000 GGG AAG AAA CCC ATA CCA GAA AAT AT ACC GTG TAT AGA TGG TCT GGC 1056 Cly Lys Lys Pro Ile Pro Giu Asn Ile Thr Vai Tyr Arg Tip Cys Cly 1005 1010 1015 ATC CCC GAA TIT GGT TAT CAA AT AGT CAT CCG TTA Cer TCI TrA AAA 1104 Met Pro Glu Phe Gly Tyr Gin Ile Ser Asp Pro Leu Pro Ser Leu Lys 1020 1025 1030 1035 CAT IIT GAA GAA CAA TIT ITA AAT ACA ATT AAA GAA GAC AAA GGG TAT 1152 Asp Phe Giu Giu Gin Phe Leu Asn Thr Ile Lys Clu Asp Lys Cly Tyr 1040 1045 1050 ATG AGT ACA AGC TA TCC AGT GAA CGT CIT CCA GCT TIT CGA TCr AGA 1200 Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg 1055 1060 1065 AAA A T ATA TrA CCC ITA CAA GIT CCC AAA GCA ACT ACG GGG GCG TAT 1248 Lys Ile Ile Leu Arg Leu Gin Val Pro Lys Giy Ser Thr Cly Ala Tyr 1070 1075 1080 TA AGT CCC ATT GGT GCA TT CCA AGT GAA AAA GAG ATC CrA CIT CAT 1296 Leu Ser Ala Ile Gly Gly Phe Ala Ser Giu Lys Glu Ile Leu Leu Asp 1085 1090 1095 AAA GAT ACT AAA TAT CAT ATT CAT AAA GCA ACA GAG GTA ATC ATr AAA 1344 Lys Asp Ser Lys Tyr His Ile Asp Lys Ala Thr Glu Val Ile Ile Lys 1100 1105 1110 1115 GGT GTT AAC CGA TAT GTA GTG GAT GCA ACA 'TA 'TA ACA AAT 1386 Gly Val Lys Arg Tyr Vai Val Asp Ala Thr Leu Leu Thr Asn 1120 1125 TAAGGAG ATG AAA AAT AIG AAC AAA AAG TA CCA AGT 'rr GTA ACC IXGr 1435 Met Lys Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys i 5 SWO 97/46105 PCT/EP97/02737 -63-

ATG

Met TTA TA GCT Leu Leu Ala CCT AMR TMP Pro Met Phe 20 TMG AAT GGA Leu Asn Gly GTG AAT OCT GTT AAC Val Asn Ala Val Asn 1483 GCG GAT ACT AAA Ala Asp Ser Lys AAT CAG APT TC ACA ACC CAG GAA AAC Asn Gin Ile Ser Thr Thr Gin Giu Asn CAA CAC Gin Gin 1531 AAA GAG ATG Lys Clu Met CAT TIT AAT Asp Phe Asn CGA AAC GCA TTA Arg Lys Gly Leu GGA TAT TAT TTC Gly Tyr Tyr Phe AAA GGA AAA Lys Giy Lys AAT ACC rr Asn Thr Leu 1579 1627 AAT CT ACT ATG Asn Leu Thr Met GCA CCC ACA CGT Ala Pro Thr Arg ATG TAT Met Tyr CAC CAA CAA ACA Asp Gin Gin Thr AAT GCA TA PTA Asn Ala Leu Leu

GAT

Asp AAA AAA CAA CAA Lys Lys Gin Gin 1675 GAA TAT CAG Glu Tyr Gin TCC ATr CGT Ser Ile Arg 100 TrG APT CT TM Irp Ile Gly Leu

AT

Ile 105 CAG CGT Gin Arg CAG CCA Gin Ala GGC CAT TIC ACA Gly Asp Phe Thr AAC PTA TCA AAG Asn Leu Ser Lys CAT GAA Asp Glu 120 AAA GAA ACO Lys Glu Thr 110 AT ATA GAA Ile Ile Glu 125 CAA GTT GIC Gin Val Val 140 TAT CAA TCA Tyr Gin Ser ATC CAT GGG Ile Asp Gly CAT PTA GAA His Leu Glu 145 CAT ACG AAA Asp Thr Lys 160 AAA ATC Lys Ile 130 ATT TCT AAT Ile Ser Asn

AAA

Lys 135 GGG AAA GAA AAG Gly Lys Giu Lys 1723 1771 1819 1867 1915 AAA GAA AAA TTA Lys Glu Lys Leu

GIT

Val 150 CCA ATC AAA Pro Ile Lys ATA GAG Ile Glu 155 PIT AAT ATT Phe Asn Ile

GAT

Asp 165 AGT AAA ACA Ser Lys Thr CAA TCT CAA Gin Ser Gin TIT AAA Phe Lys 170 GAA CIT AAA PTA Glu Leu Lys Leu

TT

Phe 175 AAA ATA CAT AGT Lys Ile Asp Ser CAA AAC Gin Asn 180

GI

T CAA CTG AGA Val Gin Leu Arg

AAC

Asn 190 CCT GAA PMT AAC Pro Glu Phe Asn

AAA

Lys 195 AAA GAA TCA CAG Lys Glu Ser Gin

GAA

Glu 200 PIT TA CCA Phe Leu Ala AAA GCA CA Lys Ala Ser 205 1963 2011 2059 2107 AAA ACA AAC Lys Thr Asn ACG GAT ACA Thr Asp Thr 225

CT

Leu 210 ITT AAG CAA AAA Phe Lys Gin Lys AAA AGA CAT Lys Arg Asp ATT CAT GAA GAT Ile Asp Glu Asp 220 GAA GAA AAT GCC Glu Glu Asn Gly 235 CAT GGA CAC TCC Asp Cly Asp Ser AT COT CAT CiT TGG Ile Pro Asp Leu Trp 230 WO 97/46105 WO 9746105PCTIEP97/02737 64 TAC AG Tyr Th 240 APr CAA AAT AAA Ile Gin Asn Lys

GT

Val 245 GCT GTC AAA TG Ala Val Lys TrP

GAT

Asp 250 GAT WCG CTA GCA Asp Ser Leu Ala 2155

AGT

Ser 255 AAG OGA TAT ACA AAA M ITr T G AAT CCA TTA GAC AGC CAC Lys Giy Tyr Thr Lys Phe Val Ser Asn Pro Leu Asp Ser His

ACA

Thr 270 2203 OPT GGC OAT Val Gly Asp TA TWA AAT Leu Ser Asn ACT c'iG AAT Ser Val Asn 305 CCC TAT ACT OAT TAT GAA MAG GCC Pro TYr 275 GCA AAG Ala Lys 290 Thr Asp Tyr Giu Ala GCA AGO GAT Ala Arg Asp PTA GAT Leu Asp 285 2251 GAA AmG TIC Giu Thr Phe CCA TIG GTA GC? Pro Leu Val Ala GOT TIT CCA Ala Phe Pro 300 2299 =T AGT ATG GAA Val Ser Met Giu OTG ATA TA TCA CCA MAT GMA MT Val Ile Leu Ser Pro Asn Giu Asn 315 2347 TA WC Leu Ser 320 MAT AGT OTA GAG Asn Ser Val Giu wr Ser 325 CAT WCA TCC AmG His Ser Ser Thr

MAT

Asn 330 TOG 'ICr TAT AG Trp Ser Tyr Thr ACA GMA GGA OCT Thr Giu Gly Ala

'ICC

Ser 340 ATr GMA OCT GOT Ile Olu Ala Gly

GGC

Gly 345 GOT CCA PTA GGC Oly Pro Leu Gly CeIr Leu 350 2395 2443 2491 'ICT T= GOC GTG Ser Phe Gly Val

AGT

Ser 355 OTT ACT TAT CMA Val Thr Tyr Gin TCT GMA ACA IT Ser Glu Thr Val OCA CAA Ala Gin 365 GMA TOO GGA Glu Trp Gly

ACA

Thr 370 'rer ACA OGA MAT Ser Thr Oly Asn MAT GCA MAT OTr Asn Ala Asn Val 390

ACT

Thr 375 TCA CAA WrC Ser Gin Phe MAT AG OCT WCA Asn Thr Ala Ser 380 OTA GOG ACT GOT Val Gly Thr Gly 395 2539 2587 OCO OGA TAT TrA Ala Gly Tyr Leu 385 cGG TAT MC MAT Arg Tyr Asn Asn 0CC ATC Ala Ie 400 TAT GAT OTA A Tyr Asp Val Lys CCT ACA Pro Thr 405 ACA AOT MI Thr Ser Phe GTA PTA MAT MAC MT Val Leu Asn Asn Asn 410 ACA OCT PTA COT ATA Thr Ala Leu Arg Ile 2635 2683

ACC

Thr 415 ATC OCA ACG Ile Ala Thr Apr ACA le Thr 420 OCA MAA WCA Ala Lys Ser MAT TWA Asn Ser 425 GOA GMA Gly Oiu 440 TOT CCG 000 OAT Ser Pro Oly Asp TAT CCA GMA ATA Tyr Pro Glu Ile AAC OCT Apr Asn Ala Ile GmG Apr Ala Ile 445 2731, ACA 'IcT ATG OAT OAT PiT MAT TcT CAT CCA Apr ACA PTA MAT AMA CM Thr Ser Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys Gin 2779 WO 97/46105 PCT/EP9702737 CAG GTA AAT Gin Val Asn 465 CAA TMI ATA PAT PAT Gin Leu Ile Asn Asn 470 AAG CCA AIT AM( Lys Pro Ile Met

CTA

Leu 475 GAG ACA GAC Glu Thr Asp 2827 CAA ACA Gin Thr 480 CAT GGT OTT TAT AAA ATA Asp Gly Val Tyr Lys Ile 485 AGA CAT ACA Arg Asp Thr

CAT

His 490 GGA PAT AT GTA Cly Asn Ile Val 2875

ACT

Thr 495 GGT GGA GAA Gly Gly Glu GG AAT GGT Trp Asn Gly 500 OTA ACA CAA Val Thr Gin CAA AIT AAA CCA AAA ACA Gin Ile Lys Ala Lys Thr 505 510 2923 GCG TCT ATT AT Ala Ser Ile Ile GCG GCA AAA CAT Ala Ala Lys Asp 530 TTA AAA CAT ACC Leu Lys Asp Thr 545 GT CAT CAC GGG Vai Asp Asp Gly 515 TAT GGT CAT CCA Tyr Cly His Pro AAA CAG Lys Gin 520 GAA CAT Glu Asp 535 GTA GCA GAA Vai Ala Glu AAA ACA CCA Lys Thr Pro AAA COT GMt Lys Arg Vai 525 CCT TA ACT Pro Leu Thr 540 AAA GAA ACT Ly's Giu Thr 2971 3019 3067 CIC AAG CT Leu Lys Leu

TCA

Ser 550 TAC CCA CAT GAA Tyr Pro Asp Glu

ATA

Ile 555 PAT GGA Asn Gly 560 TIM TMC TAC TAT Leu Leu Tyr Tyr

GAT

Asp 565 GAC AAA CCA ATC Asp Lys Pro Ile

TAT

Tyr 570 GAA W AT GITC Glu Ser Ser Val 3115 3163

ATG

Met 575 ACT TAT CI GAT Thr Tyr Leu Asp

GAA

Glu 580 PAT ACG GCA AAA Asn Thr Ala Lys GAA GTC AAA AAA Glu Vai Lys Lys 585 CAA ATA Gin Ile 590 PAT CAT ACA ACC Asn Asp Thr Thr

GGA

Gly 595 AAA MF AAG Lys Phe Lys CAT OTA PAT Asp Vai Asn 600 CAC TA TAT CAT OTA His Leu Tyr Asp Vai 605 3211 AAA CIG ACT Lys Leu Thr CAT GGG GCT Asp Cly Ala 625

CCA

Pro 610 AAA ATG PAT T ACG ATT AAA AIG CCT Lys Met Asn Phe Thr Ile Lys Met Ala 615 TCC PIG TAT Ser Leu Tyr 620 TAT TA ACA Tyr Leu Thr 3259 GAA PAT PAT CAT Glu Asn Asn His =T TTA GCA ACC Ser Leu Gly Thr

TGG

Trp 635 3307 TAT PAT Tyr Asn 640 CAT TCT His Ser 655 OT GCT GGT CGA Val Ala Gly Gly ACT GOG PAG AGA Thr Gly Lys Arg

CAA

Gin 650 TAT COT TCA GCT Tyr Arg Ser Ala 3355 3403 IT CCA CAT Cys Ala His GCT CTA =r TCA Ala Leu Ser Ser GCG AAA PAG AAA Ala Lys Lys Lys

CTA

Leu 670 PAT CPA PAT GCG AAT TAC TAT CIT AGC ATG TAT ATG AAG GCT CAT TT 3 3451 WO97/46105 PCT/EP97/02737 -66- Asn

ACT

Thr

AGT

Ser

'TA

Leu

GGA

Gly 735

GTA

Val

ATA

Ile

GAT

Asp

GAA

Glu

TT

Phe 815

AAG

Lys Asn

GAA

Glu

AAA

Lys 705

AAA

Lys

GGC

Gly

GCT

Ala

AAA

Lys

GTA

Val 785

GAA

Glu

ATC

Ile

GCC

Ala Ala Asn Tyr 675 CCr ACA ATA Pro Thr Ile 690 GTA AAA TrA Val Lys Leu AAT TCT GAA Asn Ser Glu ACG ACA AAT Thr Thr Asn 740 ATA AAT CCG Ile Asn Pro 755 GAC TCA ACT Asp Ser Thr 770 ACA TTT AAA Thr Phe Lys ATA TAT CAT Ile Tyr His ATG GGT Grr Met Gly Val 820 GCA TAATIT Tyr Leu GAA GTA Glu Vai AAT AAT Asn Asn 710 AGA AAT Arg Asn 725 GTT TAT Val Tyr GCT AGT Ala Ser ATT GAA Ile Glu AAT ATA Asn Ile 790 AAA TCT Lys Ser 805 CAT TAT His Tyr Ser

GCT

Ala 695

CAA

Gin

CCA

Pro

GGG

Gly

CTA

Leu

TAT

Tyr 775

AAA

Lys

CAT

His

GAG

Glu Met Tyr 680 GGG GAA Gly Glu AAT TAT Asn .Tyr AT GAT Met Asp CAT CAT Asp Asp 745 TCA CAT Ser Asp 760 GGA AAT Gly Asn CCT TTA Pro Leu CGA TAT Arg Tyr TAT AGT Tyr Ser 825 Lys Ala Asp 685 TCT GCA ATA Ser Ala Ile 700 AGA GIT CAT Arg Val Asp 715 ATA TAT ATA Ile Tyr Ile ACT ATC CCA Thr Ile Pro GAA ATT CAA Glu Ile Gin 765 AGT TTC GUT Ser Phe Val 780 AAT TAT CTA Asn Tyr Val 795 AAG AAA ACG Lys Lys Thr GCT AGG GAA Ala Arg Glu Ser

ACA

Thr

AT

Ile

AGA

Arg

GAG

Glu 750

GAA

Glu

GCT

Ala

AAG

Lys

GTC

Val

CAA

Gin 830 3499 3547 3595 3643 3691 3739 3787 3835 3883 3935 3995 4055 4074 IAA MTAAAACI GC AGAT ATITAGCATG Ala GTATrTAA GAATAATAA TAIGrTGMS CGTTAGC WTIgTGGMrAA GGCAATICA i TITAT=ICG TCTAAGT TGATGGGCAT GGGATATGT CACCATCCAA GCTINGGG GGTANAAAA TCCAATIT INFORMATION FOR SDQ ID SEQUENCE CHARACERISTICS: LENGTH: 462 amino acids TYPE: amino acid WO 97/46105 PCT/EP97/02737 -67- TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Gin Arg Met Glu 1 5 Val Val Thr Arg Thr Leu Asn Asn Val Val Ser Lys Tyr Thr Asn Asp Phe Lys Glu Asp Gly Glu Glu Trp Arg Phe Leu Asp Asn Lys 100 Phe Ser Met Ala Gly 115 Ile Asp Lys Ile Phe 130 Tyr Lys Asn Val Glu 145 Glu Gly Asn Thr Ile 165 Phe Leu Gly Lys Asp 180 Thr Ala Gin Gin Val 195 Val Pro Ser Gly Lys 210 Leu Asn Asn Asn Glu 225 His Val Asp Lys Val 245 Gin Val Glu Gly Thr DESCRIPTION: SEQ ID Gly Lys Leu Phe Val Val Ser Lys Thr Leu Val Ile Leu Lys 70 Pro Asn Ser Asp Pro 150 Asn Met Ser Gly Tyr 230 Ser Leu Leu Lys Gin 55 Gly Pro Asp Cys Lys 135 Ala Ser Lys Ser Ser 215 Lys Lys Lys Leu Ala 40 Asn Lys Ala Ile Glu 120 Ala Thr Asp Phe Lys 200 Thr Met Val Lys Ser 25 Asp Leu Ala Thr Lys 105 Asp Asn Ile Ala Asp 185 Lys Thr Leu Val Ser Thr Val Gin Leu Lys Ile Lys Glu 75 Glu Lys Thr Asn Glu Ile Leu Ser Gly Phe 155 Met Ala 170 Ser Tyr Arg Val Pro Thr Ile Asp 235 Lys Lys 250 Leu Asp Tyr Asn Pro Trp Gly Tyr Lys Ser 140 Asn Gin Leu Ile Lys 220 Asn Gly Phe Ser Ile Ile Asn Asp Asn Gly Lys Glu Met Lys Glu 110 Asp Leu 125 Ser Ile Lys Ser Phe Lys Asp Thr 190 Leu Lys 205 Ala Gly Gly Tyr Met Glu Lys Asn Gin Leu Gin Glu Lys Asn Thr Glu Thr Thr 160 Gin Leu Thr Ile Leu 240 Leu Ile j, WO 97/46105 PCTEP97/02737 -68- 260 265 270 Asn Ala Giu Ala His Ser Trp Gly Met Lys Ile Tyr Glu Asp Trp Ala 275 280 285 Lys Asn Leu Thr Ala Ser Gn Arg Glu Ala Leu Asp Gly Tyr Ala Arg 290 295 300 Gin Asp Tyr Lys Glu ie Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 305 310 315 320 Gly Asn Glu Lys Leu Asp Ala Gln Leu Lys Asn Ile Ser Asp Ala Leu 325 330 335 Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Val Tyr Arg Trp Cys Gly 340 345 350 Met Pro Glu Phe Gly ITyr Gin le Ser Asp Pro Leu Pro Ser Leu Lys 365 355 360 365 Asp Phe Glu Glu Gin Phe Leu Asn Thr Ile Lys Glu Asp Lys Gly Tyr 370 375 380 Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg 385 390 395 400 Lys Ile Ile Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 405 410 415 Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Lys Glu Ile Leu Leu Asp 420 425 430 Lys Asp Ser Lys Tyr His lie Asp Lys Ala Thr Glu Val Ile Ile Lys 445 435 440 445 Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 450 455 460 INFORMATION FOR SEQ ID NO:21: SEQUENCE

CHARACTERISTICS:

LENGTH: 834 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Lys Asn Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Met Leu 1 5 10 Leu Ala Pro Met Phe Leu Asn Gly Asn Val Asn Ala Val Asn Ala Asp 25 WO 97/46105 PCT/EP97/02737 -69- Ser Lys Ile Asn Gin Ile Ser Thr Thr Gin Giu Asn Gin Gin Lys Glu Met Asn Asp Gin Phe Gly Glu 145 Lys Ile Phe Asn Thr.

225 Ile Gly Asp Asn 4 Asp Asn Gin Ser Thr Lys 130 Lys Phe Asp Asn Leu 210 Asp Gln Tyr Pro Ala 290 Arg Leu Gin Ile Phe 115 Ile Glu Asn Ser Lys 195 Phe.

Gly Asn Thr Tyr' 275 Lys Lys Thr Thr Arg 100 Asn Ile Lys Ile Gin 180 Lys Lys Asp Lys Lys 260 Thr 3lu Gly Met Ala Trp Leu Ser Leu Asp 165 Asn Glu Gin Ser Vai 245 Phe Asp' Thr Leu Phe 70 Asn Ile Ser Asn Vai 150 Ser Gin Ser Lys Ile 230 Ala Vai Tyr Phe Leu 55 Ala Ala Gly Lys Lys 135 Pro Lys Ser Gin Met 215 Pro.

Val Ser Glu Asn.

295 Gly Pro Leu Leu Asp 120 Gly Ile Thr Gin Glu 200 Lys Asp Lys Asn Lys 280 Pro Tyr Thr Leu Ile 105 Glu Lys Lys Phe Gin 185 Phe Arg Leu Trp Pro 265 Ala Leu Tyr Phe Lys Arg Asp Asn 75 Asp Lys Lys 90 Gin Arg Lys Gin Ala Ile Glu Lys Gin 140 Ile Giu Tyr 155 Lys Giu Leu 170 Vai Gin Leu Leu Ala Lys Asp Ile Asp 220 Trp Giu Glu 235 Asp Asp Ser 250 Leu Asp Ser Ala Arg Asp Val Ala Ala 300 Gly Thr Phe Tyr Gin Gin Giu Tyr Glu Thr Gly Asp 110 Ile Giu Ile Asp 125 Val Val His Leu Gin Ser Asp Thr 160 Lys Leu Phe Lys 175 Arg Asn Pro Glu 190 Ala Ser Lys Thr 205 3iu Asp Thr Asp Asn Gly Tyr Thr 240 Leu Ala Ser Lys 255 His Thr Val Gly 270 Leu Asp Leu Ser 285 ?he Pro Ser Val Asn 305 Vai Ser Met Glu Lys Val Ile Leu Ser Pro Asn Giu Asn 310 315 Leu Ser 320 WO 97/46105 PCT/EP97/02737 Asn Ser Val Glu Gly Ala Gly Val Ser 355 Gly Thr Ser 370 Tyr Leu Asn 385 Tyr Asp Val Ala Thr Ile Gly Asp Ser 435 Met Asp Asp 450 Asn Gin Leu 465 Asp Gly Val Gly Glu Trp Ile Ile Val 515 Lys Asp Tyr 530 Asp Thr Leu 545 Leu Leu Tyr Tyr Leu Asp Thr Thr Gly 595 His Ser Glu Ala Tyr Gin Asn Thr 375 Val Arg 390 Thr Thr Lys Ser Glu Ile Ser His 455 Asn Lys 470 Ile Arg Val Thr Gly Lys Pro Glu 535 Ser Tyr 550 Asp Lys Thr Ala Lys Asp Ser Gly His 360 Ser Tyr Ser Asn Gly 440 Pro Pro Asp Gin Gin 520 Asp Pro Pro Lys Val Thr Asn 330 Gly Gly 345 Ser Glu Gin Phe Asn Asn Phe Val 410 Ser Thr 425 Glu Asn Ile Thr Ile Met Thr His 490 Gin Ile 505 Val Ala Lys Thr Asp Glu Ile Tyr 570 Glu Val 585 Asn His Trp Ser Tyr Thr Asn Thr Gly Leu 350 Ala Gin 365 Ala Ser Thr Gly Asn Asn Arg Ile 430 Ala Ile 445 Lys Gin Thr Asp Ile Val Lys Thr 510 Arg Val 525 Leu Thr Glu Thr Ser Val Gin Ile 590 Asp Val Phe Trp Gly Ile 400 Ile Pro Ser Val Thr 480 Gly Ser Ala Lys Gly 560 Thr Asp Leu 600 605 Thr Pro Lys Met Asn Phe Thr Ile Lys Met Ala Ser Leu Tyr Asp Gly WO 97/46105 PCT/EP97/02737 -71 610 615 620 Ala Glu Asn Asn His Asn Ser Leu Gly Thr Trp Tyr Leu Thr Tyr Asn 625 630 635 640 Val Ala Gly Gly Asn Thr Gly Lys Arg Gin Tyr Arg Ser Ala His Ser 645 650 655 Cys Ala His Val Ala Leu Ser Ser Glu Ala Lys Lys Lys Leu Asn Gin 660 665 670 Asn Ala Asn Tyr Tyr Leu Ser Met Tyr Met Lys Ala Asp Ser Thr Thr 675 680 685 Glu Pro Thr Ile Glu Val Ala Gly Glu Lys Ser Ala Ile Thr Ser Lys 690 695 700 Lys Val Lys Leu Asn Asn Gin Asn Tyr Gin Arg Val Asp Ile Leu Val 705 710 715 720 Lys Asn Ser Glu Arg Asn Pro Met Asp Lys Ile Tyr Ile Arg Gly Asn 725 730 735 Gly Thr Thr Asn Val Tyr Gly Asp Asp Val Thr Ile Pro Glu Val Ser 740 745 750 Ala Ile Asn Pro Ala Ser Leu Ser Asp Glu Glu Ile Gin Glu Ile Phe 755 760 765 Lys Asp Ser Thr Ile Glu Tyr Gly Asn Pro Ser Phe Val Ala Asp Ala 770 775 780 Val Thr Phe Lys Asn Ile Lys Pro Leu Gin Asn Tyr Val Lys Glu Tyr 785 790 795 800 Glu Ile Tyr His Lys Ser His Arg Tyr Glu Lys Lys Thr Val Phe Asp 805 810 815 Ile Met Gly Val His Tyr Glu Tyr Ser Ile Ala Arg Glu Gin Lys Lys 820 825 830 Ala Ala INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 4041 base pairs TYPE: nucleic acid STRANDENJESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 97/46105 PCT/EP97/02737 -72- (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 1..4038 OTH IFORMATION: /prOduct= "VIP1A(a)/VIp2A(a) fusion product" (xi) SEQUENCE DESCRIPrION: SEQ ID NO:22: ATG AAA Met Lys 835 AGA ATG GAG Arg Met Glu AAG TI3 TT ATG GMO Lys Leu Phe Met Val 845 TI CIT ACT ACA CIT Leu Leu Ser Thr Val 860 GTA CIT ACT AAA ACT GTA Val Val Thr Lys Thr Val 855 TCA AAA AAA TrA CAA Ser Lys Lys Leu Gin 850 TICI TCT ATA TCT ITA Phe Ser Ile Ser Leu 865 PTA AAT AAT Leu Asn Asn

GAA

Glu 870 GIT ATA AAA GCT Val Ile Lys Ala AAC TM (AA AAT Asn Leu Gin Asn 890

GAA

Glu 875 CAA TA AAT Gin Leu Asn AGT AAA TAT ACT Ser Lys Tyr Thr 885 CTA AAA ATC ACT Leu Lys Ile Thr ATA AAT TCT CAA Ile Asn Ser Gin 880 GAC AAG CTA GAG Asp Lys Vai Glu 895 GGG AAA GAA AAA Gly Lys Giu Lys GAT TIT Asp Phe 900 GAA AAA Glu Lys 915 AAA GAA GAT AAG Lys Glu Asp Lys

GAA

Glu 905 AAA GCG AAA GAA Lys Ala Lys Glu

TGG

91p GAG TGG AAA Glu Trp Lys CTA ACT Leu Thr 920 GCT ACT GAA Ala Thr Glu

AAA

LyS 925 GGA AM AI3 MAT Gly Lys Met Asn PIT PTA GAT AAT Phe Leu Asp Asn AAA AAT Lys Asn 935 GAT ATA AAG Asp Ile Lys

ACA

Thr 940 AAT TAT AAA Asn Tyr Lys GAA ATT ACT Glu Ile Thr 945 'rrr TcT ATG Phe Ser Met AIT GAT AAG Ile Asp Lys 965

GCA

Ala 950 GGC TCA PIT Gly Ser Phe GAA GAT GAA Glu Asp Glu 955 ATA AAA GAT TTA AAA GAA Ile Lys Asp Leu Lys Glu 960 336 384 432 ATG IT GAT AAA Met Phe Asp Lys

ACC

Thr 970 AAT CTA TCA AAT Asn Leu Ser Asn

'TCT

Ser 975 AIT ATc ACC Ile Ile Thr TAT AAA Tyr Lys 980 AAT GTGl GAA CCG Asn Vai Glu Pro ACA APT GGA Thr Ile Gly GAT GCA ATG Asp Ala Met PIT AAT Phe Asn 990 GCA CAG Ala Gin 1005

GAA

Glu 995 GGT AAT ACG AIT Gly Asn Thr Ile AAA TCT TA ACA Lys Ser Leu Thr T AAA GAA CAA Phe Lys Giu Gin 1010 480 528 AAT TCT Asn Ser 1000 PT PTA GAT AGG GAT ATT MG PIT GAT ACT TAT CTA GAT AmO CAT PTA Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Tyr Leu Asp Thr His Leu t. WO 97/46105 PCT/EP97/02737 -73- 1015 ACT GCT CAA CAA GT TCC Thr Ala Gin Gin Val Ser 1030 1020 AGT AAA GAA AGA GIT AT Ser Lys Giu Arg Val Ile 1035 1025 TMX AAG GTr ACG Leu Lys Val Thr 1040 GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA ACA Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr 1045 1050 TA AAT AAT AGT GAA TAC AAA ATG CTC ATT GAT Leu Asn Asn Ser Giu Tyr Lys Met Leu Ile Asp 1060 1065 CAT GTA GAT AAE GTA TCA AAA GIG GTG AAA AAA His Val Asp Lys Val Ser Lys Val Val Lys Lys 1075 1080 1085 CAA ATr GAA GGG ACT TA AAA AAG AGT CT GAC Gin Ile Glu Gly Thr Leu Lys Lys Ser Leu Asp 1095 1100 AAT GCT GAA GCG CAT AGC TGG GGT ATG AAG AAT Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn 1110 1115 AAA GAT 'TA ACC GAT TOG CAA AGG GAA GCr TTA Lys Asp Leu Thr Asp Ser Gin Arg Giu Ala Leu 1125 1130 CAA GAT TAT AAA GAA ATC AAT AAT TAT TrA AGA Gin Asp Tyr Lys Giu Ile Asn Asn Tyr Leu Arg 1140 1145 GGA AAT GAA AAA CTA CAT GCT CAA ATA AAA AAT Gly Asn Glu Lys Leu Asp Ala Gin Ile Lys Asn 1155 1160 116f GGG AAG AAA CCA ATA CCG GAA AAT AT ACT GT1 Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Val 1175 1180 ATG CCG GAA TT GGT TAT CAA AT AGT CAT CCG Met Pro Glu Phe Gly Tyr Gin Ile Ser Asp Pro 1190 1195 CAT 'Tr GAA GAA CAA TTr TTA AAT ACA ATC AAA Asp Phe Glu Giu Gin Phe Leu Asn Thr Ile Lys 1205 1210 ATG AGT ACA AGC TrA TCG AT GAA CGT CT GCA Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala AAA GCA GGT GIC NT Lys Ala Gly Val 1055 AAT GGG TAT ATG Asn Gly Tyr Met 1070 GGG GIG CAG TGC Gly Val Glu Cys Trr AAA AAT GAT Ile

GTC

Val

TA

Leu 1090

ATA

Phe Lys Asn Asp Ile 1105 TAT GAA GAG TGG GCT Tyr Glu Glu Trp Ala 1120 CAT GGG TAT GCT AGG Asp Gly Tyr Ala Arg 1135 AAT CAA GGC GCA AGT Asn Gin Gly Gly Ser 1150 AT T=T CAT GCT TTA Ile Ser Asp Ala Leu 5 1170 TAT AGA TGG XT GGC Tyr Arg Trp Cys Gly 1185 TrA CCT TOT TA AAA Leu Pro Ser Leu Lys 1200 GAA GAC AAA GGA TAT Glu Asp Lys Gly Tyr 1215 GCT 'IT GGA T AGA Ala Phe Gly Ser Arg 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1220 AAA AT ATA ITA 1225 CGA TA CAA GIT CCC 1230 AAA GGA ACT ACG GGT GCG TAT c WO 97/46105 PCTEP97/02737 -74- Lys Ile Ile Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 1235 1240 1245 1250 TPA AGT GCC AT Leu Ser Ala Ile GGT GGA Gly Gly 1255 TIT GCA AGT GAA AAA Phe Ala Ser Glu Lys 1260 GAG ATC CTA CTT GAT Glu Ile Leu Leu Asp 1265 1296 AAA GAT AGT AAA TAT Lys Asp Ser Lys Tyr 1270 CAT ATT GAT His Ile Asp AAA GTA Lys Val 1275 ACA GAG GTA Thr Glu Val AT ATT AAA Ile Ile Lys 1280 1344 GGT GT AAG CGA Gly Val Lys Arg 1285 TAT GTA GTG GAT GCA ACA TA TA Tyr Val Val Asp Ala Thr Leu Leu 1290 ACA AAT ATG AAA Thr Asn Met Lys 1295 1392 AAT AIG AAG Asn Met Lys 1300 AAA AAG TTA Lys Lys Leu GCA AGT Ala Ser 1305 GTT GTA ACG Val Val Thr 'IGT ACG TTA TA GCT Cys Thr Leu Leu Ala 1310 1440 CCT ATG Pro Met 1315 TPIT TIG AAT GGA AAT GITG AAT GCT GIT TAC GCA GAC AGC Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp Ser

AAA

Lys 1330 1488 1320 1325 ACA AAT CAA ATT TCT ACA ACA CAG Thr Asn Gin Ile Ser Thr Thr Gin 1335 AAA AAT CAA CAG Lys Ash Gin Gin 1340 AAA GAG ATG GAC Lys Glu Met Asp 1345 1536 CGA AAA GGA Arg Lys Gly TTA CT GGG TAT TAT Leu Leu Gly Tyr Tyr 1350 TIC AAA Phe Lys 1355 GGA AAA GAT Gly Lys Asp TIT AGT AAT Phe Ser Asn 1360 1584 CIT ACT AG TIT GCA CCG ACA Leu Thr Met Phe Ala Pro Thr 1365 CGT GAT Arg Asp 1370 AGT ACT CIT AT TAT GAT CAA Ser Thr Leu Ile Tyr Asp Gin 1375 1632 CAA ACA GCA Gin Thr Ala 1380 AAT AAA CTA Asn Lys Leu TTA GAT AAA Leu Asp Lys 1385 AAA CAA CAA GAA Lys Gin Gin Glu 1390 TAT CAG TT Tyr Gin Ser 1680 ATT CGT Ile Arg 1395 TGG AT GGT Trp Ile Gly TIM AT Leu Ile 1400 CAG AGT AAA Gin Ser Lys GAA ACG Glu Thr 1405 GGA GAT TC Gly Asp Phe

ACA

Thr 1410 1728 TIT AAC TTA ICT GAG GAT GAA CAG GCA AT ATA GAA AC AAT GGG AAA Phe Asn Leu Ser Glu Asp Glu Gin Ala Ile Ile Glu Ile Asn Gly Lys 1776 1415 1420 1425 ATp A'T TTr Ile Ile Ser AAT AAA GGG AAA Asn Lys Gly Lys 1430 GAA AAG CAA GIT G'IC Glu Lys Gin Val Val 1435 ATA GAG TAT CAA 'ITCA Ile Glu Tyr Gin Ser 1450 CAT TA GAA AAA His Leu Glu Lys 1440 GAT ACA AAA IT Asp Thr Lys Phe 1455 1824 GGA AAA TTA GIT CCA ATC AAA Gly Lys Leu Val Pro Ile Lys 1445 1872 WO W97/46105 PCT/EP97/02737 AAT NIT GAC Asn Ile Asp 1460 AGT CAA AAC Ser Gin Asn 1475 GAA T AAC Glu Phe Asn AGT AAA ACA TIT AAA Ser Lys Thr Phe Lys 1465 CAA CIT AAA TTA T AAA ATA CAT Glu Leu Lys Leu Phe Lys Ile Asp 1470 1920 CAA CCC CAC CAA GTC CAG Gin Pro Gin Gin Val Gin 1480 CAA GAT GAA Gin Asp Glu 1485 CTc AGA AAT CCT Leu Arg Asn Pro 1490 1968 AAG AAA GAA Lys Lys Glu 1495 TCA CAG GAA Ser Gin Glu TIC TA Phe Leu 1500 GCG AAA CCA Ala Lys Pro TCG AAA Ser Lys 1505 2016 ATA AAT CIT TI ACT CAA AAA ATG Ile Asn Leu Phe Thr Gin Lys Met 1510 AAA AGG GAA ATT Lys Arg Glu Ile 1515 GAT GAA GAC ACG Asp Giu Asp Thr 1520 GAA AAT GGG TAT Glu Asn Gly Tyr 1535 2064 GAT ACG GAT GGG Asp Thr Asp Gly 1525 CAC TCT AT Asp Ser Ile CCr GAC CT TGG GAA Pro Asp Leu Trp Glu 1530 2112 ACG NIT CAA AAT AGA ATC GCT GTA AAG TG GAC Thr Ile Gin Asn Arg Ile Ala Val Lys Trp Asp CAT TCT Asp Ser 1550 CIA GCA AGT Leu Ala Ser 2160 1540 1545 AAA GGG TAT ACG Lys Gly Tyr Thr 1555 CGT CAT COT TAT Gly Asp Pro Tyr AAA PIT GTr Lys Phe Val 1560 TCA AAT CCA Ser Asn Pro CrA GAA ACT CAC ACA Leu Giu Ser His Thr 1565 ar Vai 1570 2208 2256 ACA CAT Thr Asp 1575 TAT GAA AAG Tyr Giu Lys GCA CCA AGA Ala Ala Arg 1580 CAT CrA CAT TTG Asp Leu Asp Leu 1585 !CA AAT CCA Ser Asn Ala AAG GAA ACG Lys Giu Thr 1590 PIT AAC CCA TI'G Phe Asn Pro Leu 1595 GTA GCT GCT Val Ala Ala TIT CCA ACT Phe Pro Ser 1600 2304 GTG AAT GT ACT ATG GAA Val Asn Val Ser Met Glu 1605 AAG GTG ATA TrA TCA Lys Val Ile Leu Ser 1610 CCA AAT GAA AAT ITA Pro Asn Giu Asn Leu 1615 2352 TCC AAT ACT GTA CAG TT CAT Ser Asn Ser Val Glu Ser His TCA TCC ACG AAT TGG TOT TAT ACA AAT 2400 Ser Ser Thr Asn Trp Ser Tyr Thr Asn 1630 1620 1625 ACA GAA Thr Glu 1635 GGT GCT TCT GTT GAA GCG GGG AT Gly Ala Ser Vai Giu Ala Gly Ile 1640 GGA CCA Cly Pro 1645 AAA CGT AT TCG Lys Gly Ile Ser 1650 2448 TIC GGA GTT AGC CTA AAC TAT CAA Phe Gly Val Ser Val Asn Tyr Gin 1655 TGG CGA ACA TCT ACA GGA AAT ACT Trp Cly Thr Ser Thr Cly Asn Thr 1670 CAC TCT GAA ACA G'T CCA CAA GAA His Ser Clu Thr Val Ala Gin Glu 1660 1665 1CC CAA TIC AAT ACG GCT ICA GCG Ser Gin Phe Asn Thr Ala Ser Ala 1675 1680 2496 2544 1. WO 97/46105 PCT/EP97/02737 GGA TAT TA AAT Gly Tyr Leu Asn 1685 GCA AAT GTT CGA TAT Ala Asn Val Arg Tyr 1690 AAC AAT GTA GGA ACT GGT GCC Asn Asn Val Gly Thr Gly Ala 1695 2592 ATC TAC GAT Ile Tyr Asp 1700 GTA AAA CCT Val Lys Pro ACA ACA Thr Thr 1705 AGT TIT GTA Ser Phe Val TrTA AAT Leu Asn 1710 AAC GAT ACT Asn Asp Thr 2640 ATC GCA Ile Ala 1715 ACT AT ACG GCG AAA TT AAT Thr Ile Thr Ala Lys Ser Asn 1720 TCT ACA GCC Ser Thr Ala 1725 TIA AAT ATA TCTr Leu Asn Ile Ser 1730 2688 CCT GGA GAA AGT TAC CCG AAA AAA GGA Pro Gly Glu Ser Tyr Pro Lys Lys Gly 1735 CAA AAT Gin Asn 1740 GGA ATC GCA Gly Ile Ala ATA ACA Ile Thr 1745 2736 2784 TCA A[G GAT GAT TIT AAT 'ICC CAT CCG AIT ACA TA AAT AAA AAA CAA Ser Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys Lys Gin 1750 1755 1760 GTA GAT AAT CTG Val Asp Asn Leu 1765 CTA AAT AAT AAA CCT AG ATG TIG Leu Asn Asn Lys Pro Met Met Leu 1770 GAA ACA AAC CAA Glu Thr Asn Gin 1775 2832 ACA GAT GGT Thr Asp Gly 1780 GT1 TAT AAG Val Tyr Lys ATA AAA Ile Lys 1785 GAT ACA CAT Asp Thr His GGA AAT Gly Asn 1790 ATA GTA ACT Ile Val Thr AAA ACA GCG Lys Thr Ala 1810 GGC GGA Gly Gly 1795 GAA TGG AAT Glu Trp Asn GGT GTC ATA Gly Val Ile 1800 CAA CAA ATC AAG GCT Gin Gin Ile Lys Ala 1805 2880 2928 2976 3024 'ITCT AT AT GTG GAT GAT GGG GAA Ser Ile Ile Val Asp Asp Gly Glu 1815 GCA AAA GAT TAT GAA AAT CCA GAA Ala Lys Asp Tyr Glu Asn Pro Glu 1830 CGT GTA GCA Arg Val Ala 1820 GAT AAA ACA Asp Lys Thr 1835 GAA AAA CGT GTA GC Glu Lys Arg Val Ala 1825 CCG TCT 'TA ACT TTA Pro Ser Leu Thr Leu 1840 AAA GAT GCC CIG AAG CTT TCA TAT CCA GAT GAA ATA AAA GAA ATA GAG Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu Ile Glu 1845 1850 1855 GGA 'TA TTA TAT TAT AAA AAC AAA CCG ATA TAC GAA TCG AGC GIT A'IG Gly Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser Val Met 1860 1865 1870 ACT TAC TA GAT GAA AAT ACA GCA AAA GAA GIG ACC AAA CAA A AAT Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gin Leu Asn 1875 1880 1885 1890 GAT ACC ACT CGGG AAA IT AAA GAT GTA AGT CAT 'TA TAT GAT GTA AAA Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp Val Lys 3072 3120 3168 3216 WO 97/46105 PCT/EP97/02737 -77- 1900 189r CIG ACT CCA AAA ATG Leu Thr Pro Lys Met 1910 AAT GCT GAG TCT AAT Asn Ala Giu Ser Asn.

1925 1905 AAT CIT ACA ATC Asn Val Thr Ile 191' GAT AAC TCA Apr Asp Asn Ser Ile 1930 PAA TTG TCT ATA CT TAT GAT Lys Leu Ser Ile Leu Tyr Asp 5 1920 GGT AAA TGG ACA AAC ACA AAT Gly Lys Trp Thr Asn Thr Asn 1935 AAA CAA TAT TCT TCr AAT AAT Lys Gin Tyr Ser Ser Asn Asn 1950 AMI GIT TCA GGT OGA AAT MC GGA Ile Val Ser Gly Gly Asn Asn Gly 1940 1945 CCG GAT GCT AAT [rG ACA 'TA AAT Pro Asp Ala Asn Leu Thr Leu Asn 1955 1960 AAA AAT CGT GAC TAT TAT ATA AGT LYs Asn Arg Asp Tyr Tyr Ile Ser 1975 3264 3312 3360 3408 3456 3504 ACA GAT GCT CAA GAA AAA Thr Asp Ala Gin Glu Lys 1965 'ITA AAT Leu Asn 1970 ITA TAT ATG AAG Leu Tyr Met Lys 1980 TCA GAA AAA AAC Ser Giu Lys Asn 1985 ACA CAA TGT GAG AqT ACT Thr Gin Cys Giu Ile Thr 1990 ATA GAT GGG GAG Ile Asp Gly Glu 1995 API TAT CCG Ile Tyr Pro ATC ACT ACA Ile Thr Thr 2000 AAA ACA GTG AAT Lys Thr Val Asn 2005 (3W AAT AAA GAC AAT TAC Val Asn Lys Asp Asn Tyr 2010 AAA AGA 'ITA GAT AIT ATA Lys Arg Leu Asp Ile Ile 2015 GCT CAT AAT Ala His Asn 2020 AAT GAT GAA Asn Asp Glu 2035 GCA WCA ATA Ala Ser Ile TAT AGT AGG Tyr Ser Arg ATA AAA AGT Ile Lys Ser AAT CCA Asn Pro 2025 ATA ACT TA TIT TG Ile Thr Leu Phe Trp 2040 AAA CCG GAA AAT ITA Lys Pro Giu Asn Leu 2055 ATT

TCA

Ile Ser Ser CAT CAT ATr Asp Asp Ile 2041 ACA GAT TCA Thr Asp Ser 2060 CIT CAT ATr AAA ACG Leu His Ile Lys Thr 2030 TCr ATA ACA GAT GrA Ser Ile Thr Asp Val 2050 GAA A'IT AAA CAG AlT Glu Ile Lys Gin Ile 2065 3552 3600 3648 3696 3744 3792 3840 TAT GGT AlT Tyr Gly Ile 2070 AAG rI' GAA GAT Lys Leu Giu Asp 2075 GGA AM CIT Gly Ile Leu AT GAT AAA Ile Asp Lys 2080 AAA GGT GGG AIT Lys Gly Giy Ile 2085 ATT GAA CCA TIM Ile Giu Pro Leu 2100 CAT TAT GGT His Tyr Gly GAA TIT Glu Phe 2090 ATT AAT GAA Ile Asn Glu GCT AGT IT AAT Ala Ser Phe Asn 2095 CAA AAT TAT GTG ACC Gin Asn Tyr Val Thr 2105 AAA TAT GAA IT ACT TAT Aar Lys Tyr Glu Vai Thr Tyr Ser 2110 AGT GAG WrA GGA CCA AAC GM AGT GAC ACA CIT GA AGT CAT AMA Ap 3888 t WO 97/46105 PCT/EP97/02737 -78- Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu 2115 2120 2125 TAC AAG GAT GGG ACA ATT AAA TPIT GAT TIT ACC AAA Tyr Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys 2135 2140 GAA CAA GGA TA TIT TAT GAC AGT GGA TTA AAT TGG Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp 2150 2155 AAT GCT ATT ACT TAT GAT GGT AAA GAG ATG AAT GTT Asn Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val 2165 2170 AAT AAA TAG Asn Lys 2180 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 1346 amino acids TYPE: amino acid TOPOLOGY: linear Ser Asp Lys Ile 2130 TAT AGT AAA AAT Tyr Ser Lys Asn 2145 GAC TIT AAA ATT Asp Phe Lys Ile 2160 TIT CAT AGA TAT Phe His Arg Tyr 2175 3936 3984 4032 4041 (ii) MOLECULE TYPE: protein Met 1 Val Leu Ser Asp Glu Phe (xi) SEQUENCE Lys Arg Met Glu 5 Val Thr Lys Thr Asn Asn Glu Val Lys Tyr Thr Asn Phe Lys Glu Asp Lys Glu Trp Lys Leu Asp Asn Lys 100 DESCRIPTION: SEQ ID Gly Lys Leu Phe Met Val Leu Leu Ser Thr Ile Lys Ala Glu Gin 40 Leu Gin Asn Leu Lys 55 Lys Glu Lys Ala Lys 70 Leu Thr Ala Thr Glu Asn Asp Ile Lys Thr NO:23: Val Ser Lys Val Phe Ser Leu Asn Ile Ile Thr Asp Glu Trp Gly Lys Gly Lys Asn Tyr Lys 105 110 Phe Ser Met 115 Ala Gly Ser Phe Glu 120 Asp Glu Ile Lys Asp Leu Lys Glu 125 S WO 97/46105 PCT/EP97/02737 -79 Ile Asp 130 Tyr Lys 145 Glu Gly Phe Leu Thr Ala Val Pro 210 Leu Asn 225 His Val Gin Ile Asn Ala Lys Asp 290 Gin Asp 305 Gly Asn Gly Lys Met Pro Asp Phe 370 Met Ser 385 Lys Ile Lys Met Asn Val Asn Thr Asp Arg 180 Gin Gin 195 Ser Gly Asn Ser Asp Lys Glu Gly 260 Glu Ala 275 Leu Thr Tyr Lys Glu Lys Lys Pro 340 Glu Phe 355 Glu Glu Thr Ser Ile Leu Phe Glu Ile 165 Asp Val Lys Glu Val 245 Thr His Asp Glu Leu 325 Ile Gly Gin Leu Arg 405 Asp Pro 150 Asn Ile Ser Gly Tyr 230 Ser Leu Ser Ser Ile 310 Asp Pro Tyr Phe Ser 390 Leu Lys Thr 135 Thr Thr Ser Asp Lys Phe.

Ser Lys 200 Ser Thr 215 Lys Met Lys Val Lys Lys Trp Gly 280 Gin Arg 295 Asn Asn Ala Gin Glu Asn Gin Ile 360 Leu Asn 375 Ser Glu Gin Val Asn Ile Ala Asp 185 Glu Thr Leu Val Ser 265 Met Glu Tyr Ile Ile 345 Ser Thr Arg Pro Leu Gly Met 170 Ser Arg Pro Ile Lys 250 Leu Lys Ala Leu Lys 330 Thr Asp Ile Leu Lys 410 Ser Asn 140 Phe Asn 155 Ala Gin Tyr Leu Val Ile Thr Lys 220 Asp Asn 235 Lys Gly Asp Phe Asn Tyr Leu Asp 300 Arg Asn 315 Asn Ile Val Tyr Pro Leu Lys Glu 380 Ala Ala 395 Gly Ser Ser Lys Phe Asp Leu 205 Ala Gly Val Lys Glu 285 Gly Gin Ser Arg Pro 365 Asp Phe Thr Ile Ser Lys Thr 190 Lys Gly Tyr Glu Asn 270 Glu Tyr Gly Asp Trp 350 Ser Lys Gly Gly Ile Leu Glu 175 His Val Val Met Cys 255 Asp Trp Ala Gly Ala 335 Cys Leu Gly Ser Ala 415 Thr Thr 160 Gin Leu Thr Ile Val 240 Leu Ile Ala Arg Ser 320 Leu Gly Lys Tyr Arg 400 Tyr Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Lys Glu Ile Leu Leu Asp o, WO 97/46105 PCT/EP97/02737 420 Lys Asp Ser Lys 435 Tyr His Ile Asp Val Thr Glu Val 440 445 Ile Lys Gly Asn 465 Pro Thr Arg Leu Gin 545 Ile Phe Ile Gly] Asn 625 Ser C Glu F Ile Aspi 6 Val Met Met Asn Lys Thr 530 Thr Arg A~sn Ile -ys 610 Ile ;In 'he sn 90 Lys Lys Phe Gin Gly 515 Met Ala Trp Leu Ser 595 Leu' Asp Asn Asn] Leu I 675 Asp C Arg Tyr Val LyS Leu Ile 500 Leu Phe Asn Ile Ser 580 Asn Val Ser 31n ys 660 ?he ly Lys Asn 485 Ser Leu Ala Lys Gly 565 Glu Lys Pro Lys' Pro 645 Lys Thr( Aspi 47C Gly Thr Gly Pro Leu 550 Leu Asp Gly Ile Thr 630 ln 3iu 3In 3er Vai 455 Ala Asn *Thr Tyr Thr 535 Leu Ile Glu Lys Lys 615 Phe Gin' Ser Lys I Ile 695 Asp Ala Thr Leu Leu Thr Asn Met Lys 460 Sex Val Gin Tyr 520 Arg Asp Gin Gin Glu 600 Ile Lys Val ln Aet 680 Pro Val Asn Lys 505 Phe Asp Lys Ser Ala 585 Lys Glu Glu Gin Glu 665 Lys Asp Val Thr 475 Ala Vai 490 Asn Gin Lys Gly Ser Thr Lys Gin 555 Lys Glu 570 Ile Ile Gin Val Tyr Gin Leu Lys 635 Gin Asp 650 Phe Leu Arg Glu Leu Trp Cys Tyr Gin Lys Leu 540 Gin Thr Glu Vai Ser 620 Leu lu Ala lie lu 700 SThr Ala Lys Asp 525 Ile Glu Gly Ile His 605 Asp' Phe Leu Lys AspC 685 Glu Leu Asp Glu 510 Phe Tyr Asp Asn 590 Leu Thr] Lys nrg2 ?ro 670 lu k.sn C Leu Ser 495 Met Ser Asp Gin Ala 480 Lys Asp Asn Gin Ser Thr Lys Lys Phe Asp 640 Pro Lys Thr Tyr Thr 705 Ile Gin Asn Arg Ile Ala Vai Lys Trp 710 Asp Ser Leu Ala WO 97/46105 PCT/EP97/02737 -81 Lys Gly Ser Val Ser 785 Thr Phe Trp Gly Ile 865 Ile Pro Ser Val Thr 945 Gly Ser Gly Asp Asn Asn 770 Asn Glu Gly Gly Tyr 850 Tyr Ala Gly Met Asp 930 Asp Gly Ile Tyr Thr Pro Tyr 740 Ala Lys 755 Val Ser Ser Val Gly Ala Val Ser 820 Thr Ser 835 Leu Asn Asp Val Thr Ile Glu Ser 900 Asp Asp 915 Asn Leu Gly Val Glu Trp Ile Val 980 Lys 725 Thr Glu Met Glu Ser 805 Val Thr Ala Lys Thr 885 Tyr Phe Leu Tyr Asn 965 Asp Phe Val Asp Tyr Thr Phe Glu Lys 775 Ser His 790 Val Glu Asn Tyr Gly Asn Asn Val 855 Pro Thr 870 Ala Lys Pro Lys Asn Ser Asn Asn 935 Lys Ile 950 Gly Val Asp Gly Ser Glu Asn 760 Val Ser Ala Gin Thr 840 Arg Thr Ser Lys His 920 Lys Lys Ile Glu Asn Pro 730 Lys Ala 745 Pro Leu Ile Leu Ser Thr Gly Ile 810 His Ser 825 Ser Gin Tyr Asn Ser Phe Asn Ser 890 Gly Gin 905 Pro Ile Pro Met Asp Thr Gin Gin 970 Arg Val 985 Leu Glu Ala Arg Val Ala Ser Pro 780 Asn Trp 795 Gly Pro Glu Thr Phe Asn Asn Val 860 Val Leu 875 Thr Ala Asn Gly Thr Leu Met Leu 940 His Gly 955 Ile Lys Ala Glu Ser Asp Ala 765 Asn Ser Lys Val Thr 845 Gly Asn Leu Ile Asn 925 Glu Asn Ala Lys His Leu 750 Phe Glu Tyr Gly Ala 830 Ala Thr Asn Asn Ala 910 Lys Thr Ile Lys Arg 990 Val Leu Ser Leu Asn 800 Ser Glu Ala Ala Thr 880 Ser Thr Gin Gin Thr 960 Ala Ala Ala Lys Asp 995 Tyr Glu Asn Pro Glu Asp 1000 Lys Thr Pro Ser Leu Thr Leu 1005 t, WO 97/46105 PCTIEP97/02737 -82 Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu Ile Glu 1010 1015 1020 Gly Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser Val Met 1025 1030 1035 1040 Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gin Leu Asn 1045 1050 1055 Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp Val Lys 1060 1065 1070 Leu Thr Pro Lys Met Asn Val Thr Ile Lys Leu Ser Ile Leu Tyr Asp 1075 1080 1085 Asn Ala Glu Ser Asn Asp Asn Ser Ile Gly Lys Trp Thr Asn Thr Asn 1090 1095 1100 Ile Val Ser Gly Gly Asn Asn Gly Lys Lys Gin Tyr Ser Ser Asn Asn 1105 1110 1115 1120 Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gin Glu Lys Leu Asn 1125 1130 1135 Lys Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Met Lys Ser Glu Lys Asn 1140 1145 1150 Thr Gin Cys Glu Ile Thr Ile Asp Gly Glu Ile Tyr Pro Ile Thr Thr 1155 1160 1165 Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp Ile Ile 1170 1175 1180 Ala His Asn Ile Lys Ser Asn Pro Ile Ser Ser Leu His Ile Lys Thr 1185 1190 1195 1200 Asn Asp Glu Ile Thr Leu Phe Trp Asp Asp Ile Ser Ile Thr Asp Val 1205 1210 1215 Ala Ser Ile Lys Pro Glu Asn Leu Thr Asp Ser Glu Ile Lys Gin Ile 1220 1225 1230 Tyr Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile Asp Lys 1235 1240 1245 Lys Gly Gly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala Ser Phe Asn 1250 1255 1260 Ile Glu Pro Leu Gin Asn Tyr Val Thr Lys Tyr Glu Val Thr Tyr Ser 1265 1270 1275 1280 Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu Ser Asp Lys Ile 1285 1290 1295 Tyr Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys 7Lr Ser Lys Asn WO097/46105 PCTIEP97/02737 83 1300 1305 1310 Giu Gin Giy Leu Phe Ty'r Asp Ser Gly Leu Asn Ttp Asp Phe Lys Ile 1315 1320 1325 Asn Ala Ile Thr Ty~r Asp Gly Lys Giu Met Asn Val Phe His Arg Tyr 1330 1335 1340 Asn Lys 1345 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 1399 base pairs TYPE: nucleic acid STRANDEEN~ESS: single EOPOLOC: linear (i i) MOLECULE TIYPE: EM~ (genom-ic) (ix) FEATURE: NME/KEY: misc-feature LOCATION: l. .1386 OTHER INFORMATION: /note= "Maize optimized A.

sequence for VIP2A(a) protein from AB78" (xi) SEQUENCE DESCRIPION: SEQ ID NO:24: ATGAAGCGCA TGGAGGGCAA. GCTTCATG GTGUAGCAAGX ACCG I GCC 'IGAGCACCGT G'ITCAGCATC

GAGCAGCTGA

GACAAGGTGG

GAGAAGGAGT

AAGAACGACA

GACGAGATCA

AGCATCATCA

GAGGGCAACA

GACAKCAAGT

GAGCGCG'rGA

GCCGGCTA

ACATCAACAG

AGGACICAA.

GGAAGCITAC

TCAAGACCAA

AGGACCTGAA

CCTACAAGAA

CCAWAACAG

W GACAGC TA ±±CC73AAK.

1 T CTGAACAA

CCAGAGCAAG

GGAGGACAAG

CGCCACCGAG

CTACAAGGAG

GGAGATCGAC

CGTGAGCCC

CGACGCCATIG

CCTGACACC

GACCGTCCC

CAGCGAGTAC

AGCCI'GC1'G

TACACCAACC

GAGAAGGCCA

AAGGGCAAGA

ATCACCFICA

AAGA'IGITC

ACCACCATCG

GCCCAGTTCA

CACCTGACCG

AGCGGCAAGG

AAGATGCTGA

AGCTCCAGGT

ACAACGAGGT

TCCAGAACCT

AGGAGWGGGG

TAACAACTT

GCATGXGCCGG

ACAAGACCAA

GCPTICAACAA

AGGAGCATr

CCCAGCAGGT

GCAGCACCAC

'ICGACAACGG

GG IGACCAAG

GATCAAGGCC

GAkAGATCACC

CAAGGAGAAG

CCTGGACAAC

CAGCITTCGAG

CC!TAGCAAC

GAGCCTGACC

CCTGACCGC

GAGCAGCAAG

CCCCACCAAG

CTACATIG

120 180 240 300 360 420 480 540 600 660 720 WO 97/46105 WO 9746105PCT/EP97/02737 84

CACGGCACA

ACCC IAAGA

ATGAAGAACT

GGCTACGCCC

GGCAACGAGA

ATCCCCGAGA

AGCGACCCCC

GACAAGGGCC

AGA ICATC

GGCGGC=TC

AAGGTGACCG

AGGTG\AGCAA

AGAGTCTNA

ACGAGGAGTG

GCCAGGAC'TA

AGC1GGACGC

ACATCACCGT

TCCCAGCCT

ACATGAGCAC

CGC)CTGCA

CCAGCGAGAA

AGG1XGATCAT GGTrG LTjAAG

CITCAGAC

GGCCAAGGAC

CAAGGAGATC

CCAGATCAAG

GTACCGCTGG

GAAGGACTTC

CAGCCqrGAGC

GGTCCCAAG

GGAGAWCCTG

CAAGGGCGTG

AAGGGCGG AGTGCCTC'C GACAT AACG CCGAGGCCCA C1XACCGACA GCCNGCGCGA AACAACTACC TGCGCAACCA AACATCAGCG ACGCCCITGG TGCGGCATGC CCGAGTICG GAGGAGCAGT T C!TAACAC AGCGAGCGCC TGGCGCT GGCAGCACCG GCGCCTACCT CTGGACAAGG ACAGCAAG'rA AAGCGC'rACG TGGTGGACYGC

GATCGAGGGC

CACIGGOC

GCCCflUGAC

GGGCGGCAGC

CAAGAAGCCC

CTACCAGAIC

CAWCAAGGAG

CGGCAGCCGC

GAGCGCCATC

CCACA'rCGAC CACCC1rCTG 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1399 ACCAACTAGA T( ITAGCTC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: am-ino acid STRANDEEXNESS: single TIOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATUVRE: NAME/KEY: Peptide LOCATION: 1. .19 OYTHER INFORMATION: /note= "Secretion signal peptide to secrete VIP2 out of a cell" (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Trp Ser Trp Ile Phe Leu Phe Leu Loeu Ser Gly Ala Ala Gly Val 1 5 10 His Cys Leu INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 2655 base pairs TYPE: nucleic acid ~WO 97/46105 85 STRANDEDNESS: single TOXPOLOXGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic ENA..

(iii) HYPOIHEICAL:

NO

PCT/EP97/02737 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .2655 OTHER INFORMATION: /note= "maize optimized MN sequence encoding VIPlA(a)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO :26: ATGXAAGAACA TAAAAGAA Gcfl~GCCAGC TTCC7TGAACG

ACCCAGAAGA

GGCAAGGACT

GACCAGCAGA

IGGATCGGCC

GAGCAGGCCA

GTGTGCACC

AAGTAACA

AACCAGCCCC

AGCCAGGAGT

GAGATCGACG

GGCTACACCA

TACACCAAGT

TACGAGAAGG

GTGGCCGCCWP

AACCIrAGCA

GGCGCCAGCG

TACCAGCACA

GCAACGTGAA

ACCAGCAGAA

TCAGCAACCT

CCGCCAACAA

TACCAGAG

TCATCGAGAT

TGAGAAGGG

TCGACAGCAA

C'GCCG'IGTAC

GGAGAGGAC

GACCATG=mx GCTI~qrGAC

CAAGGAGACC

CAACGGCAAG

CAAGCIGT

GACC'ITCAAG

G'I)~G

T

DACcr

GCCGACAGCA

CGCAAGGGCC

GCCCCCACGC

AAGAAGCAGC

GGCGAC1rC

ATCATCAGJCA

CCCATCAAGA

GAGCTGAAGC

CTCGCAACC

ATCAACC0T

GACAGCATICC

AAG7GCGACG GCACCC GGCCCCCArpG AGACCAACCA GAWCAGCACC

T

ftCTGGGCrA CTACTI2AAG G-IMCAGCAC

CCTGATCAC

AGGAGTACCA GAGCAWCcGC CCICAACCF GAGCGAGGAC ACAAGGGCAA GGAGAAGCAG T CGAGTACr-A GAGCGACACC AGCAGG'IGCA

GCAGGACGAG

TCCTGGCCAA GCCCAGCAAG3 Ar.GACACCGA CACCGACGGC WCAGAACCG CACGCCT

TCGTGAGCAA

CCGCCCGCGA

W CCCAGCGT

ACAGCGTGGA

TGAGGCCGG

GCGAGACCGT~

CCCCCIGGAG AGCCACACCG CCIGGACCTG

AGCAACGCCA

GAACGTGAGC A RGGAGAAGG GAGCCACICG AGCACCAACr CATCGG-CC AAGGGCATCA GGCCCAGGAG

TGGGGCACCA

TIICAAGAT

CCGAG'mCAA

TICACCCAGCA

CCGAC

ACAGCCTGGC

TGGCGACCC

AGGAGACC 1T

'IGATCCTGAG

GGAGCTACAC

GIC71vGGCG

GCACCGGCAA

CGACAGCCAG

CAAGAAGGAG

GATAAGCGC

GGAGGAGAAC

TAGCAAGGGC

CTACACCGAC

CAACCCCT

CCCCAACGAG

CAACACCGAG

GAGCGT 3AAC

CACCAGCCAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 o WO 97/46105 ~.WO97/4105PCT/EP97/02737 86

TICAACACCG

GGCGCCA~r

ACCATCACCG

AAGAAGGGC

ACCCTGAACA

AACCAGACCG

GAGTGGAACG

GGCGAGCGCG

ACCCCAGCC

ATGAG3GGCT

CTAGAGGAGA

AAGGACTA

C1X3AGCATCC

ACCAACAWCG

GCCAACCTGA

AWCAGCCTG

ATATACCCCA

ATCA'ICGCCC

GAGATCACCC

AACCIT3ACCG

GGCATCCTGA

TqVAACATCG

CCAGCGCCGG

ACGACG'PGAA

CCAAGWCGAA

AGAACGGCAT

AGAAGCAGGT

ACGGCGTCA

GCTATCCA

TGCCGAGAA

TACCCTGAA

'1XYI=ACTA

ACACCGCCAA

GCCACCI=A

TG~TACGACAA

TGAGCGGCGG

CCCTGAACAC

ACATGAAGAG

CTACCTGAAC GCCAACGTIMC GCCCACCACC AGCFiqCG'IGC TTCCACCGCC CIXAACATCA CGCCATCACC AGCA'IGGACG

GGACAACCTG

CAAGATCAAG

GCAGATCAAG

GCGCGTGCC

GGACGCCCTG

CAAGAACAAG

GGAGGTGACC

CGAC IAAG

CGCCGAGAGC

CAACAACGGC

CGACGCCCAG

CGAGAAGAAC

CTGAACAACA

GACACCCACG

GCCAAGACCG

GCCAAGGACT

AAGCTGAGCT

CCCATCTACG

AAGCAGCTGA

CTGACCCCCA

AACGACAACA

AAGAAGCAGT

GAGAAGUMG

ACCCAG'IGCG

GrAACAAGG

ATAGCAGCC

ATrACCGACG

TACAG'ICG=

CACTACGGCG

ACCAAGTrA

GCTACAACAA

TAACAACGA

GCCC~dGCGA

ACTCAACAG

AGCCCATGAT

GCAAOAWT

CCAGCATCAT

ACGAGAACCC

ACCCCGACGA

AGAGCAGCGT

ACGACACCAC

AGATGAACGT

GCATCGGCAA

ACAGCAGCAA

ACAAGAACCG

AGATCACCAT

ACAACTACAA

=CCATCAA

CGCCAGCAT

ACGGCATCAA

AG7I'A CAA

AGGIGACCTA

TITACAAGGA

TGT1TCTACGA

AGGAGATGAA

CGGCACC

CACCATCGCC

GAGCI'ACCCC

CCACCCCAWC

GCTGGAGACC

GACGGGCGGC

CGTCGACGAC

CGAGGACAAG

GA ECAAGGAG

GAM~ACCAT

CGGCAAG=1

GACCATCAAG

G'rGGACCAAC

CAACCCGAC

CGACTACTAC

CGACGGCGAG

GCGCCTGGAC

GACCAACGAC

CAAGCCCGAG

GC1IGGAGGAC

CGAGGCCAGC

CAGCAGCGAG

CGGCACCATC

CAGCGGCCMG

CGGI3'

T

CCAC

1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2655 TCACCACCAA GACCGIGAAC ACAACATCAA GAGCAACCCC T±'±vIVIGG' CGACATATCG ACAGCGAGAT CMSGCAGATA TCGACAAGAA. AGGCGGCAWC AGCCCCI=C GAACI'ACGTG CIGGGCCCCA ACGWIGAGCGA CACCCIGGAG AGCGACAAGA AAGT1X2GACT TCACCAAGTA CAGCAAGAAC G.AGCAGGGCC AACIGJGGACT TCAAGATCAA ~CCATCACC TACGACGGCA CGCTACAACA AGTAG INFORMATION FOR SEQ ID NO:27: k, WO 97/46105 W097/6105PCT/EP97/02737 87 Wi SEQUENCE CHARACTERISTICS: LENGTH: 1389 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic IM"A (iii) HYPTHTETICAL: NO (ix) FEATURE: N7VMEKEY: misc-feature LOCATION: 1. .1389 OTHER INFORMATION: /note= "maize optimized EMI sequence encoding VIP2A(a)" (xi) SEQUENCE DESCRIPTION: SE)Q ID NO:27: ATGAAGCGCA 'IGAGGGCAA GC TI'CTG GIGAGCAAGA AGC-C=GGT ACCG IGCTGC 'GAGCACCGT GAGCAGCI'GA ACATCAACAG GACAAGGTG~G AGGACFI'AA GAGAAGGAGT GGAAGCITAC AAGAACGACA TCAAGACCAA GACGAGA'rCA AGGACCTGAA AGCAWCAWA CCTACAAG3AA GAGGGCAACA CCATCAACAG GACATCAAGT WGACAGCTA GAGCGCTA TCCTGAAGGT GCCGGCTA TCCTGAACAA CACGTGACA AGGTGAGCAA ACCCIGAAGA AGiAGTCTAGA A'IGAAGAACr ACGAGGAGTG GGCTACGCCC GCCAGGACTA GGCAACGAGA AGC IGGACGC G'FrCAGCATC AGcCICGCTA CCAGAGCAAG TACACCAACC GGAGGACAAG GAGAAGGCCA COCCACCOAG AAGGGCAASA CTACAAGGAG A ECACCrIr-A

GGAGA'ICGAC

CGTGGAGCC

CGACGCCATG

CC)XGGACACC

GACCGTCCC

CAGCGAGTAC

GGTGTGAAG

Cq1IAASAAC

GGCCAAGGAC

CAAGGAGAWC

CCAGATCAAG

AAGA'TGFrC

ACCACCATCG

GCCCAG'PICA

CAC1CCG

AGCGGCAAGG

AAGATGCI7A

AAGGGCGTGG

GACATAACG

ACAACGAGGI

TCAGAACCT

AGGAGT)GGGG

IGMACAACT

GCATAGCCGG

ACAAGACCAA

GC1IrAACAA AGGAGCAGTr

CCCAGCAGGT

GCAGCACCAC

TY-ACAACGG

AGIXGCCTCCA

CCGAGGCCCA

GG IGACCAAG

GA'TCAAGGCC

GAAGATCACC

CAAGGAGAAG

CCTGGACAAC

CAGCTCG

CCTGAflCAAC

GAGCCIGACC

CCTGGACCGC

GAGCAGCAAG

CCCCACCAAG

C TACA EGGTG GA'1XGAGGGC CAGC 1GGGC GGCCCIrGAC

GGGCGGCAGC

CAAGAAGCCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 C'TGACCGACA GCCAGCGCGA AACAACTACC TGCGCAACCA AACATCAGCG ACGCCCTGW t, WO 97/46105 WO 9746105PCTIEP97/02737 88 ATCCCCGAGA ACAWCACCGT GTACCGCIX-=GCXGGC-AtC AGCGACCCCC TCCCAGCCT GAAGGACTTC GAGOAGCAGT GACAAGGGCr ACATG-4AGCAC CAGCC PrAGC AGCGAGC1GCC AAGAWCATCC !flCGCCIGCA GGTGCCCAAG GGCAGCAC'C GC=FC CCAGCGAGAA GGAGATC=T C1XG4,ATAAG AAGC'DACCG AGGIGATCAT CAAGGCCGTG AAGCrACG

ACCAACTAG

INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 2378 base pairs TYPE: nucleic acid STRANDEENSESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: EN~A (genoinc) (iii) HYPOTHETICAL: NO

CCGAG'ITCO

'ICCTfl3MCAC

TGGCCGCCIT

a'IGcCACCr

ACAGCAAGTA

TGGTGGACC

CTACCAGATC

CATCAAGGAG

CGGCACCGC

GAGCGCCATC

CCACATCGAC

CACCCTCrG 1080 1140 1200 1260 1320 1380 1389 (ix) FEATURE: N7A4E/KFX: CDS LO:CATION: 9. .2375 OTIHER INFORMATION: /note= "Native DNA sequence encoding VIP3A(a) protein from AB88 as contained in pCIB7lO4" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AGATGAAC AT1X AAC AAG3 AAT AAT ACT AAA 'ITA AGC ACA AGA CCC TTA CCA Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu pro 1 5 ACT Tr' ATT GAT Ser Phe Ile Asp TAT PIT MAT CCC APT' TAT OCA Tyr Phe Asn Cly Ile Tyr Oly 20 25 PTr CC ACT Phe Ala Thr ACA OCTr OCT Thr Cly Cly OCT AWC Cly Ile CAT CTA Asp Leu AAA GAC Apr A IG AAC ATG~ Lys Asp Ile Met Asn Met Apr PTr AAA ACC GAT Ile Phe Lys Thr Asp ACC CTA CAC CAA Thr Loeu Asp Olu OCT MAA PIG GAT Oly Lys Leu Asp Apr TA MG AAT CAG CAG PTA CIA, AAT Ile Leu Lys Asn Gln Gin Leu Leu Asn 55 COG GTG AAT OGA ACC 'TA AAT OAT CT Cly Val Asn Cly Ser Leu Asn Asp Leu 70 OAT ArT TcT Asp Ile Ser ATC GCA CAO Ile Ala Gin i'

C

WO 97/46105 PCT/EP97/02737 -89-

GGA.

Gly.

GAA

Glu

AAT.

Asn GAT I Asp'

AGT

Ser AAT I Asn'

AGO.

Arg 175

GAA.

Glu

GAT

Asp!

GAT

Asp

OTA

Val TrA Leu 255

GTT

Val AAC TA AAT Asn Leu Asn CAA AAT CAA Glh Asn Gin ACG ATG CTT Thr Met Leu Sm ATG AAA Val Met Lys 130 AAA CAA TTG Lys Gin Leu 145 GTA CT ATT Val Leu Ile 160 AT AAA TAT Ile Lys Tyr ACT AmT TCA Thr Ser Ser GAG TTA ACT Glu Leu Thr 210 GTM GAT GGT Val Asp Gly 225 OGA AAT AAT Gly Asn Asn 240 AT ACT AAA Ile Thr Lys TAT AAC TWr Tyr Asn Phe TA TCT Leu Ser AAT GAT Asn Asp TAT TA Tyr Leu TAT GCG Tyr Ala AT TOT Ile Ser 150 ACA CIT Thr Leu 165 GAA AAA Glu Lys AAA AAG Lys Lys ACT GAA Thr Glu AAG GAA Lys Glu OTT AAT Val Asn Car AAA Pro Lys 120 OTA AGT Leu Ser 135 GAT AAG Asp Lys ACT GAA Thr Glu TIT GAG Phe Glu GAT GGC Asp Gly 200 OTA GCG Leu Ala 215 CT AAT Leu Asn TCA GCT Ser Ala ACA AGT Thr Ser ACA OCT Thr Ala 280 AAA AT GCA AAT Lys Ile Ala Asn CIC GAT GCG ATA Leu Asp Ala Ile 110 TOF ATG TIG AGT Ser Met Leu Ser 125 ATA GAA TAC TTA Ile Giu Tyr Leu 140 ATT NT AAT GTA Ile Ile Asn Val 155 CT GCG TAT CAA Pro Ala Tyr Gin ACT PT GOT ACA Thr Phe Ala Thr 190 GCA GAT MIT CIT Ala Asp Ile Leu 205 GTA ACA AAA AAT Val Thr Lys Asn 220 CAC GAT CA ATG His Asp Val Met 235 ACT GCA TG GAA Thr Ala Ser Glu GAG TCI GGA AAT Glu Val Gly Asn 270 GCC CAA GOT TT Ala Gin Ala Phe 285 290 338 386 434 482 530 578 626 674 722 770 818 866 T GAA Phe Glu PTA =1X Leu Phe GAA AAT Glu Asn 260 TA AT Leu Ile 275 CIT ACT TA ACA ACA TGC CGA AAA TA TA GGC PTA GCA OAT AT OAT Leu Thr Leu Thr Thr Oys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp WO 97/46105 PCT/EP97/02737

TAT

Tyr

AGA

Arg

TAT

Tyr 335

GCT

Ala

AT

Ile

GTC

Val

TIA

Leu

GTA

Vai 415

ATG

Met

ACA

Thr

GAG

Glu

GGT

Gly

CAA

Gin 495

TTA

TCT

Ser 305

AAC

Asn

AAA

Lys

CCA

Pro

GTA

Vai

AAG

Lys 385

TGC

Cys

CCA

Pro

AC?

Thr

GAA

Glu

AGA

Arg 465

ATC

Ile

GAT

Asp

GAA

290

ATT

Ile

ATC

Ile GTr Vai

GGA

Gly

TA

Leu 370

GAT

Asp

CCA

Pro

AAT

Asn ATG AAT Met Asn CTC CCT Leu Pro AAA GGA Lys Gly 340 CAT GCA His Ala 355 AAA GTA Lys Vai TCC TA Ser Leu GAT CAA Asp Gin GAA TAT Glu Tyr 420

GAA

Glu

ACA

Thr 325

AGT

Ser

TM

Leu

TAT

Tyr

TCG

Ser Ser 405

OTA

Vai

GAG

Glu

AAT

Asn GCr Ala 7n.

Phe 485

AGA

Arg

GCA

CAT

His 310

CIT

Leu

GAT

Asp

ATT

Ile

GAG

Glu

GAA

Glu 390

GAA

Glu ATr Ile

GTA

Val

AAG

Lys

AAT

Asn 470

TI

Leu

TA

Leu

ACA

295 TTA AAT Leu Asn TCT AAT Ser Asn GAA GAT Glu Asp GGG 'Tr Gly Phe 360 GCT AAG Ala Lys 375 GT ATr Val Ile CAA ATO Gin Ile ACT AAA Thr Lys ACA GCG Thr Ala 440 AAA AAA Lys Lys 455 GAT GAT Asp Asp ACT CCG Thr Pro NT ACT Ile Thr GAC TrA

AAG

Lys

ACT

Thr

GCA

Ala 345

GAA

Glu

CTA

Leu

TAT

Tyr

TAT

Tyr ATr Ile 425

AAT

Asn

GTA

Val

GGG

Gly

AIT

Ile

TA

Leu 505

AGC

GAA

Glu Trr Phe 330

AAG

Lys ATr Ile

AAA

Lys

GGT

Gly

TAT

Tyr 410

GAT

Asp Phe

GAA

Glu

GIG

Val

AAT

Asn 490

ACA

Thr

AAT

AAA

Lys 315

TOT

Ser

ATG

Met

AGT

Ser

CAA

Gin

GAT

Asp 395

ACA

Thr

TIC

Phe

TAT

Tyr

TCA

Ser

TAT

Tyr 475

GGG

Gly

TGT

Cys

AAA

GAA TIT Glu Phe CCT AAT Pro Asn GG GAA Val Glu 350 GAT TCA Asp Ser 365 TAT CAA Tyr Gin GAT AAA Asp Lys AAC ATA Asn Ile AAA AAA Lys Lys 430 TCT Tcr Ser Ser 445 GAA GCG Glu Ala CCG TTA Pro Leu GGC CTC Gly Leu TCA TAT Ser Iyr 510 ACT AAA 962 1010 1058 1106 1154 1202 1250 1298 1346 1394 1442 1490 1538 1586

AAA

Lys

GGA

Gly

TAT

Tyr

GTC

Val 480

GCT

Ala

AGA

TrA AGA TAT Leu Arg Tyr 435 ATT CAC TrA Ile Asp Leu 450 ACG TA AGT Thr Leu Ser AGT GMA ACA Ser Giu Thr GAA AAT TCA Glu Asn Ser 500 CTA CIT CTA WO 97/46105 PCT/EP97/02737 -91 Leu TIr Leu

TCC

Ser

GCG

Ala GTr Val 575

CCG

Pro

AT

Ile Glu Leu Leu Leu Ala 515 GTC CCG CCA AGT GGT Val Pro Pro Ser Gly 530 GAA GAG GAC AAT 'TA Glu Giu Asp Asn Leu 545 GTA CAT CAT ACA GGC Val Asp His Thr Gly 565 AAG GAC GGA GGA AT Lys Asp Gly Gly Ile 580 ACT GAG TAT GTA ATC Thr Giu Tyr Val Ile 595 'TA AAA CAT GAA AAT Leu Lys Asp Giu Asn Thr Asp Leu 520 IT NT AGC Phe Ile Ser 535 GAG CCC TGG Glu Pro Trp 550 GGA GTG AAT Gly Val Asn TCA CAA IT Ser Gin Phe CAA TAT ACT Gin Tyr Thr 600 ACT GGA TAT Thr Gly Tyr 615 CAA ACT ATr Gin Thr Ile 630 TAT TIA ATI Tyr Leu Ile T ATr ATT Phe Ile Ile 610 Ser

AAT

Asn

AAA

Lys

GGA

Gly ATr Ile 585 arr Val

AT

Ile

AAT

Asn

ITA

Leu

TIC

Leu 665

ACA

Thr crc Leu

ITA

Leu Asn Lys Giu Thr Lys 525 AT GTA GAG AAC GGG Ile Val Giu Asn Cly 540 OCA AAT AAT AAG AAT Ala Asn Asn Lys Asn 555 ACT AAA GCT TrA TAT Thr Lys Ala Leu Tyr 570 GGA CAT AAG TA AAA Gly Asp Lys Leu Lys 590 AAA GGA AAA CCT TC Lys Gly Lys Pro Ser 605 CAT TAT GAA GAT ACA His Tyr Glu Asp Thr 620 AAA CGT IT ACT ACA Lys Arg Phe Thr Thr 635 AAA AGT CAA AAT GGA Lys Ser Gin Asn Gly 650 GAA A T ACT CCT Wr Glu Ile Ser Pro Ser 670 AAT AAT TGG ACG AGT Asn Asn Trp Thr Ser 685 ACT CIT TAT CAG GGA Thr Leu Tyr Gin Gly 700 CAT AGT 'Ir TCA ACT Asp Ser Phe Ser Thr 715 1634 1682 1730 1778 1826 1874 1922 1970 2018 2066 2114 2162 AAT AAT AAT TTA GAA GAT Asn

GGA

Gly

GAT

Asp 655

GAA

Glu

ACG

Thr

GGA

Gly Asn Asn 625 ACT CAT Thr Asp 640 GAA GCT Glu Ala AAG TTA Lys Leu OGA TCA Oly Ser CGA GGG Arg Gly 705 Glu Asp AAG GGA Lys Gly GGA CAT Cly Asp 660 AGT CCA Ser Pro 675 AAT ATr Asn Ile CTA AAA Leu Lys

TAT

Tyr

GTG

Val 645

AAC

Asn

GAA

Glu

AGC

Ser

CAA

Gin

TA

Leu

GGT

Gly

AAC

Asn 710

AAT

Asn 680

ACA

Thr

CAA

Gin TAT AGA G=G TAT =FT TCT G=G WC GGA CAT GCT AAT CTA AG T AGA 2210 Iyr Arg 720 Val Tyr Phe Ser Val Ser Oly Asp Ala Asn Val Arg Ile Arg 725 730 WO 97/46105 PCTEP97/02737 -92- AAT TCT AGG GAA GTG TTA TI GAA AAA AGA Asn Ser Arg Glu Val Leu Phe Glu Lys Arg 735 740 GAT GTT'TCT GAA ATG TTC ACT ACA AAA TIT Asp Val Ser Glu Met Phe Thr Thr Lys Phe 755 760 ATA GAG CTrr ITCT CAA GGG AAT AAT TTA TAT Ile Glu Leu Ser Gin Gly Asn Asn Leu Tyr 770 775 TT TAC GAT GTC TCT ATT AAG TAA Phe Tyr Asp Val Ser Ile Lys 785 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 789 amino acids TYPE: amino acid TOPOLOGY: linear [AT ATG AGC GGT GCT AAA 'yr Met Ser Gly Ala Lys 745 750 GAG AAA GAT AAC IT TAT Glu Lys Asp Asn Phe Tyr 765 GGT GGT CCT AT GTA CAT ly Gly Pro Ile Val His 780 2258 2306 2354 2378 (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Asn Lys Asn Asn 1 5 Ile Asp Tyr Phe Asn Ile Met Asn Met Ile Asp Glu Ile Leu Lys Leu Asp Gly Val Asn Leu Asn Thr Glu Leu Asn Gin Val Leu Asn 100 Met Leu Arg Val Tyr 115 Met Lys Gin Asn Tyr 130 DESCRIPTION: SEQ ID NO:29: Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe Gly Asp Leu Asn Ile Asn 105 Ile Leu 10 Phe Ala Thr Gly Leu Asn Asp Leu 75 Leu Lys 90 Lys Leu Thr Ser Gin Ile Thr Gly Asp Ile Ile Asp Met Glu 140 Lys Thr Gly Gly Glu Asn Asp Ser Asp Leu Lys Asn Gin Thr Val Lys 4, WO 97/46105 W097/6105PCT/EP97/02737 93 Gin Leu Gin Giu Ile Ser Asp Lys Leu Asp Ilie Ile Asn Vai Asn Val Leu Ile Asn Ser Thr

LYS

Ser Lieu Asp 225 Asn Thr Asn Loeu Ser 305 Asn

LYS

Pro Vai

LYS.

385 Cys Pro Tyr Ser Thr 210 Gly Asn Lys Phe Thr 290 Ile Ile Vai Gly Leu 370 Asp Pro Asn Leu Vai

LYS

195 Giu Phe Leu Giu Leu 275 Thr Met Leu Lys His.

355 Lys Ser Asp Giu' Arg Asn 180 Val Leu Giu Phe Asn 260 Ile Cys Asn Pro Gly 340 Ala Val Leu Gin 420 TyrI 165 Giu

LYS

Thr Phe Giy 245 Val Val Arg Glu Thr 325 Ser Leu Tyr Ser 405 Val Glu Leu

LYS

LYS

Giu 230 Arg

LYS

Leu

LYS

His 310 Leu Asp Ile Giu.

Glu 390 Giu Ile Val Thr Phe Asp Leu 215 Leu Ser Thr Thr Leu 295 Leu Ser Giu Gly Ala 375 Val Gin Thr Thr Giu Giu Giy 200 Ala Asn Ala Ser Ala 280 Leu Asn Asn Asp Phe 360

LYS

Ile le

LYS

Ala Ile Giu 185 Ser

LYS

Thr Leu Gly 265 Loeu Gly

LYS

Thr Ala 345 Giu Leu le 425 Asn Thr Pro 170 Loeu Thr Pro Ala Ser Val Phe His 235 Lys Thr 250 Ser Giu Gin Ala Loeu Ala Glu Lys 31i5 Phe Ser 330 Lys Met le Ser Lys Gin Gly Asp 395 Tyr Thr.

410 Asp Phe Phe Tyr Ala Phe Asp Thr 220 Asp Ala Val Gin Asp 300 Giu Asn Ile Asn Asn 380 Met Asn Thr Asp Tyr Gin Ala Thr 190 le Leu 205 Lys Asn Val Met Ser Giu Giy Asn 270 Ala Phe 285 le Asp Giu Phe Pro Asn Val Glu 350 Asp Ser 365 Tyr Gin* Asp Lys Asn Ile Lys Lys 430 Ser Ser Arg i7 Giu Asp Asp Val Leu 255 Vai Leu Arg 335 Ala le Val Leu Val 415 Met Thr le .Thr Glu Val Gly 240 le Thr Thr Val 320 Ala

LYS

Thr Asp Loeu 400 Phe

LYS

Gly WO 97/46105 PCTEP97/02737 -94 Glu Arg 465 Ile Asp Glu Val Glu 545 Val Lys Thr Leu Asn 625 Asp Ala Leu Ser Gly 705 435 Ile Asp 450 Thr Leu Ser Glu Glu Asn Leu Leu 515 Pro Pro 530 Glu Asp Asp His Asp Gly Glu Tyr 595 Lys Asp 610 Leu Glu Leu Lys Trp Gly Leu Ser 675 Thr Asn 690 Ile Leu 440 Leu Asn Lys Lys Lys 455 Ser Ala Asn Asp Asp 470 Thr Phe Leu Thr Pro 485 Ser Arg Leu Ile Thr 500 Leu Ala Thr Asp Leu 520 Ser Gly Phe Ile Ser 535 Asn Leu Giu Pro Trp 550 Thr Gly Gly Val Asn 565 Gly Ile Ser Gin Phe 580 Val Ile Gin Tyr Thr 600 Glu Asn Thr Giy Tyr 615 Asp Tyr Gin Thr Ile 630 Gly Val Tyr Leu Ile 645 Asp Asn Phe Ile Ile 660 Pro Giu Leu Ile Asn 680 Ile Ser Gly Asn Thr 695 Lys Gin Asn Leu Gin 710 Vai Giu Ser Ser 460 Gly Vai Tyr Met 475 Ile Asn Gly Phe 490 Leu Thr Cys Lys 505 Ser Asn Lys Glu Asn Ile Vai Glu 540 Lys Ala Asn Asn 555 Gly Thr Lys Ala 570 Ile Giy Asp Lys 585 Val Lys Gly Lys Ile His Tyr Glu 620 Asn Lys Arg Phe 635 Leu Lys Ser Gin 650 Leu Giu Ile Ser 665 Thr Asn Asn Trp Leu Thr Leu Tyr 700 Leu Asp Ser Phe 715 445 Glu Ala Glu Pro Leu Gly Gly Leu Gin 495 Ser Tyr Leu 510 Thr Lys Leu 525 Asn Giy Ser Lys Asn Ala Leu Tyr Val 575 Leu Lys Pro 590 Pro Ser Ile 605 Asp Thr Asn Thr Thr Gly Asn Gly Asp 655 Pro Ser Glu 670 Thr Ser Thr 685 Gin Gly Gly Ser Thr Tyr Tyr Val 480 Ala Arg Ile Ile Tyr 560 His Lys His Asn Thr 640 Glu Lys Gly Arg Arg 720 Vai Tyr Phe Ser Vai Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser 735 WO 97/46105 W09746105PCT/EP97/02737 Arg Glu Val Ser Giu Met 755 Leu Ser Gin 770 Asp Val Ser 785 Leu 740 Phe Glu Lys Arg Tyr 745 Phe Thr T-u Gly Asn Asn Lys Phe Giu 760 Leu. Tyr Giy 775 Met Ser Gly Ala Lys Asp Val 750 Lys Asp Asn Phe Tlyr Ile Giu 765 Gly Pro Ile Val His Phe Tyr 780 Ile Lys INFORMATION FOR SEQ ID SEQUENCE CHARACT'ERIST2ICS: LENGTH: 2403 base pairs TYPE: nucleic acid STRANDEEN>ESS: single TOPOLOGY: linear (i)MOLECZULE TYPE: other nucieic acid DESCRIPTION: /desc "Synthetic 11WIA (i)HYPOTFHEICAL: NO (ix) FEATURE: NAME/KEY: misc-feature LOCATION: 1i. .2389 OTHER INFORMATION: /note= "maize optimized ENA sequence encoding VIP3A(a)" (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCACCA A IGAACATGA ACAAGAACAA CFIrCATCGAC TACriCAAG GCATcrACGG C~AIcwrr AAG3ACCGACA CCGGCGGCGA.

GCAGCIGCTG AACGACAcA GCGGCAAGCT GA IOGCCCAG GGCAACCIGA ACACCGAGCT GCAGAACCAG GTGCTGAACG ACGTGAACAA CGTGTACCTG CCGAAGATCA CCAGCAT13CT GAGCCIGCAG AWCGAGTACC! TGAGCAAGCA CATCAACGTG AACcI'CCIXIA WAACAGCAC CATCAAGTAC GIKAACGAGA AG PI9GA CACCAAC AGCACCCGCG CICGOCCACC GGCA'rCAAGG CCI ACCCIO GACGAGATCC GGACGGCGIM AACGGCAGCC GAGCAAXX3AG ATCC!TTANA CAAGCTG)GAC GCCAWAACI.

GAGCGACGTG ATAAGCAGA CGCAGGAG AWAGCGACA CCTGACCGAG AWACCCCGG GC1XGACCFIC GCCACCGAGA

CCCTG~CCGAG

ACATCATGAA

TAAGAACCA

TAACGACCT

TCGCCAACGA

CCATGC=

ACTACGCCCT

AGCI'GGACAT

CCTACCAGCG

CCAGCAGCAA

120 180 240 300 360 420 480 540 600 (,WO 97/46105 WO 9746105PCTIEP97/02737 96

GGTGAAGAAG

GCCAAGAGC

CGCG'DGAG

GATCACCAAG

GA'IXY3GC1X3 GCrGGCII

GGAGGAG=I

CGCCAAGGG

GACGCCAGCC CGGCCGACAT GTACCAAGA ACGACG IGGA GTGCGCAACA ACCIrGICG GAGAACGTGA AGACCAGCGG ACCGCCTC AGGCCCAGGC GCCGACATOG ACrACACCAG CGCGTGAACA TCCI C AAGGGCAGCG ACGAGGACGC CGCG7TCr\ GGL± ±Z2'A

CAAGCTGAAG

CATGACAAG

G'IYCCCGATAC

CrACGAGGIT3 GAAGG1XGGAG

CATGCCACTG

GGCCGACGAG

GCTAGCCACC

CAGCAACATC

CAACAAGAAC

GCACAAGGAC

CGTGATCCAG

CTACATCCAC

CFTCACCACC

CGAGGCCTG

CCCGGAGCTG

CACCCIMACC

C=ICGCACC

CAGAACrACC

GAGTACG'IGA

ACCGCCAACr

AGCACGAGG

GGCGTGATCA

AACAGCCGCC

GACCICAGCA

GTGAGAACG

CCCrACG

WCAGCAACGA

AGGTGGACAA

CGGACCAGAG

TICACCAAGAT

TCrACGACAG

CCGAGTACCG

GCGAGACC~r TATCACCCr ACAAGGAI3AC

GCAGCATCGA

ACCACACCGG

CCTGXGACGAG

CGGCI=G

CCGCAGOGCC

CAGCGAGGTG

CIYICCIGACC

CAMATGXAAC

CC'IGAGCAAC

CAAGATGAT1C

CAGCATCACC

GGACAGC=~

CGAGCAAAWC

CGACITCACC

CAGCACCGGC

CACCCIGAGC

CCTGACCCOG

GACCI'GrAAG

CAAGCTGAC

GGAGGACAAC

CrGCGTGAAC

CGGCGACAAG

ATCGATICAC

CCITGGAGGAC

GTACCTGATC

GGAGATCAGC

CAGCACCGGC

CATCCTGAAG

GAGCGGCGAC

CTGACCGAGC

=ACCXGA

CIT3AAGACCG

GGCAACGG'

C1Gr.ACCACar

GAGCATIA

ACCFICAGCA

G'IGGAGGCrA

GTGXCTGAAGG

AGCGAGGTGA

TACrACACCA

AGAAGATGA

GAGATCGACC

GCGAACGACG

ATCAACGG=

AGCrACCI'GC

GTCCACCGA

CTGGAGCOGT

GGCACCAAGG

CIAAGCCGA

C'IGAAGGACG

TACCAGACCA

CrGAGAGCC TGXACCGAGCTr

ACACCTI'CC

CCAGCGAGCT

ACAACTCC

GCGYCAAGCPr

ACAAGGAGAA

ACCCGAACTA

AGCCGGGCCA

TGXTACGAGGC

IXrACGGCGA

ACAACAT

AGACCCIG

TAACAAGAA

ACGGC=~A

IG3GCCI~cA

CGCGAGCI=C

GCriGCI'=T

GGAAGCCAA

CCC'J=ACGT

AGACCGA~'rA

AGAACACCGG

TCACAGCG

AGAACGGCGA

660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 GGCGGCATCA GCCAGTICAT TACACC~lrUA AGGGCAAGCC TACGAGGACA CCAACAACAA GGCACCGACC TGAAGGGCGT~ GGCGACAACr WAWCCT AWCAACACCA ACAACI'GGAC C1=ACCAGG GCGGCCGGG TACCGCG=B~ ACI'X2GCGr CCGAGCGAGA AGCIGCvMAG AGCACCAACA TCACCGGCAA CAGAAC=I~ AGCrGGACAG GCCAAC~rC GCAMXCGCAA t, WO 97/46105 t' W 97/6105PCT/EP97/02737 97 CAGCCGCGAG GTGC X1T2G AGAAGAGGTA CATGAGCGGC GCCAAGGACG TG~AGCGAGAT GTIr-CcCACC AAGTTCGAGA AGGACAACTT CPACATX;AG CIGAGCCAGG GCAACAACCT GTACGGCGGC CCGATCGTGC ACTTCTACGA. CGTGlAGCA Ic AA=TAACGT ;AGCTCWGA INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 2612 base pairs TYPE: nucleic acid STRANDEENESS: single TIOPOLOGY: linear (ii) MOLECULE TYPE: DNiA (genomic) (iii) HYPO)THETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: -118. .2484 OTIHER INFORMATION: /note= "Native DNA. sequence encoding VIP3A(b) from AB424" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: ATIGAAATIG ATAAAAAGIT ATGAGIGT AATAATCAGT AATACCAAT AANITrAA GAATACAAGT 'TACAAGAAA TAAGI=1Ac AAAAAATAGC 'IGAAAASGAA GA'ICAAC A B3 AAC AAG AAT AAT ACT AAA 'ITA AGC ACA AGA GCC TTA CCA, ACT TIT 2280 2340 2400 2403 Met Asn Lys Asn Asn Thr 790 795 T GAT TAT TI'C AAT GGC Ile Asp Tyr Phe Asn Gly 810 Lys Leu Ser Thr Arg Ala Leu. Pro Ser Phe 805 AlT A EG AAC le Met Asn GAC GAA T Asp Glu Ile 840 ITG GAT GGG Leu Asp Gly 855 'TrA AAT ACA ATO ArlT TIT Met Ile Phe 825 'ETA AAG AAT Leu Lys Asn GTG AAT GGA Val Asn Gly ATT TAT GGA TIT GCC ACT GGT Ile T'yr Gly Phe Ala Thr Gly 815 AAA ACG, GAT ACA GGT GGT GAT Lys Thr Asp Thr Gly Gly Asp 830 CAG GAG CTA CTA AAT GAT AT Gin Gin Leu. Leu Asn Asp Ile 845 850 AGC 'ITA AAT GAT CIT ATC GCA Ser Leu Asn Asp Leu Ile Ala 860 865 AW MAA GAC Ile Lys Asp 820 CTA ACC CTA Leu Thr Leu 835 TCT GGT A Ser Gly Lys CAG GGA MAC Gln Giy Asn GMA WIA ITCT MG GAA ATA ITA MAA ATT GCA MAT GAA CAA40 405 SWO 97/46105 PCT/EP97/02737 -98- Leu Asn Thr 870 AAT CAA GT Asn Gin Val ATG CT CGG Met Leu Arg A1X AAA CAA Met Lys Gin 920 CAA TMG CAA Gin Leu Gin 935 CIT Ar AAC Leu Ile Asn 950 AAA TAT GIG Lys Tyr Val AGT TCA AAA Ser Ser Lys TA ACT GAG Leu Thr Glu 100C GAT GGT TIT Asp Gly Phe 1015 AAT AAT rTA Asn Asn Leu 1030 ACT AAA GAA Thr Lys Glu AAC TC CTA Asn Phe Leu Glu Leu Ser Lys Giu Ile 875 TA PAT GAT =T AAT AAC Leu Asn Asp Val Asn Asn 890 GTA TAT CTA CCT AAA Apr Vai Tyr Leu Pro Lys Ile 905 910 AAT TAT GCG CA AGT CTG Asn Tyr Ala Leu Ser Leu 925 GAG ATr TCr GAT AAG TIM Glu Ile Ser Asp Lys Leu 940 TCT ACA CIT ACT GAA Apr Ser Thr Leu Thr Giu Ile 955 AAC GAA AAA IT GAG GAA Asn Giu Lys Phe Giu Glu 970 GTA AAA AAG GAT GGC TCr Val Lys Lys Asp Giy Ser 985 990 prA ACT GAA CrA GCG AAA Leu Thr Giu Leu Ala Lys 1005 GAA TIT TAC CIT AAT ACA Glu Phe Tyr Leu Asn Thr 1020 TIC GGG CGT. TCA GCT TTA Phe Gly Arg Ser Ala Leu 1035 AAT GTGX AAA ACA AGT GGC Asn Val Lys Thr Ser Gly 1050 ATr GTA ITA ACA GCT CIMG Ile Val Leu Thr Ala Leu 1065 107( Leu Lys Ile Ala Asn Glu 880 AAA CIC GAT GCG ATA AAT Lys Leu Asp Ala Ile Asn 895 900 ACC TT ATG TG AGT GAT Thr Ser Met Leu Ser Asp 915 CAA ATA GAA TAC TrA AGT Gin Ile Giu Tyr Leu Ser 930 GAT Ar Apr AAT GTA AAT Asp Ile Ile Asn Val Asn 945 ACA CCT GCG TAT CAA AGG Thr Pro Ala Tyr Gin Arg 960 TrA ACT PT GCT ACA GAA Leu Thr Phe Ala Thr Glu 975 980 CCT GCA GAT ATT CGT GAT Pro Ala Asp Ile Arg Asp 995 AGT GrA ACA AAA AAT GAT Ser Val Thr Lys Asn Asp 1010 TIC CAC GAT GTA ATG GTA Phe His Asp Val Met Val 1025 AAA ACT GCA TCG GAA TrA Lys Thr Ala Ser Giu Leu 1040 AGT GAG GC GGA AAT rIT Ser Giu Vai Giy Asn Val 1055 1060 CAA GCA AAA GCT PT CIT Gin Ala Lys Ala Phe Leu 1075 Gin 885

ACG

Thr

OTA

Val

AAA

Lys

GTA

Val ATr Ile 965

ACT

Thr

GAG

Glu

GIG

Vai

GGA

Gly ATr Ile 1045

TAT

Tyr

ACT

Thr 453 501 549 597 645 693 741 789 837 885 933 981 1029 prA ACA CCA 1(C CGA AAA TTA WrA GGC TA OCA GAT Apr GAT TAT ACT Leu Thr Pro Cys Arg Lys Leu Leu Giy Leu Ala Asp Ile Asp Tyr Thr 1080 1085 1090 t WO 97/46105 PCTIEP97/02737 -99- TCT ATT ATG AAT GAA CAT TTA AAT AAG CPA AAA GAG GAA FT AGA GTA 1077 Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 1095 1100 1105 PAC ATC CTC COT ACA CT TCT PAT ACT =IT TCT PAT C AAT TAT GCA 1125 Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala 1110 1115 1120 1125 AAA GIT AAA GGA ACT CAT GAA GAT GCA PAG ATG AT GTG GAA GCT AAA 1173 Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Giu Ala Lys 1130 1135 1140 CCA GGA CAT GCA I ATT GGG ITT GAA AIT AGT PAT GAT TA ATr ACA 1221 Pro Gly His Ala Leu Ile Gly Phe Glu lie Ser Asn Asp Ser lie Thr 1145 1150 1155 GTA qTA AAA GTA TAT GAG GCT AAG CIA AAA CAA PAT TAT CAA =IC GAT 1269 Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gin Asn Tyr Gin Val Asp 1160 1165 1170 AAG AT TCC TA TCG GAA rr ATT TAT GCC GAT ATG CAT AAA qTA Tc 1317 Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu 1175 1180 1185 TGC CCA CAT CAA TCT GGA CAA ATC TAT TAT ACA PAT AAC ATA GTA PIT 1365 Cys Pro Asp Gin Ser Gly Gin Ile Tyr Tyr Thr Asn Asn Ile Val Phe 1190 1195 1200 1205 CCA PAT GAA TAT GTA ATr ACT AAA AT CAT Tr ACT AAA AAA AT's AAA 1413 Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys 1210 1215 1220 ACT TTA ACA TAT GAG GTA ACA GCG PAT ITT TAT CAT WI? TT ACA GCA 1461 Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly 1225 1230 1235 GAA APT CAC TA PAT PAG AAA AAA GrA GAA TCA AGT GAA GCG GAG TAT 1509 Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Giu Ala Glu Tyr 1240 1245 1250 AGA ACG TrA ACT GOT PAT CAT CAT GGG CGI TAT ATO CCG TA GGT GTC 1557 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val 1255 1260 1265 ATC ACT GAA ACA PIT PTG ACT CCC ATr AAT CGG TIT GGC CM CAA CT 1605 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gin Ala 1270 1275 1280 1285 CAT GAA AAT 'CA AGA TrA NT ACT TTA ACA TGT AAA TCA TAT TA AGA 1653 Asp Clu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg 1290 1295 1300 GAA CTA CIG cTA GCA ACA CAC TA AGO AAT AAA GAA ACT AAA 'IG ATO 1701 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Giu Thr Lys Leu Ile 1305 1310 1315 "WO 97/46105 PCTEP97/02737 -100- CIC CCG CCA AGT GGT TT AT AGC AAT ATT OTA GAG AAC GGG TCC ATA 1749 Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile 1320 1325 1330 GAA GAG GAC PAT TA GAG CCG TGG AAA GCA AAT AAT AAG AAT GCG TAT 1797 Glu Glu Asp Asn Leu Giu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr 1335 1340 1345 GTA GAT CAT ACA GGC GGA GTG AAT GGA ACT AAA GCr TrA TAT OTT CAT 1845 Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Vai His 1350 1355 1360 1365 AAG GAC GGA GGA AT TCA CAA TT ATT GGA GAT MAG TrA AAA CCG AAA 1893 Lys Asp Gly Gly Ile Ser Gin Phe Ile Gly Asp Lys Leu Lys Pro Lys 1370 1375 1380 ACT GAG TAT GTA ATC CAA TAT ACT Gpr AAA GGA AAA CCT TCT ATr CAT 1941 Thr Glu Tyr Val Ile Gin Tyr Thr Val Lys Gly Lys Pro Ser Ile His 1385 1390 1395 TA AAA GAT GAA AAT ACT GGA TAT ATT CAT TAT GAA GAT ACA AAT AAT 1989 Leu Lys Asp Giu Asn Thr Gly Tyr Ile His Tyr dlu Asp Thr Asn Asn 1400 1405 1410 AAT TrA GM GAT TAT CAA ACT ATr AAT AAA CGT T ACT ACA GGA ACT 2037 Asn Leu Giu Asp Tyr Gin Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr 1415 1420 1425 CAT TA AAG GCA GTG TAT TPA AT TA AAA ACT CAA AAT GGA CAT GAA 2085 Asp Leu Lys Giy Val Tyr Leu Ile Leu Lys Ser Gin Asn Gly Asp Glu 1430 1435 1440 1445 GCT TGG GCA CAT AAC TIT AT Alr TIG GAA ATT AGT CCT WTT GAA AAG 2133 Ala Trp Giy Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Giu Lys 1450 1455 1460 TA TA AGT CCA GAA TA NIT PAT ACA PAT MT TGG ACO AGT ACG GGA 2181 Leu Leu Ser Pro Giu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly 1465 1470 1475 WCA ACT AAT ATT AGC GGT AAT ACA CTC ACT CIT TAT CAG GCA GGA CGA 2229 Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly Gly Arg 1480 1485 1490 GGG ATT CTA AAA CAA AAC CIT CAA TA CAT ACT TIT TCA ACT TAT AGA 2277 Gly Ile Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr Tyr Arg 1495 1500 1505 GTG TAT TC TOT GTG TCC GGA CAT GCT AAT GTA AGG AT AGA AAT TcT 2325 Val Tyr Phe Ser Vai Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser 1510 1515 1520 1525 AGG GAA GTG ITA TIT GAA AAA AGA TAT ATG AGC GOT dCT AAA GAT alT 2373 Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val (WO 97/46105 PCTIEP97/02737 -101 1530 1535 1540 TCT GAA ATG TC ACT ACA AAA TIT GAG AAA GAT AAC TIC TAT ATA GAG 2421 Ser Glu Met Phe Thx Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu 1545 1550 1555 CIT TCT CAA GGG AAT AAT TPA TAT GGT GGT CCT ATr GTA CAT PIT TAC 2469 Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr 1560 1565 1570 GAT GTC TCT ATT AAG TAAGA'CGGG ATCTAATATT AACAGIT AGAAGC-AAT 2524 Asp Val Ser Ile Lys 1575 TCTATAA TG1w'CCAT TATGGAAAAA CACAAET=IG TITGCAAGA TGTATATATA 2584 GCICACICAT TAAAAGGCAA TCAAGCIT 2612 =INFOEATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 789 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe 1 5 10 Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp 25 Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu 40 Asp Giu Ile Leu Lys Asn Gin Gin Leu Leu Asn Asp Ile Ser Gly Lys 55 Leu Asp Gly Val Asn Giy Ser Leu Asn Asp Leu Ile Ala Gin Giy Asn 70 75 Leu Asn Phr Giu Leu Ser Lys Giu Ile Leu Lys Ile Ala Asn Giu Gin 90 Asn Gin Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr 100 105 110 Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val 115 120 125 Met Lys Gin Asn Tyr Ala Leu Ser Leu Gin Ile Giu Tyr Leu Ser Lys WO 97/46105 PCT/EP97/02737 -102 130 135 140 Gin Leu Gin Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val 145 150 155 160 Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gin Arg Ile 165 170 175 Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr 180 185 190 Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Arg Asp Glu 195 200 205 Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val 210 215 220 Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly 225 230 235 240 Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile 245 250 255 Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr 260 265 270 Asn Phe Leu Ile Val Leu Thr Ala Leu Gin Ala Lys Ala Phe Leu Thr 275 280 285 Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr 290 295 300 Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 305 310 315 320 Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala 325 330 335 Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys 340 345 350 Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr 355 360 365 Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gin Asn Tyr Gin Val Asp 370 375 380 Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400 Cys Pro Asp Gin Ser Gly Gin Ile Tyr Tyr Thr Asn Asn Ile Val Phe 405 410 415 Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys 420 425 430 V WO 97/46105 PCTEP97/02737 -103- Thr Leu Arg Tyr Giu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly 435 440 445 Glu Ile Asp Leu ksn Lys Lys Lys Val Giu Ser Ser Giu Ala Glu Tyr 450 455 460 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val 465 470 475 480 Ile Ser Giu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gin Ala 485 490 495 Asp Giu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg 500 505 510 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile 515 520 525 Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Vai Giu Asn Gly Ser Ile 530 535 540 Glu Giu Asp Asn Leu Giu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr 545 550 555 560 Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His 565 570 575 Lys Asp Giy Gly Ile Ser Gin Phe Ile Giy Asp Lys Leu Lys Pro Lys 580 585 590 Thr Glu Tyr Val Ile Gin Tyr Thr Vai Lys Gly Lys Pro Ser Ile His 595 600 605 Leu Lys Asp Giu Asn Thr Giy Tyr Ile His Tyr Glu Asp Thr Asn Asn 610 615 620 Asn Leu Glu Asp Tyr Gin Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr 625 630 635 640 Asp Leu Lys Gly Vai Tyr Leu Ile Leu Lys Ser Gin Asn Gly Asp Glu 645 650 655 Ala Trp Gly Asp Asn Phe Ile Ile Leu Giu Ile Ser Pro Ser Giu Lys 660 665 670 Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly 675 680 685 Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly Gly Arg 690 695 700 Gly Ile Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr Tyr Arg 705 710 715 720

'I

WO 97/46105 PCTEP97/02737 -104- Val Tyr Phe Arg Glu Val Ser Glu Met 755 Ser Val 725 Leu Phe 740 Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser 730 735 Met Ser Gly Ala Lys Asp Val 750 Glu Lys Arg Tyr 745 Phe Thr Thr Lys Phe 760 Leu Tyr 775 Glu Lys Asp Asn Phe 765 Tyr Ile Glu Leu Ser 770 Gin Gly Asn Asn Gly Gly Pro Ile 780 Val His Phe Tyr Val Ser Ile Lys INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEENESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "forward primer used to make pCIB5526" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GGATCCACCA TGAAGACCAA CCAGATCAGC INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEENESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "reverse primer used to make pCIB5526 (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: n I' C WO 97/46105 PCT/EP97/02737 -105- AAGCITICAGC TCCT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2576 base pairs TYPE: nucleic acid STRANDEEI\IESS: single TOPOLX3Y: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic ENA" (iii) HYPOTHETICAL:

NO

(ix) FEATURE: NAME/KEY: CDS LOCATION: 9..2564 OTHER INFORMATION: /note= "Maize optimized sequence encoding VIPiA(a) with the Bacillus'secretion signal removed as contained in pCIB5526" (xi) SEQUENCE DESCRIPTION: SEQ ID GACCACC ATIG AAG ACC AAC CAG ATC AGC ACC ACC CAG AAG AAC CAG CAG Met Lys Thr Asn Gin Ile Ser Thr Thr Gin Lys Asn Gin Gin AAG GAG ATG GAC CGC AAG Lys Glu Met Asp Arg Lys 840 GGC CTG CTG Gly Leu Leu

GGC

Gly 845 TAC TAC TIC AAG Tyr Tyr Phe Lys GGC AAG Gly Lys 850 GAC TIC AGC AAC Asp Phe Ser Asn 855 ATC TAC GAC CAG Ile Tyr Asp Gin 870 CTG ACC ATG TIC Leu Thr Met Phe

GCC

Ala 860 CCC ACG CGT GAC Pro Thr Arg Asp AGC ACC CTG Ser Thr Leu 865 AAG CAG CAG Lys Gin Gin CAG ACC GCC Gin Thr Ala

AAC

Asn 875 AAG CIG CIXG GAC Lys Leu Leu Asp GAG TAC Glu Tyr 885 CAG AGC ATC CGC Gin Ser Ile Arg

TGG

Trp 890 ATC GGC CTG ATC lie Gly Leu Ile

CAG

Gin 895 AGC AAG GAG ACC Ser Lys Glu Thr

GGC

Gly 900 GAC TIC ACC TIC Asp Phe Thr Phe CTG AGC GAG GAG Leu Ser Giu Asp CAG GCC ATC ATC Gin Ala Ile Ile

GAG

Glu 915 ATC AAC GGC AAG Ile Asn Giy Lys ATC AGC AAC AAG Ile Ser Asn Lys

GGC

Gly 925 AAG GAG AAG GAG Lys Giu Lys Gin cTG GTG Val Val 930 CAC CTG GAG AAG GGC AAG C GM CC ATC AAG AIC GAG TAC CAG AGC "WO 97/46105 ~WO 9746105PCTIEP97/02737 106- His Leu Giu Ly's Gly Lys Leu Val. Pro Ile Lys Ile Giu Tyr Gin Ser 935 940 945 GAC ACC AAG Asp Thr Lys 950 =I AAC A'IC GAC The Asn Ile Asp

AGG

Ser 955 AAG ACC TTC AAG Lys Thr Phe Lys CIG MAG MI Leu LYS Leu TIC AAG Phe Lys 965 CiT: CGC Leu Arg 980 ATC GAC AGC CAG Ile Asp Ser Gin AAG CCC GAG 7MC Asn Pro Giu Phe 985

AAC

Asn 970 GAG CCC GAG GAG Gin Pro Gin Gi-n Val 975 GAG GAG GAC GAG Gin Gin Asp Giu AAC AAG AAG Asn Lys Lys GAG AGC Giu Ser 990 GAG GAG TIC =I Gin Glu Phe Leu 482 530 578 626 AAG CCC AGC AAG Lys Pro Ser Lys Ile Asn 1000 =r TIC Leu Phe ACC GAG GAG ATG Thr Gin Gin Met 1005 GAG AGG ATC CCC Asp Ser Ile Pro 1020 AAG CG GAG ATC LYS Arg Giu Ile 1010 GAGCIX TGG GAG Asp Leu Trp Glu GAG GAG GAG Asp Giu Asp AGG GAG Thr Asp 1015 ACG GAG GGC Thr Asp Giy GAG AAG GGC TAG Giu Asn Gly Tyr 1030 ACC AWc GAG AAC Thr Ile Gin Asn 1035 GGC AMC GCC GM~ MG TP3G GAG GAG Arg Ile Ala Val Lys Trp Asp Asp 1040 MAG TIC GMG AGC MAC CCC CM GAG Lys Phe Val Ser Asn Pro Leu Glu 1055 AGG GIG GCT Ser Leu Ala 1045 AGG MAG GGC Ser Lys Gly TAG ACC TyrThr 1050 AGG GAG Ser His 1060 ACC GTG GGC Thr Val Giy GAG CCC Asp Pro 1065 TAG ACC GAG Tyr Thr Asp TAG GAG TYr Giu 1070 MG GCc CC Lys Ala Ala

CGG

Arg GAG CTGl GAG CG Asp Leu Asp Leu AGC MGC GC Ser Asn Ala 1080 MAG GAG ACC TC MGC Lys Glu Thr Phe Asn 1085 CCC GG G CC Pro Leu Val Ala 1090 770 818 866 GCG TIC CCC Ala Phe pro AGG G1XG Ser Val.

1095 MGC G'1G AGG Asn Val Ser A I( GAG Met Glu 1100 AAG Gr AWC LYS Val le GIG AGC CCC Leu Ser Pro WC GAG WMC I AGX MC AGG Asn Glu Asn Leu Ser Asn Ser 1110 cGIG GAG AGC Val. Glu Ser 1115 GAGC IOG AGG ACC MA G G His Ser Ser Thr Asn Trp 1120 GAG GCC GGC AWC GGT CCC Glu Ala Gly Ile Gly Pro 1135 AGG TAC ACC MGC Ser Ty~r Thr Asn 1125 ACC GAG GGC GCC AGC GTG Thr Giu Gly Ala Ser Val 1130 MAG GGG ATC AGG TIM Lys Gly Ile Ser Phe 1140 GGC M~ AGG MI MGC TAG CAG GAC AGG GAG ACC Gly Val Ser Val Asn Tyr Gin His Ser Giu Thr 1145 1150 1155 1010 '.WO 97/46105 PCTIEP97/02737 -107- CTG CCC CAG GAG TGG GGC ACC AGC Val Ala Gin Glu Trp Cly Thr Ser 1160 ACC CCC ACC CCC GGC TAC CTG AAC Thr Ala Ser Ala Gly Tyr Leu Asn 1175 ACC CCCM ,C GC AAC Thr Gly Asn 1165 ACC ACC CAG TTC AAC Thr Ser Gin Phe Asn 1170 1058 CCC AAC Ala Asn 1180 G'IY CCC TAC AAC AAC GIT Val Arg Tyr Asn Asn Val 1185 ACC ACC ACC TIC GTG CI Thr Thr Ser Phe Val Leu 1200 1106 GGC ACC GGC GCC Cly Thr Cly Ala 1190 ATC TAC GAC Ile Tyr Asp GIG AAG CCC Val Lys Pro 1195 1154 AAC AAC CAC Asn Asn Asp 1205 ACC ATC GCC ACC ATC ACC CCC AAG Thr Ile Ala Thr Ile Thr Ala Lys 1210 TCG AAT Ser Asn 1215 ICC ACC GCC Ser Thr Ala CTG AAC Leu Asn 1220 ATC ACC CCC Ile Ser Pro GGC GAG Gly Glu 1225 AGC TAC CCC Ser Tyr Pro AAG AAG Lys Lys 1230 GGC CAG AAC Gly Gin Asn

GGC

Gly 1235 1202 1250 1298 1346 ATC CCC ATC ACC Ile Ala Ile Thr AGC ATG Ser Met 1240 GAC GAC =rr Asp Asp Phe AAC AGC Asn Ser 1245 CAC CCC ATC His Pro Ile ACC CTG Thr Leu 1250 AAC AAG AAG Asn Lys Lys CAG GM GAC AAC CTG Gin Val Asp Asn Leu 1255 C= AAC Leu Asn 1260 AAC AAG CCC AM AIG CTG Asn Lys Pro Met Met Leu 1265 GAG ACC AAC GAG ACC GAC GGC Glu Thr Asn Gin Thr Asp Gly 1270 GTC TAC Val Tyr 1275 AAG ATC AAG Lys Ile Lys GAC ACC CAC GGC Asp Thr His Gly 1280 1394 AAC ATC GTI Asn Ile Val 1285 ACG GGC GGC Thr Gly Gly GAG T(GG AAC GGC GM3 Glu Trp Asn Gly Val 1290 ATC CAG GAG ATC AAG Ile Gin Gin Ile Lys 1295 1442 CCC AAG Ala Lys 1300 ACC CCC AGC ATC ATC GIC GAC GAC Thr Ala Ser Ile Ile Val Asp Asp 1305 GGC GAG CGC GIG CCC GAG Gly Glu Arg Val Ala Clu 1310 1315 1490 AAG CCC GTG GCC Lys Arg Val Ala CCC AAG GAC Ala Lys Asp 1320 TAC GAG AAC CCC GAG GAC AAG Tyr Giu Asn Pro Giu Asp Lys 1325 ACC CCC Thr Pro 1330 1538 AGC CIG ACC Ser Leu Thr C=G AAG Leu Lys 1335 GAC CCC CIG Asp Ala Leu AAG CG AGC Lys Leu Ser 1340 TAC CCC GAC GAG ATC Tyr Pro Asp Glu Ile 1345 1586 AAG GAG ATC GAG GGC IG IG TAC TAC AAG AAC AAG Lys Clu Ile Giu Gly Leu Leu Tyr LYr Lys Asn Lys CCC ATC TAC GAG Pro Ile Tyr Glu 1360 1634 1350 1355 AGC ACC GIG ATG ACC TAT Ser Ser Val Met Thr Tyr 1365 CTA GAC GAG AAC ACC Leu Asp Glu Asn Thr 1370 CCC AAG GAG GIG ACC Ala Lys Clu Val Thr 1375 1682 'WO 97/46105 PCT/EP97/02737 -108- AAG CAG Lys Gin 1380 C1X AAC GAG ACC ACC Leu Asn Asp Thr Thr 1385 GGC AAG =TI AAG GAG Gly Lys Phe Lys Asp 1390 GTG AGC CAC CG Val Ser His Leu 1395 1730 TAC GAC GTG AAG Tyr Asp Val Lys G ACC CCC Leu Thr Pro 1400 AAG AIG AAC GG ACC ATC AAG Lys Met Asn Val Thr Ile Lys 1405 CTG AGC Leu Ser 1410 1778 1826 ATC CIG TAC Ile Leu Tyr GAC AAG Asp Asn 1415 GCC GAG AGC AAC GAC AAC AGC Ala Giu Ser Asn Asp Asn Ser 1420 ATC GGC AAG GG Ile Gly Lys Trp 1425 AAG AAG GAG TAC Lys Lys Gin Tyr 1440 ACC AAG ACC AAG Thr Asn Thr Asn 1430 AGG AGC AAC AAG Ser Ser Asn Asn 1445 ATC GIG AGG Ile Val Ser GGG GGG Gly Gly 1435 AAC AAC GGG Asn Asn Gly 1874 1922 CCC GAC GCC AAC Pro Asp Ala Asn 1450 GIG ACC Cc Leu Thr Leu AAC ACC Asn Thr 1455 GAG GGC GAG Asp Ala Gin GAG AAG Glu Lys 1460 CTG AAG AAG Leu Asn Lys AAG CGC Asn Arg 1465 GAG TAG TAC ATC AGG Asp Tyr Tyr Ile Ser 1470 IG TAG ATG Leu Tyr Met

AAG

Lys 1475 AGG GAG AAG AAG Ser Glu Lys Asn ACC CAG Thr Gin 1480 TGG GAG ATG ACC ATC Cys Giu Ile Thr Ile 1485 GAG GGG GAG Asp Gly Glu ATA TAG Ile Tyr 1490 1970 2018 2066 CC ATC AGG ACC AAG ACC GIG AAG Pro Ile Thr Thr Lys Thr Val Asn 1495 GIG AAG AAG Val Asn Lys 1500 GAG AAG TAG AAG CGC Asp Asn Tyr Lys Arg 1505 CTG GAG ATG ATC Leu Asp Ile Ile 1510 GCC CAC AAG Ala His Asn AC AAG AGG AAC CCC Ile Lys Ser Asn Pro 1515 ATC AGG AGC MIG Ile Ser Ser Leu 1520 2114 CAC ATC AAG ACC AAG GAG His Ile Lys Thr Asn Asp 1525 GAG ATC ACC GIG TIC Glu Ile Thr Leu Phe 1530 GG GAG GAG ATA TCG Trp Asp Asp Ile Ser 1535 2162 ATT ACC GAG GIC GCC Ile Thr Asp Val Ala 1540 AGG ATC Ser Ile 1545 AAG CCC GAG AAC IG ACC GAG AGG Lys Pro Giu Asn Leu Thr Asp Ser 1550

GAG

Glu 1555 2210 2258 ATC AAG GAG ATA TAG ACT CGC TAG GGG Ile Lys Gin Ile Tyr Ser Arg Tyr Cly 1560 ATC AAG Ile Lys 1565 CTG GAG GAG GGC ATC Leu Giu Asp Gly Ile 1570 GAG TIC ATC AAC GAG Glu Phe Ile Asn Glu 1585 CrT ATC GAG Leu Ile Asp AAG AAA Lys Lys 1575 GGG GGG ATC Gly Gly Ile CAC TAC GGC His Tyr Gly 1580 2306 GCG AGC T AAC ATC GAG CCC IG GAG AA TA GIG ACC AAG TAG GAG Ala Ser Phe Asn Ile Giu Pro Leu Gin Asn Tvr Val Thr Lys TYr Glu 2354 WO 97/46105 PCTIEP97/02737 -109- 1590 1595 GTG ACC TAC AGC AGC GAG CTG GGC CCC AAC GTM AGC Val Thr Tyr Ser Ser Giu Leu Gly Pro Asn Val Ser 1605 1610 161 AGC GAC AAG ATT TAC AAG GAC GGC ACC ATC AAG TTC Ser Asp Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe 1620 1625 1630 TAC AGC AAG AAC GAG CAG GCC CIT TTC TAC GAC AGC Tyr Ser Lys Asn Giu Gin Giy Leu Phe Tyr Asp Ser 1640 1645 GAC TIC AAG ATC AAC GCC ATC ACC TAC GAC GGC AAG Asp Phe Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys 1655 1660 TIC CAC CGC TAC AAC AAG TAGATCTIGAG CT Phe His Arg Tyr Asn Lys 1670 INFORMATION FOR SEQ ID N:36: SEQUENCE CHARACTERISUCS: LENGTH: 852 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Met Lys Thr Asn Gin Ile Ser Thr Thr Gin Lys Asn 1 5 10 Met Asp Arg Lys Giy Leu Leu Gly Tyr Tyr Phe Lys 25 Ser Asn Leu Thr Met Phe Ala Pro Thr Arg Asp Ser 40 Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp Lys Lys 55 Gin Ser Ile Arg Trp Ile Giy Leu Ile Gin Ser Lys 70 Phe Thr Phe Asn Leu Ser Giu Asp Giu Gin Ala Ile 90 Gly Lys Ile Ile Ser Asn Lys Gly Lys Glu Lys Gin 100 105 1600 GAC ACC CM( GAG Asp Thr Leu Glu GAC TC ACC MG Asp Phe Thr Lys 1635 GGC C'G AAC TGG Gly Leu Asn Trp 1650 GAG ATG AAC GTI Glu Met Asn Val 1665 2402 2450 2498 2546 2576 Gin Gin Lys Gly Lys Asp Thr Leu Ile Gin Gin Glu Glu Thr Gly Ile Giu Ile Val Val His 110 WO 97/46105 PCT/EP97/02737 -110- Glu Lys Gly Lys Leu Val Pro Ile Lys Ile Glu Tyr Gin Ser Asp Thr 115 120 125 Lys Phe Asn Ile Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys 130 135 140 Ile Asp Ser Gin Asn Gin Pro Gin Gin Val Gin Gin Asp Glu Leu Arg 145 150 155 160 Asn Pro Glu Phe Asn Lys Lys Glu Ser Gin Glu Phe Leu Ala Lys Pro 165 170 175 Ser Lys Ile Asn Leu Phe Thr Gin Gin Met Lys Arg Glu Ile Asp Glu 180 185 190 Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu Glu Asn 195 200 205 Gly Tyr Thr Ile Gin Asn Arg Ile Ala Val Lys Trp Asp Asp Ser Leu 210 215 220 Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His 225 230 235 240 Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu 245 250 255 Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe 260 265 270 Pro Ser Val Asn Val Ser Met Glu Lys Val Ile Leu Ser Pro Asn Glu 275 280 285 Asn Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr 290 295 300 Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gly Ile Gly Pro Lys Gly 305 310 315 320 Ile Ser Phe Gly Val Ser Val Asn Tyr Gin His Ser Glu Thr Val Ala 325 330 335 Gin Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gin Phe Asn Thr Ala 340 345 350 Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr 355 360 365 Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn 370 375 380 Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn 385 390 395 400 Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gin Asn Gly Ile Ala S 'WO 97/46105 PCTIEP97/02737 -111 405 410 415 Ile Thr Ser Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys 420 425 430 Lys Gin Val Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr 435 440 445 Asn Gin Thr Asp Gly Val Tyr Lys Ile Lys Asp Thr His Gly Asn Ile 450 455 460 Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gin Gin Ile Lys Ala Lys 465 470 475 480 Thr Ala Ser Ile Ile Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg 485 490 495 Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu 500 505 510 Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu 515 520 525 Ile Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser 530 535 540 Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gin 545 550 555 -560 Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp 565 570 575 Val Lys Leu Thr Pro Lys Met Asn Val Thr Ile Lys Leu Ser Ile Leu 580 585 590 Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser Ile Gly Lys Trp Thr Asn 595 600 605 Thr Asn Ile Val Ser Gly Gly Asn Asn Gly Lys Lys Gin Tyr Ser Ser 610 615 620 Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gin Glu Lys 625 630 635 640 Leu Asn Lys Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Met Lys Ser Glu 645 650 655 Lys Asn Thr Gin Cys Glu Ile Thr Ile Asp Gly Glu Ile Tyr Pro Ile 660 665 670 Thr Thr Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp 675 680 685 Ile Ile Ala His Asn Ile Lys Ser Asn Pro Ile Ser Ser Leu His Ile 690 695 700 WO 97/46105 PCTEP97/02737 -112- Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp Asp Asp Ile Ser Ile Thr 705 710 715 720 Asp Val Ala Ser Ile Lys Pro Glu Asn Leu Thr Asp Ser Glu Ile Lys 725 730 735 Gin Ile Tyr Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile 740 745 750 Asp Lys Lys Gly Gly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala Ser 755 760 765 Phe Asn Ile Glu Pro Leu Gin Asn Tyr Val Thr Lys Tyr Glu Val Thr 770 775 780 Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu Ser Asp 785 790 795 800 Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys Tyr Ser 805 810 815 Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser Gly Leu Asn Tp Asp Phe 820 825 830 Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His 835 840 845 Arg Tyr Asn Lys 850 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEONESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "forward primer used to make pCIB5527 (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GGATCCACCA TGCIGCAGAA CCTGAAGATC AC 32 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: WO 97/46105 PCT/EP97/02737 -113- LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single ITOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "reverse primer used to make pCIB5527" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: AAGCTTCCAC TCCTCTC 18 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 1241 base pairs TYPE: nucleic acid STRANDEONESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic IA" (iii) HYPOTHEICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 9..1238 OTHER INFORMATION: /note= "Maize optimized DNA sequence encoding VIP2A(a) with the Bacillus secretion signal removed as contained in pCIB5527" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GA'ICCACC ATG CTG CAG AAC CTG AAG AIC ACC GAC AAG GTG GAG GAC TIC Met Leu Gin Asn Leu Lys Ile Thr Asp Lys Val Glu Asp Phe 855 860 865 AAG GAG GAC AAG GAG AAG GCC AAG GAG TGG GGC AAG GAG AAG GAG AAG 98 Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys Glu Lys 870 875 880 GAG TGG AAG CTI ACC GCC ACC GAG AAG GGC AAG ATG AAC AAC TIC CTG 146 Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu 885 890 895 GAC AAC AAG AAC GAC ATC AAG ACC AAC TAC AAG GAG ATC ACC 'TC AGC i' i' SWO 97/46105 PCTEP97/02737 -114- Asp Asn Lys Asn Asp Ile Lys Thr Asn Tyr Lys 900 905 Glu Ile Thr Phe Ser 910

ATA

Ile 915 CCC GGC AGC TIC Ala Gly Ser Phe GAG GAC Glu Asp 920 GAG ATC AAG Glu Ile Lys CTG ACC AAC Leu Ser Asn 940 CTG AAG GAG ATC Leu Lys Glu Ile

GAC

Asp 930 AAG ATG TIC GAC Lys Met Phe Asp AAC GIG GAG CCC Asn Val Glu Pro 950 AAC ACC ATC AAC Asn Thr Ile Asn 965 AAG ACC AAC Lys Thr Asn 935 AGC ATC ATC ACC Ser Ile Ile Thr TAC AAG Tyr Lys 945 ACC ACC ATC GGC Thr Thr Ile Gly

TIC

Phe 955 AAC AAC AGC CTG Asn Lys Ser Leu ACC GAG GGC Thr Giu Cly 960 GAG TC CTG Gin Phe Leu 290 338 386 AGC GAC GCC Ser Asp Ala CCC CAG TIC AAG Ala Gin Phe Lys

GAG

Glu 975 GAC CGC Asp Arg 980 GAC ATC AAG ITC Asp Ile Lys Phe

GAC

Asp 985 AGC TAC CTG GAC Ser Tyr Leu Asp

ACC

Thr 990 CAC CTG ACC GCC His Leu Thr Ala

GAG

Gin 995 GAG GIG ACC AGC Gin Val Ser Ser AAC GAG Lys Glu 1000 CGC GIG ATC Arg Val Ile CMC AAC Leu Lys 1005 GIG ACC GIC Val Thr Val

CCC

Pro 1010 AGC GGC AAG Ser Gly Lys GGC AGC ACC ACC Gly Ser Thr Thr 1015 CCC ACC AAG Pro Thr Lys 1020 GCC GGC GIG ATC Ala Cly Val Ile CMC AAC Leu Asn 1025 AAC AGe GAG TAC AAG Asn Ser Giu Tyr Lys 1030 ATG CrC ATC Met Leu Ile GAC AAC Asp Asn 1035 GGC TAC ATG Gly Tyr Met GIG CAC GTG Val His Val 1040 GAC AAG GIG AGe Asp Lys Val Ser 1045 GAG GGC ACC CIG Glu Gly Thr Leu 1060 AAC GTG GTG Lys Val Val AAG AAG Lys Lys 1050 GGC GTG GAG Cly Val Glu IGC CTrC CAG ATC Cys Leu Gin Ile 1055 GAC ATC AAC GCC Asp Ile Asn Ala 578 626 674 AAG AAC ACT CrA Lys Lys Ser Leu 1065 GAC TI AAG Asp Phe Lys

AAC

Asn 1070 GAG GCC Glu Ala 1075 CAC ACC IGG His Ser Trp GGC ATG Gly Met 1080 AAG AAC TAC Lys Asn Tyr GAG GAG TIG GCC AAG Glu Giu Trp Ala Lys 1085

GAC

Asp 1090 CC ACC GAC ACC GAG CGC GAG CCC CTG GAC GGC TAC GCC Leu Thr Asp Ser Gin Arg Clu Ala Leu Asp Cly Tyr Ala 1095 1100 CGC CAG CAC Arg Gin Asp 1105 AGC GGC AAC Ser Cly Asn 1120 TAC AAG GAG Tyr Lys Glu ATC AAC AAC TAC CTG Ile Asn Asn Lyr Leu 1110 CGC AAC GAG GGC GGC Arg Asn Gin Gly Cly 1115

I,

"WO 97/46105 PCT/EP97/02737 -115- GAG AAG CTG Glu Lys Leu 1125 AAG CCC ATC Lys Pro Ile 1140 GAC GCC CAG AIC AAG AAC ATC AGC Asp Ala Gin Ile Lys Asn Ile Ser 1130 CCC GAG AAC ATC ACC GM1X TAC CGC Pro Giu Asn Ile Thr Val Tyr Arg GAC GCC CIG GGC AAG Asp Ala Leu Gly Lys 1135 '1XG TGC Trp Cys 1150 GGC ATG CCC Gly Met Pro 866 914 962 1145 GAG TIC Glu Phe 1155 GGC TAC GAG ATC AGC Cly Tyr Gin Ile Ser 1160 GAC CCC CTG Asp Pro Leu CCC AGC Pro Ser 1165 CIG AAG GAC Leu Lys Asp

TICI

Phe 1170 GAG GAG GAG T'TC Glu Clu Gin Phe CIG AAC Leu Asn 1175 ACC AM AAG GAG GAC AAG GCC TAC Thr Ile Lys Giu Asp Lys Cly Tyr 1180 ATG AGC Met Ser 1185 1010 1058 ACC AGC CTG AGC AGC GAG CGC CIG CCC CC TTC C AGC Thr Ser Leu Ser Ser Giu Arg Leu Ala Ala Phe Gly Ser CGC AAG ATC Arg Lys Ile 1200 1190 1195 ATC CTG CCC CTG Ile Leu Arg Leu 1205 GAG GTG CCC Gin Val Pro AAG GGC Lys Gly 1210 AGC ACT COT CCC TAC CMC AGC Ser Thr Gly Ala Tyr Leu Ser 1215 1106 GCC ATC GGC Ala Ile Gly 1220 GGC TIC GCC Gly Phe Ala AGC GAG Ser Glu 1225 AAG GAG ATC Lys Glu Ile CI CTG Leu Leu 1230 GAT AAG GAC Asp Lys Asp AAG GGC GIG Lys Cly Val 1250 1154 1202 AGC AAC Ser Lys 1235 TAC CAC ATC Tyr His Ile GAC AAG GTG Asp Lys Val 1240 ACC GAG GIG ATC ATC Thr Glu Val Ile Ile 1245 CC CTG ACC AAC TAG Leu Leu Thr Asn 1260 AAG CCC TAC GIG Lys Arg Tyr Vai GTG GAC CCC ACC Val Asp Ala Thr 1255 1241 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 410 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Leu Gin Asn Leu Lys lie Thr Asp Lys Val Glu Asp Phe Lys Glu 1 5 10 Asp Lys Giu Lys Ala Lys Giu Trp Cly Lys Glu Lys Glu Lys Glu TRp 25 Lys Leu Thr Ala Thr Giu Lys Gly Lys Met Asn Asn Phe Leu Asp Asn r C "WO 97/46105 PCT/EP97/02737 -116- Lys Gly Phe Glu Ile Asp Val 145 Lys Glu Val Thr His 225 Asp Glu Leu Ile Gly 305 Gin Asn Ser Asp Pro Asn Ile 130 Ser Gly Tyr Ser Leu 210 Ser Ser Ile Asp Pro 290 Tyr Phe Lys Asp Asn Ile Ala Asp Glu Thr 165 Leu Val Ser Met Glu 245 Tyr Ile Ile Ser Thr 325 Thr Glu 70 Leu Gly Met Ser Arg 150 Pro Ile Lys Leu Lys 230 Ala Leu Lys Thr Asp 310 Ile Tyr Lys Lys Asp Asn Ser Asn Lys 105 Gin Phe 120 Leu Asp Ile Leu Lys Ala Asn Gly 185 Gly Val 200 Phe Lys Tyr Glu Asp Gly Asn Gin 265 Ile Ser 280 Tyr Arg Leu Pro Glu Asp Glu Leu Ile 90 Ser Lys Thr Lys Gly 170 Tyr Glu Asn Glu Tyr 250 Gly Asp Trp Ser Lys 330 Ile Thr Phe Lys Glu Ile 75 Ile Thr Tyr Leu Thr Glu Glu Gin 'Phe 125 His Leu Thr 140 Val Thr Val 155 Val Ile Leu Met Val His Cys Leu Gin 205 Asp Ile Asn 220 Trp Ala Lys 235 Ala Arg Gin Gly Ser Gly Ala Leu Gly 285 Cys Gly Met 300 Leu Lys Asp 315 Gly Tyr Met Ser Ile Ala Asp Lys Met Lys Asn Val Gly Asn Thr 110 Leu Asp Arg Ala Gin Gin Pro Ser Gly 160 Asn Asn Ser 175 Val Asp Lys 190 Ile Glu Gly Ala Glu Ala Asp Leu Thr 240 Asp Tyr Lys 255 Asn Glu Lys 270 Lys Lys Pro Pro Glu Phe Phe Glu Glu 320 Ser Thr Ser 335

C

WO 97/46105 PCT/EP97/02737 -117- Leu Ser Ser Glu 340 Arg Leu Ala Ala Phe Gly Ser Arg Lys 345 Ile Ile Leu 350 Arg Leu Gin Val 355 Pro Lys Gly Ser Thr 360 Gly Ala Tyr Ser Ala Ile Gly Gly 370 Phe Ala Ser Glu Glu Ile Leu Leu Lys Asp Ser Lys Tyr 385 His Ile Asp Lys Val 390 Thr Glu Val Ile Ile 395 Lys Gly Val Lys Tyr Val Val Asp Thr Leu Leu Thr Asn 410 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide encoding eukaryotic secretion signal used to construct pCIB5527" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: GGATCCACCA 'IGGGCTGGAG CTGGATCTIC CIGTCCIG C TGAGCGGCGC CGCGGGCGTG CACTGCCIGC AG INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 1241 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic INA" (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS 1, WO 97/46105 PCTEP97/02737 -118- LOCATION: 9..1238 OTHER INFORMATION: /note= "Maize optimized DNA sequence encoding VIP2A(a) with the Bacillus secretion signal removed and the eukazyotic secretion signal inserted as contained in pCIB5528" (xi) SEQUENCE DESCRIPTION: SFQ ID NO:42: GATCCACC ATG CMX CAG AAC CTG AAG ATC ACC GAC AAG GTG GAG GAG TIC Met Leu Gin Asn Leu Lys Ile Thr Asp Lys Val Giu Asp Phe 415 420 AAG GAG Lys Glu 425 GAC AAG GAG Asp Lys Glu

AAG

Lys 430 GCG AAG GAG TGG Ala Lys Giu Trp

GGC

Gly 435 AAG GAG AAG GAG Lys Glu Lys Glu

AAG

Lys 440 GAG TGG AAG CIT Glu Trp Lys Leu

ACC

Thr 445 GCG AGC GAG AAG Ala Thr Giu Lys

GGG

Gly 450 AAG ATG AAG AAC TIC CTG Lys Met Asn Asn Phe Leu 455 GAG AAC AAG Asp Asn Lys ATA GGC GGG Ile Ala Gly 475

AAC

Asn 460 GAG ATG AAG ACG Asp Ile Lys Thr

AAC

Asn 465 TAC AAG GAG Tyr Lys Glu AGG TIC GAG GAG Ser Phe Giu Asp

GAG

Glu 480 ATC AAG GAC M1X Ile Lys Asp Leu ATC ACC TI AGG Ile Thr Phe Ser 470 AAG GAG ATC GAG Lys Giu Ile Asp 485 ATC ACC TAG AAG Ile Thr Tyr Lys 194 242 AAG ATG Lys Met 490 TIC GAC AAG ACC Phe Asp Lys Thr

AAG

Asn 495 CTG AGC AAC AGC Leu Ser Asn Ser 290

AAG

Asn 505 GTG GAG CCC Val Glu Pro ACC ACC Thr Thr 510 ATC GGC TIC AAC AAG Ile Gly Phe Asn Lys 515 AGG CIG ACC GAG Ser Leu Thr Glu

GGG

Gly 520 AAG ACC NrC AAG AGG GAG GCC AMIG GCC Asn Thr Ile Asn Ser Asp Ala Met Ala 525 GAG TTC Gin Phe 530 AAG GAG GAG T1 C-T Lys Glu Gin Phe Leu 535 GAC CGC GAG Asp Arg Asp GAG GAG GTG Gin Gin Val 555 ATC AAG Ile Lys 540 TIC GAG AGG Phe Asp Ser

TAG

Pyr 545 CMG GAC ACC CAC Leu Asp Thr His CTG AGG GCC Leu Thr Ala 550 ACC GTC CCC Thr Val Pro AGG AGG AAG GAG Ser Ser Lys Glu

CGC

Arg 560 GTG AIC 0RG AAG Val Ile Leu Lys AGC GGC Ser Gly 570 AAG GGG AGG ACC Lys Gly Ser Thr

ACC

Thr 575 CCC ACC AAG GCC Pro Thr Lys Ala

GGC

Gly 580 GMh ATC CTG AAC Val Ile Leu Asn AAC AGG GAG TAC AAG AM M AC GAC AAC GGC TAG AM1GM CAC GI= Asn Ser Giu Tyr Lys Met Leu Ile Asp Asn Gly Tyr Met Val His Val WO 97/46105 W097/6105PCT/EP97/02737 -119- CAC AAG CTG AGC Asp Lys Val Ser

AAG

Lys 605 GIG GTGX AAC AAC Val Val Lys Lys GAG TGC =~i Val Giu Cys Leu CAG AWC Gin Ie 615 GAG CCC ACC Giu Gly Thr GAG CCC CAC Glu Ala His 635 AAG AAC ACT CTA Lys Lys Ser Leu TCAAG AAC CAC ATC AAC CC Phe Lys Asn Asp Ile Asn Ala 630 ACC TGG CCC ATGl Ser Trp Cly Met AAC TAC GAG Asn Tyr Ciu GAG =,.,GCCC AAG GAC Glu Thp Ala Lys Asp 645 TAC CCC CCC CAC GAC Tyr Ala Arg Gin Asp 660 CT0 ACC Leu Thr 650 CAC ACC CAG Asp Ser Gin CCC GAG Arg Giu 655 CCC CTG CAC GC Ala Leu Asp Giy

TAC

T'yr 665 AAG GAG AWI Lys Ciu Ile AAC AAC TAC Asn Asn Tyr 670 CTG CCC AAC Leu Arg Asn CCC CCC ACC GC Gly Gly Ser Gly GAG AAG CTG GAC Ciu Lys Leu Asp

CC

Ala 685 CAG ATC AAG AAC Gin Ile Lys Asn ACC GAC CCC CM0 Ser Asp Ala Leu CCC AAC Giy Lys 695 AAG CCC AEC Lys Pro Ile GAG TIC GC Ciu Phe Giy 7125 GAG AAC ATC ACC Giu Asn Ile Thr TAC CCC rRM G 1C Tyr Arg Trp Cys CCC A'TG CCC Ciy Met Pro 710 TAC CAG ATC AGC Tyr Gin Ile Ser

GAC

Asp 720 CCC CTG CCC Pro Leu Pro AGC cIT3 AAG CAC TITC Ser Leu Lys Asp Phe 725 AAC CCC TAC ATG AC Lys Gly Tyr Met Ser 740 GAG GAG Giu Giu 730 GAG TIC CIG AAC Gin Phe Loeu Asn

ACC

Thr 735 A'IC AAG GAG GAC Ile Lys Giu Asp

ACC

Thr 745 ACC CTC ACC AC Ser Leu Ser Ser CCC CTG CCC CC Arg Leu Ala Ala GCC ACC CCC AAC Giy Ser Arg Lys

ATC

le 760 1010 1058 1106 A[C Mr CCC Mr le Lieu Arg Leu G'TG CCC AAG GCC Vai Pro Lys Giy

A~C

Ser 770 Acr GGT CC TAC Tr~l Giy Ala Tyr CTG AGC Leu Ser 775 CCC ATC GC Ala Ile Giy TIC CCC ACC GAG Phe Ala Ser Giu GAG AWC CTG CTG Ciu Ile Leu Leu GAT AAC GAC Asp Lys Asp 790 MG CCC GM~ Lys Giy Vai 11i54 ACC AAG TAC CAC A IC GAC AAG Ser Lys Tyr His Ile Asp Lys 795 ACC GAG GM0 ATC Thr Ciu Vai le A EC Ile 805 1202 AAG CCC TAC GTG GTG GAC CC ACC MI Cr0 ACC AAC TAG 14 1241 WO 97/46105 PCT/EP97/02737 -120- Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 810 815 820 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 410 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Met Leu Gin Asn Leu Lys Ile Thr Asp Lys Asp Lys Glu Lys Leu Thr Lys Asn Asp Gly Ser Phe Phe Asp Lys Glu Pro Thr Ile Asn Ser 115 Asp Ile Lys 130 Val Ser Ser 145 Lys Gly Ser Glu Tyr Lys Val Ser Lys 195 Glu Lys Asn 55 Ile Ser Phe Ala Tyr 135 Val Thr Asp Lys Trp Gly Tyr Lys Asn Asn Gin 120 Leu Ile Lys Asn Gly 200 Val Glu Glu Lys Asn Asn Ile Thr Lys Glu Ile Thr Leu Thr Glu Gin His Leu 140 Val Thr 155 Val Ile Met Val Cys Leu Phe Lys Leu Ser Asp Lys Gly 110 Leu Ala Pro Asn Val 190 Ile Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp Ile Asn Ala Glu Ala wVO97/46105 PCTEP97/02737 -121- 210 215 220 His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp Ala Lys Asp Leu Thr 225 230 235 240 Asp Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala Arg Gin Asp Tyr Lys 245 250 255 Glu Ile Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser Gly Asn Glu Lys 260 265 270 Leu Asp Ala Gin Ile Lys Asn Ile Ser Asp Ala Leu Gly Lys Lys Pro 275 280 285 Ile Pro Glu Asn Ile Thr Val Tyr Arg Trp Cys Gly Met Pro Glu Phe 290 295 300 Gly Tyr Gin Ile Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe Glu Glu 305 310 315 320 Gin Phe Leu Asn Thr Ile Lys Glu Asp Lys Gly Tyr Met Ser Thr Ser 325 330 335 Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg Lys Ile Ile Leu 340 345 350 Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser Ala Ile 355 360 365 Gly Gly Phe Ala Ser Glu Lys Glu Ile Leu Leu Asp Lys Asp Ser Lys 370 375 380 Tyr His Ile Asp Lys Val Thr Glu Val Ile Ile Lys Gly Val Lys Arg 385 390 395 400 Tyr Val Val Asp Ala Thr Leu Leu Thr Asn 405 410 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 86 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide encoding vacuolar targetting peptide used to construct pCIB5533" (iii) HYPOTHETICAL: NO 1 d WO 97/46105 PCT/EP97/02737 -122- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CCGCGGGCGT GCACTGCCIC ACCAGCAGCA CCTICGCCGA CACCAACCCC ATCCGCTGA CCGACCCGC CGCCAGCACC CT]CAG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1358 base pairs TYPE: nucleic acid STRANDEE]NESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic INA" (iii) HYPOTHEIICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 9..1355 OTHER INFORMATION: /note= "Maize optimized VIP2A(a) with the Bacillus secretion signal removed and the vacuolar targetting signal inserted as contained in pCIB5533" (xi) SEQUENCE DESCRIPTION: SEQ ID GATCCACC ATG GGC TGG AGC TGG ATC TIC CTG TIC CTG CTG AGC GGC GCC Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Ala 415 420 GGC GTG CAC TGC Gly Val His Cys

CTC

Leu 430 AGC AGC AGC AGC Ser Ser Ser Ser

TTC

Phe 435 GCC GAC AGC AAC Ala Asp Ser Asn

CCC

Pro 440 ATC CGC GTG Ile Arg Val ACC GAC AAG Thr Asp Lys TGG GGC AAC Trp Gly Lys 475 ACC GAC Thr Asp 445 GTG GAG Val Glu 460 CGC CCC CCC AGC Arg Ala Ala Ser CTG CAG AAC Leu Gln Asn AAG GAG AAG Lys Glu Lys C AAG ATC Leu Lys Ile 455 CCC AAG GAG Ala Lys Clu 470 GAC TIC AAG Asp Phe Lys GAG GAC Glu Asp 465 GAG AAG GAG AAG Glu Lys Glu Lys

GAG

Glu 480 TGG AAG CIT ACC CCC ACC GAG AAG Trp Lys Leu Thr Ala Thr Glu Lys 485 GGC AAG Gly Lys 490 ATG AAC AAC TIC Met Asn Asn Phe

CTG

Leu 495 GAC AAC AAG AAC GAC ATC AAC ACC AAC Asp Asn Lys Asn Asp Ile Lys Thr Asn 500 TAC AAC GAG AC ACC TC ACC ATA CCC GGC ACC TTC GAG GAC GAG ATC "WO 97/46105 ~~WO97/4105PCT/EP97/02737 -123- Tyr Lys Giu Ie Thr 505 Phe Ser Ile Ala 510 ATC GAG AAG ATG Ile Asp Lys Met Gly Ser 515 TIC GAG Phe Asp 530 Phe Glu Asp Giu AAG GAG C'iM Lys Asp Leu AAG AGG AWC Asn Ser Ile AAG AAG AGG Asn Lys Ser 555 AAG GAG Lys Giu 525 AAG ACC AAG Lys Thr Asn CMG AGG Leu Ser 535 ACC TAG AAG AAG Thr TYr Lys Asn Val 545 GAG CCC AC Glu Pro Thr AGG ATC GGC TIC Thr Ile Gly Phe 550 CTG AGG GAG GGC Leu Thr Giu Giy

AAC

Asn 560 AGG A'IC AAG AGG Thr le Asn Ser GCC A EG GGG Ala Met Ala GAG TIC Gln Phe 570 AAG GAG GAG TIC Lys Giu Gin Phe Mr Leu 575 GAG CGC GAG ATC Asp Arg Asp le

AAG

Lys 580 TI GAG AGC TAG Phe Asp Ser Tyr GAG AGG GAG =I Asp Thr His Leu AGG GGG Thr Ala 590 GAG GAG GM AIG AGC AAG GAG CC Gin Gin Val Ser Ser Lys Giu Arg 595 ATC CTG MAG GTG Ile Leu Lys Val

ACC

Thr 605 GWI CGC AGC GGC Val Pro Ser Gly GGG AGC AGC ACG Gly Ser Thr Thr CCG ACC Pro Thr 615 AAG GC GGC Lys Ala Giy AAC GGG TAG Asn Gly Tyr 635 Val 620 ATC CTG AAC AAC AGG le Leu Asn Asn Ser 625 GAG TAG AAG ATG~ Giu Tyr Lys Met OrG ATC GAG Leu Ile Asp 630 GTG AAG AAG Val Lys Lys ATEJG G AG GTG GAC AAG GG AGG AAG Met Vai His Val Asp Lys Val Ser Lys GGG GIG Gly Vai 650 GAG TG CIC GAG Giu Gys Leu Gin

ATC

le 655 GAG GGG AGG Giu Gly Thr CTG AAG Leu Lys 660 AGG TG Ser Trp 675 MAG ACT CTA GAG Lys Ser Leu Asp

TIC

Phe 665 MAG MAC GAG ATC Lys Asn Asp Ile

MGC

Asn 670 GGG GAG GGG CAG Ala Giu Ala His GGG A 3 MAG Giy Met Lys

MGC

Asn 680 TAG GAG GAG TG Tyr Giu Giu Trp

GGG

Ala 685 MAG GAG CIG AGG Lys Asp Leu Thr AGG GAG CG GAG Ser Gin Arg Giu GGG cTG Ala Leu 695 GAG GGC TAG Asp Gly Tyr AAG GAG GGG Asn Gin Gly 715

GGG

Ala 700 CG GAG GAG Arg Gin Asp TAG MAG GAG AMI MAC TIyr Lys 705 Glu Ile Asn MGC TAG CMG GGC Asn Tyr Leu Arg 710 GAG A IC MG MGC Gin Ile Lys Asn 725 GGG AGC GGC MGC Giy Ser Giy Asn

GAG

Giu 720 MAG OrG GAG GCC Lys Leu Asp Ala WO 97/46105 PCT/EP97/02737 -124- ATC AGC Ile Ser 730 GAC GCC CTG GGC Asp Ala Leu Gly AAG CCC ATC CCC Lys Pro Ile Pro

GAG

Glu 740 AAC ATC ACC GIG Asn Ile Thr Val

TAC

7yr 745 CGC TGG TGC Arg Trp Cys GGC AIG Gly Met 750 CCC GAG TIC GGC Pro Giu Phe Gly CAG ATC AGC GAC Gin Ile Ser Asp

CCC

Pro 760 1010 1058 1106 CTG CCC AGC CTG Leu Pro Ser Leu GAC TIC GAG GAG Asp Phe Giu Glu

CAG

Gin 770 TIC CIG AAC ACC Phe Leu Asn Thr ATC AAG Ile Lys 775 GAG GAC AAG Glu Asp Lys GCC 'FTC GGC Ala Phe Gly 795 GGC TAC Gly Tyr 780 ATO AGC ACC Met Ser Thr

AGC

Ser 785 CTG AGC AGC GAG Leu Ser Ser Glu AGC CGC AAG ATC Ser Arg Lys Ile

ATC

Ile 800 CIG CGC CG CAG Leu Arg Leu Gin

GTG

Val 805 CGC CTG CC Arg Leu Ala 790 CCC AAG GGC Pro Lys Gly AGC GAG AAG Ser Glu Lys 1154 1202 AGC ACr Ser Thr 810 GGT GCC TAC CIG Gly Ala Tyr Leu

AGC

Ser 815 GC ATC GGC GGC Ala Ile Gly Gly TC CC Phe Ala 820 1250 1298

GAG

Glu 825 ATC CrG CTG GAT Ile Leu Leu Asp GAC AGC AAG TAC Asp Ser Lys Tyr AIC GAC AAG GIG Ile Asp Lys Val

ACC

Thr 840 GAG GMO AC ATC AAG GGC GT AAG Glu Val Ile Ile Lys Gly Val Lys CGC TAC Arg Tyr 850 GX GI GAC GCC Val Val Asp Ala ACC CCT Thr Leu 855 1346 1358 CIG ACC AAC TAG Leu Thr Asn INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 449 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N:46: Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Ala Ala Gly Val His Cys Leu Ser Ser Ser Ser Phe Ala Asp Ser Asn 25 Pro Ile Arg Val Thr Asp Arg Ala Ala Ser Thr Leu Gin Asn Leu Lys Ile Thr Asp "WO 97/46105 PCT/EP97/02737 -125- 40 Lys Val Glu Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly 55 Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys 70 75 Met Asn Asn Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr Asn Tyr Lys 90 Glu Ile Thr Phe Ser Ile Ala Gly Ser Phe Glu Asp Glu Ile Lys Asp 100 105 110 Leu Lys Glu Ile Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser 115 120 125 Ile Ile Thr Tyr Lys Asn Val Glu Pro Thr Thr Ile Gly Phe Asn Lys 130 135 140 Ser Leu Thr Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Ala Gin Phe 145 150 155 160 Lys Glu Gin Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Tyr Leu Asp 165 170 175 Thr His Leu Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val Ile Leu 180 185 190 Lys Val Thr Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala 195 200 205 Gly Val Ile Leu Asn Asn Ser Glu Tyr Lys Met Leu Ile Asp Asn Gly 210 215 220 Tyr Met Val His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val 225 230 235 240 Glu Cys Leu Gin Ile Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys 245 250 255 Asn Asp Ile Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu 260 265 270 Glu Trp Ala Lys Asp Leu Thr Asp Ser Gin Arg Glu Ala Leu Asp Gly 275 280 285 Tyr Ala Arg Gin Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Arg Asn Gin 290 295 300 Gly Gly Ser Gly Asn Glu Lys Leu Asp Ala Gin Ile Lys Asn Ile Ser 305 310 315 320 Asp Ala Leu Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Val Tyr Arg 325 330 335 WO 97/46105 PCT/EP97/02737 -126- Trp Cys Gly Met Pro Glu Phe Gly Tyr Gin Ile Ser Asp Pro Leu Pro 340 345 350 Ser Leu Lys Asp Phe Glu Glu Gin Phe Leu Asn Thr Ile Lys Glu Asp 355 360 365 Lys Gly Tyr Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe 370 375 380 Gly Ser Arg Lys Ile Ile Leu Arg Leu Gin Val Pro Lys Gly Ser Thr 385 390 395 400 Gly Ala Tyr Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Lys Glu Ile 405 410 415 Leu Leu Asp Lys Asp Ser Lys Tyr His Ile Asp Lys Val Thr Glu Val 420 425 430 Ile Ile Lys Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr 435 440 445 Asn INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEENESS: single 'IOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOI'HETICAL: NO (ix) FEATURE: NAME/KEY: Peptide LOCATION: 1..16 OTHER INFORMATION: /note= "linker peptide for fusion of VIPA(a) and VIP2A(a) used to construct pCIB5533" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser 1 5 10 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid WO 97/46105 PCTIEP97/02737 -127- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA encoding linker peptide used to construct pCIB5533" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CCCGGGCCT CTACTCCCCC AACICCCICT CCTAGCACGC CTCCGACACC TAGCGATATC GGAICC 66 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 4031 base pairs TYPE: nucleic acid STRANDEDENESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA" (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 6..4019 OTHER INFORMATION: /note= "Maize optimized DNA sequence encoding a VIP2A(a) VIPA(a) fusion protein as contained in pCIB5531" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GATCC ATG AAG CGC ATG GAG GGC AAG CTG TIC ATG GIG AGC AAG AAG 47 Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys 450 455 460 CIC CAG GTG GTG ACC AAG ACC GIG CIG CG AGC ACC GTG TIC AGC ATC Leu Gin Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser Ile 465 470 475 AGC CIG CG AAC AAC GAG GIG A'IC AAG GCC GAG CAG CIG AAC ATC AAC 143 Ser Leu Leu Asn Asn Glu Val Ile Lys Ala Glu Gin Leu Asn Ile Asn 480 485 490 495 AGC CAG AGC AAG TAC ACC AAC CTC CAG AAC CTG AAG ATC ACC GAC AAG WO 97/46105 WO 9746105PCT/EP97/02737 128 Ser Gin Ser G IG GAG GAC Val Giu Asp GAG AAG GAG Giu Lys Giu 530 Lys Tyr Thr Asn Leu Gin Asn Leu 500 505 Lys Ile Thr Asp Lys 510 =I AAG GAG GAC AAG GAG Phe 515 Lys Giu Asp AAG GAG TGG AAG Lys Glu Trp Lys Lys Giu 520 CYIT ACC Leu Thr 535 AAG GCC AAG GAG fCG GGC AAG Lys Ala Lys Giu Trp Gly Lys 525 GCC ACC GAG Ala Thr Giu

AAG

Lys 540 GGC AAG ATIx Gly Lys Met AAC AAC Asn Asn 545 TIC MI~ GAC AAC Phe Loeu Asp Asn AAC GAC A'lC AAG Asn Asp Ilie Lys

ACC

Thr 555 AAC TAC AAG GAG Asn Tyrr Lys Giu

ATC

Ile 560 ACC TIC AGC ATA Thr Phe Ser le GGC AGC TIC GAG Gly Ser Phe Glu GAG ATC AAG GAC Giu Ile Lys Asp AAG GAG A'lC GAC Lys Glu Ile Asp AI( TIC GAC AAG Met Phe Asp Lys AAC =I AGC AAC Asn Leu Ser Asn AGC A IC Ser Ile 590 ATC ACC TAC Ile Thr Tyr CTG: ACC GAG Leu Thr Giu 610

AAG

Lys 595 AAC GIY GAG CCC Asn Val Giu Pro

ACC

Thr 600 ACC A'IC GGC TIc Thr Ile Gly Phe AAC AAG AGC Asn Lys Ser 605 CAG TIC AAG Gin Phe Lys GGC AAC ACC ATC Giy Asn Thr Ile

AAC

Asn 615 AGC GAC GCC ATGX Ser Asp Ala Met GAG GAG Giu Gin 625 TIC CTG GAC CGC Phe Leu Asp Arg

GAC

Asp 630 ATC AAG TIC GAC le Lys Phe Asp TAC MI GAC ACC Tyr Leu Asp Thr

CAC

His 640 CTG ACC GCC Leu Thr Ala GAG GAG Gin Gin 645 AGC GGC Ser Giy 660 G'IG AGC AGC AAG Vai Ser Ser Lys

GAG

Giu 650 CGC G1TG ATC MI Arg Val Ile Loeu

AAG

Lys 655 cix; ACC GTC CCC Val Thr Vai Pro AAG GGC AGC Lys Giy Ser

ACC

Thr 665 ACC CCC ACC AAG Thr Pro Thr Lys GCC GCc Ala Giy 670 G'IG ATC CTG Vai Ile Leu ATG GTG GAC Met Val His 690 TGC CTC GAG Cys Leu Gin 705 AAC AGC GAG Asn Ser Giu TAC AAG A1X Tyr Lys Met 680 C1XG ATC GAC AAC CGC TAC Loeu Ile Asp Asn Gly Tyr 685 GMG GAC AAG GTG Vai Asp Lys Val ATC GAG GGC ACC Ile Giu Giy Thr 710 AGC AAG Ser Lys 695 Mix Gix; AAG Val Val Lys AAG GOC cix; GAG Lys Giy Val Giu 700 GAC TIC MAG AAC Asp Phe Lys Asn CPG MG AAG AGT CTA Leu Lys Lys Ser Leu 715 WO 97/46105 WO 9746105PCT/EP97/02737 129

GAG

Asp 720 ATC AAC GCC GAG Ile Asn Ala Giu

CC

Ala 725 GAC AGG TGO His Ser Trp GGC A!IG Giy Met 730 AAG AAC TAG GAG Lys Asn Tyr Glu

GAG

Glu 735 rGG GCC AAG GAC Trp Ala LYS Asp

GM

Leu 740 ACC GAG AGC GAG Thr Asp Ser Gin

GG

Arg 745 GAG GCC GIG GAC Glu Ala Lieu Asp GGG TAC Gly Tyr 750 CCC CGG GAG Ala Arg Gin

GAG

Asp 755 TAG AAG GAG A~TC Tyr Lys Giu Ile

AAC

Asn 760 AAG TAG CMG Asn Tyr Leu CG AAG GAG GGG Arg Asn Gin Oly 765 959 1007 GGG AGG GGC AAG Gly Ser Gly Asn 770 GAG AAG CIG Giu Lys Leu

GAC

Asp 775 GGG GAG ATC AAG Ala Gin Ile Lys AWC AGG GAC Ile Ser Asp GGG CMx Ala Leu 785 GGC AAG AAG GGG Gly Lys Lys Pro A EC Ile 790 CCC GAG AAC AWC Pro Giu Asn Ile

AGG

Thr 795 GIG TAG CG TG Val Ty~r Arg Trp GGG ATG GGG GAG Giy Met Pro Glu GGC TAG GAG AWC Gly Tyr Gin Ile

AGG

Ser 810 GAG CGG CMG GC Asp Pro Leu Pro 1055 1103 1151 GIG AAG GAG TIC Leu Lys Asp Phe GAG GAG TrC Mlx Giu Gin Phe Leu AAG AC Asn Thr 825 ATC AAG GAG GAG AAG Ile Lys Oiu Asp Lys 830 GGG TAG ATG Oly Tyr Met AGG CG AAG Ser Arg Lys 850 AGG AGG Ser Thr 835 AGG Clx; AGG Ser Leu Ser

AGG

Ser 840 GAG CG CIG GGG Giu Arg Leu Ala GGG m~ GG Ala Phe Oly 845 AGC ACT OCT Ser Thr Giy 1199 1247 ATC AWC Clx; GG Ile Ile Leu Arg cT0 Leu 855 GAG GIG GGG AAG Gin Val Pro Lys

GGG

Oly 860 GGG TAG Ala Ty~r 865 GIG AGG GGG AWC Leu Ser Ala Ile GC TM' GGG AGG Giy Phe Ala Ser

GAG

Giu 875 AAG GAG AWC Ml Lys Glu Ile Leu GAT AAG GAG AGG Asp Lys Asp Ser TAG GAG ATO Tyr His Ile GAG AAG Asp Lys 890 GAG GCG Asp Ala 905 GIG AGG GAG GIG ATC Val Thr Giu Vai Ile 895 1295 1343 1391 ATC AAG GGG I Ile Lys Gly Val CGC TAG GIG GIG Arg Tyr Val Vai ACC CTG CG Thr Leu Leu ACG AAC Thr Asn 910 TcGGGG Cod Ser Arg Giy cci' Pro 915 TCI' ACT CC GGA Ser Thr Pro Pro

ACT

Thr 920 GGG E(T AGG Pro Ser Pro Ser AG COT cG Thr Pro Pro 925 ATC ACC AGG Ile Ser Thr 1439 ACA COT AGG GAT ATC GGA TIXC ACC Thr Pro Ser Asp Ile Giy Ser Thr 930 935 A IG AAG ACC AAG Met Lys Thr Asn 1487 WO 97/46105 WO 9746105PCT/EP97/02737 130- ACC GAG Thr Gin 945 AAG AAC GAG GAG Lys Asn Gin Gin

AAG

Lys 950 GAG A 1X GAC CGC Giu Met Asp Arg GOC GIG CTG GGG Gly Leu Leu Gly

TAC

TIyr 960 TAC =I AAG GGG Tyr Phe Lys Gly AAG GAC Lys Asp 965 TI AGC AAG Phe Ser Asn Loeu 970 ACG A B3 7m GCC Thr Met Phe Ala 1535 1583 1631 ACG CxT GAG AGC Thr Arg Asp Ser =I AWC TAC GAG Leu Ie Tyr Asp

GAG

Gin 985 GAG AGG GCC AAG Gin Thr Ala Asn AAG CG Lys Leu 990 GIG GAG AAG Leu Asp Lys GAG GAG GAG TAG Gin Gin Giu Tyr GAG AGG Gin Ser 1000 AIC CGG IGG Ile Arg Trp AIC GGG cTG Ile Gly Leu 1005 1679 ATW GAG AGG AAG Ile Gin Ser Lys 1010 GAG AGG GGG GAG TIC AGG TIC AAG Giu Thr Gly Asp Phe Thr Phe Asn 1015 cTG AGG GAG GAG Leu Ser Glu Asp 1020 1727 GAG GAG GGG Giu Gin Ala 1025 AWC ATC GAG ATC AAG GGG AAG A IC A IC AGC MGC AAG GGG Ile Ile Giu Ile Asn Gly Lys Ile Ile Ser Asn Lys Gly 1775 1030 1035 MAG GAG Lys Giu 1040 MAG GAG GTG Lys Gin Val GIG GAG GIG GAG MAG Vai His Leu Giu Lys 1045 GGG MAG Gly Lys 1050 CIG GIG GGG Leu Val Pro

ATG

le 1055 1823 MAG A'IC GAG TAG Lys Ile Giu Tyr GAG AGG Gin Ser 1060 GAG AGG MAG Asp Thr Lys TIC AAG AW Phe Asn Ile 1065 GAG AGG MAG AGG Asp Ser Lys Thr 1070 TIC MG GAG Phe Lys Giu GAG GTG~ GAG Gin Val Gin 1090 1075 MAG CT TIC Lys Leu Phe MAG AWC GAG AGG Lys Ile Asp Ser 1080 GAG AAC GAG GGC GAG Gin Asn Gin Pro Gin 1085 1871 1919 1967 CAG GAG GAG CG Glm Asp Giu Leu CGG MGC Arg Asn 1095 GGG GAG TIC Pro Giu Phe MGC MG AAG GAG Asn Lys Lys Glu 1100 AGG GAG Ser Gin 1105 GAG ATG Gin Met 1120 GAG TIC GIG GGG AAG GGG AGG MG ATC MG GIM TIC AGG GAG Giu Phe Leu Ala Lys Pro Ser Lys Ilie Asn Leu Phe Thr Gin 2015 1110 MAG GG GAG ATC GAG GAG GAG Lys Arg Giu Ile Asp Giu Asp 1125 AGG GAG AGG Thr Asp Thr 1130 GAC GGG GAG Asp Gly Asp

AGG

Ser 1135 2063 ATC CCC GAG CGG Ile Pro Asp Leu GCC GTG MAG 'IGG Ala Val Lys Trp

TGG

Trp 1140C GAG GAG MGC GGC TAG ACG ATC GAG AAG GGG A IC Giu Giu Asn Gly Tjyr Thr le Gin Asn Arg Ile 1145 1150 2111 2159 GAG GAG AGG GIG C AGC MAG GGG TAG ACC AAG TIC Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thx Lys Phe WO 97/46105 PCT/EP97/02737 -131 1155 1160 1165 GTG AGC AAC CCC Val Ser Asn Pro 1170 CTG GAG AGC CAC ACC Leu Glu Ser His Thr 1175 GM3 GGC GAC CCC TAC ACC GAC Val Gly Asp Pro Tyr Thr Asp 1180 2207 TAC GAG AAG Tyr Glu Lys 1185 CCC GCC CGC Ala Ala Arg GAC CTc Asp Leu 1190 GAC CTG AGC Asp Leu Ser AAC CCC AAG GAG ACC Asn Ala Lys Glu Thr 1195 2255 TI AAC Phe Asn 1200 CCC CIG GTG Pro Leu Val CCC CC Ala Ala 1205 TIC CCC AGC Phe Pro Ser GTG AAC Val Asn 1210 GTG ACC ATG Val Ser Met

GAG

Glu 1215 2303 AAG GTG ATC CIG AGC CCC AAC GAG AAC CTG AGC AAC AGC GIG GAG AGC Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu Ser 2351 1220 1225 1230 CAC TCG AGC His Ser Ser ACC AAC Thr Asn 1235 [GG AGC TAC Trp Ser Tyr ACC AAC Thr Asn 1240 ACC GAG GGC CCC AGC GTG Thr Glu Gly Ala Ser Val 1245 2399 GAG CCC GGC ATC Glu Ala Gly Ile 1250 GGT CCC AAG Gly Pro Lys GGC ATC Cly Ile 1255 AGC TIC GGC Ser Phe Gly GIG AC GIG AAC Val Ser Val Asn 1260 2447 2495 TAC CAC CAC Tyr Gin His 1265 AGC GAG ACC Ser Glu Thr GI GCC Val Ala 1270 CAG GAG IGG Gin Glu Trp GGC ACC AGC ACC GGC Gly Thr Ser Thr Gly 1275 AAC ACC AGC CAG TIC Asn Thr Ser Gin Phe 1280 AAC ACC GCC AGC GCC Asn Thr Ala Ser Ala 1285 GGC TAC CTG AAC GCC Gly Tyr Leu Asn Ala 1290

AAC

Asn 1295 2543 2591 GTG CCC TAC AAC Val Arg Tyr Asn AAC CG GGC ACC GGC Asn Val Gly Thr Gly 1300 CCC AIC TAC GAC GIG AAG CCC Ala Ile Tyr Asp Val Lys Pro 1305 1310 ACC ACC AGC Thr Thr Ser TIC GI CTG AAC AAC Phe Val Leu Asn Asn 1315 GAC ACC ATC GCC Asp Thr Ile Ala 1320 ACC ATO ACC GCC Thr Ile Thr Ala 1325 GAG AGC TAC CCC Glu Ser Tyr Pro 1340 2639 AAG TCG AAT CC ACC CCC CMC AAC ATC AGC CCC GGC Lys Ser Asn Ser Thr Ala Leu Asn Ile Ser Pro Cly 1330 1335 2687 2735 AAG AAG Lys Lys 1345 GGC CAG AAC GGC ATC CCC ATC ACC AGC ATG GAC GAC TIC AAC Gly Gin Asn Gly Ile Ala Ile Thr Ser Met Asp Asp Phe Asn 1350 1355 AGC CAC CCC ATIC ACC CTG AAC AAG AAG CAG GG Ser His Pro Ile Thr Leu Asn Lys Lys Gin Val GAC AAC CIG C=G AAC Asp Asn Leu Leu Asn 2783 1360 1365 1370 1375 AAC AAG CCC ATG AEG CTG GAG ACC AAC CAG ACC GAC GGC GC TAC AAG 2831 WO 97/46105 PCT/EP97/02737 -132- Asn Lys Pro Met Met Leu Giu Thr Asn Gin Thr Asp Gly Val Tyr Lys 1380 1385 1390 AM12 MG GAC ACC CAC GGC WC AMT GT ACG Ile Lys Asp Thr His Gly Asn Ile Val Thr GGC GGC GAG IXG AAC GGC Gly Gly Glu Trp Asn Gly 1405 2879 1395 1400 GTG NATC GAG GAG Val Ile Gin Gin 1410 ATC AAG GCC Ile Lys Ala AAG ACC Lys Thr 1415 CCC AGC AW AC GTC GAC GAC Ala Ser Ile Ile Val Asp Asp 1420 2927 GGC GAG CGC Gly Giu Arg 1425 GIG GCC GAG Val Ala Glu AAG CGC Lys Arg 1430 GTG GCC GCC AAG GAC Val Ala Ala Lys Asp 1435 TAC GAG AAC Tyr Glu Asn 2975 CCC GAG Pro Glu 1440 GAC AAG ACC Asp Lys Thr CCC AGC Pro Ser 1445 CTG ACC CMG Leu Thr Leu AAG GAC Lys Asp 1450 AGC TAC CCC GAC Ser Tyr Pro Asp GAG ATC Glu Ile 1460 AAG GAG ATC Lys Giu Ile GAG GGC TTG Glu Gly Leu 1465 GCC C-M AAG CTG Ala Leu Lys Leu 1455 CMG TAC TAC AAG Leu Tyr Tyr Lys 1470 CTA GAC GAG AAC Leu Asp Giu Asn 1485 3023 3071 AAC AAG CCC Asn Lys Pro ATC TAC GAG Ile Tyr Glu 1475 AGC AGC GMATG ACC TAT Ser Ser Val Met Thr Tyr 1480 3119 ACC GCC AAG GAG GTC ACC AAG Thr Ala Lys Giu Val Thr Lys 1490 GAG CC AAC GAC ACC ACC GGC AAG TIC Gin Leu Asn Asp Thr Thr Gly Lys Phe 1495 1500 3167 AAG CAC GMI AGC Lys Asp Val Ser 1505 CAC CrC TAC GAC I'GM AAG His Leu Tyr Asp Val Lys 1510 CrC ACC CCC AAG ATG AAC Leu Thr Pro Lys Met Asn 1515 GrC ACC Val Thr 1520 ATC AAG CTG Ile Lys Leu AGC ATC Ser Ile 1525 MC AGC ATC GGC AAG TG ACC Asn Ser Ile Cly Lys Trp Thr 1540 AAC GGC AAG AAG GAG TAC AGC Asn Gly Lys Lys Gin Tyr Ser 1555 CTG AAC ACC GAC GCC CAG GAG Leu Asn Thr Asp Ala Gin Glu 1570 ATC AGC CrC TAC ATG AAG AGC Ile Ser Leu Tyr Met Lys Ser 1585 159( CTG TAC GAC Leu Tyr Asp AAC ACC AAC Asn Thr Asn 1545 AGC AAC AAC Ser Asn Asn 1560 AAG CTG AAC Iys Leu Asn 1575 AAC GCC Asn Ala 1530 GAG AGC AAC Clu Ser Asn

GAC

Asp 1535

ATC

Ile =rC ACC GGC Val Ser Gly CGC AAC Gly Asn 1550 3215 3263 3311 3359 3407 3455 CCC GAC GCC AAC CTG ACC Pro Asp Ala Asn Leu Thr 1565 AAG AAC CCC GAC TAC TAC Lys Asn Arg Asp Tyr Tyr 1580 ACC GAG TGC GAG ATC ACC Thr Gin Cys Glu Ile Thr 1595 GAG AAG AAC Glu Lys Asn WO 97/46105 PCT/EP97/02737 -133- ATC GAC Ile Asp 1600 GGC GAG ATA TAC CCC Gly Giu Ile Tyr Pro 1605 ATC ACC ACC AAG ACC G=X Ile Thr Thr Lys Thr Val 1610 GAG ATC ATC GCC CAC AAG Asp Ile Ile Ala His Asn 1625 AAG GAC AAC TAC Lys Asp Asn Tyr AAG CGC Lys Arg 1620 AAC GTG AAC Asn Val Asn 1615 ATC AAG AGC Ile Lys Ser 1630 ATC ACC CIG Ile Thr Leu 1645 3503 3551 AAC CCC ATC Asn Pro Ile TIC TGG GAC Phe Trp Asp 165( AGC AGC Ser Ser 1635 CTG CAC ATC Leu His Ile AAG ACC Lys Thr 1640 AAC GAG GAG Asn Asp Glu 3599 GAG ATA W G AT ACC GAG GIC GCC AGG AC MG CCC GAG Asp Ile Ser Ile Thr Asp Val Ala Ser Ile Lys Pro Glu 3647 1655 1660 AAC CGI Asn Leu' 1665 ACC GAG AGG IThr Asp Ser GAG ATO AAG Glu Ile Lys 1670 GAG ATA TAC Gin Ile Tyr AGT CGC TAC GGC ATC Ser Arg Tyr Gly Ile 1675 3695 3743 AAG CTG GAG GAG Lys Leu Giu Asp 1680 GGC GAG TIC ATC Gly Giu Phe Ile GGC ATC CIG Gly Ile Leu 1685 ATC GAG AAG Ile Asp Lys AAA GGC GGC ATC Lys Gly Gly Ile 1690 CAC TAG His Tyr 1695 GAG AAG Gin Asn 1710 AAG GAG Asn Glu 1700 CCC AGC TTC Ala Ser Phe AAC ATC Asn Ile 1705 GAG CCC GTG Glu Pro Leu TAC GIG ACC Tyr Val Thr AAG TAG Lys Tyr 1715 GAG GIG ACC Clu Val Thr TAG AGC Tyr Ser 1720 AGC GAG CG GGGc Ccc AA Ser Giu Leu Gly Pro Asn 1725 3791 3839 3887 GIG ACC GAG ACC Val Ser Asp Thr 1730 CIG GAG AGG Leu Glu Ser GAG AAC Asp Lys 1735 ATT TAC AAG GAC GGC ACC AMC Ile Tyr Lys Asp Gly Thr Ile 1740 AAG TIC GAG Lys Phe Asp 1745 GAG AGC GGC Asp Ser Cly 1760 TIC ACC AAC Phe Thr Lys CTG AAC TGG Leu Asn Trp 1765 TAG AGG AAG AAG GAG Tyr Ser Lys Asn Glu 1750 GAG GGC Gin Gly 1755 CTG TIC TAG Leu Phe Tyr 3935 GAC TIC AAG AT AAC GCC ATC ACC TAG Asp Phe Lys Ile Asn Ala Ile Thr Tyr

GAG

Asp 1775 1770 3983 4029 GGC AAG GAG ATG Gly Lys Glu Met AAG GTG TIC CAC CGC Asn Val Phe His Arg 1780 TAG AAC AAG TAGATCIGAG Tyr Asn Lys 1785 4031 TIFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LEN=H: 1338 amino acids WO 97/46105 PCT/EP97/02737 -134- TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Val Leu Ser Asp Glu Phe Phe Ile Tyr 145 Glu Phe Thr Val Leu 225 His (xi) SEQUENCE Lys Arg Met Glu 5 Val Thr Lys Thr Asn Asn Glu Val Lys Tyr Thr Asn Phe Lys Glu Asp Lys Glu Trp Lys Leu Asp Asn Lys 100 Ser Ile Ala Gly 115 Asp Lys Met Phe 130 Lys Asn Val Glu Gly Asn Thr Ile 165 Leu Asp Arg Asp 180 Ala Gin Gin Val 195 Pro Ser Gly Lys 210 Asn Asn Ser Glu Val Asp Lys Val 245 DESCRIPTION: SEQ ID Gly Lys Leu Phe Met Val Ser Lys Lys Leu Gin Val Ile Leu Lys 70 Leu Asn Ser Asp Pro 150 Asn Ile Ser Gly Tyr 230 Ser Leu Lys Gin 55 Glu Thr Asp Phe Lys 135 Thr Ser Lys Ser Ser 215 Lys Lys Leu Ala 40 Asn Lys Ala Ile Glu 120 Thr Thr Asp Phe Lys 200 Thr Met Val Ser 25 Glu Leu Ala Thr Lys 105 Asp Asn Ile Ala Asp 185 Glu Thr Leu Val Thr Val Gin Leu Lys Ile Lys Glu 75 Glu Lys 90 Thr Asn Glu Ile Leu Ser Gly Phe 155 Met Ala 170 Ser Tyr Arg Val Pro Thr Ile Asp 235 Lys Lys 250 Phe Ser Asn Ile Thr Asp Trp Gly Gly Lys Tyr Lys Lys Asp 125 Asn Ser 140 Asn Lys Gin Phe Leu Asp Ile Leu 205 Lys Ala 220 Asn Gly Gly Val Ile Asn Lys Lys Met Glu 110 Leu Ile Ser Lys Thr 190 Lys Gly Tyr Glu Ser Ser Val Glu Asn Ile Lys Ile Leu Glu 175 His Val Val Met Cys 255 Leu Gin Glu Lys Asn Thr Glu Thr Thr 160 Gin Leu Thr Ile Val 240 Leu WO 97/46105 PCT/EP97/02737 -135- Gln Ile Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp Ile 260 265 270 Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp Ala 275 280 285 Lys Asp Leu Thr Asp Ser Gin Arg Glu Ala Leu Asp Gly Tyr Ala Arg 290 295 300 Gin Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Arg Asn Gin Gly Gly Ser 305 310 315 320 Gly Asn Glu Lys Leu Asp Ala Gin Ile Lys Asn Ile Ser Asp Ala Leu 325 330 335 Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Val Tyr Arg Tip Cys Gly 340 345 350 Met Pro Glu Phe Gly Tyr Gin Ile Ser Asp Pro Leu Pro Ser Leu Lys 355 360 365 Asp Phe Glu Glu Gin Phe Leu Asn Thr Ile Lys Glu Asp Lys Gly Tyr 370 375 380 Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg 385 390 395 400 Lys Ile Ile Leu Arg Leu Gin Val Pro Lys Gly Ser Thr Gly Ala Tyr 405 410 415 Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Lys Glu Ile Leu Leu Asp 420 425 430 Lys Asp Ser Lys Tyr His Ile Asp Lys Val Thr Glu Val Ile Ile Lys 435 440 445 Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn Ser Arg 450 455 460 Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro 465 470 475 480 Ser Asp Ile Gly Ser Thr Met Lys Thr Asn Gin Ile Ser Thr Thr Gin 485 490 495 Lys Asn Gin Gin Lys Glu Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr 500 505 510 Phe Lys Gly Lys Asp Phe Ser Asn Leu Thr Met Phe Ala Pro Thr Arg 515 520 525 Asp Ser Thr Leu Ile Tyr Asp Gin Gin Thr Ala Asn Lys Leu Leu Asp 530 535 540 Lys Lys Gin Gin Glu Tyr Gin Ser Ile Arg Trp Ile Gly Leu Ile Gin WO 97/46105 PCT/EP97/02737 -136 545 Ser Lys Ala Ile Lys Gin Glu Tyr 610 Glu Leu 625 Gin Gin Glu Phe Lys Arg Asp Leu 690 Lys Trp 705 Asn Pro Lys Ala Pro Leu Ile Leu 770 Ser Thr 785 Gly Ile His Ser Thr Lys Lys 600 Phe Asp Pro Lys Thr 680 Tyr Ser Val Leu Ser 760 Leu Asn Ser Glu Ala 840 555 Leu Ser Ser Asn Leu Val Asp Ser 620 Asn Gin 635 Asn Lys Leu Phe Asp Gly Gin Asn 700 Tyr Thr 715 Pro Tyr Ala Lys Val Ser Ser Val 780 Gly Ala 795 Val Ser Thr Ser Glu Asp Lys Gly 590 Pro Ile 605 Lys Thr Pro Gin Lys Glu Thr Gin 670 Asp Ser 685 Arg Ile Lys Phe Thr Asp Glu Thr 750 Met Glu 765 Glu Ser Ser Val Val Asn Thr Gly 830 Ala Asn 845 560 Glu Gin 575 Lys Glu Lys Ile Phe Lys Gin Val 640 Ser Gin 655 Gin Met Ile Pro Ala Val Val Ser 720 Tyr Glu 735 Phe Asn Lys Val His Ser Glu Ala 800 Tyr Gin 815 Asn Thr Val Arg Ser Gin Phe 835 Asn Thr Ala Ser Gly Tyr Leu Asn WO 97/46105 PCTIEP97/02737 -137- Tyr Asn Asn Val Gly Thr Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr 850 855 860 Ser Phe Val Leu Asn Asn Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser 865 870 875 880 Asn Ser Thr Ala Leu Asn Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys 885 890 895 Gly Gin Asn Gly Ile Ala Ile Thr Ser Met Asp Asp Phe Asn Ser His 900 905 910 Pro Ile Thr Leu Asn Lys Lys Gin Val Asp Asn Leu Leu Asn Asn Lys 915 920 925 Pro Met Met Leu Glu Thr Asn Gin Thr Asp Gly Val Tyr Lys Ile Lys 930 935 940 Asp Thr His Gly Asn Ile Val Thr Gly Gly Glu Trp Asn Gly Val Ile 945 950 955 960 Gin Gin Ile Lys Ala Lys Thr Ala Ser Ile Ile Val Asp Asp Gly Glu 965 970 975 Arg Val Ala Glu Lys Arg Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu 980 985 990 Asp Lys Thr Pro Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr 995 1000 1005 Pro Asp Glu Ile Lys Glu Ile Glu Gly Leu Leu Tyr Tyr Lys Asn Lys 1010 1015 1020 Pro Ile Tyr Glu Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Thr Ala 1025 1030 1035 1040 Lys Glu Val Thr Lys Gin Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp 1045 1050 1055 Val Ser His Leu Tyr Asp Val Lys Leu Thr Pro Lys Met Asn Val Thr 1060 1065 1070 Ile Lys Leu Ser Ile Leu Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser 1075 1080 1085 Ile Gly Lys Trp Thr Asn Thr Asn Ile Val Ser Gly Gly Asn Asn Gly 1090 1095 1100 Lys Lys Gin Tyr Ser Ser Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn 1105 1110 1115 1120 Thr Asp Ala Gin Glu Lys Leu Asn Lys Asn Arg Asp Tyr Tyr Ile Ser 1125 1130 1135 WO 97/46105 PCTEP97/02737 -138- Leu Tyr Met Lys Ser Glu Lys Asn Thr Gin Cys Glu Ile Thr Ile Asp 1140 1145 1150 Gly Glu Ile Tyr Pro Ile Thr Thr Lys Thr Val Asn Val Asn Lys Asp 1155 1160 1165 Asn Tyr Lys Arg Leu Asp Ile Ile Ala His Asn Ile Lys Ser Asn Pro 1170 1175 1180 Ile Ser Ser Leu His Ile Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp 1185 1190 1195 1200 Asp Asp Ile Ser Ile Thr Asp Val Ala Ser Ile Lys Pro Glu Asn Leu 1205 1210 1215 Thr Asp Ser Glu Ile Lys Gin Ile Tyr Ser Arg Tyr Gly Ile Lys Leu 1220 1225 1230 Glu Asp Gly Ile Leu Ile Asp Lys Lys Gly Gly Ile His Tyr Gly Glu 1235 1240 1245 Phe Ile Asn Glu Ala Ser Phe Asn Ile Glu Pro Leu Gin Asn Tyr Val 1250 1255 1260 Thr Lys Tyr Glu Val Thr Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser 1265 1270 1275 1280 Asp Thr Leu Glu Ser Asp Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe 1285 1290 1295 Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gin Gly Leu Phe Tyr Asp Ser 1300 1305 1310 Gly Leu Asn Trp Asp Phe Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys 1315 1320 1325 Glu Met Asn Val Phe His Arg Tyr Asn Lys 1330 1335 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 2444 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 17..2444 WO 97/46105 WO 9746105PCT/EP97/02737 -139- OTHER INFORmATION: /product= "3A(a) synthetic:native fusion" (xi) SEQUJENCE DESCRIPTION: SEQ ID NO: 51: GGAWICACCA A I AC A EG AAC AAG AAC AAC ACC AAG dCT AGC ACC CGC Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg GCC CIG CCG Ala Leu Pro ACC GCc ATC Thr Gly Ile

AGC

Ser TjVC AM GAC TAC =FT AC GGC AM TAC Phe le Asp T~yr Phe Asn Gly Ile Tyr GGC TTC GCC Gly Phe Ala GAC ACC GGC Asp Thr Gly AAG WA AM AM1 AAC ATM AMTC'TC AAG Lys Asp le Met Asn Met Ile Phe Lys GGC GAC Gly Asp CTG ACC CIG Leu Thr Leu GAC GAG ATC Asp Giu Ile CTG GAC GGC Leu Asp Giy 65

GAC

Asp AWC AGC GGC AAG le Ser Giy Lys CTG MAG AAC Leu Lys Asn Gr'G AAC GCc Val Asn Gly GAG rCT AGC Giu lieu Ser 85 CAG Ci\G C1X3 AAC Gin Loeu Leu Asn AGC CI'G AAC GAC Ser Leu Asn Asp AMTC Cc CAG GCc AAC CM~ MC AMC Ile Ala Gin Gly Asn Lieu Asn Thr AAG GAG ATC Lys Giu Ile crr MAG Leu. Lys AMTC Cc AAC GAG CAG MAC CAG M'I CTG WC GAC =T Ile Ala Asn Giu Gin Asn Gin Vai Leu Asn Asp Vai AAC AAC AAG dIG Asn Asn Lys Leu.

105 GCc GCC AWC Asp Ala Ile 110 AAC ACC A1X Asn Thr Met dIG CGC Leu Arg 115 =I TAC CCG MAG AMc ACC AGC Val Tyr Leu Pro Lys Ile Thr Ser 120 ATGl dIG Met Leu 12 5 AGC GAC GIG ATG Ser Asp Vai Met MAG CAG AAC Lys Gin Asn 130 TAC GCC MI AGC TYr Ala Leu Ser 135 dIG CAG ATC Leu Gin Ile

GAG

Giu 140 TAC CTG AGe AAG Tyr Leu Ser Lys

CAG

Gin 145 CIG CAG GAG ATC Leu Gin Giu le

AGC

Ser 150 GAC AAG dIG GAC Asp Lys Leu Asp

ATC

le 155 ATC AAC =I MAC le Asn Val Asn MI AMI AAC AGe ACC CMG ACC GAG AM~ Leu Ile Asn Ser Thr Leu Thr Glu Ile 165 ACC CCG Thr Pro 170 GCC TAC CAG Ala Tyr Gin AWC AG TAC GIG Ile Lys Tyr Val

AAC

Asn 180 GAG AAG TIrC GMA Giu Lys Phe Giu GAG CMG ACC Giu Leu Thr 185 'FTC CC ACC GAG ACC AGC AGC MG GMG MG AAG GAC cc GC cc CM CC WO 97/46105 WO 9746105PCT/EP97/02737 140 Phe Ala TI-i Glu Thr Ser Ser 190 Lys Val Lys LYS 195 GAG CTG ACC GAG Glu Leu Thr Glu GAG ATC GIG GAG GAG G Asp Ile 205 Leu Asp Glu Leu AcG Thr 210 Asp Gly 200 CG CC Leu Ala 215 Ser Pro Ala AAG AGC I Lys Ser Val

ACC

Thr 220 AAG AAC GAG GTG Lys Asn Asp Val

GAG

Asp 225 GCC T~iC GAG TIC Gly Phe Glu Phe

TAC

230 CTG MC ACC TIC Leu Asn Thr Phe

GAG

His 235 673 721 769 GACG cm A IC GTG Asp Val Met Val

GC

Cly 240 MGC AC CmG IC Asn Asn Leu Phe

GC

Cly 245 CG AGCGCCC

CG

Arg Ser Ala Leu MAG AGG Lys Thr 250 CCC AGC GAG Ala Ser Clu GTG CCC MGC Val. Cly Asn 270 CmG Leu 255 A IC ACC MAG GAG Ile Thr Lys Clu

AAG

Asn 260 GIG AAG ACC AC Vai LYS Thr Ser CCC ACC GAG Cly Ser Clu 265 CM GAG CC Leu Gin Ala GTG TAG MGC TIC Val Tyr Asn Phe ATC Mix cm AGc Ile Val Leu Thr GAG GGG Gin Ala 285 TI CcM ACC cM Phe Leu Thr ILeu

ACC

Thr 290 ACC TGXT CGC AAG Thr Gys Arg Lys

CTG

Leu 295 cM GC C cmCC Leu Gly Leu Ala A'IC GAG TAG ACC Ile Asp Tryr Thr ATC A'IG MGC GAG Ile Met Asn Ciu TMG C GAG Leu Asn Lys Ciu 961 1009 GAG GAG TIC CGG Giu Ciu Phe Arg

GM

Val 320 M~C A'I m =GGG Asn Ile Leu Pro AcG Thr 325 cmG AC MG AGG Leu Ser Asn Thr TIC AGG Phe Ser 330 MAC CGC Asn Pro Asn ATC GTG GAG Ile Val. Clu 350

TAG

335 GGG AAG C'm Ala Lys Val AAG GC Lys Cly 340 AC GAG GAG GAG Ser Asp Giu Asp CCC AAG ATG Ala Lys Met 345 GAG ATC AC iu Ile Ser 1057 1105 GCT MG CCG GCC Ala Lys Pro Cly CC TM' A'IC GC Ala Leu Ile Cly MGC GAG Asn Asp 365 A~C ATC ACC GTG Ser Ile Thr Val AAG GIM TAG GAG LYS Val Tyr Giu

GCC

Ala 375 MAG cmG MC GAG Lys Leu Lys Gin

MGC

Asn 380 TAG GAG GIG GAG Tyr Gin Val Asp GAG ACC TMC AGG Asp Ser Leu Ser GIG ATC TACG C Vai Ile Tyr Gly

GAG

Asp 395 1153 1201 1249 ATG GAG MAG CTG CMG Met Asp LYS Leu Leu 400 IGT CCC GAG GAG AGG GAG GMA A'IC Gys Pro Asp Gin Ser Giu Gin Ile 405 TAG TAG ACG Tyr Tyr Thr 410 WO 97/46105 WO 9746105PCT/EP97/02737 .141- AAC AAC AWC ac TITC CCG AAC GAG Asn Asn Ile Val Phe Pro Asn Clu 415 GliG AWC ACC AAG Val Ile Thr Lys ACC AAG AAG Thr Lys Lys 430 ATG AAC ACC Met Lys Thr Cix; CGC Leu Arg 435 TAC GAG =ix ACC Tyr Glu Val Thr

CC

Ala 440 ATC GAC TCr Ile Asp Phe 425 AAC TMC TAC Asn Phe Tlyr aix; GAG AGC Val Glu Ser 1297 1345 GAC AGC Asp Ser 445 AGC ACC GCC GAG Ser Thr Cly Giu GAC CIx; AAC MAG Asp Loeu Asn Lys AAG MAG Lys Lys 455

AGC

Ser 460 GAG GCC GAG TAC Clu Ala Ciu Tlyr

CC

Arg 465 ACC Cix; AC Thr Lieu Ser C AAC Ala Asn 470

CT

Phe Leu 485 GAC GAC GCCa Asp Asp Giy Val 1393 1441 1489 ATC CCA Cix; GGC Met Pro Leu Gly Val 480 AWC AGC GAG ACC Ile Ser Ciu Thr ACC CCC ATC AAC GGC Thr Pro Ile Asn Gly 490 TIT CCC CIG Phe Gly Leu AAG ACC TAC Lys Ser Tryr 510

CAG

Gin 495 CCC GAC GAG MAC Ala Asp Clu Asn CCC CMx A'IC ACC Arg Leu Ile Thr CMC ACC EGT Leu Thr Cys 505 1537 Cix; CCC GAG Cix; aCT CrA CCC ACC GAC Leu Arg Glu Leu Leu Leu Ala Thr Asp CIG ACC AC MAG Leu Ser Asn Lys 520 ACC MAC ATC G'lG Ser Asn le Val GAG ACC Giu Thr 525 MAG Cix AWC GrC Lys Leu Ile Val CCC ACC GCC TrrC Pro Ser Gly Phe 1585 1633 1681 1729

GAG

Giu 540 MAC CCC AGC AliC Asn Gly Ser Ile

GAG

Giu 545 GAG GAC AAC Glu Asp Asn Cix; GAG Leu Ciu 550 GCC GC Gly Cly 565 CCG EGC MG CC Pro Trp Lys Ala

MAC

Asn 555 MAC MAG MC CC Asn Lys Asn Ala

TAC

560 GTG GAC CAC ACC Val Asp His Thr GrC M~C GC Val Asn Cly ACC MAG Thr Lys 570 CCC CrC TAC Ala Leu Tyr MAGCT AA; G Lys Leu Lys 590 GTIG CAC MCG GAC Val His Lys Asp 575 CCC MAG ACC GAG Pro Lys Thr Ciu CCC CCC AWC AGC Gly Gly Ile Ser 580 CAG T= ATC CCC GAC Gin Phe Ile Cly Asp 585 1777 1825

TAC

Tyr 595 aix; AWC CAG TAC Val Ile Gin Ty~r

ACC

Thr 600 GMx MCG GC Val Lys Gly MCG CCA Lys Pro 605 'mx; AlT CAC Cix; Ser Ile His Leu

MAG

Lys 610 GAC GAG AMC Acc Asp Ciu Asn Thr

GCC

Gly 615 TAC ATC C-AC TAC Tyr Ile His TrI, 1873 GAC ACC MAC MAC Asp Thr Asn Asn MAC C'rC GAG GAC TAC CAG ACC ATC MAC MCG CC Asn Leu Ciu Asp TIyr Gin Thr le Asn Lys Arg 625 630 635 1921 WO 97/46105 PCTIEP97/02737 -142 TI ACC ACC GGC Phe Thr Thr Gly GAC CTG AAG GGC Asp Leu Lys Gly GT3 Val 645 TAC CIG ATC CMC Tyr Leu Ile Leu AAG AGC Lys Ser 650 1969 CAG AAC GGC Gin Asn Gly AGC CCG AGC Ser Pro Ser 670 GAG CCC TGG GGC Glu Ala Trp Gly

CAC

Asp 660 AAC TM ATC ATC Asn Phe Ile Ile CYi GAG ATC Leu Glu Ile 665 ACC AAC AAC Thr Asn Asn 2017 2065 GAG AAG CTG CTr Glu Lys Ieu Leu

AGC

Ser 675 CCC GAG CG ATC Pro Glu Leu Ile

AAC

Asn 680 TGG ACC Trp Thr 685 AGC ACC GGC Ser Thr Gly AGC ACC Ser Thr 690 AAC ATC ACC GGC Asn Ile Ser Cly

AAC

Asn 695 ACC CTG ACC CrC Thr Leu Thr Leu

TAC

700 700 CAG GGC GGC CGG Cmn Gly Cly Arg

GGG

Cly 705 ATT CTA AAA CAA Ile Leu Lys Gin

AAC

Asn 710 CTr CAA 'ITA GAT Leu Gin Leu Asp

AGT

Ser 715 2113 2161 2209 TIT TCA ACT TAT Phe Ser Thr Tyr AGA GTG Arg Vai 720 TAT TIT TCT Tyr Phe Ser

GTG

Val 725 TCC GCA GAT CCT Ser Gly Asp Ala AAT GTA Asn Val 730 AGG AT AGA Arg Ile Arg GGT GCT AAA Gly Ala Lys 750

PAT

Asn 735 TCT AGG GAA GTG 'ITA Ser Arg Giu Val Leu 740 T GAA AAA AGA Phe Giu Lys Arg ACT ACA AAA TIT Thr Thr Lys Phe 760 CAT ITT TT GAA ATG TI'C Asp Val Ser Glu Met Phe 755 TAT ATG AGC Tyr Met Ser 745 GAG AAA GAT Glu Lys Asp GGT GGT CCT Gly Gly Pro CAT CIA ATA Asp Leu Ile 795 2257 2305 AAC TIT Asn Phe 765 TAT ATA GAG Tyr Ile Glu CIT TCT Leu Ser 770 CAA GGG AAT PAT Gin Gly Asn Asn TIA TAT Leu Tyr 775 2353

ATT

Ile 780 GTA CAT ITT Vai His Phe TAC CAT GIC TCT ATT Tyr Asp Vai Ser Ile 785 AAA ACC NAA TX TMC Lys Ser Xaa Phe Leu AAG NMA GAT CGG Lys Xaa Asp Arg 790 2401 TTA ACA GTT TT Leu Thr Vai Phe PAT G=C CPT GAT P Asn Vai Leu Asp 2444 INFORMATION FOR SE) ID NO:52: SEQUENCE CHARACERISTICS: LENGTH: 809 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 97/46105 PCT/EP97/02737 -143- (xi) SEQUENCE DESCRIPTION: SEQ Met Asn Lys Asn Asn Thr Lys Leu Ser i Ile Asp Tyr Phe Asn Ile Met Asp Glu Leu Asp Leu Asn Asn Gin Met Leu Met Lys 130 Gin Leu 145 Leu Ile Lys Tyr Ser Ser Leu Thr 210 Asp Gly 225 Asn Asn Thr Lys Asn Phe Ile Lys Asn Leu Asn Tyr Tyr Ile Thr 165 Glu Lys Thr Phe Gly 245 Val Val Tyr Thr Gin Leu Glu Asn Lys 120 Ser Lys Glu Glu Gly 200 Ala Asn Ala Ser Ala 280

F

1

L

A

L

L

IT

G

A

T

1

L

P

S

F

L

2

S

C

ID NO:52: bhr Arg Ala 'he Ala Thr Thr Gly Gly ou Asn Asp sp Leu Ile 75 eu Lys Ile 90 ys Leu Asp hr Ser Met in Ile Glu 140 sp Ile Ile 155 hr Pro Ala 70 eu Thr Phe ro Ala Asp er Val Thr 220 he His Asp 235 ys Thr Ala 50 er Giu Vai in Ala Gin Leu Gly Asp Ile Ala Ala Ala Leu 125 Tyr Asn Tyr Ala Ile 205 Lys Val Ser Gly Ala 285 Ser Lys Thr Gly Gly Glu Asn Asp Ser Asn Arg 175 Glu Asp Asp Val Leu 255 Val Leu Phe Asp Leu Lys Asn Gin Thr Val Lys Val 160 Ile Thr Glu Val Gly 240 Ile Tyr Thr WO 97/46105 PCT/EP97/02737 -144 Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr 290 295 300 Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val 305 310 315 320 Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala 325 330 335 Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys 340 345 350 Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr 355 360 365 Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gin Asn Tyr Gin Val Asp 370 375 380 Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu 385 390 395 400 Cys Pro Asp Gin Ser Glu Gin Ile Tyr Tyr Thr Asn Asn Ile Val Phe 405 410 415 Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys 420 425 430 Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly 435 440 445 Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr 450 455 460 Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val 465 470 475 480 Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gin Ala 485 490 495 Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg 500 505 510 Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile 515 520 525 Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile 530 535 540 Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr 545 550 555 560 Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His 565 570 575 "WVO 97/46105 PCT/EP97/02737 -145 Lys Thr Leu Asn 625 Asp Ala Leu Ser Gly 705 Val Arg Ser Leu Asp 785 Ser Asp Gly Glu Tyr 595 Lys Asp 610 Leu Glu Leu Lys Trp Gly Leu Ser 675 Thr Asn 690 Ile Leu Tyr Phe Glu Vai Glu Met 755 Ser Gin 770 Val Ser Xaa Phe Gly 580 Vai Glu Asp Gly Asp 660 Pro Ile Lys Ser Leu 740 Phe Gly Ile Leu Ile Ser Ile Gin Asn Thr Tyr Gin 630 Val Tyr 645 Asn Phe Glu Leu Ser Gly Gin Asn 710 Val Ser 725 Phe Glu Thr Thr Asn Asn Lys Xaa 790 Tyr Asn 805 Gin Tyr Gly 615 Thr Lieu Ile Ile Asn 695 Leu Gly Lys Lys Leu 775 Asp Vai Phe Thr 600 Tyr Ile Ile Ile Asn 680 Thr Gin Asp Arg Phe 760 Arg Leu Ile Gly 585 Val Lys Ile His Asn Lys Leu Lys 650 Leu Glu 665 Thr Asn Leu Thr Leu Asp Ala Asn 730 Tyr Met 745 Glu Lys Gly Gly Asp Leu Asp Asp Gly Tyr Arg 635 Ser Ile Asn Leu Ser 715 Val Ser Asp Pro Ile 795 Lys Leu Lys Pro 605 Glu Asp 620 Phe Thr Gin Asn Ser Pro Trp Thr 685 Tyr Gin 700 Phe Ser Arg Ile Gly Ala Asn Phe 765 Ile Vai 780 Leu Thr Lys Pro Lys 590 Ser Ile His Thr Asn Asn Thr Giy Thr 640 Gly Asp Glu 655 Ser Giu Lys 670 Ser Thr Gly Gly Giy Arg Thr Tyr Arg 720 Arg Asn Ser 735 Lys Asp Val 750 Tyr Ile Glu His Phe Tyr Val Phe Lys 800 WO 97/46105 PCTIEP97/02737 -146- INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 3474 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Pure maize optimized synthetic BT CryIA(b) gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: ATGGACAACA ACCCCAACAT CAACGAGTGC ATCCCCTACA ACTGCCTGAG CAACCCCGAG GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ACCGGCTACA CCCCCATCGA CATCAGCCTG 120 AGCCTGACCC AGTTCCTGCT GAGCGAGTTC GTGCCCGGCG CCGGCTTCGT GCTGGGCCTG 180 GTGGACATCA TCTGGGGCAT CTTCGGCCCC AGCCAGTGGG ACGCCTTCCT GGTGCAGATC 240 GAGCAGCTGA TCAACCAGCG CATCGAGGAG TTCGCCCGCA ACCAGGCCAT CAGCCGCCTG 300 GAGGGCCTGA GCAACCTGTA CCAGATCTAC GCCGAGAGCT TCCGCGAGTG GGAGGCCGAC 360 WO 97/46105 WO 9746105PCT/EP97/02737 -147- CCCACCAACC CCGCCCTGCG CGAGGAGATG CGCATCCAGT TCAACGACAT GAACAGCGCC 420 CTGACCACCG CCATCCCCCT GTTCGCCGTG CAGAACTACC AGGTGCCCCT GCTGAGCGTG 480 TACGTGCAGG CCGCCAACCT GCACCTGAGC GTGCTGCGCG ACGTGAGCGT GTTCGGCCAG 540 CGCTGGGGCT TCGACGCCGC CACCATCAAC AGCCGCTACA ACGACCTGAC CCGCCTGATC 600 GGCAACTACA CCGACCACGC CGTGCGCTCG TACAACACCG GCCTGGAGCG CGTCTGGGGC 660 CCCGACAGCC GCGACTGGAT CCGCTACAAC CAGTTCCGCC GCGAGCTGAC CCTGACCGTG 720 CTGGACATCG TGAGCCTGTT CCCCAACTAC GACAGCCGCA CCTACCCCAT CCGCACCGTG 780 AGCCAGCTGA CCCGCGAGAT CTACACCAAC CCCGTGCTGG AGAACTTCGA CGGCAGCTTC 840 CGCGGCAGCG CCCAGGGCAT CGAGGGCAGC ATCCGCAGCC CCCACCTGAT GGACATCCTG 900 AACAGCATCA CCATCTACAC CGACGCCCAC CGCGGCGAGT ACTACTGGAG CGGCCACCAG 960 ATCATGGCCA GCCCCGTGGG CTTCAGCGGC CCCGAGTTCA CCTTCCCCCT GTACGGCACC 1020

ATGGGCAACG

1080 CCGCCCCCCA GCAGCGCATC GTGGCCCAGC TGGGCCAGGG CGTGTACCGC WO 97/46105 WO 9746105PCT/EP97/02737 -148 ACCCTGAGCA GCACCCTGTA CCGCCGCCCC TTCAACATCG GCATCAACAA CCAGCAGCTG 1140 AGCGTGCTGG ACGGCACCGA GTTCGCCTAC GGCACCAGCA GCAACCTGCC CAGCGCCGTG 1200 TACCGCAAGA GCGGCACCGT GGACAGCCTG GACGAGATCC CCCCCCAGAA CAACAACGTG 1260 CCCCCCCGCC AGGGCTTCAG CCACCGCCTG AGCCACGTGA GCATGTTCCG CACCGGCTTC 1320 AGCAACAGCA GCGTGAGCAT CATCCGCGCC CCCATGTTCA GCTGGATCCA CCGCAGCGCC 1380 GAGTTCAACA ACATCATCCC CAGCAGCCAG ATCACCCAGA TCCCCCTGAC CAAGAGCACC 1440 AACCTGGGCA GCGGCACCAG CGTGGTGAAG GGCCCCGGCT TCACCGGCGG CGACATCCTG 1500 CGCCGCACCA GCCCCGGCCA GATCAGCACC CTGCGCGTGA ACATCACCGC CCCCCTGAGC 1560 CAGCGCTACC GCGTGCGCAT CCGCTACGCC AGCACCACCA ACCTGCAGTT CCACACCAGC 1620 ATCCACGGCC GCCCCATCAA CCAGGGCAAC TTCAGCGCCA CCATGAGCAG CGGCAGCAAC 1680 CTGCAGAGCG GCAGCTTCCG CACCGTGGGC TTCACCACCC CCTTCAACTT CAGCAACGGC 1740 AGCAGCGTGT TCACCCTGAG 1800 CGCCCACGTG TTCAACAGCG GCAACGAGGT GTACATCGAC WO 97/46105 WO 9746105PCT/EP97/02737 -149 CGCATCGAGT TCGTGCCCGC CGAGGTGACC TTCGAGGCCG AGTACGACCT GGAGCGCGC 1860 CAGAAGGCCG TGAACGACCT GTTCACCAGC AGCAACCAGA TCGGCCTGAA

GACCGACGTG

1920 ACCGACTACC ACATCGACCA GGTGAGCAAC CTGGTGGAGT GCCTGAGCGA CGAGTTCTGC 1980 CTGGACGAGA AGAAGGAGCT GAGCGAGAAG GTGAAGCACG CCAAGCGCCT GAGCGACGAG 2040 CGCAACCTGC TGCAGGACCC CAACTTCCGC GGCATCAACC GCCAGCTGGA

CCGCGGCTGG

2100 CGCGGCAGCA CCGACATCAC CATCCAGGGC GGCGACGACG TGTTCAAGGA GAACTACGTG 2160 ACCCTGCTGG GCACCTTCGA CGAGTGCTAC CCCACCTACC TGTACCAGAA GATCGACGAG 2220 AGCAAGCTGA AGGCCTACAC CCGCTACCAG CTGCGCGGCT ACATCGAGGA CAGCCAGGAC 2280 CTGGAGATCT ACCTGATCCG CTACAACGCC AAGCACGAGA CCGTGAACGT GCCCGGCACC 2340 GGCAGCCTGT GGCCCCTGAG CGCCCCCAGC CCCATCGGCA AGTGCGCCCA CCACAGCCAC 2400 CACTTCAGCC TGGACATCGA CGTGGGCTGC ACCGACCTGA ACGAGGACCT GGGCGTGTGG 2460

GTGATCTTCA

2520 AGATCAAGAC CCAGGACGGC CACGCCCGCC TGGGCAACCT

GGAGTTCCTG

"I 4, WO 97/46105 W097/6105PCT/EP97/02737 -150- GAGGAGAAGC CCCTGGTGGG CGAGGCCCTG GCCCGCGTGA AGCGCGCCGA GAAGAAGTGG 2580 CGCGACAACC CCGAGAAGCT GGAGTGGCAG ACCAACATCG TGTACAAGGA GGCCAAGGAG 2640 AGCGTGGACG CCCTGTTCGT GAACAGCCAG TACGACCGCC TGCAGGCCGA CACCAACATC 2700 GCCATGATCC ACGCCGCCGA CAAGCGCGTG CACAGCATCC GCGAGGCCTA CCTGCCCGAG 2760 CTGAGCGTGA TCCCCGGCGT GAACGCCGCC ATCTTCGAGG AGCTGGAGGG CCGCATCTTC 2820 ACCGCCTTCA GCCTGTACGA CGCCCGCAAC GTGATCAAGA ACGCCGACTT CAACAACGGC 2880 CTGAGCTGCT GGAACGTGAA GGGCCACGTG GACGTGGAGG AGCAGAACAA CCACCGCAGC 2940 GTGCTGGTGG TGCCCGAGTG GGAGGCCGAG GTGAGCCAGG AGGTGCGCGT GTGCCCCGGC 3000 CGCGGCTACA TCCTGCGCGT GACCGCCTAC AAGGAGGGCT ACGGCGAGGG CTGCGTGACC 3060 ATCCACGAGA TCGAGAACAA CACCGACGAG CTGAAGTTCA GCAACTGCGT GGAGGAGGAG 3120 GTGTACCCCA ACAACACCGT GACCTGCAAC GACTACACCG CCACCCAGGA GGAGTACGAG 3180

GGCACCTACA

3240 CCAGCCGCAA CCGCGGCTAC GACGGCGCCT ACGAGAGCAA CAGCAGCGTG VOo 97/46105 PCT/EP97/02737 -151 CCCGCCGACT ACGCCAGCGC CTACGAGGAG 3300 CCCTGCGAGA GCAACCGCGG CTACGGCGAC 3360 AAGGAGCTGG AGTACTTCCC CGAGACCGAC 3420 GGCACCTTCA TCGTGGACAG CGTGGAGCTG 3474 AAGGCCTACA CCGACGGCCG CCGCGACAAC TACACCCCCC TGCCCGCCGG CTACGTGACC AAGGTGTGGA TCGAGATCGG CGAGACCGAG CTGCTGATGG AGGAGTAGTA CATG INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 3508 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Full length synthetic maize optimized BT CryIA(b) gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: GATCCAACAA TGGACAACAA CCCCAACATC AACGAGTGCA TCCCCTACAA CTGCCTGAGC AACCCCGAGG TGGAGGTGCT GGGCGGCGAG CGCATCGAGA CCGGCTACAC CCCCATCGAC 120 ATCAGCCTGA GCCTGACCCA GTTCCTGCTG AGCGAGTTCG TGCCCGGCGC CGGCTTCGTG 180 WO 97/46105 WO 9746105PCT/EP97/02737 152 CTGGGCCTGG TGGACATCAT CTGGGGCATC TTCGGCCCCA GCCAGTGGGA CGCCTTCCTG 240 GTGCAGATCG AGCAGCTGAT CAACCAGCGC ATCGAGGAGT TCGCCCCCAA

CCAGGCCATC

300 AGCCGCCTGG AGGGCCTGAG CAACCTGTAC CAAATCTACG CCGAGAGCTT CCGCGAGTGG 360 GAGGCCGACC CCACCAACCC CGCCCTGCGC GAGGAGATGC GCATCCAGTT CAACGACATG 420 AACAGCGCCC TGACCACCGC CATCCCCCTG TTCGCCGTGC AGAACTACCA GGTGCCCCTG 480 CTGAGCGTGT ACGTGCAGGC CGCCAACCTG CACCTGAGCG TGCTGCGCGA CGTCAGCGTG 540 TTCGGCCAGC GCTGGGGCTT CGACGCCGCC ACCATCAACA GCCGCTACAA CGACCTGACC 600 CGCCTGATCG GCAACTACAC CGACCACGCC GTGCGCTGGT ACAACACCGG CCTGGAGCGC 660 GTGTGGGGTC CCGACAGCCG CGACTGGATC AGGTACAACC AGTTCCGCCG CGAGCTGACC 720 CTGACCGTGC TGGACATCGT GAGCCTGTTC CCCAACTACG ACAGCCGCAC CTACCCCATC 780 CGCACCGTGA GCCAGCTGAC CCGCGAGATT TACACCAACC CCGTGCTGGA GAACTTCGAC 840 GGCAGCTTCC GCGGCAGCGC CCAGGGCATC GAGGGCAGCA TCCGCAGCCC CCACCTGATG 900 WO 97/46105 WO 9746105PCT/EP97/02737 GACATCCTGA ACAGCATCAC CATCTACACC GACGCCCACC GCGGCGAGTA CTACTGGAGC 960 GGCCACCAGA TCATGGCCAG CCCCGTCGGC TTCAGCGGCC CCGAGTTCAC CTTCCCCCTG 1020 TACGGCACCA TGGGCAACGC TGCACCTCAG CAGCGCATCG TGGCACAGCT GGGCCAGGGA 1080 GTGTACCGCA CCCTGACCAG CACCCTGTAC CGTCGACCTT TCAACATCGG CATCAACAAC 1140 CAGCAGCTGA GCGTGCTGGA CGGCACCGAG TTCGCCTACG GCACCAGCAG CAACCTGCCC 1200 AGCGCCGTGT ACCGCAAGAG CGGCACCGTG GACAGCCTGG ACGAGATCCC CCCTCAGAAC 1260 AACAACGTGC CACCTCGACA GGGCTTCAGC CACCGTCTGA GCCACGTGAG CATGTTCCGC 1320 AGTGGCTTCA GCAACAGCAG CGTGAGCATC ATCCGTGCAC CTATGTTCAG CTGGATTCAC 1380 CGCAGTGCCG AGTTCAACAA CATCATCCCC AGCAGCCAGA TCACCCAGAT CCCCCTGACC 1440 AAGAGCACCA ACCTGGGCAG CGGCACCAGC GTGGTGAAGG GCCCCGGCTT CACCGGCGGC 1500 GACATCCTGC GCCGCACCAG CCCCGGCCAG ATCAGCACCC TGCGCGTGAA CATCACCGCC 1560

CCCCTGAGCC

1620 AGCGCTACCG CGTCCGCATC CGCTACGCCA GCACCACCAA CCTGCAGTTC WO 97/46105 WO 9746105PCT/EP97/02737 -154- CACACCAGCA TCGACGGCCG CCCCATCAAC CAGGGCAACT TCAGCGCCAC

CATGAGCAGC

1680 GGCAGCAACC TGCAGAGCGG CAGCTTCCGC ACCGTGGGCT TCACCACCCC

CTTCAACTTC

1740 AGCAACGGCA GCAGCGTGTT CACCCTGAGC GCCCACGTGT TCAACAGCGG

CAACGAGGTG

1800 TACATCGACC GCATCGAGTT CGTGCCCGCC GAGCTGACCT TCGAGGCCGA

GTACGACCTG

1860 GAGAGGGCTC AGAAGGCCGT GAACGAGCTG TTCACCAGCA GCAACCAGAT

CGGCCTGAAG

1920 ACCGACGTGA CCGACTACCA CATCGATCAG GTGAGCAACC TGGTGGAGTG

CCTGAGCGAC

1980 GAGTTCTGCC TGGACGAGAA GAAGGAGCTG AGCGAGAAGG TGAAGCACGC CAAGCGCCTG 2040 AGCGACGAGC GCAACCTCCT GCAGGACCCC AACTTCCGCG GCATCAACCG

CCAGCTGGAC

2100 CGCGGCTGGC GCGGCAGCAC CGACATCACC ATCCAGGGCG GCGACGACGT

GTTCAAGGAG

2160 AACTACGTGA CCCTGCTGGG CACCTTCGAC GAGTGCTACC CCACCTACCT

GTACCAGAAG

2220 ATCGACGAGA GCAAGCTGAA GGCCTACACC CGCTACCACC TGCGCGGCTA

CATCGAGGAC

2280 AGCCAGGACC TGGAGATCTA CCTGATCCGC TACAACGCCA AGCACGAGAC

CGTGAACGTG

2340 WO 97/46105 WO 9746105PCT/EP97/02737 155 CCCGGCACCG GCAGCCTGTG GCCCCTGAGC GCCCCCAGCC CCATCGGCAA GTGCGCCCAC 2400 CACAGCCACC ACTTCAGCCT GGACATCGAC GTGGGCTGCA CCGACCTGAA CGAGGACCTG 2460 GGCGTGTGGG TGATCTTCAA GATCAAGACC CAGGACGGCC ACGCCCGCCT GGGCAACCTG 2520 GAGTTCCTGG AGGAGAAGCC CCTGGTGGGC GAGGCCCTGG CCCGCGTGAA GCCCGCCGAG 2580 AAGAAGTGGC CCGACAAGCG CGAGAAGCTG GAGTGGGAGA CCAACATCGT GTACAAGGAG 2640 GCCAAGGAGA GCGTGGACGC CCTGTTCGTG AACAGCCAGT ACGACCGCCT GCAGGCCGAC 2700 ACCAACATCG CCATGATCCA CGCCGCCGAC AAGCGCGTGC ACAGCATTCG CGAGGCCTAC 2760 CTGCCCGAGC TGAGCGTGAT CCCCGGCGTG AACGCCGCCA TCTTCGAGGA GCTGGAGGGC 2820 CGCATCTTCA CCGCCTTCAG CCTGTACGAC GCCCGCAACG TGATCAAGAA CGGCGACTTC 2880 AACAACGGCC TGAGCTGCTG GAACGTGAAG GGCCACGTGG ACGTGGAGGA GCAGAACAAC 2940 CACCGCAGCG TGCTGGTGGT GCCCGAGTGG GAGGCCGAGG TGAGCCAGGA GGTGCGCGTG 3000 TGCCCCGGCC GCGGCTACAT CCTGCGCGTG ACCGCCTACA AGGAGGGCTA CGGCGAGGGC 3060 WO 97/46105 WO 9746105PCTIEP97/02737 156

TGCGTGACCA

3120

GAGGAGGAGG

3180

GAGTACGAGG

3240

AGCAGCGTGC

3300 TCCACGAGAT CGAGAACAAC ACCGACGAGC TCAAGTTCAG CAACTGCGTG TGTACCCCAA CAACACCGTG ACCTGCAACG ACTACACCGC CACCCAGGAG GCACCTACAC CAGCCGCAAC CGCGGCTACG ACGGCGCCTA CGAGAGCAAC CCGCCGACTA CGCCAGCGCC TACGAGGAGA AGGCCTACAC CGACGGCCGC CGCGACAACC CCTGCGAGAG CAACCGCGGC TACGGCGACT ACACCCCCCT GCCCGCCGGC 3360

TACGTGACCA

3420

GAGACCGAGG

3480 AGGACCTGGA GTACTTCCCC GAGACCGACA AGGTGTGGAT CGAGATCGGC GCACCTTCAT CGTGGACAGC GTGGAGCTGC TGCTGATGGA GGAGTAGTAC ATGTGATAGT ACGTAAGCTC GAGGATCT INFOPNATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1961 base pairs TYPE: nucleic acid STRANDEINESS: single TIOPOLOGY: linear (ii) MOLECULE TYPE: EN~A 3508 (iii) HYPOTHETICAL: NO WO 97/46105 WO 9746105PCT/EP97/02737 157 (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: ORGANISMl: Truncated synthetic ma~ize optimized BT CryIA gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCCAACAA. 'GGACAACAA CCCCAACATC AACGAGIGCA TCCCCTACAA. CrGCCYIG-AGC AACCCCGAGG EMGAGaI'GCT GGGCGGCGAG CGCArICGAGA CCGGCTACAC CCCCA ICGAC 120 ATCAGCCIMA GCCIXIACCCA G'PIlCCIGCTG AGCGAG=TCG CCCGGCGC CGCTCI 180 CIrGGCCXGG TGACATCAT C7 GGGGCAIIC ICGGCCCCA. GCCAG IGGGA CGLCCTCC~T 240 GMCAGATCG AGCAGCTGAT CAAOCAGCGC ATCGAGGAGT TIX2GCCCGCAA. CCAGGCCATC 300 AGCCGCCIG AGGCIGAG CAACCIMTAC CAAATCTACG CCGAGAGCTT CCGCGAGIGG 360 GAGGCCGACC CCACCAACDC 0GCCCIr-CGC GAGGAGATGC GCATCCAGTT CAACGACA'IG 420 AACAGCGCCC IGACCACCGC CATCCCCCTG TICGCL t AGAACTACCA GG~TCCCCI 480 CKIAGCGTGT ACGIG3CAGGC -CCCAACCI'G CACCTGAGCG TrGC-'IGCGCGA CGTCAGCcI'G 540 T'ICGGCCAGC WTGGGCTTo~ CGACGCCGCC ACCATCAACA GCCGC~rACAA. CGACVMGCC 600 CGCC-MGATG GCAACTACAC CGACCACGC MflCGCI=G ACAACACCG3G CCI'GGAGCGC 660 G-IGGG=CCGAAGCG CAGGAT2 AGGTACAACC AG71CCGCCG CGAGCTGACC 720 CTGCCTG TGACA TMT GAGCr,=~ CCCAACTACG ACAGCCGCAC CTACCCCATC 780 CGCACCGTGA GCCAGC1GAC CCGCGAGATT TACACCAACC L-CGI1rTGA GAAC'PI CGAC 840 WO 97/46105 W097/6105PCT/EP97/02737 158 GCACo=

GACATCCXGA

GGCCACCAGA

TACGGCACCA

GTGACCGCA

CAGCAGCTGA

AGCGCCG=

AACAACGGC

AGTGGCIT-A

CGCAGTGCCG

AAGAGCACCA

GACA~C

CCCCarGAGC

CACACCAGCA

GGCAGCAACC

AGCAACGGCA

TACATCGACC

.GCGGCAGCGC

ACAGCATCAC

TCATGCCAG

TIGGGCAACGC

~CCCTAGCAG

GCGTGCTGGA

ACCGCAAGAG

CACCTCGACA

GCAACAGCAC

AGCIIWAACAA

ACCIGGGCAG

GCCGCACCAG

AGCOCACCG

TCGACGGCCG

TCAGAGCG

GCAGCGrr

GCATCGAGT

CCAGGGCATC

CA TrACACC

CCCCGTCGC

TGCACCTICAG

CACCCI'GAC

CGGCACCGAG

CGGCACCGTX2 GGGCrrCAGC

CGTGAGCATC

CATCATCCCC

CGGCACCAGC

CCCCGGCCAG

CGTCGCATW

CCCCATCAAC

CAGCTTCCG

CACCCTGAGC

Ccq)3CCGCC

GAGGGCAGCA

GACGCCCACC

IvAGCGGCC

CAGCGCATICG

CGTCACC~r

TIC'GC=ACG

GACAGCCI=

CACCM71A

ATCCGTGCAC

AGCAGCCAA

GTGAAGG

AWAGCACCC

CGCTACGCCA

CAGGGCAACr ACCGTGGGCr GCCCACG=t

GAGGI=CCT

rCACCAGCA

TCCGCAGCCC

GCGGCGAGTA

CCGAGTICAC

TGCACAGCr

TAACATCGG

GCACCAGCAG

ACGAGATCC

GCCACGTG

CrT IrCAG

TCACCCAGAT

GCCCCGG=~

TGYCGCG'IGAA

GCACCACCAA

WCAGCGCCAC

TACCACCC

TCAACAGCGG

CCAC'PAG

CTACTGGAGC

crrlccccac

GGGCCAGGGA

CATCAACAAC

CAACCTGCCC

CCC7VAGAAC

CATCCGC

CCIGGT~r-AC

CCCCCTGACC

CACCGGCGGC

CATCACCGCC

CC'IGC~Arr

CATAGCAGC

Crrcm~crrc

CAACGAGT

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1961 ICGAGGCCGA GTACOG.CCIT GCAACCAGAT CGCCIGAAG GAGAGGOCIC AGAAGGCCGT GAACGAGCTG ACCGACG'IGA CCGACTACCA CA'fCGA

T

ICAG GTGTAGGAGC T WO 97/46105 PCT/EP97/02737 -159-

DEPOSITS

with the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A.

Strain designation Bacillus cereus AB78 Escherichia coli pCIB4431 Deposition Number NRRL B-21058 NRRL B-18998 Deposition Date March 18, 1998 September 21, 1992

Claims (4)

160- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method for protecting plants including progeny thereof against damage caused by Ostrinia fumacalis species comprising directly or indirectly applying to the plant or the plant seed or the growing area of the plant as an active ingredient a toxin protein of Bacillus species, wherein the toxin protein is a VIP-type protein. 2. The method according to claim 1 wherein said VIP-type protein is a VIP1- type protein, a VIP2-type protein, a VIP3-type protein or a combination of a VIP1- type protein and a VIP2-type protein or a VIP3-type protein. 3. The method according to claim 2 wherein said VIP-type protein/s is/are selected from VIP1A(a) protein, VIP1A(b) protein, VIP2A(a) protein, VIP2A(b) protein, VIP3A(a) protein and VIP3A(b) protein. S 4. A method according to claim 1, wherein the toxin protein is a VIP-type protein according to SEQ ID Nos. 1, 2, 4-7, 17-24, 26-32, 35, 36, 39, 40, 42, 43, 45, 46, 49, 50, 51 or 52. 20 5. A method according to any one of claims 1 to 4, wherein at least one Cry- type toxin protein is directly or indirectly applied to the plant or the plant seed or the growing area of the plant to be protected in combination with a VIP-type toxin protein in an amount sufficient to provide control against Asian Corn Borer (Ostrinia fumacalis) pests. 6. The method according to claim 5, wherein the Cry-type toxin protein is a Cryl-type protein. 7. The method according to claim 6 wherein the Cryl-type protein is a CrylA- type protein. P:\OPER\MKR\SPECI30296-97 235.doc-2M2/08a 161 8. The method according to claim 7 wherein the CrylA-type protein is a CrylA(b) protein. 9. The method according to any one of claims 1 to 8, wherein the toxin protein is applied to the plant in the form of an entomocidal composition. The method according to claim 9, wherein the entomocidal composition comprises a microorganism. 11. The method according to claim 10, wherein the entomocidal composition comprises at least one VIP-type toxin protein or a microorganism containing at least one gene encoding said toxin protein together with a suitable carrier. **oo 12. The method according to claim 11, wherein the microorganism is a Bacillus thuringiensis and/or a Bacillus cereus strain. 13. A method according to either claim 11 or claim 12, wherein the microorganism is a naturally-occurring organism containing at least one VIP-type S: protein gene encoding the said toxin protein. 14. The method according to claim 13 wherein said VIP-type protein is a VIP1- type protein, a VIP2- type protein, a VIP3-type protein or a combination of a VIP1- type protein and a VIP2-type protein or a VIP3-type protein. 15. The method according to claim 14 wherein said VIP-type protein/s is/are selected from VIP1A(a) protein, VIP1A(b) protein, VIP2A(a) protein, VIP2A(b) protein, VIP3A(a) protein and VIP3A(b) protein. 16. A method according to any one of claims 11 to 15, wherein the toxin protein is a VIP-type protein according to SEQ ID Nos. 1, 2, 4-7, 17-24, 26-32, 35, 36, 39, 1, 0, 42, 43, 45, 46, 49, 50 51 or 52. P:\OPER\MKR\SPEC30296-97 235.doc-22/AM80 -162- 17. The method according to any one of claims 11 to 16, wherein the entomocidal composition comprises at least one Cry-type toxin protein or a microorganism containing at least one gene encoding said toxin protein in combination with a VIP-type toxin protein together with a suitable carrier. 18. The method according to claim 17, wherein the microorganism is a Bacillus thuringiensis and/or a Bacillus cereus strain. 10 19. The method according to either claim 17 or claim 18, wherein the microorganism is a naturally-occurring organism containing at least one cry-type toxin gene. 20. The method according to claim 19, wherein the cry-type toxin gene is a cryl- type toxin gene. 21. The method according to claim 20 wherein the cryl-type toxin gene is a crylA-type toxin gene. 20 22. The method according to claim 21 wherein the crylA-type toxin gene is a crylA(b) toxin gene. 23. A method according to any one of claims 17 to 22, wherein the Cry-type protein is a toxin protein according to SEQ ID Nos. 53 to 24. The method according to any one of claims 10 to 23, wherein the microorganism is a recombinant organism. A method for protecting plants including progeny thereof against damage caused by Ostrinia fumacalis species comprising directly or indirectly applying to ,t the plant or the plant seed or the growing area of the plant as an active ingredient P:\OPER\MKR\SPECI\30296-97 235.doc-22080

163- a toxin protein of Bacillus species, wherein the toxin protein is selected from the group consisting of a Cryl-type protein according to SEQ ID Nos. 53 to 26. The method according to any one of claims 1 to 25, wherein the toxin protein is indirectly applied to the plant, by transforming said plant with a toxin gene encoding a toxin protein Bacillus species and expressing said toxin protein in an amount sufficient to provide control against Ostrinia fumacalis (Asian Corn Borer) species upon planting the so transformed plant in an area where said insect pest may occur. 27. A method according to claim 26, wherein the toxin gene encodes a toxin :protein of Bacillus thuringiensis or Bacillus cereus. 28. The method according to either of claims 26 or 27, wherein the toxin gene 0 is a synthetic gene the codon usage of which is optimized by using the codons which are most preferred in plants. 29. The method according to any one of claims 1 to 28, wherein the plant to be protected is a cereal plant. S :30. The method according to claim 29, wherein the plant to be protected is a maize plant. 31. The method according to any one of claims 5 to 20, wherein the active ingredient is a CrylA(b) protein. 32. Use of an entomocidal composition as defined in any one of claims 9 to 24 for controlling Ostrinia fumacalis (Asian Corn Borer) pests in crop plants. 0 33. Use of a transgenic plant as defined any one of claims 26 to 30 for controlling Ostrinia fumacalis (Asian Corn Borer) pests in crop plants. P:'OPERMKR\SPECI\30296-97 235.doc-14/11 /0

164- 34. Use of recombinant microorganisms or transgenic plants according to any one of claims 24 or 26 to 30 comprising a DNA molecule which hybridizes to a cry- type or a VIP-type gene encoding the respective toxin protein under moderate stringent conditions for controlling Ostrinia furnacalis (Asian Corn Borer) pests in crop plants. 35. A seed of a transgenic plant comprising at least a toxin gene encoding a toxin protein as defined previously in any one of claims 1 to 30, and expressing the said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species, when used for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants. 36. A commercial bag comprising seed of a transgenic plant or a microorganism comprising at lest a toxin gene encoding a toxin protein as defined previously in any one of claims 1 to 30 and expressing the said toxin protein in an 00 *amount sufficient to provide control against Ostrinia furacalis (Asian Corn Borer) species, when used for control of Ostrinia furnacalis (Asian Corn Borer) pests in crop plants. 0*0 0 37. The commercial bag according to claim 36, wherein the microorganism is a Bacillus thuringiensis and/or a Bacillus cereus strain. i 38. An agricultural method for control against Ostinia furnacalis (Asian Corn Borer) comprising growing a transgenic plant or the progeny thereof wherein said plant or progeny thereof comprises a toxin gene encoding a VIP-type toxin protein of a Bacillus species and expressing the said toxin protein in an amount sufficient to provide control against Ostrinia furnacalis (Asian Corn Borer) species in relation to the plant or transgenic seed thereof, as defined in any one of claims 26 to 38. A composition comprising as an active ingredient at least a Cry-type toxin P:\OPER\MKR\SPEC\30296-97 235 doc-14/1 1/00

165- protein and a VIP-type protein in an insecticidally effective amount together with an agronomically acceptable carrier when used to provide control against Ostrinia furnacalis (Asian Corn Borer). 39. Methods of protecting plants including progeny thereof against damage caused by Ostrinia furnacalis species according to claim 1, substantially as hereinbefore described with reference to the examples. DATED this 14 t h day of November, 2000 S 10 NOVARTIS AG by its Patent Attorneys S DAVIES COLLISON CAVE 002 0 0 0*